Clinical Hematology and Fundamentals of Hemostasis

Description

Efnisyfirlit

Front Matter
Dedication
Preface
Contributors
Reviewers
PART 1 INTRODUCTION TO CLINICAL HEMATOLOGY
Chapter 1 Morphology of Human Blood and Marrow Cells: Hematopoiesis
OBJECTIVES
Basic Morphology and Basic Concepts
Table 1–1 History of Hematology
Figure 1-1 Centrifuged whole blood depicting plasma, buffy coat, and RBC layers.
Morphology of Cells on the Normal Blood Smear
Erythrocytes (Red Blood Cells)
Figure 1-2 Cell types found in smears of peripheral blood from normal individuals. A. Red blood cells (RBCs). B. Large lymphocyte. C. Segmented neutrophil. D. Eosinophil. E. Segmented neutrophil. F. Monocyte. G. Platelets. H. Small lymphocyte. I. Neutrophilic band. J. Basophil.
Platelets (Thrombocytes)
Figure 1-3 Normal peripheral blood smear; normal erythrocytes and platelets.
Leukocytes (White Blood Cells)
SEGMENTED NEUTROPHILS (FILAMENTED NEUTROPHILS, POLYMORPHONUCLEAR NEUTROPHILS)
Table 1–2 Peripheral Blood Cells: Normal Adult Values
BAND NEUTROPHILS (NONSEGMENTED NEUTROPHILS, NONFILAMENTED NEUTROPHILS)
Figure 1-4 Two segmented neutrophils.
Figure 1-5 Neutrophilic band.
EOSINOPHILS
BASOPHILS
Figure 1-6 Eosinophil (segmented).
LYMPHOCYTES
Figure 1-7 Basophil.
Figure 1-8 Lymphocytes. A. Small mature lymphocyte. B. Lymphocyte of intermediate size. C. Lymphocyte with indented nucleus. D. Lymphocyte of intermediate size. E. Lymphocyte with pointed cytoplasmic projections (frayed cytoplasm); typical nucleus. F. Spindle-shaped and pointed cytoplasmic projections. G. Large lymphocyte with indented nucleus and pointed cytoplasmic projections. H. Large lymphocyte. I. Large lymphocyte with purplish-red (azurophilic) granules. J. Large lymphocyte with irregular cytoplasmic contours. K. Large lymphocyte with purplish-red (azurophilic) granules and with indentations caused by pressure of erythrocytes. L. Large lymphocyte with purplish-red (azurophilic) granules.
MONOCYTES
Figure 1-9 A. Segmented neutrophil. B. Lymphocyte with azurophilic granules.
Figure 1-10 Monocytes. A. Monocyte with “ground-glass” appearance, evenly distributed fine granules, occasional azurophilic granules, and vacuoles in cytoplasm. B. Monocyte with opaque cytoplasm and granules and with lobulation of nucleus and linear chromatin. C. Monocyte with prominent granules and deeply indented nucleus. D. Monocyte without nuclear indentations. E. Monocyte with gray-blue color, band type of nucleus linear chromatin, blunt pseudopods, and granules. F. Monocyte with gray-blue color, irregular shape, and multilobulated nucleus. G. Monocyte with segmented nucleus. H. Monocyte with multiple blunt nongranular pseudopods, nuclear indentations, and folds. I. Monocyte with vacuoles and with nongranular ectoplasm and granular endoplasm.
Figure 1-11 Monocytes.
LARGE LYMPHOCYTES VERSUS MONOCYTES
Figure 1-12 A. Lymphocytes with azure granules. B. Monocyte.
Figure 1-13 Two monocytes.
Figure 1-14 A. Lymphocyte. B. Monocyte.
Hematopoiesis
Definition
Ontogeny (Origin) of Hematopoiesis
Table 1–3 Morphological Comparison of Large Lymphocytes and Monocytes
Table 1–4 Hematopoietic Cell Function
Figure 1-15 Diagram of hematopoietic cell differentiation. Hematopoietic stem cells can duplicate themselves during cell division (self-replicate), as indicated by the curved arrow. Most descendants of the stem cells are committed to differentiate. This commitment process occurs through a series of steps or stages, each of which leads to further restriction of lineage choice, until finally the descendant cells are limited to a single lineage. After lineage commitment, the progenitor cells continue to differentiate and mature into the terminally differentiated cells found in the blood. The diagram shows only the steps of commitment and does not depict the proliferation of cells that occurs throughout the process. The amplification of cell numbers accompanying differentiation is very large.
Figure 1-16 Location of active marrow growth in the fetus and adult. During fetal development, hematopoiesis is first established in the yolk sac mesenchyme, later moves to the liver and spleen, and finally is limited to the body skeleton. From infancy to adulthood, there is a progressive restriction of productive marrow to the axial skeleton and proximal ends of the long bones, shown as the shaded areas on the drawing of the skeleton.
Figure 1-17 Hematopoiesis.
Erythropoiesis
Pronormoblast (Rubriblast, Proerythroblast)
Figure 1-18 Erythrocytic system. Erythropoiesis.
Basophilic Normoblast (Prorubricyte, Basophilic Erythroblast)
Figure 1-19 A. Two pronormoblasts (note the perinuclear halo). B. Two polychromatophilic normoblasts. C. Neutrophilic band. D. Segmented neutrophil. E. Smudge cell.
Polychromatophilic Normoblast (Rubricyte, Polychromatophilic Erythroblast)
Figure 1-20 A. Pronormoblast. B. Orthochromatic normoblast. C. Two polychromatophilic normoblasts.
Figure 1-21 Center: pronormoblasts; upper center: plasmacyte.
Figure 1-22 Center: pronormoblasts; lower center: lymphocyte.
Figure 1-23 A. Pronormoblasts. B. Neutrophilic myelocyte. C. Neutrophilic metamyelocyte. D. Segmented neutrophil.
Orthochromatic Normoblast (Metarubricyte, Orthochromatic Erythroblast)
Figure 1-24 Basophilic normoblasts.
Table 1–5 Bone Marrow Cells: Normal Adult Values
Reticulocyte (Diffusely Basophilic Erythrocyte, Polychromatophilic Erythrocyte)
Figure 1-25 A. Basophilic normoblast. B. Three polychromatophilic normoblasts. C. Orthochromatic normoblast.
Figure 1-26 Left: Basophilic normoblast; center: plasmacyte.
Figure 1-27 Center: Basophilic normoblast; right: Orthochromatic normoblast.
Figure 1-28 Polychromatophilic normoblasts: early and late stages.
Erythrocyte (Red Blood Cell, Discocyte)
Myelopoiesis (Granulocytopoiesis)
Figure 1-29 A. Polychromatophilic normoblasts. B. Lymphocyte. C. Segmented neutrophil.
Table 1–6 Morphological Characteristics of the Erythrocytic Series
Figure 1-30 Reticulocytes. New methylene blue stain of peripheral blood. Note reticulocytes with varying amounts of stained reticulum (RNA). Reticulocytosis is associated with increased erythropoietic activity reflected by polychromasia on the Wright’s stain of the peripheral blood.
Figure 1-31 Granulocytopoiesis: myelocytic (granulocytic) system. A. Myeloblast. B. Promyelocyte (progranulocyte). C. Basophilic myelocyte. D. Basophilic metamyelocyte. E. Basophilic band. F. Segmented basophil. G. Neutrophilic myelocyte. H. Neutrophilic metamyelocyte. I. Neutrophilic band. J. Segmented neutrophil. K. Eosinophilic myelocyte. L. Eosinophilic metamyelocyte. M. Eosinophilic band. N. Segmented eosinophil.
Morphological Changes
Table 1–7 Morphological Characteristics of the Granulocytic (Neutrophilic) Series
Stages of Differentiation and Maturation
NEUTROPHILS (GRANULOCYTES)
MYELOBLASTS
PROMYELOCYTE (PROGRANULOCYTE)
Figure 1-32 Center: myeloblast: right: segmented neutrophil; left: disintegrated neutrophil.
Figure 1-33 Promyelocyte.
NEUTROPHILIC MYELOCYTES
Figure 1-34 Center: promyelocyte.
Figure 1-35 A. Neutrophilic myelocyte. B. Neutrophilic metamyelocyte. C. Plasmacyte. D. Orthochromatic normoblasts. E. Segmented neutrophils. F. Nucleus of a degenerated cell.
NEUTROPHILIC METAMYELOCYTES
Figure 1-36 Terminology based on indentation of nuclei: (left to right) myelocyte, metamyelocyte, band, segmented.
Figure 1-37 A. Two neutrophilic metamyelocytes. B. Three neutrophilic bands. C. Two segmented neutrophils.
BAND NEUTROPHILS
SEGMENTED NEUTROPHILS
TISSUE NEUTROPHILS
Figure 1-38 Tissue neutrophil (large center cell).
Figure 1-39 Center: eosinophilic myelocyte.
EOSINOPHILS
Figure 1-40 A. Eosinophilic metamyelocyte. B. Neutrophilic band. C. Polychromatophilic normoblast.
Figure 1-41 A. Eosinophilic band. B. Neutrophilic band. C. Lymphocyte. D. NRBC (orthochromatic normoblast). E. Segmented neutrophil.
Figure 1-42 A. Segmented eosinophil. B. Lymphocyte. C. Neutrophilic band. D. Neutrophilic metamyelocyte. E. Plasmacyte. F. Two diffusely basophilic red cells.
Figure 1-43 Arrow: tissue eosinophil; A. binucleated orthochromatic normoblast. B. Orthochromatic normoblast.
BASOPHILS
Figure 1-44 Tissue basophil (arrow).
Table 1–8 Morphological Characterization of the Granulocytic (Eosinophilic) Series
Table 1–9 Morphological Characteristics of the Granulocytic (Basophilic) Series
Monopoiesis
Monoblasts and Promonocytes
Monocytes and Macrophages
Figure 1-45 Lymphocytic, monocytic, and plasmacytic systems. A. Lymphoblast. B. Monoblast. C. Plasmablast. D. Prolymphocyte. E. Promonocyte. F. Proplasmacyte. G. Lymphocyte with clumped chromatin. H. Monocyte. I. Plasmacyte.
Lymphopoiesis
Lymphoblasts and Prolymphocytes
Lymphocytes
Plasmablasts and Proplasmacytes
Plasmacytes (Plasma Cells)
Figure 1-46 Center: plasmacyte; upper right: segmented neutrophil; lower left: resting monocyte.
Table 1–10 Morphological Characteristics of the Monocytic Series
Table 1–11 Morphological Characteristics of the Lymphocytic Series
Figure 1-47 Plasmacyte.
Megakaryocytopoiesis
Table 1–12 Morphological Characteristics of the Plasmacytic Series
Figure 1-48 Megakaryocytic system. A. Megakaryoblast with single oval nucleus, nucleoli, and bluish foamy marginal cytoplasmic structures. B. Promegakaryocyte with two nuclei, granular blue cytoplasm, and marginal bubbly cytoplasmic structures. C. Megakaryocyte with granular cytoplasm and without discrete thrombocytes (platelets). D. Megakaryocyte with multiple nuclei and with thrombocytes (platelets). E. Megakaryocyte nucleus with attached thrombocytes. F. Thrombocytes (platelets).
Figure 1-49 Center: early megakaryocyte; top left: segmented neutrophil; bottom center: neutrophilic metamyelocyte.
Figure 1-50 Center: early megakaryocyte.
Figure 1-51 Megakaryocytes without platelets.
Bone-Derived Cells
Figure 1-52 Megakaryocytes tend to be in small groups with multilobulated single nuclei. Mature megakaryocytes have numerous fine cytoplasmic granules, and occasionally platelet units can be seen at their periphery. (magnification ×640)
Figure 1-53 Megakaryocyte with platelets.
Osteoblasts
Table 1–13 Morphological Characteristics of the Megakaryocytic Series n/a = Not applicable.
Figure 1-54 Naked nuclei, megakaryocyte.
Figure 1-55 Three osteoblasts.
Osteoclasts
Figure 1-56 Group of osteoblasts (center) aspirated from the marrow of a child. (magnification ×400)
Figure 1-57 The osteoclast is usually seen as a single giant cell with multiple and separated nuclei and basophilic granular cytoplasm (center). (magnification ×640)
Figure 1-58 Osteoclast versus a megakaryocyte.
Molecular Hematology and Advanced Concepts
Introduction to the Cell Cycle
THE GENERATIVE (G) CELL CYCLE KINETICS
Specific Cell Line Ontogeny
MULTIPOTENTIAL STEM CELLS—COLONY-FORMING UNITS
Table 1–14 Morphological Characteristics of Osteoclasts and Megakaryocytes
Table 1–15 Morphological Characteristics of Osteoblasts and Osteoclasts
Figure 1-59 Cell cycle kinetics. Tg = one complete mitotic cycle; G0 = resting or dormant phase; G1 = postmitotic rest period; S = active DNA synthesis phases; G2 = premitotic rest period; M = mitotic period; GND = nondividing cell.
COLONY-STIMULATING FACTORS AND INTERLEUKINS
Table 1–16 Hematopoietic Progenitor Cells
Figure 1-60 CFU-GM at 14 days (×50 magnification). Colony-forming unit that makes colonies of granulocytes, monocytes, and/or macrophages under appropriate growth conditions.
Trends in Therapeutic Manipulation of Hematopoiesis
Recombinant Cytokines
Figure 1-61 BFU-E at 18 days (×75 magnification). Early burst-forming unit, an erythroid progenitor committed to making colonies of erythroid cells.
Table 1–17 Functions of Lymphocytes
Clinical Trials of Recombinant Cytokines
Figure 1-62 Regulation of hematopoiesis by cytokines. BFU-E = burst-forming unit-erythroid; CFU-Bas = colony-forming unit-basophil; CFU-E = colony-forming unit-erythroid; CFU-Eo = colony-forming unit-eosinophil; CFU-G = colony-forming unit-granulocyte; CFU-GEMM = colony-forming unit-granulocyte, erythroid, monocyte macrophage, megakaryocyte; CFU-M = colony-forming unit-monocyte; CFU-Meg = colony-forming unit-megakaryocyte; EPO = erythropoietin; G-CSF = granulocyte colony-stimulating factor; M-CSF = monocyte-colony-stimulating factor.
Table 1–18 Cellular Cytokine Production
CD Nomenclature
Table 1–19 Cytokine Characteristics
Clinical Applications of Cell Surface Markers
Table 1–20 Cytokines Involved in Hematopoietic Cytokines Involved in Hematopoietic Blood Cell Development
Questions
SUMMARY CHART
REFERENCES
Chapter 2 Bone Marrow
OBJECTIVES
Introduction
Bone Marrow Structure
Erythropoiesis
Granulopoiesis
Figure 2-1 Graphic presentation of hematopoietic tissue. The vascular compartment consists of arteriole (A) and central sinus (CV). The venous sinusoids are lined by endothelial cells (End), and their wall outside is supported by adventitial-reticulum cells (Adv). Fat tissue (F) is part of the marrow. The compartmentalization of the hematopoiesis is represented by areas of granulopoiesis (GP), areas of erythropoiesis (RCP), and erythropoietic islands (EI) with their nutrient histiocyte (Hist). The megakaryocytes protrude with small cytoplasmic projections through the vascular wall (Meg). Lymphocytes (Lym) are randomly scattered among the hemapoietic cells, whereas plasma cells (Pla) are usually situated along the vascular wall.
Figure 2-2 Erythropoietic island composed mainly of polychromatophilic normoblasts. The nutrient-histiocyte (arrow) is slightly displaced off its central position by smearing of the particle. Its cytoplasmic slender processes envelop a basophilic normoblast, establishing intimate contact with the maturing red cell precursor. (Wright–Giemsa, magnification ×600)
Figure 2-3 Compartment of granulopoiesis. A reticulum cell (arrow) with open reticulated chromatin and light blue cytoplasm containing dustlike fine granules is situated among numerous granulocytic precursors, especially myelocytes. (Wright–Giemsa, magnification ×600)
Megakaryopoiesis
Lymphopoiesis
Stem Cells
Figure 2-4 Mature megakaryocytes releasing proplatelets (packages of platelets). (Wright–Giemsa, magnification ×600)
Figure 2-5 A lymphocytic nodule (follicle) in bone marrow as shown here may alter very significantly the marrow differential count when aspirated and give a false impression of lymphocytic malignancy. (H&E, magnification ×200)
Hematogones
Figure 2-6 An arrow points toward a hematogone showing high nuclear-to-cytoplasmic ratio, homogeneous chromatin, and scant cytoplasm. It can be confused with a blast of lymphoblastic leukemia. (Wright–Giemsa, magnification ×1000)
Marrow Stromal Cells
Figure 2-7 An alkaline phosphatase-stained reticulum cell extends its slender cytoplasmic projections deep in the hemopoietic cord, maintaining an intimate contact with granulopoiesis. The background cells are stained with neutral red. (magnification ×600)
Mast Cells
Figure 2-8 Two acid phosphatase–positive macrophages in bone marrow of a patient treated with chemotherapeutic agents. Macrophages are also scavengers and cleaners of the hematopoietic tissue, so they increase in number during massive destruction of hematopoietic cells. (magnification ×600)
Figure 2-9 A string of endothelial cells aspirated from hypocellular marrow. The nuclei are elongated and slightly tapered. The cytoplasm is transparent and barely visible. (Wright–Giemsa, magnification ×600)
Bone-Forming Cells
Figure 2-10 Three mast cells, known also as tissue basophils, are shown in this marrow aspirate in a background of erythroid hyperplasia. Numerous regular round granules fill their cytoplasm and obscure the nuclear details. (Wright-Giemsa, magnification ×600)
Bone Marrow Function
Indications for Bone Marrow Studies
Table 2–1 Indications for Bone Marrow Study
Figure 2-11 Bone marrow biopsy showing a collection of Gaucher cells. (H&E, magnification ×600)
Obtaining and Preparing Bone Marrow for Hematologic Studies
Figure 2-12 Common sites from which bone marrow is obtained for studies.
Equipment
Table 2–2 Example of a Tray for Bone Marrow Aspiration and Biopsy
Aspiration
Figure 2-13 Adult-sized Jamshidi 11-gauge × 4-inch biopsy/aspiration needle showing stylet (left), biopsy needle (center), and probe (right).
Preparation of Bone Marrow Aspirate
Histologic Marrow Particle Preparation
Figure 2-14 Distribution of bone marrow sample. EDTA = ethylene diaminetetraacetic acid; M:E = myeloid-to-erythroid ratio.
Bone Marrow Core Biopsy
Figure 2-15 Bone marrow biopsy specimen showing aspiration artifact that can affect the cellularity and alter the relationship of cells to each other. (H&E, magnification ×200)
Preparation of Trephine Biopsy
TOUCH PREPARATION
HISTOLOGIC BONE MARROW BIOPSY PREPARATION
Figure 2-16 Wright-stained bone marrow touch preparation from a patient with hairy cell leukemia. Aspirate was a dry tap. A few diagnostic hairy cells are seen with abundant, fluffy, light blue cytoplasm and inconspicuous nucleoli. (magnification ×1000)
Figure 2-17 Tartrate-resistant acid phosphatase (TRAP) stain showing a strong positive reaction in neoplastic cells which is useful in the diagnosis of hairy cell leukemia. (magnification ×1000)
Figure 2-18 Bone marrow biopsy specimen from an HIV-positive patient with tuberculosis shows a well-formed granuloma. (H&E, magnification ×200)
Figure 2-19 Acid-fast stain on a bone marrow biopsy specimen from the same patient as in Figure 2–18 shows acid-fast organisms, suggesting infection with Mycobacterium. (magnification ×1000)
Figure 2-20 GMS (Gomori’s methenamine silver) stain on bone marrow biopsy specimen from an HIV-positive patient shows multiple budding yeasts consistent with histoplasmosis. (magnification ×1000)
Figure 2-21 Bone marrow biopsy specimen from a patient with metastatic carcinoma shows glandular formation, a morphological feature of adenocarcinoma. (H&E, magnification ×600)
Bone Marrow Examination
Figure 2-22 Immunohistochemical stain for prostate-specific antigen (PSA) performed on a marrow biopsy specimen from the same patient as in Figure 2–21. The specimen shows positive staining with PSA, thus confirming that the metastatic tumor is from prostate. (magnification ×600)
Figure 2-23 Aspirate smear from a patient with metastatic prostate carcinoma to the bone marrow. Note the cohesive crowded groups of large neoplastic cells (A) and glandular formation (B) suggesting adenocarcinoma. (Wright–Giemsa, magnification ×600)
Figure 2-24 H&E stained bone marrow biopsy specimen from a patient with acute lymphoblastic leukemia shows an increased number of blasts. Note that the blasts are not cohesive and are individually scattered. This is a morphological feature of hematolymphoid malignancy. Fine cellular details are lost in biopsy sections. (magnification ×600)
Figure 2-25 Wright–Giemsa-stained aspirate smear of acute lymphoblastic leukemia showing cytoplasmic and nuclear details useful in the diagnosis of acute leukemias. As compared to the metastatic tumor cells seen in Figure 2–23, the blasts are not cohesive and scattered.
Estimation of Bone Marrow Cellularity
Figure 2-26 Smear of normal cellular marrow with normal maturation of erythropoietic, granulocytic, and megakaryocytic cells. (Wright–Giemsa, magnification ×200)
Figure 2-27 Normal bone marrow biopsy specimen from a 50-year-old patient shows approximately 50% cellularity. (H&E, low magnification)
Figure 2-28 Markedly hypocellular bone marrow biopsy specimen from a 20-year-old patient. (H&E, low magnification)
Figure 2-29 Hypercellular bone marrow biopsy specimen from a 60-year-old patient with 80% cellular marrow. This patient was receiving growth factor therapy. (H&E, low magnification)
Bone Marrow Differential Count
Figure 2-30 Bone marrow biopsy specimen showing normal bony trabeculae. (H&E, low magnification)
Figure 2-31 Bone marrow biopsy specimen shows marked thickening of bony trabeculae (osteosclerosis). (H&E, magnification ×200)
Figure 2-32 Bone marrow biopsy specimen shows thinning of bony trabeculae (osteopenia). (H&E, magnification ×200)
Figure 2-33 High-power view of the bone marrow biopsy from a 60-year-old patient shows lymphoid aggregate (low-power view is seen in Fig. 2–5). Note the presence of blood vessel and plasma cells at the periphery of the lymphoid aggregate. (H&E, magnification ×400)
Figure 2-34 Bone marrow biopsy specimen shows a paratrabecular lymphoid aggregate. This pattern of infiltration is most likely indicative of involvement of the marrow by malignant lymphoma. (H&E, magnification ×200)
Figure 2-35 Reticulin stain on a marrow biopsy specimen from a patient with hairy cell leukemia (same patient as Figs. 2–16 and 2–17) showing diffuse and severe fibrosis. Dilated sinusoids are present. As expected, the aspirate was a dry tap. (magnification ×200)
Bone Marrow and Peripheral Blood Interpretation Based on Cellularity and M:E Ratio Changes
Bone Marrow Iron Stores
Table 2–3 Differential Cell Count of Bone Marrow in Percentage of Total Nucleated Cells*
Table 2–4 Marrow and Blood Interpretation Based on Cellularity and M:E Ratio
Figure 2-36 Iron stain of a bone marrow smear shows a marked increase in marrow storage iron, thus ruling out the possibility of iron-deficiency anemia. (magnification ×200)
Bone Marrow Report
Case Study 1
BONE MARROW EXAMINATION GROSS DESCRIPTION
MICROSCOPIC DESCRIPTION
DIAGNOSIS
COMMENT
Case Study 2
BONE MARROW EXAMINATION GROSS DESCRIPTION
MICROSCOPIC DESCRIPTION
DIAGNOSIS
COMMENT
Questions
SUMMARY CHART
REFERENCES
Chapter 3 The Red Blood Cell: Structure and Function
OBJECTIVES
Introduction
The Red Blood Cell Membrane
Table 3–1 Areas of Red Cell Metabolism Important in Normal RBC Survival and Function
Figure 3-1 Transmission electron microscopy (TEM) of plasma membrane.
Red Blood Cell Membrane Proteins
Figure 3-2 Schematic illustration of red blood cell membrane depicting the composition and arrangement of red cell membrane proteins. GP-A = glycophorin A; GP-B = glycophorin B; GP-C = glycophorin C. Numbers refer to pattern of migration of sodium dodecyl-polyacrylamide gel pattern stained with Coomassie brilliant blue. Relations of proteins to each other and to lipids are purely hypothetical positions of the proteins relative to the inside or outside of the lipid bilayer are accurate.
Table 3–2 Subcellular Localization of Identified RBC Membrane Proteins
Table 3–3 Red Cell Membrane Integral and Peripheral Proteins
Deformability
Table 3–4 Characteristics of the Major Components of the Red Cell Membrane
Permeability
Figure 3-3 Scanning electron micrograph (SEM) of red cells (3 to 6) squeezing through fenestrated wall in transit from splenic cords to sinus. Epithelial linings of sinus wall to which platelets (P) adhere, along with “hairy” white cells (W), probably macrophages, are shown.
Figure 3-4 Spherocytes.
Figure 3-5 Bite cells.
Red Blood Cell Membrane Lipids
PHOSPHOLIPIDS
GLYCOLIPIDS AND CHOLESTEROL
Figure 3-6 Target cells.
Figure 3-7 Acanthocytes.
Hemoglobin Structure and Function
Hemoglobin Synthesis
IRON DELIVERY AND SUPPLY
Table 3–5 Abnormalities That Can Lead to a Change in RBC Morphology
Figure 3-8 Hemoglobin A molecule comprised of two alpha, two beta, and four iron-containing heme groups. Beta chains have 146 amino acids and alpha chains have 141 amino acids.
SYNTHESIS OF PROTOPORPHYRINS
GLOBIN SYNTHESIS
Figure 3-9 Hemoglobin synthesis in the reticulocyte. δ-ALA = delta-aminolevulinic acid; URO = uroporphyrinogen; COPRO = coproporphyrinogen; PROTO = protoporphyrin.
Figure 3-10 Synthesis of heme. The heme biosynthetic pathway, showing the distribution of enzymes between the mitochondria and the cytoplasm. Condensation of glycine and succinyl coenzyme A yields δALA, which is irreversible; two molecules of ALA undergo condensation by the enzyme ALA dehydrase to yield porphobilinogen (PBG). In the presence of uroporphyrinogen III cosynthase and uroporphyrinogen I synthase, PBG yields uroporphyrinogen III. Uroporphyrinogen III undergoes four decarboxylation steps, catalyzed by the enzyme uroporphyrinogen decarboxylase, to yield coproporphyrinogen III. Coproporphyrinogen III is transported from the cytosol into the mitochondria, where the enzyme coproporphyrinogen oxidase acts on the propionic acid side chains to yield protoporphyrinogen IX. Catalyzed by protoporphyrinogen IX oxidase, protoporphyrinogen IX is oxidized to protoporphyrin IX. Protoporphyrin IX combines with ferrous iron to yield heme (catalyzed by heme synthase). (Intermediates between uroporphyrinogen and coproporphyrinogen, designated by X, remain unidentified.) B6PO4 = pyridoxal phosphate.
Figure 3-11 Genetic control and formation of human hemoglobins.
Figure 3-12 Changes in globin chain synthesis during fetal development, birth, and infancy.
Table 3–6 Composition of Hemoglobin Found in Normal Human Development
HEMOGLOBIN FUNCTION
Figure 3-13 Ringed sideroblast and siderocytes as detected by Prussian blue staining of a bone marrow aspirate. (Prussian blue stain, ×1000)
Figure 3-14 Normal hemoglobin-oxygen dissociation curve.
Figure 3-15 Hemoglobin-oxygen dissociation curve. Blue line is the normal curve. The green line represents a “shift to the left” that can occur with an increase in abnormal hemoglobins, increase in pH (alkalosis), a decrease in 2,3-DPG, and a decrease in body temperature. The red line represents a “shift to the right” that can occur with a decrease in pH (acidosis), an increase in 2,3-DPG, and an increase in body temperature.
Abnormal Hemoglobins of Clinical Importance
Maintenance of Hemoglobin Function: Active Red Blood Cell Metabolic Pathways
Table 3–7 Toxic Levels for Abnormal Hemoglobins of Clinical Importance
Table 3–8 Comparison of Red Blood Cell Metabolic Activities During Various Stages of Maturation
Figure 3-16 Red cell metabolism.
Erythrocyte Senescence and Hemolysis
Figure 3-17 Scanning electron micrograph (SEM) of a normal red cell.
Extravascular Hemolysis
Table 3–9 Changes Occurring During Aging of RBCs
Figure 3-18 Normal extravascular hemolysis.
Intravascular Hemolysis
Figure 3-19 Intravascular hemolysis.
Table 3–10 Protein Carriers
Questions
SUMMARY CHART
REFERENCES
Chapter 4 Anemia: Diagnosis and Clinical Considerations
OBJECTIVES
Definition of Anemia
Considerations by Age, Sex, and Other Factors
Table 4–1 Reference Range Values for Hemoglobin
Causes of Anemia
Table 4–2 Categories of Anemia by Cause
Significance of Anemia and Compensatory Mechanisms
Red Blood Cell and Hemoglobin Production
Clinical Diagnosis of Anemia
Classification of Anemia
Hemoglobin and Hematocrit
Red Blood Cell Indices
Red Blood Cell Indices and Other Tests
Table 4–3 Classification of Anemias by RBC Indices
Overview of the Treatment of Anemias
Figure 4-1 Decision-making flowchart (algorithm) for symptoms of anemia.
Table 4–4 Clinical Symptoms of Anemia
Other Factors Involved in Red Blood Cell Production
Tests in the Diagnosis of Anemia
Hemoglobin
Hematocrit
Red Blood Cell Indices
Peripheral Blood Smear
Reticulocyte Count
Figure 4-2 Reticulocytosis (new methylene blue stain).
Figure 4-3 Ringed sideroblast (center) and siderocytes (surrounding cells).
Figure 4-4 Heinz bodies.
Bone Marrow Smear and Biopsy
Patient Studies of Anemia
Table 4–5 Other Tests That May Be Performed in the Diagnosis of Anemias
Questions
SUMMARY CHART
REFERENCES
Chapter 5 Evaluation of Cell Morphology and Introduction to Platelet and White Blood Cell Morphology
OBJECTIVES
Introduction
Examination of the Peripheral Blood Smear
The Normal Red Blood Cell
Table 5–1 Estimation of Total WBC Count from the Peripheral Blood Smear
Assessment of Red Cell Abnormality
Figure 5-1 Blood smear.
Figure 5-2 Normal red blood cells.
Table 5–2 Grading Scale for Red Cell Morphology (Anisocytosis/Poikilocytosis)
Variations in Red Cell Distribution
Normal Distribution
Abnormal Distribution
AGGLUTINATION
Figure 5-3 Normal and abnormal red blood cell morphology.
Figure 5-4 Note the agglutination on the smear from a patient with cold hemagglutinin disease.
ROULEAUX
Figure 5-5 Peripheral blood showing marked rouleaux formation. Note the “stacked coin” appearance of the red cells.
Variations in Size
Anisocytosis
Normocytes
Figure 5-6 Note the different size (anisocytosis) and shape (poikilocytosis) of the red cells. Compare the largest (macrocytic) cell below the arrow in the center of the field with the smaller (microcytic) cells.
Macrocytes
Figure 5-7 Correlation of macrocytes to pathologic processes.
Microcytes
Hemoglobin Content—Color Variations
Normochromia
Hypochromia
Figure 5-8 Correlation of microcytes to pathologic processes.
Figure 5-9 Note the large central pallor in many of the red cells depicting hypochromia.
Table 5–3 Hypochromia Grading
Hyperchromia
Polychromasia
Figure 5-10 Note polychromasia in the cell with the arrow.
Variations in Shape
Poikilocytosis
Table 5–4 Polychromasia Grading
Target Cells (Codocytes)
Spherocytes
Figure 5-11 Note the target cell at the arrow.
Stomatocytes
Figure 5-12 Correlation of target cells to pathologic processes.
Figure 5-13 Note the spherocyte at arrow in a blood smear from a patient with hereditary spherocytosis.
Ovalocytes and Elliptocytes
Figure 5-14 Correlation of spherocytes to pathologic processes.
Figure 5-15 Stomatocytes in peripheral blood.
Figure 5-16 Note the high percentage of elliptocytes in this blood smear from a patient with hereditary elliptocytosis.
Figure 5-17 Note the oval macrocyte (microovalocyte) at the arrow. Smear from a patient with pernicious anemia.
Sickle Cells (Drepanocytes)
Figure 5-18 Correlation of ovalocytes and elliptocytes to pathologic processes.
Fragmented Cells (Schistocytes, Helmet Cells, Keratocytes)
Figure 5-19 Irreversibly sickled cells.
Figure 5-20 Reversible, oat-shaped sickle cell.
Figure 5-21 Correlation of sickle cells to pathologic processes.
Figure 5-22 Peripheral blood from a patient with renal disease. Note the presence of fragmented cells: A. burr cells; B. acanthocyte; C. blister/pocketbook cells; D. schistocyte.
Figure 5-23 Note the bite cell at the arrow.
Burr Cells (Echinocytes)
Figure 5-24 Correlation of fragmented cells to pathologic processes. HA = hemolytic anemia; DIC = disseminated intravascular coagulation; HUS = hemolytic uremic syndrome; TTP = thrombotic thrombocytopenic purpura.
Acanthocytes (Thorn Cells, Spur Cells)
Figure 5-25 Note the acanthocytes on this peripheral smear.
Teardrop Cells (Dacrocytes)
Figure 5-26 Correlation of acanthocytes to pathologic processes.
Red Cell Inclusions
Howell–Jolly Bodies
Figure 5-27 Teardrop cells (peripheral blood).
Basophilic Stippling
Figure 5-28 Howell–Jolly body.
Figure 5-29 Note the cells with red cell inclusions: basophilic stippling seen on a peripheral smear in a patient with lead poisoning.
Pappenheimer Bodies and Siderotic Granules
Figure 5-30 Pappenheimer bodies (Wright stain).
Heinz Bodies
Cabot Rings
Figure 5-31 Heinz body prep; note the appearance of Heinz body inclusions.
Figure 5-32 Note the appearance of a Cabot’s ring in the cell at the arrow.
Hemoglobin CC Crystals
Hemoglobin SC Crystals
Table 5–5 Summary of Abnormal Red Cell Morphologies and Disease States That May Be Associated with These Abnormal Morphologies
Figure 5-33 Note the hexagonal shaped crystal inclusions in a peripheral smear from a patient with HbC disease. These HbC crystals leave the remainder of the cellular cytoplasm to appear as “empty.”
Protozoan Inclusions
Figure 5-34 Note the “fingerlike projections” in this peripheral smear from a patient with HbSC disease. These HbSC crystals are said to resemble the Washington Monument.
Examination of Platelet Morphology
Figure 5-35 Comparison of babesiosis (left) and malarial forms (right).
Figure 5-36 Normal platelet at arrow.
White Blood Cell Morphology
Figure 5-37 A, Normal neutrophil, B, normal lymphocyte.
Case Study 1
DISCUSSION
QUESTIONS
ANSWERS
Figure 5-38 Case study; note abnormal red cell morphology.
Questions
SUMMARY CHART
REFERENCES
PART 2 ANEMIAS
Chapter 6 Iron Metabolism and Hypochromic Anemias
OBJECTIVES
Introduction
Normal Iron Metabolism
Distribution and Requirements
Daily Iron Requirements and Absorption
Figure 6-1 Daily iron turnover and body iron distribution.
Table 6–1 Minimum Daily Requirement (MDR) for Iron
Table 6–2 Iron-Containing Foods
Table 6–3 Substances that Increase and Decrease the Absorption of Iron
Iron Transport
Figure 6-2 Schematic of iron intake and absorption through the mucosal cells in the small intestine. Absorbed iron is converted to ferritin for storage or transported bound to transferrin for distribution to body tissues.
Table 6–4 Iron (Fe) Molecules and Compounds
Table 6–5 Factors Affecting Iron Absorption
Iron Storage
Table 6–6 Proteins Involved in Iron Metabolism
Table 6–7 Iron Status: Normal Values*
Laboratory Evaluation of Iron Status
Serum Iron
Total Iron-Binding Capacity
Transferrin Saturation
Ferritin
Transferrin Receptor
Free Erythrocyte Protoporphyrin and Zinc Protoporphyrin
Iron-Deficiency Anemia
Etiology
Table 6–8 High-Risk Groups
DIET AND INCREASED NEED
BLOOD LOSS
Table 6–9 Causes of Iron-Deficiency Anemia
MALABSORPTION
Pathophysiology
STAGE 1: IRON DEPLETION
Table 6–10 Stages of Iron-Deficiency Anemia
STAGE 2: IRON-DEFICIENT ERYTHROPOIESIS
Figure 6-3 Bone marrow aspirate from a patient with iron-deficiency anemia stained with Prussian blue. Note the negative staining indicated by a lack of any blue stain indicating an absence of iron.
STAGE 3: IRON-DEFICIENCY ANEMIA
Clinical Features
Figure 6-4 Iron-deficiency anemia characterized by microcytic, hypochromic red blood cells.
Laboratory Findings
Figure 6-5 Bone marrow aspirate in iron-deficiency anemia showing ineffective erythropoiesis, “ragged” erythroid precursors.
Figure 6-6 Koilonychias or spooning of the nails, characteristic of iron-deficiency anemia.
PERIPHERAL BLOOD
Figure 6-7 Clinical manifestations of iron-deficiency anemia. A. Cheilitis, before (top) and after (bottom) therapy. B. Glossitis before (top) and after (bottom) therapy.
Table 6–11 Indices and Features of Iron-Deficiency Anemia
IRON STUDIES
BONE MARROW
Treatment
Anemia of Chronic Disease
Pathophysiology
Table 6–12 Disorders Associated with Anemia of Chronic Disease (ACD)
Figure 6-8 Peripheral blood of a patient with iron-deficiency anemia after therapy. Note the two populations of red blood cells: microcytic hypochromic and normal cells.
Clinical Features
Laboratory Findings
PERIPHERAL BLOOD
IRON STUDIES
Table 6–13 Indices and Features of Anemia of Chronic Disease
Figure 6-9 Normochromic, normocytic red blood cells in a patient with anemia of chronic disease. (Wright’s stain, ×200)
BONE MARROW
Treatment
Sideroblastic Anemia
Figure 6-10 Increased reticuloendothelial iron in a patient with anemia of chronic disease. (Prussian blue stain, ×200)
Table 6–14 Common Causes of Sideroblastic Anemia
Pathophysiology
Figure 6-11 Ringed sideroblast as detected by Prussian blue staining of a bone marrow aspirate. (Prussian blue stain, ×1000)
Laboratory Findings
PERIPHERAL BLOOD
IRON STUDIES
Figure 6-12 Prominent basophilic stippling is found with defective heme synthesis.
Table 6–15 Clinical and Morphological Features of Refractory Anemia with Ringed Sideroblasts (RARS)
Figure 6-13 Dimorphic population of red blood cells with a striking hypochromic component in a patient with sideroblastic anemia. Moderate anisopoikilocytosis is also present. (Wright’s stain, ×200)
BONE MARROW
Treatment
Table 6–16 Differential Diagnosis Comparing RBC Indices and Peripheral Blood and Bone Marrow Features of IDA, ACD, and Sideroblastic Anemia
The Porphyrias
Iron Overload and Hemochromatosis
Figure 6-14 Erythropoietic porphyria. Note the precipitated porphyrins in the cytoplasm.
Table 6–17 Classification and Characteristics of the Porphyrias
Hereditary Hemochromatosis
Pathophysiology
Table 6–18 Classification of Hemochromatosis
Figure 6-15 Liver biopsy from a patient with idiopathic hemochromatosis and cirrhosis. Note the excess deposits of iron. (Ferric ferricyanide stain)
Clinical Features
Laboratory Findings
IRON STUDIES
Treatment
Table 6–19 Summary and Comparison of Iron Studies in Microcytic–Hypochromic Anemia’s and Hemochromatosis
Secondary Hemochromatosis
African Iron Overload
Case Study 1
QUESTIONS
ANSWERS
Case Study 2
QUESTIONS
ANSWERS
Case Study 3
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 7 Megaloblastic Anemias
OBJECTIVES
Introduction
Biochemical Aspects
Clinical Manifestations of Megaloblastic Anemia
Hematologic Features
Ineffective Hematopoiesis
Bone Marrow Morphology
Figure 7-1 Thymidine synthesis pathway from uridine nucleotide. Uracil is incorporated into DNA in the absence of thymine. UDP = uridine diphosphate; dUDP = deoxyuridine diphosphate; dUTP = deoxyuridine triphosphate; dUMP = deoxyuridine monophosphate; dTMP = deoxythymidine monophosphate; dTDP = deoxythymidine diphosphate; dTTP = deoxythymidine triphosphate: CH2 THF = methylene tetrahydrofolate.
Figure 7-2 A. Mitotic figures in megaloblastic marrow. B. Large megaloblastic band neutrophil. C. Megaloblastic pronormoblast with open, sievelike chromatin. The myeloid-to-erythroid (M:E) ratio is decreased because of the increase in megaloblastic erythroid precursors labeled A and C.
Peripheral Blood Morphology
Figure 7-3 Bone marrow. A. Polychromatophilic megaloblasts. B. Orthochromic megaloblast with multiple Howell–Jolly bodies.
Figure 7-4 Extreme degree of anisocytosis (+4) and poikilocytosis (+4) with oval macrocytosis (arrow) in a patient with severe pernicious anemia.
Figure 7-5 Howell–Jolly body in an RBC in pernicious anemia (arrow).
Figure 7-6 Cabot ring in pernicious anemia (arrow).
Figure 7-7 Neutrophil hypersegmentation in pernicious anemia.
Etiology of Megaloblastic Anemia
Table 7–1 Clinical Features of Megaloblastic Anemia
Vitamin B12 Deficiency
SOURCES AND REQUIREMENTS
STRUCTURE
Figure 7-8 Structure of vitamin B12 (cobalamin). When R is adenosyl, the compound is adenosylcobalamin (AdoCb); when R is methyl, it is methylcobalamin (MeCb); when R is cyanide, it is cyanocobalamin (CNCb); and when R is hydroxy, it is hydroxocobalamin (OHCb).
TRANSPORT AND METABOLISM
Figure 7-9 Transportation path of vitamin B12 from the diet to the tissues. IF = intrinsic factor; TC II = transcobalamin II.
Figure 7-10 The role of vitamin B12 and folate in DNA synthesis. CH2THF = methylene tetrahydrofolate; THF = tetrahydrofolate; DHF = dihydrofolate; CH3THF = methyl tetrahydrofolate; dUMP = deoxyuridine monophosphate; dTMP = deoxythymidine monophosphate.
CAUSES OF VITAMIN B12 DEFICIENCY
Table 7–2 Causes of Vitamin B12 Deficiency
DIETARY VITAMIN B12 DEFICIENCY
PERNICIOUS ANEMIA
DEFINITION
Figure 7-11 Conversion of methylmalonyl CoA to succinyl CoA. AdoCo = adenosylcobalamin.
PATHOPHYSIOLOGY
CLINICAL MANIFESTATIONS OF VITAMIN B12 DEFICIENCY
NEUROLOGIC MANIFESTATIONS
Table 7–3 Neurologic Manifestations in Pernicious Anemia
OTHER CAUSES OF VITAMIN B12 DEFICIENCY
GASTRECTOMY
BLIND LOOP SYNDROME
FISH TAPEWORM
DISEASES OF ILEUM
CHRONIC PANCREATIC DISEASE
OTHER DISORDERS
DRUG-INDUCED VITAMIN DEFICIENCY
Folic Acid Deficiency
SOURCES AND REQUIREMENTS
STRUCTURE
Figure 7-12 Structure of folic acid and its derivative. A. Folic acid (pteroylglutamic acid). The three components are defined by vertical lines. B. Tetrapteroyltriglutamic acid (tetrahydrofolate triglutamate), the active form of folate present in the tissues.
ABSORPTION AND METABOLISM
CAUSES OF FOLIC ACID DEFICIENCY
DIETARY DEFICIENCY
MALABSORPTION
Figure 7-13 Absorption and metabolism of folic acid. CH3THF = methyl tetrahydrofolate; Glu = glutamic acid; THF = tetrahydrofolate.
DRUG-INDUCED FOLATE DEFICIENCY
Figure 7-14 Intestinal absorption of the folate derivatives of food.
CLINICAL MANIFESTATIONS OF FOLIC ACID DEFICIENCY
Table 7–4 Causes of Folic Acid Deficiency
Laboratory Diagnosis of Megaloblastic Anemia
Laboratory Tests for the Diagnosis of Vitamin B12 and Folic Acid Deficiencies
Table 7–5 Laboratory Evaluation for the Diagnosis of Macrocytic Anemia
SERUM B12 (SERUM COBALAMIN) LEVEL
SERUM AND RED CELL FOLATE
Other Laboratory Tests
GASTRIC AUTOANTIBODIES
BLOOD TEST FOR GASTRIC ATROPHY
SERUM COBALAMIN BINDING PROTEINS
SCHILLING TEST
Figure 7-15 The two-part Schilling test. IF = intrinsic factor.
SERUM AND URINE METHYLMALONIC ACID
SERUM HOMOCYSTEINE
DEOXYURIDINE SUPPRESSION TEST
Treatment
Vitamin B12 Deficiency
Folic Acid Deficiency
Response to Therapy
Vitamin-Independent Megaloblastic Changes
Inherited
Table 7–6 Vitamin-Independent Megaloblastic Anemias
Acquired
Drug and Toxin Induced
Macrocytic Nonmegaloblastic Anemias
Causes of Macrocytic Nonmegaloblastic Anemias
Table 7–7 Causes of Macrocytic Nonmegaloblastic Anemias
Case Study
QUESTIONS
COMMENTS
Questions
SUMMARY CHART
REFERENCES
Chapter 8 Aplastic Anemia Including Pure Red Cell Aplasia, Congenital Dyserythropoietic Anemia, and Paroxysmal Nocturnal Hemoglobinuria
OBJECTIVES
Definition
Pathogenesis
Table 8–1 Postulated Pathogenic Mechanisms in Development of Aplastic Anemia
Etiology
Figure 8-1 Schematic representation of possible defects in hematopoiesis that may give rise to aplastic anemia. It is postulated that decreased numbers of bone marrow stem cells and/or changes in the bone marrow microenvironment that alter cytokine levels, or both, may cause aplasia to develop. Most evidence points to decreased stem cells caused by the lack of self-replication (1) or direct destruction of stem cells (2), rather than changes in the bone marrow microenvironment (3), as pathogenetic mechanisms for development of aplastic anemia.
Table 8–2 Etiologies Associated with Development of Aplastic Anemia
Acquired Aplastic Anemia
Idiopathic or Primary
Secondary Causes
CHEMICAL AGENTS
Table 8–3 Causes of Secondary Acquired Aplastic Anemia
DRUGS
Table 8–4 Agents Associated with Aplastic Anemia
Figure 8-2 Vacuolization of bone marrow hematopoietic precursor cells indicating toxicity in a patient being treated with chloramphenicol. (Wright–Giemsa stain, ×1000 magnification)
IONIZING RADIATION
INFECTIONS
MISCELLANEOUS CAUSES
Congenital Aplastic Anemia
Fanconi’s Anemia
Clinical Manifestations of Aplastic Anemia
Laboratory Evaluation
Table 8–5 Laboratory Evaluation for Aplastic Anemia
Table 8–6 Characteristic Abnormal CBC Values Seen in Severe Aplastic Anemia
Table 8–7 Differential Diagnosis for Pancytopenia
Figure 8-3 Hypocellular bone marrow aspirate containing primarily lymphocytes and plasma cells, reflecting bone marrow aplasia. (Wright–Giemsa stain, ×500 magnification)
Figure 8-4 A. Normocellular bone marrow. B. Markedly hypocellular bone marrow biopsy specimen from a patient with aplastic anemia. (H & E stain, ×500 magnification)
Figure 8-5 Residual lymphocytes, plasma cells, and bone marrow stroma with marked decrease in hematopoietic cells in a bone marrow biopsy specimen from a patient with aplastic anemia. (H & E stain, ×200 magnification)
Figure 8-6 Lymphoid aggregate seen in a bone marrow biopsy specimen from a patient with aplastic anemia. (H & E stain, ×100 magnification)
Figure 8-7 Focal area of bone marrow hyperplasia adjacent to a hypoplastic area in early aplastic anemia. (H & E stain, ×100 magnification)
Treatment, Clinical Course, and Prognosis
Related Disorders
Pure Red Cell Aplasia
Table 8–8 Causes of Pure Red Cell Aplasia
Figure 8-8 Erythroid precursors containing parvovirus B19 viral inclusions. (Wright–Giemsa stain, ×500 magnification)
Congenital Dyserythropoietic Anemias
Paroxysmal Nocturnal Hemoglobinuria
Table 8–9 Common Types of Congenital Dyserythropoietic Anemias
Figure 8-9 Multinucleated erythroid precursors in type 2 congenital dyserythropoietic anemia (HEMPAS). (Wright–Giemsa stain, ×1000 magnification)
Table 8–10 Characteristics of Less Common Congenital Dyserythropoietic Anemias
Figure 8-10 Binucleated erythroid precursor with chromatin bridge seen in a patient with type I congenital dyserythropoietic anemia. (Wright–Giemsa stain × 1000 magnification)
Table 8–11 Hematopoietic Cell Surface Proteins Decreased or Absent in Paroxysmal Nocturnal Hemoglobinuria Patients
Figure 8-11 A. The normal cell is protected from C3b binding and complement-mediated hemolysis because the PIG-A anchored proteins CD59 and CD55 are present and thus complement fixation cannot occur. B. The PNH cell has decreased CD55 and CD59 protein on the cell surface due to lack of GPI anchoring proteins, allowing for C3b binding and complement fixation that leads to red cell lysis.
Table 8–12 Types of Cells Observed in Paroxysmal Nocturnal Hemoglobinuria
Laboratory Evaluation of PNH
Figure 8-12 Peripheral blood smear from a patient with PNH (Wright–Giemsa stain, ×600 magnification) demonstrating normochromic, normocytic red cells, as well as occasional hypochromic, microcytic, and polychromatophilic cells. A nucleated red cell is also seen.
Figure 8-13 Bone marrow aspirate smear from a patient with paroxysmal nocturnal hemoglobinuria demonstrating erythroid hyperplasia. (Wright–Giemsa stain, ×500 magnification)
Figure 8-14 Sugar water test. The tube on the left represents the control (C) and the tube on the right represents the patient (P) with a positive sugar water test demonstrating 10% to 80% hemolysis associated with PNH.
Figure 8-15 Ham’s test. Positive results occur in patients with PNH. A positive test is reported when hemolysis occurs in tube 1, containing fresh normal serum and patient cells; tube 2, containing acidified normal serum and patient cells; and tube 3, containing acidified patient serum and patient cells.
Table 8–13 Acidified Serum Lysis Test
Figure 8-16 Flow cytometry diagnosis of PNH. The flow cytometric histograms from a patient with PNH (A, B) show decreased expression of CD55 (A, arrow) and CD59 (B, arrow) in addition to populations of red cells containing relatively normal levels of CD55 and CD59. For comparison, a normal control showing single red cell populations for both CD55 (C) and CD59 (D).
Case Study 1
QUESTIONS
ANSWERS
Case Study 2
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 9 Introduction to Hemolytic Anemias: Intracorpuscular Defects: I. Hereditary Defects of the Red Cell Membrane
OBJECTIVES
Classification of Hemolytic Anemias
Approach to Diagnosis of a Hemolytic State
Establishing the Presence of Hemolysis
TESTS REFLECTING INCREASED RED CELL DESTRUCTION
Figure 9-1 Diagrammatic representation of the degradation of hemoglobin after intravascular or extravascular destruction of red cells. Fe = iron; Hb = hemoglobin; RBC = red blood cell; R.E. = reticuloendothelial cell.
TESTS REFLECTING INCREASED RED CELL PRODUCTION
Establishing the Cause of Hemolysis
Figure 9-2 Diagnostic approach to hemolytic anemias.
Hereditary Defects of the Red Cell Membrane
Red Cell Membrane Structure
Box 9–1 Predominant Red Cell Morphology Commonly Associated with Nonimmune Hemolytic Disorders
Classification
Figure 9-3 Transmission electron micrographs of negatively stained red blood cell membrane skeletons. A. An area of spread skeleton network. B, C. The hexagonal lattice made up of spectrin tetramers (Sp4), hexamers (Sp6), or double tetramers (2Sp4). Cross-linking junctional complexes contain short F-actin filaments and protein 4.1. Globular ankyrin structures are bound to spectrin filaments about 80 nm from their distal ends.
Table 9–1 Properties of Selected Red Cell Membrane Proteins Implicated in Hemolytic Anemia
Figure 9-4 ▪ Schematic representation of the structure of four of the red blood cell membrane proteins involved in hereditary hemolytic anemias. The molecular masses of the various domains are given in kilodaltons (kD). A. Spectrin. The subunit structure of α- and β-spectrin, showing the antiparallel arrangement of the N- and C-terminals, the homologous triple helical spectrin repeat units (indicated above the α and β chains), and the trypsin-resistant domains (open squares). α-Spectrin has five domains (αI to αV) and 21 repeats. β-Spectrin has four domains (βI to βIV) and 17 repeats. Proteins interacting with spectrin are indicated below the relevant domains. B. Band 3. The 52-kD membrane domain contains 14 transmembrane segments and is responsible for anion exchange. The 43-kD N-terminal cytoplasmic domain and the proteins binding to this domain are shown. C. Ankyrin. The N-terminal 89-kD domain consists of 24 ankyrin repeat units, which bind to band 3. The 62-kD domain interacts with spectrin and the C-terminal 55-kD regulatory domain modulates the activity of the protein. D. Protein 4.1. The four structural domains of the protein are depicted as open rectangles and the proteins binding to each domain are shown below.
Figure 9-5 Diagrammatic illustration of the vertical and horizontal interactions between the red cell membrane components (top). The bottom section illustrates the pathophysiology of the red cell lesion in hereditary spherocytosis (HS), hereditary elliptocytosis (HE), and hereditary pyropoikilocytosis (HPP). A defect in a vertical interaction resulting in spherocytes and HS is illustrated at the bottom left. A defect in a horizontal interaction resulting in elliptocytes and poikilocytes (HE and HPP) is illustrated at the bottom right.
Hereditary Spherocytosis
MODE OF INHERITANCE
MOLECULAR DEFECTS
Figure 9-6 Photomicrograph of peripheral blood smear from a patient with hereditary spherocytosis (HS). Note the microspherocytes (small condensed spherocytes with no central pallor), indicated by the black arrow and the pincered (mushroom-shaped) cell, indicated by the white arrow.
Box 9–2 Defects of Red Cell Membrane Proteins in Hereditary Spherocytosis
PATHOPHYSIOLOGY
CLINICAL MANIFESTATIONS
Figure 9-7 Schematic representation of postulated mechanisms of “conditioning” and destruction of HS red cells in the spleen.
CLINICAL LABORATORY FINDINGS
EVIDENCE OF HEMOLYTIC PROCESS
RED CELL INDICES
MORPHOLOGY OF PERIPHERAL SMEAR
Figure 9-8 Laboratory diagnosis of HS using a Technicon H1 laser scattering automated blood counter. A. The histogram indicates a subpopulation of microcytes with a low mean cell volume (MCV) in a patient with HS. B. The histogram depicts a subpopulation of dehydrated HS red cells with a high mean cell hemoglobin concentration (MCHC).
SPECIAL LABORATORY TESTS
OSMOTIC FRAGILITY TEST
Figure 9-9 Osmotic fragility curves of fresh blood (top) and incubated blood (bottom) obtained from a patient with HS. The normal range is shown by blue areas. Note the increased fragility of the HS red cells to osmotic lysis.
AUTOHEMOLYSIS TEST
ACIDIFIED GLYCEROL LYSIS
RED CELL MEMBRANE STUDIES
TREATMENT
Case Study 1
QUESTIONS
Hereditary Elliptocytosis
MODE OF INHERITANCE
Clinical Phenotypes
Figure 9-10 Photomicrograph of peripheral blood smear from a patient with mild hereditary elliptocytosis (HE). Note the high percentage of elliptocytes.
Figure 9-11 Photomicrograph of peripheral blood smear from a patient with hereditary pyropoikilocytosis (HPP). Note the bizarre micropoikilocytosis, red cell budding, and very few elliptocytes.
COMMON HEREDITARY ELLIPTOCYTOSIS
MOLECULAR DEFECTS
Box 9–3 Defects of Red Cell Membrane Proteins in Hereditary Elliptocytosis
Table 9–2 Characteristics of Hereditary Elliptocytosis Phenotypes
HEREDITARY PYROPOIKILOCYTOSIS
PATHOPHYSIOLOGY OF COMMON HE AND HPP
SOUTHEAST ASIAN OVALOCYTOSIS
Figure 9-12 Photomicrograph of peripheral blood smear from a patient with Southeast Asian ovalocytosis (SAO). Note the characteristic spoon-shaped oval cells with a band across the central area.
CLINICAL LABORATORY FINDINGS
EVIDENCE OF HEMOLYTIC PROCESS
MORPHOLOGY OF PERIPHERAL SMEAR
Figure 9-13 Photomicrograph of peripheral blood smear from a patient with mild HE and poikilocytosis of infancy. Note the poikilocytosis and fragmentation.
RED CELL INDICES
SPECIAL LABORATORY TESTS
OSMOTIC FRAGILITY AND AUTOHEMOLYSIS
RED CELL MEMBRANE STUDIES
TREATMENT
Case Study 2
QUESTIONS
Disorders of Membrane Cation Permeability
Hereditary Stomatocytosis and Hereditary Xerocytosis
MODE OF INHERITANCE
ETIOLOGY AND PATHOPHYSIOLOGY
Figure 9-14 Photomicrograph of peripheral blood smear from a patient with hereditary stomatocytosis. Note the high percentage of red cells with a central slit of pallor.
Figure 9-15 Photomicrograph of peripheral blood smear from a patient with hereditary xerocytosis. Note the characteristic target cells and cells with hemoglobin concentrated on one side of the cell.
CLINICAL LABORATORY FINDINGS
MORPHOLOGY OF PERIPHERAL SMEAR
RED CELL INDICES
SPECIAL LABORATORY TESTS
TREATMENT
Case Study 3
QUESTIONS
Questions
SUMMARY CHART
REFERENCES
Chapter 10 Hemolytic Anemias: Intracorpuscular Defects: II. Hereditary Enzyme Deficiencies
OBJECTIVES
History
Enzyme Deficiencies: Hexose Monophosphate Pathway
Glucose-6-Phosphate Dehydrogenase Deficiency
Mode of Inheritance
Pathogenesis
Table 10–1 Distribution of Some Common G6PD Variants
Figure 10-1 Red cell metabolic pathways. The nucleated red cell depends almost exclusively on the breakdown of glucose for energy requirements. The Embden–Meyerhof (nonoxidative or anaerobic) pathway is responsible for most of the glucose utilization and generation of ATP. In addition, this pathway plays an essential role in maintaining pyridine nucleotides in a reduced state to support methemoglobin reduction (the methemoglobin reductase pathway) and 2,3-diphosphoglycerate synthesis (the Luebering–Rapaport pathway). The phosphogluconate pathway couples oxidative metabolism with pyridine nucleotide and glutathione reduction. It serves to protect red cells from environmental oxidants.
Figure 10-2 Reactions with erythrocytes to prevent accumulation of oxidants.
Table 10–2 Drugs and Chemicals Associated with Hemolytic Anemia in G6PD Deficiency
Clinical Manifestations
Figure 10-3 Heinz bodies, using peripheral blood from a patient with G6PD deficiency, stained with the supravital stain, crystal violet.
Figure 10-4 Peripheral blood smear from a patient with a G6PD deficiency. Note the small, condensed “bite” or “helmet” cells.
Figure 10-5 Fava beans.
Table 10–3 Comparison of Clinical Features of Gd A– and Gd Med (Gd B–)
Laboratory Testing
Enzyme Deficiencies: Glycolytic Pathway
Pyruvate Kinase Deficiency
MODE OF INHERITANCE
PATHOGENESIS
CLINICAL MANIFESTATIONS
LABORATORY TESTING
Other Enzyme Deficiencies of the Glycolytic Pathway
Enzyme Deficiencies: Methemoglobin Reductase Pathway
Methemoglobin Reductase Deficiency
Table 10–4 Other Glycolytic Enzyme Deficiencies
Acknowledgment
Case Study
QUESTIONS TO CONSIDER
Table 10–5 Laboratory Differentiation of Methemoglobinemia
Questions
SUMMARY CHART
REFERENCES
Chapter 11 Hemolytic Anemias: Intracorpuscular Defects: III. The Hemoglobinopathies
OBJECTIVES
Introduction and Review of Normal Hemoglobin Structure
Figure 11-1 Inheritance of abnormal hemoglobins. A. With one parent heterozygous for an abnormal hemoglobin, the offspring have a one-in-two chance of carrying the trait. B. With one parent homozygous for an abnormal hemoglobin, all offspring will carry the trait because that parent can contribute only an abnormal gene. C. With both parents heterozygous for the abnormality, the chances are one in four for normal, two in four for heterozygous, and one in four for homozygous. D. With both parents carrying the same abnormal hemoglobin—one homozygous and one heterozygous—the offspring have a 50–50 chance of being either homozygous or heterozygous. E. With parents carrying two different abnormal hemoglobins, offspring have a one-in-four chance of not inheriting an abnormality, a one-in-two chance of carrying the trait for one or the other abnormality, and a one-in-four chance of carrying both abnormalities in codominance.
Figure 11-2 Location of the globin genes on chromosomes 16 and 11.
Table 11–1 Composition of Normal Physiologic Hemoglobins
Classification
Table 11–2 Classification of Hemoglobinopathies
Hemoglobinopathies
Nomenclature
Sickle Cell Anemia
HISTORIC OVERVIEW
DEFINITION
Figure 11-3 Amino acid substitution in hemoglobin S.
PATHOPHYSIOLOGY
Table 11–3 Sickle Cell Disease (a Group of Genetic Disorders Characterized by the Production of HbS)
CLINICAL FEATURES
Figure 11-4 Scanning electron micrograph (SEM) of sickle cells.
Figure 11-5 Sickle cell disease (peripheral blood). Note the sickle-shaped red cells and target cells.
Table 11–4 Factors Affecting the Severity of HbS
Figure 11-6 Asthenic physique with mild jaundice.
VASCULOPATHY
Table 11–5 Clinical Manifestations of Sickle Cell Anemia
Table 11–6 Clinical Features of Sickle Cell Anemia by Category
Figure 11-7 Leg ulcers in a patient with sickle cell anemia.
INFECTIONS
Figure 11-8 Hand–foot syndrome in a patient with sickle cell anemia.
Table 11–7 Organisms Implicated in Causing Infections in Patients with Sickle Cell Anemia
SICKLE CELL NEPHROPATHIES
Table 11–8 Factors Responsible for the Increased Susceptibility of Patients with Sickle Cell Anemia to Infections
STROKE
Sickle Cell Trait
LABORATORY DIAGNOSIS
Figure 11-9 Sickle trait (peripheral blood). Note the normal appearing smear.
LABORATORY SCREENING FOR SICKLE CELL DISEASE
Table 11–9 Screening Methods
Figure 11-10 Electrophoretic patterns of hemoglobin on (A) cellulose acetate, run at pH 8.4, and (B) citrate agar, run at pH 6.0 to 6.5. apl = point of application.
Figure 11-11 Tube solubility screening test for sickle cell anemia.
TREATMENT
Table 11–10 Examples of Rare Hemoglobins That Sickle and Give a Positive Tube Solubility Test
Table 11–11 Treatment of Sickle Cell Anemia: Goals of Therapeutic Approaches
Table 11–12 Effects of Approaches to Specific Therapy
Table 11–13 General Considerations and Indications for Blood Transfusion in Patients with Sickle Cell Anemia
Hemoglobin C Disease and Trait
Figure 11-12 Amino acid substitution in hemoglobin C.
Figure 11-13 Hemoglobin C disease (presplenectomy). Note the numerous target and folded cells.
Figure 11-14 Hemoglobin C disease (peripheral blood). Note the particular crystals: “bar of gold” and numerous target cells (postsplenectomy).
Hemoglobin D Disease and Trait
Hemoglobin E Disease and Trait
Figure 11-15 SEM of hemoglobin C crystals.
Hemoglobin OArab Disease and Trait
Hemoglobin S with Other Abnormal Hemoglobins
HEMOGLOBIN SC DISEASE
Table 11–14 Common and Uncommon Forms of Sickle Cell Disease
Figure 11-16 Hemoglobin SC disease (peripheral blood). Note the formation of the SC crystal (yellow arrow) and the polychromasia (black arrow).
HEMOGLOBIN SD DISEASE
Figure 11-17 Hemoglobin SC disease (peripheral blood). Note the type of “Washington Monument” crystals and target cells.
Figure 11-18 Hemoglobin electrophoretic patterns: (1) HbAC, (2) HbAS, (3) commercial control, (4) HbSC, (5) HbA-Lepore (see Chap. 12), and (6) HbAA normal control.
Table 11–15 Incidence of Common Hemoglobinopathies in American Blacks
HEMOGLOBIN SOArab AND S-Oman DISEASE
HEMOGLOBIN S/β THALASSEMIA COMBINATION
LABORATORY DIAGNOSIS OF HbS WITH OTHER ABNORMAL HEMOGLOBINS
Hemoglobin Variants with Altered Oxygen Affinity
Table 11–16 Clinical and Hematologic Findings in the Common Variants of Sickle Cell Disease After the Age of 5 Years
Table 11–17 Hemoglobins Associated with Altered Oxygen Affinity
Unstable Hemoglobins
Figure 11-19 Oxygen equilibrium curve of whole blood from subjects with Hb Rainier, Hb Seattle, Hb Kansas and normal control (HBA).
Figure 11-20 Unstable hemoglobin: Hb Zurich (peripheral blood).
Table 11–18 Unstable Hemoglobins*
Table 11–19 Hemoglobins Associated with Methemoglobinemia and Cyanosis
Methemoglobinemia
General Summary of Laboratory Diagnosis
Case Study
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 12 Hemolytic Anemias: Intracorpuscular Defects: IV. Thalassemia
OBJECTIVES
Introduction
Genetics of Hemoglobin Synthesis
Figure 12-1 World distribution of alpha (a) and beta (β) thalassemia.
Table 12–1 Composition of Hemoglobins Found in Normal Human Development and Abnormal Hemoglobins Found in Thalassemia
Pathophysiology of Thalassemia
Figure 12-2 The areas of homology (x, y, and z) can lead to mispairing.
Figure 12-3 Diagram of the β-globin gene expression: (top) schematic of the location of the β-like genes on chromosome 11 with the locus control region (LCR) on the 5′ end; (bottom) exploded β-globin gene depicting the three exons and two introns with the three promoter areas upstream.
Thalassemia Syndromes
Beta Thalassemia
Figure 12-4 Diagram of globin chain imbalance. Excess globin chains precipitate and damage red blood cells and their precursors. Destruction of red blood cells within the bone marrow (ineffective erythropoiesis) predominates in severe β thalassemia. In contrast, hemoglobin H precipitates and damages circulating red blood cells (hemolysis) in severe α thalassemia. Notice the changes in hemoglobin constitution of the peripheral blood with thalassemia: hemoglobin F is increased in severe β thalassemia and hemoglobin H is detectable in severe α thalassemia.
β0 THALASSEMIA HAPLOTYPES
β+ THALASSEMIA HAPLOTYPES
CLINICAL EXPRESSION OF THE DIFFERENT GENE COMBINATIONS (β THALASSEMIA)
Figure 12-5 Peripheral smear from a patient with β-thalassemia major. Note the nucleated red cells, Howell–Jolly body in the hypochromic microcyte (arrow), numerous target cells, and moderate anisocytosis and poikilocytosis (Wright’s stain).
CLINICAL COURSE AND THERAPY OF THE β THALASSEMIA SYNDROMES
β THALASSEMIA MAJOR
Figure 12-6 Peripheral smear from a patient with thalassemia minor. Note the microcytosis and hypochromia with mild anisocytosis and poikilocytosis. A few target cells and basophilic stippling are present. (Wright’s stain, magnification ×400)
Figure 12-7 Skull x-ray film of a 5-year-old child with homozygous β thalassemia. Note the dilation of the diploic space and the typical “hair-on-end” appearance caused by subperiosteal bone growth in radiating striations.
Figure 12-8 Face (A) and profile (B) of an 11-year-old child, with homozygous β thalassemia who is receiving hypertransfusion. The characteristic facial changes are not as prominent as those in an untransfused child, but are still present. Note the bossing of the skull, hypertrophy of the maxilla with prominent malar eminences, depression of the bridge of the nose, and mongoloid slant of the eyes.
β THALASSEMIA INTERMEDIA
β THALASSEMIA MINOR
Alpha Thalassemia
α0 THALASSEMIA (α THALASSEMIA 1) HAPLOTYPES
α+ THALASSEMIA (α THALASSEMIA 2) HAPLOTYPES
HEMOGLOBIN CONSTANT SPRING
CLINICAL EXPRESSION OF THE DIFFERENT GENE COMBINATIONS (α THALASSEMIAS)
Figure 12-9 Diagram of the migration of the different hemoglobins at different pH: (1) normal adult (A, A2); (2) homozygous Hb Lepore (F, Lepore); (3) HbH/Constant Spring disease αCSα/(Constant Spring, A2, A, Bart’s H); (4) compound heterozygous HbE/β-thalassemia (E, F); (5) Hb Bart’s hydrops fetalis syndrome (Portland, Bart’s); (6) HbS/C disease (S, C).
Figure 12-10 Hemoglobin H inclusions (supravital stain).
CLINICAL COURSE AND THERAPY OF α THALASSEMIA SYNDROMES
HEMOGLOBIN BART’S HYDROPS FETALIS
HEMOGLOBIN H DISEASE
Figure 12-11 Simplistic look at the interactions of deletional mutations of α thalassemia. The severity of disease is predictable and depends on the number deleted α-globin genes.
Table 12–2 Genetic Background of α Thalassemia Clinical Syndromes (Mating Combinations)
α THALASSEMIA MINOR (HOMOZYGOUS α+ THALASSEMIA OR HETEROZYGOUS α0 THALASSEMIA)
SILENT CARRIER OF α+ THALASSEMIA (HETEROZYGOUS α+ THALASSEMIA)
Delta–Beta Thalassemia and Hemoglobin Lepore Syndrome
Figure 12-12 Hemoglobin Lepore formation. An abnormal crossing over between β and δ-globin genes gives rise to hemoglobin Lepore and to hemoglobin anti-Lepore.
Hereditary Persistence of Fetal Hemoglobin
Figure 12-13 Mutations seen in HPFH with hemoglobin F in 20% range. Mutations producing HPFH in this range have common themes. One common molecular mechanism (left) is the deletion of the δ and β-globin genes, (δβ)0. This eliminates the competition of the locus control region for the δ and β promoters and allows increased γ transcription. Another common theme is a point mutation in the Gγ or Aγ promoters which increases the association of the locus control region with the respective γ-globin gene and increases its transcription.
Figure 12-14 (δβ)0 Deletions can be found in both HPFH and δβ thalassemia. A greater amount of hemoglobin F is seen in HPFH than in δβ thalassemia. Two hypotheses for increased production of γ globin expression in HPFH are shown above. The δβ deletion along with an intergenic sequence responsible for γ silencing during development is shown at the left. A second hypothesis in which a deletion results in the juxtaposition of a downstream enhancer with the γ-globin gene, where it can have a positive effect on γ transcription, is shown at the right. The mechanisms are not exclusive of one another and may coexist. Complete compensation for loss of δ and β globins with increased γ globin prevents a globin imbalance between α and non-α globin chains in HPFH.
Pancellular and Heterocellular HPFH
PANCELLULAR HPFH
Figure 12-15 Kleihauer–Betke stain of blood from a patient with hereditary persistence of fetal hemoglobin (HPFH). Note that all red cells stain red, owing to the varying amounts of hemoglobin F.
HETEROCELLULAR HPFH
Thalassemia Associated with Hemoglobin Variants
β THALASSEMIA WITH HEMOGLOBIN S
β THALASSEMIA WITH HEMOGLOBIN C
β THALASSEMIA WITH HEMOGLOBIN E
α THALASSEMIA WITH SICKLE CELLANEMIA
A Broad Clinical Classification of Thalassemia Syndromes
Laboratory Diagnosis of Thalassemia
Table 12–3 Genetic Background of the Different Clinical Courses of Thalassemia
Routine Hematology Procedures
AUTOMATED BLOOD CELL ANALYZER
PERIPHERAL SMEAR EXAMINATION
WRIGHT’S STAIN
SUPRAVITAL STAINS
ACID ELUTION STAIN
OSMOTIC FRAGILITY
Flow Cytometry
Hemoglobin Electrophoresis
Figure 12-16 Flow cytometric analysis to determine hemoglobin F distribution. The x-axis represents the amount of hemoglobin in red cells, and the y-axis represents the amount of red blood cells. A pancellular distribution of hemoglobin F in an individual with HPFH is shown in the diagram at left. A heterocellular distribution of hemoglobin in an individual with heterozygous (δβ)0 thalassemia is shown in the diagram at right.
Table 12–4 Levels of Hemoglobins A, A2, and F in the Different Non-α Thalassemias
CELLULOSE ACETATE
OTHER GELS
Hemoglobin Quantitation
Figure 12-17 Electrophoresis at alkaline pH. Lanes 1 and 7 are controls with hemoglobins C, S, F, and A. Lanes 3 and 4 are normal individuals (A2 = 2.5%). Lane 5 is an individual with β thalassemia minor (A2 = 4.7%). Lane 6 is an individual with α thalassemia minor; α thalassemia minor has a pattern with a normal A2. Lane 2 is an individual with β thalassemia minor (A2 = 5%) and “Swiss HPFH” (F2 = 7%).
HEMOGLOBIN A2 QUANTITATION
HEMOGLOBIN F QUANTITATION
Routine Chemistry
Differential Diagnosis of Microcytic, Hypochromic Anemia
Treatment of Thalassemia
Blood Transfusion in Thalassemia
Table 12–5 Differential Diagnosis of Microcytic Hypochromic Anemia
Curative Treatment of Thalassemia
Prevention of Thalassemia
Case Study 1
QUESTIONS
ANSWERS
Case Study 2
QUESTIONS
ANSWERS
Case Study 3
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 13 Hemolytic Anemias: Extracorpuscular Defects
OBJECTIVES
Immune Hemolytic Anemia
Definition
Immune Hemolysis
ROLE OF COMPLEMENT
CLASSICAL PATHWAY
Figure 13-1 Classical pathway of complement activation.
ALTERNATE (PROPERDIN) PATHWAY
Figure 13-2 Alternate pathway of complement activation.
MECHANISMS OF IMMUNE HEMOLYSIS
INTRAVASCULAR HEMOLYSIS
Table 13–1 Biologic Properties of IgG Isotypes
Table 13–2 Mechanisms of Immune Hemolysis
EXTRAVASCULAR IMMUNE HEMOLYSIS
Figure 13-3 Indicators of acute intravascular hemolysis. Within a few hours of an acute hemolytic event, free hemoglobin is cleared from plasma and the serum haptoglobin falls to undetectable levels: hemoglobinuria ceases soon after. If no further hemolysis occurs, the serum haptoglobin level recovers, and methemalbumin disappears within several days. The urinary hemosiderin can provide more lasting evidence of the hemolytic event.
Figure 13-4 Autoimmune hemolytic anemia (peripheral blood). Note (A) spherocytes and (B) polychromasia.
Classification of Immune Hemolytic Anemia
Table 13–3 Factors Influencing the Presence and Extent of Immune Hemolysis
ALLOIMMUNE HEMOLYTIC ANEMIA
ACUTE HEMOLYTIC TRANSFUSION REACTIONS
DELAYED HEMOLYTIC TRANSFUSION REACTIONS
Table 13–4 Clinical Features of Acute Hemolytic Transfusion Reactions
HEMOLYTIC DISEASE OF THE NEWBORN
Table 13–5 Antibodies Most Commonly Implicated in Delayed Hemolytic Transfusion Reactions (DHTRs)
Table 13–6 Differential Diagnosis of Hemolytic Anemia
AUTOIMMUNE HEMOLYTIC ANEMIA
Table 13–7 Frequency of Types of Hemolytic Disease of the Newborn
WARM AUTOIMMUNE HEMOLYTIC ANEMIA
Table 13–8 Comparison of ABO and Rh Hemolytic Disease of the Newborn
Table 13–9 Types of Autoimmune Hemolytic Anemia (by Percent Age)
Table 13–10 Disorders Associated with Warm Autoimmune Hemolytic Anemia
COLD AUTOAGGLUTININS
Table 13–11 Comparison of Characteristics of Normal and Pathologic Cold Autoantibody
Figure 13-5 Cold hemagglutinin disease (peripheral blood). Note the autoagglutination of red cells.
Table 13–12 Clinical Criteria for the Diagnosis of Cold Agglutinin Disease
Table 13–13 Secondary Cold Autoimmune Hemolytic Anemia
Table 13–14 Comparison of Paroxysmal Cold Hemoglobinuria and Cold Agglutinin Syndrome
MIXED AUTOIMMUNE HEMOLYTIC ANEMIA
Table 13–15 Donath–Landsteiner Test
Table 13–16 Comparison of Warm and Cold Autoimmune Hemolytic Anemias
DRUG-INDUCED IMMUNE HEMOLYTIC ANEMIA
AUTOIMMUNE MECHANISM
DRUG ADSORPTION (HAPTEN) MECHANISM
Table 13–17 Partial List of Drugs Associated with Positive DAT or Hemolytic Anemia by Mechanism
Figure 13-6 Drug adsorption mechanism.
IMMUNE COMPLEX MECHANISM
Figure 13-7 Immune complex mechanism.
MEMBRANE MODIFICATION MECHANISM (PROTEIN ADSORPTION)
Figure 13-8 Membrane modification mechanism.
Table 13–18 Mechanism Leading to Development of Drug-Related Antibodies
Nonimmune Hemolytic Anemia
Intracellular Infections
MALARIA
Table 13–19 Summary of Antibody Characteristics in Autoimmune Hemolytic Anemia
LIFE CYCLE
CLINICAL PRESENTATION
LABORATORY DIAGNOSIS
Table 13–20 Classification of Nonimmune Acquired Hemolytic Anemias
BABESIOSIS
LABORATORY DIAGNOSIS
Figure 13-9 Malarial life cycle in humans and mosquitoes. Beginning of cycle is indicated by an asterisk.
Figure 13-10 Ringed forms of Plasmodium falciparum in red blood cells (RBCs). Note that the same RBCs may be infected with more than one ring.
Figure 13-11 Late stages of Plasmodium vivax malaria. Schüffner’s dots. Note and contrast the platelet on the RBC (center) with the ring form of malaria toward the periphery (arrow).
Table 13–21 Characteristics of Malarial Parasites Infecting Humans
Extracellular Infections
BARTONELLOSIS (OROYA FEVER)
Figure 13-12 Comparison of babesiosis (left) and malaria (right).
CLOSTRIDIUM PERFRINGENS (WELCHII)
Mechanical Etiologies
CARDIAC PROSTHESIS
Figure 13-13 Peripheral blood showing red cell fragmentation with thrombocytopenia. (A) Polychromasia and (B) nucleated RBCs from a patient with thrombotic thrombocytopenia purpura (TTP).
MARCH HEMOGLOBINURIA
Table 13–22 Organisms Associated with Hemolytic Anemia
Figure 13-14 RBC fragmentation in microangiopathic hemolysis from a patient with a prosthetic cardiac valve (mechanical hemolysis); note the presence of schistocytes (arrows).
MICROANGIOPATHIC HEMOLYTIC ANEMIA
Chemical and Physical Agents
OXIDATIVE HEMOLYSIS
NONOXIDATIVE HEMOLYSIS
ARSENIC
LEAD
Figure 13-15 Peripheral blood from a patient with lead poisoning. Note the normocytic, hypochromic red cells, with the classic punctate basophilic stippling.
COPPER
VENOMS
OSMOTIC EFFECTS
BURNS
DROWNING AND OTHER WATER-RELATED OSMOTIC INJURY
Figure 13-16 Peripheral blood from a patient with extensive burns. Note the typical (A) microspherocytes and (B) membranous fragments.
Figure 13-17 Spur-cell anemia (acanthocytosis) associated with severe liver disease.
Acquired Membrane Disorders
Figure 13-18 Acanthocytosis from a patient with abetaliproteinemia.
Figure 13-19 Renal disease (peripheral blood). Note the presence of (A) burr cells, (B) thorn cell, (C) blister cell, and (D) shistocyte.
Case Study 1
QUESTIONS
ANSWERS
Case Study 2
QUESTIONS
ANSWERS
Case Study 3
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 14 Hypoproliferative Anemia: Anemia Associated with Systemic Diseases
OBJECTIVES
Introduction
Anemia of Chronic Disease
The Inflammatory Response and Body Defense Mechanisms
THE GENERAL INFLAMMATORY RESPONSE (FIRST LINE OF DEFENSE)
Etiology and Pathophysiology
DYSREGULATION OF IRON HOMEOSTASIS
Figure 14-1 Mechanism of humoral and cellular immunity. A. The antigen phagocytized by the APC is digested, and small antigenic fragments or epitopes are associated with the class II MHC and presented to a T cell with a receptor specific for the antigen. Formation of the antigen-receptor complex between the two cells and IL-1 secreted by the APC provide the signals for the T cell to be activated and secrete IL-2 for its autostimulation and proliferation to effector T-helper cells. B. Humoral immunity. The T-helper effector cell (CD4) and some of its lymphokines provide the necessary signals for the B cell with the same antigen specificity to be activated and proliferate to B memory and antibody-producing plasma cells. C. Cellular immunity. Some of the lymphokines from the activated CD4 cells and the complex formed between antigens associated with class I MHC on altered self-cell and T-cytotoxic cell (CD8) receptors cause the activation of the CD8 cells, which mediate the cytotoxic killing of the altered self-cells. Some of the other lymphokines produced also play significant roles in hematopoiesis and activation of phagocytic cells.
SUPPRESSION OF ERYTHROPOIESIS AND THE ROLE OF CYTOKINES
Table 14–1 Thymus-Derived Lymphocytes
DECREASED ERYTHROPOIETIN RESPONSE
Table 14–2 Cytokines That Contribute to Inflammation
Table 14–3 Functions of Macrophages
Table 14–4 Diseases That Cause Anemia of Chronic Disease
Table 14–5 Mechanisms Involved in Anemia of Chronic Disease
DECREASED RED BLOOD CELL SURVIVAL AND LIFE SPAN
Characteristics
Figure 14-2 Pathophysiological mechanisms underlying anemia of chronic disease. In A, the invasion of microorganisms, the emergence of malignant cells or autoimmune dysregulation leads to activation of T cells (CD3+) and monocytes. These cells induce immune effector mechanisms, thereby producing cytokines such as interferon-γ (from T cells) and tumor necrosis factor-α (TNF-α), interleukin-1, interleukin-6 and interleukin-10 (from monocytes or macrophages). In B, interleukin-6 and lipopolysaccharide stimulate the hepatic expression of the acute-phase protein hepcidin, which inhibits duodenal absorption of iron. In C, interferon-γ, lipopolysaccharide, or both increase the expression of divalent metal transporter 1 on macrophages and stimulate the uptake of ferrous iron (Fe2+). The anti-inflammatory cytokine interleukin-10 upregulates transferrin receptor expression and increases transferrin-receptor-mediated uptake of transferrin-bound iron into monocytes. In addition, activated macrophages phagocytose and degrade senescent erythrocytes for the recycling of iron, a process that is further induced by TNF-α through damaging of erythrocyte membranes and stimulation of phagocytosis. Interferon-γ and lipopolysaccharide down-regulate the expression of the macrophage iron transporter ferroportin 1, thus inhibiting iron export from macrophages, a process that is also affected by hepcidin. At the same time, TNF-α, interleukin-1, interleukin-6 and interleukin-10 induce ferritin expression and stimulate the storage and retention of iron within macrophages. In summary, these mechanisms lead to a decreased iron concentration in the circulation and thus to a limited availability of iron for erythroid cells. In D, TNF-α and interferon-γ inhibit the production of erythropoietin in the kidney. In E, TNF-α, interferon-γ and interleukin-1 directly inhibit the differentiation and proliferation of erythroid progenitor cells. In addition, the limited availability of iron and the decreased biologic activity of erythropoietin lead to inhibition of erythropoiesis and the development of anemia. Plus signs represent stimulation and minus signs inhibition.
Treatment
Table 14–6 Comparison of Anemia of Chronic Disease with Iron-Deficiency Anemia
Anemia Associated with Renal Disease and Renal Failure
Etiology and Pathophysiology
Table 14–7 Mechanisms Involved in Anemia of Renal Insufficiency
Characteristics
Figure 14-3 Peripheral blood from a patient with renal disease. Note the burr cells (arrows).
Treatment
Anemia Associated with Liver Disease
Etiology and Pathophysiology
Table 14–8 Mechanisms of Anemia in Liver Disease
Figure 14-4 Peripheral blood smear from an individual with severe liver disease. Target cells (arrows) are caused by altered lipid metabolism with subsequent effects on red blood cell membrane.
Table 14–9 RBC Morphology in Liver Disease
Characteristics
Treatment
Anemia Associated with Alcoholism/Alcohol Abuse
Etiology and Pathophysiology
Characteristics
Treatment
Anemia Associated with Endocrine Disease/Disorders
Adrenal Insufficiency
Thyroid Disease
Hyperparathyroidism
Hypogonadism
Pituitary Dysfunction
Anemia Associated with Malignancy
Etiology and Pathophysiology
DIRECT EFFECTS
Table 14–10 Endocrine Disorders/Diseases
Figure 14-5 Peripheral blood from a patient with disseminated carcinoma. Note presence of (A) schistocytes and (B) helmet cells.
Table 14–11 Mechanisms of Anemia in Malignancy
Figure 14-6 Leukoerythroblastosis, a peripheral blood picture that often accompanies marrow infiltration by tumors (myelophthisic anemia). Note the presence of immature (A) red and (B) white cells.
INDIRECT EFFECTS
TREATMENT/THERAPY-ASSOCIATED EFFECTS
Figure 14-7 Peripheral blood smear from an individual with myelophthisic anemia. Note left-shifted granulocyte precursors (solid arrow). Teardrop-shaped red blood cells (open arrows) are indicative of marrow fibrosis in this type of leukoerythroblastic reaction. Nucleated red blood cell precursors were seen in other fields
Figure 14-8 Peripheral blood smear from a neonate with leukoerythroblastosis caused by severe blood loss associated with birth (a reactive condition). (A) Left-shifted granulocyte precursors and (B) nucleated red blood cell precursors are seen. Although some (C) “burr” cells are seen, no teardrop-shaped red blood cells are identified.
Table 14–12 RBC Morphology in Anemia of Malignancies
Characteristics
Treatment
Anemia Associated with Human Immunodeficiency Virus Infection and the Acquired Immunodeficiency Syndrome
Pathophysiology and Characteristics
Treatment
Anemia of Infancy
Etiology and Pathophysiology
Table 14–13 Pathophysiology of Anemia in HIV-Positive Individuals
Characteristics
Treatment
Table 14–14 Contributing Factors in the Anemia of Infancy
Anemia Associated with Prematurity
Etiology and Pathophysiology
Characteristics
Treatment
“Anemia” Associated with Improperly Collected Laboratory Samples: Preanalytical Anemia
Etiology and Pathogenesis
Characteristics
Prevention
Summary
Case Study 1
QUESTIONS
Case Study 2
QUESTIONS
Case Study 3
QUESTIONS
Questions
SUMMARY CHART
REFERENCES
PART 3 WHITE BLOOD CELL DISORDERS
Chapter 15 Cell Biology, Disorders of Neutrophils, Infectious Mononucleosis, and Reactive Lymphocytosis
OBJECTIVES
Neutrophils
Neutrophil Function
MIGRATION AND DIAPEDESIS
Table 15–1 Contents of Neutrophil Granules
Figure 15-1 Illustration of the phases of neutrophilic phagocytosis, which include activation, diapedesis, chemotaxis, opsonization, ingestion, killing, and digestion. PMN = polymorphonuclear neutrophil.
Table 15–2 Chemical Factors that Signal Neutrophil Activation
Table 15–3 Three Modes of Neutrophil Migration
OPSONIZATION AND RECOGNITION
PHAGOCYTOSIS: INGESTION, KILLING, AND DIGESTION
Figure 15-2 The shape change of the neutrophil, from smooth and round to ruffled and flat with pseudopods, is a result of stimulation by chemoattractants.
Figure 15-3 Electron microscopy of phagolysosome formation. (A) Staphylococci lie within phagocytic vesicles limited by sacs formed from inverted pieces of the neutrophil membrane. Cytoplasmic granules are approaching the phagocytic vesicles. (B) Higher magnification shows degranulation with the discharge of granule contents into the vicinity of the staphylococcus.
Disorders of Neutrophils
QUANTITATIVE DISORDERS
NEUTROPHILIA
Table 15–4 Causes of Secondary Reactive Neutrophilia (Absolute Count > 6000/mm3 or 6.0 × 109/L)
Figure 15-4 Toxic granulation (peripheral blood). Note the prominent dark-staining granules.
Figure 15-5 Döhle bodies (arrows). Note the large bluish bodies in the periphery of the cytoplasm.
NEUTROPENIA
Figure 15-6 Vacuolated neutrophils suggesting the presence of infection or a severe inflammation.
Table 15–5 Characteristics of Leukemoid Reaction
Table 15–6 Classification of Neutropenia
Table 15–7 Pathogenesis of Neutropenia
ACQUIRED NEUTROPENIA
Table 15–8 Drugs Associated with Causing Neutropenia
Table 15–9 Causes of Acquired Neutropenia (Absolute Count < 1500/mm3 or 1.5 × 109/L)
QUALITATIVE DISORDERS OF NEUTROPHILS
DISORDERS OF NEUTROPHIL FUNCTION
Table 15–10 Disorders of Congenital Neutropenia
Table 15–11 Classes of Qualitative Neutrophil Disorders and Related Conditions
Table 15–12 Classification of Disorders of Neutrophil Functions that Can Be Inherited or Acquired
Table 15–13 Acquired Disorders of Neutrophil Function
Table 15–14 Inherited Disorders of Neutrophil Function
Figure 15-7 Peripheral blood from a patient with Chediak–Higashi syndrome. (Right) Lymphocyte. (Left) Neutrophil.
Figure 15-8 Neutrophil from a patient with Chediak–Higashi syndrome. The cytoplasm is filled with strikingly large primary (azurophilic) granules.
Table 15–15 Molecular Basis of Chronic Granulomatous Disease
DISORDERS OF ABNORMAL NEUTROPHIL MORPHOLOGY
Table 15–16 Acquired Disorders of Neutrophil Morphology
Table 15–17 Inherited Disorders of Neutrophil Morphology
Figure 15-9 Pelger–Huët anomaly (peripheral blood).
Figure 15-10 Alder–Reilly anomaly. (Left and middle) Note azurophilic granulation in cells from peripheral blood. (Right) Bone marrow.
Figure 15-11 May–Hegglin anomaly. Note the Döhle body present in each neutrophil (arrows). Not shown in the slide but associated with May–Hegglin anomaly is the presence of giant platelets.
Eosinophils
Table 15–18 Classification of Eosinophilia
Table 15–19 Causes of Secondary (Nonmalignant) Reactive Eosinophilia (Absolute Count > 600/mm3 or 0.6 × 109/L)
Table 15–20 Characteristics of Acquired Secondary Eosinophilia
Basophils
Monocytes
Table 15–21 Causes of Secondary Reactive Basophilia (Absolute Count > 200/mm3 or 0.2 × 109/L)
Absolute Monocytosis: Reactive versus Malignant Causes
Table 15–22 Enzymes Found in the Granules of Monocytes
Table 15–23 Functions of Proteins Secreted by Monocytes
Table 15–24 Causes of Secondary Reactive Absolute Monocytosis (Adults: Absolute Counts >1.0 × 109/L; Newborns: >1.2 × 109/L)
Lymphocytes
Definition of Lymphocytosis
Lymphocyte Morphology
Figure 15-12 A large, mature-appearing lymphocyte with azurophilic granules.
Table 15–25 Lymphocyte Morphologies
Figure 15-13 Three lymphocytes are present, one with abundant cytoplasm (low N:C ratio) and two with moderate amounts of cytoplasm, from a patient with infectious mononucleosis. Note the variation in the chromatin coarseness. The larger or reactive-appearing lymphocyte has prominent cytoplasm indentations (center arrow).
Figure 15-14 Lymphocyte with nuclear indentation.
Figure 15-15 A small plasmacytoid lymphocyte with coarse chromatin and an eccentric nucleus.
Causes of Reactive Lymphocytosis
INFECTIOUS MONONUCLEOSIS
HISTORY
Figure 15-16 A large reactive lymphocyte with prominent nucleolus (immunoblast).
Table 15–26 Causes of Reactive Lymphocytosis
Table 15–27 Malignant Conditions that May Be Confused with Reactive Lymphocytosis
CLINICAL MANIFESTATIONS
DIFFERENTIAL DIAGNOSIS
TREATMENT, CLINICAL COURSE, AND PROGNOSIS
CYTOMEGALOVIRUS INFECTION
CLINICAL MANIFESTATIONS
OTHER VIRAL INFECTIONS
BACTERIAL INFECTIONS
MALIGNANT CONDITIONS
Figure 15-17 L2 leukemia. The L2 lymphoblasts have fine chromatin with a moderate amount of cytoplasm and nucleoli. Note the prominent nucleolus in one of the lymphoblasts (arrow).
Figure 15-18 L1 leukemia. These L1 lymphoblasts have a high N:C ratio (scant cytoplasm), fine chromatin, and indistinct nucleoli. Except for the fine chromatin, note the similarity to mature lymphocytes. These cells should not be confused with reactive lymphocytes. Note the large “smudge cells” (arrows).
Laboratory Examination
Figure 15-19 L3 leukemia. Note the abundant cytoplasmic vacuoles and clumped chromatin.
Figure 15-20 Prolymphocytes with moderate amounts of cytoplasm and prominent nucleoli.
Figure 15-21 Lymphocytes from a patient with follicular small cleaved-cell lymphoma. Note the prominent nuclear clefting.
Figure 15-22 This monocyte has very fine granular cytoplasm, cerebriform nucleus, linear condensation of chromatin, and no nucleolus.
Figure 15-23 M5a leukemia. These monoblasts have abundant cytoplasm (low N:C ratio), fine chromatin with some chromatin clumping, and prominent nucleoli.
Table 15–28 Summary of Antibody Tests Useful in the Differential Diagnosis of Infectious Mononucleosis
Figure 15-24 Time-course relationship between heterophile antibodies, various anti-Epstein–Barr antibodies, and the mean total and reactive lymphocyte counts. VCA = viral capsid antigen. EBNA = Epstein-Barr nuclear antigen, EA = early antigen, D = diffuse component.
Lymphocytopenia
Figure 15-25 Testing strategies for infectious mononucleosis differential diagnosis.
Table 15–29 Causes of Secondary Reactive Lymphocytopenia (Absolute Counts < 1.0 × 109/L in Adults; < 2.0 × 109/L in Children)
Case Study 1
HISTORY OF PRESENT ILLNESS
LABORATORY DATA
DISCUSSION
Case Study 2
HISTORY OF PRESENT ILLNESS
LABORATORY DATA
DISCUSSION
Questions
SUMMARY CHART
REFERENCES
Chapter 16 Introduction to Leukemia and the Acute Leukemias
OBJECTIVES
Introduction
Definition and Overview
Table 16–1 Comparison of Acute and Chronic Leukemia
Table 16–2 Estimated Percentage of New Cases of Each Type of Leukemia (Includes Adults and Children)
Comparison of Acute and Chronic Leukemia
Historical Perspectives
Etiology and Risk Factors
Classifications
Table 16–3 Host and Environmental Factors Associated with an Increased Risk of Leukemia
Table 16–4 Classification of Leukemia
Introduction to Acute Leukemia
Incidence
Clinical Features
Table 16–5 Clinical Features of Acute Leukemia
Laboratory Evaluation of Acute Leukemia
Table 16–6 Comparison of Acute Myeloblastic and Acute Lymphoblastic Leukemia
SPECIMENS
EVALUATION OF MORPHOLOGY
Table 16–7 Morphological Features of Blasts in Acute Myeloid and Acute Lymphoblastic Leukemias
CYTOCHEMISTRY
Figure 16-1 Myeloblast (note the Auer rod).
Figure 16-2 Lymphoblasts (peripheral blood).
Table 16–8 Summary of Cytochemical Reactions Useful in Diagnosing Acute Leukemia
MYELOPEROXIDASE (MPO)
SUDAN BLACK B (SBB)
Figure 16-3 Myeloperoxidase positivity in acute promyelocytic leukemia.
SPECIFIC ESTERASE (NAPHTHOLAS-D CHLOROACETATE)
Figure 16-4 Sudan black B positivity in acute myeloblastic leukemia (AML), M2.
Figure 16-5 Specific esterase (naphthol AS-D chloroacetate) positivity in AML, M2.
NONSPECIFIC ESTERASE (ALPHA-NAPHTHYLACETATE OR BUTYRATE)
PERIODIC ACID–SCHIFF
Figure 16-6 Nonspecific esterase (alpha-naphthyl butyrate) positivity in acute monocytic leukemia (M5).
IMMUNOLOGIC MARKER STUDIES
Figure 16-7 Periodic acid–Schiff positivity in acute lymphoblastic leukemia (ALL). Note the “block” staining pattern.
CELL SURFACE MARKERS
Table 16–9 Monoclonal Antibodies Used for Study of Leukemia and Lymphoma
Figure 16-8 Distribution of myeloid and monocytic surface antigens with bars depicting the strength of antigen reactivity. The wider the bar, the stronger the reaction, the narrowing indicating a weaker reaction. PMN = polymorphonuclear neutrophil; CFU-GM = colony-forming unit granulocyte macrophage/monocyte.
CYTOPLASMIC MARKERS
Figure 16-9 Surface immunoglobulin (slg)-positive cells in B-cell ALL, L3.
TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE
CYTOGENETICS
Figure 16-10 TdT positivity in ALL using immunofluorescence method.
MOLECULAR GENETICS
Table 16–10 Common Chromosome Abnormalities and Molecular Correlates Associated with Acute Leukemia
Acute Myeloid Leukemia
FAB Classification of AML
AML FAB M0
AML FAB M
Table 16–11 FAB Classification of Acute Myeloblastic Leukemia
Table 16–12 Cytochemistry Markers for Subclassification of Nonlymphocytic Leukemia
AML FAB M2
Figure 16-11 Acute myeloblastic leukemia (AML) without maturation, M0, bone marrow.
AML FAB M3 AND M3m
Figure 16-12 AML, M1, peripheral blood.
Figure 16-13 AML with maturation, M2, bone marrow.
Figure 16-14 AML, M2.
Figure 16-15 AML, M2 (myeloperoxidase stain).
Figure 16-16 Acute promyelocytic leukemia (APL), M3, bone marrow.
Figure 16-17 APL, M3 (Sudan black B stain).
Figure 16-18 Acute “microgranular” promyelocytic leukemia, M3m, peripheral blood.
AML FAB M4
Figure 16-19 Acute myelomonocytic leukemia (AMML), M4, bone marrow.
AML FAB M4Eo
Figure 16-20 AMML, M4, peripheral blood.
Figure 16-21 Acute monocytic leukemia (AMoL), poorly differentiated, M5a, peripheral blood.
AML FAB M5a AND M5b
Figure 16-22 AMoL, well-differentiated, M5b, peripheral blood.
Figure 16-23 Gum hypertrophy: A clinical manifestation of acute leukemia (M5).
AML FAB M6
Figure 16-24 Erythroleukemia, M6, bone marrow.
Figure 16-25 Erythroleukemia, M6, peripheral blood.
AML FAB M7
Figure 16-26 Acute megakaryoblastic leukemia (AMegL), M7.
FAB REVISED CRITERIA
Figure 16-27 Suggested steps in the analysis of a bone marrow (BM) aspirate to reach a diagnosis of acute myeloid leukemia (AML, M1 to M6) or myelodysplastic syndrome (MDS). BL = blast cells; ANC = all nucleated bone marrow cells; NEC = nonerythroid cells, bone marrow cells excluding erythroblasts.
WHO Classification of AML
AML WITH RECURRENT GENETIC ABNORMALITIES
AML WITH t(8;21)(q22;q22);(ETO–AML1)
AML WITH inv(16)(p13q22) OR t(16;16)(p13;q22);(CBFβ/MYH11)
ACUTE PROMYELOCYTIC LEUKEMIA WITH t(15;17) (q22;q12); (PML/RARα AND VARIANTS)
AML WITH 11q23 (MLL) ABNORMALITIES
AML WITH MULTILINEAGE DYSPLASIA
AMLs AND MYELODYSPLASTIC SYNDROMES, THERAPY RELATED
ACUTE MYELOID LEUKEMIA NOT OTHERWISE CATEGORIZED
ACUTE MYELOBLASTIC LEUKEMIA WITHOUT MATURATION
ACUTE MYELOBLASTIC LEUKEMIA WITH MINIMAL MATURATION
ACUTE MYELOBLASTIC LEUKEMIA WITH MATURATION
ACUTE MYELOMONOCYTIC LEUKEMIA
ACUTE MONOBLASTIC LEUKEMIA AND ACUTE MONOCYTIC LEUKEMIA
ACUTE ERYTHROID LEUKEMIA
ACUTE MEGAKARYOBLASTIC LEUKEMIA (M7)
ACUTE BASOPHILIC LEUKEMIA
ACUTE PANMYELOSIS WITH MYELOFIBROSIS
MYELOID SARCOMA
ACUTE LEUKEMIAS OF AMBIGUOUS LINEAGE
Acute Lymphoblastic Leukemia
Classification of ALL and Introduction
Review of Lymphocyte Ontogeny
B-LYMPHOCYTE DEVELOPMENT
Figure 16-28 B-cell development. Heavy chain (H) and light chain (L) are designated as H°, L° if in embryonic form, and H+, L+ if rearranged. > = cytoplasmic mu; Y = immunoglobulin.
Figure 16-29 Schematic of immunoglobulin μ-heavy chain gene rearrangement. The variable (V), diversity (D), and joining (J) regions of germline DNA are linked through rearrangement and loss of intervening sequences. The VDJ complex, intervening sequences, and a constant (Cμ) region are then transcribed. The resulting RNA is spliced, linking the VDJ and Cμ regions and creating a template for the Ig μ-heavy chain.
Figure 16-30 E-rosette formation in a T-cell.
T-LYMPHOCYTE DEVELOPMENT
Figure 16-31 Normal T-cell maturation. T-cell receptor (TCR) β and a genes are designated as B0 and α0 if in the embryonic form, and B+ and α+ if rearranged. The colored blue bars indicate where the antigens are present in the stages of maturation. No color indicates the antigens are not present. Please note that the CD1 antigen disappears on mature thymocytes. The orange color indicates that as the cells mature they lose the expression of either CD4 or CD8, with only one of these antigens remaining on each mature thymocyte or T-cell, and not both.
FAB Classification of ALL
Figure 16-32 ALL, L1, bone marrow.
Table 16–13 FAB Classification of Acute Lymphoblastic Leukemia
Figure 16-33 ALL, L2, bone marrow.
Figure 16-34 ALL, L2, peripheral blood.
Figure 16-35 ALL, L3, bone marrow.
Table 16–14 Comparison of Immunologic and FAB Classifications for Acute Lymphoblastic Leukemia (ALL)
WHO Classification of ALL
Table 16–15 Immunologic Classification of Acute Lymphoblastic Leukemia
PRECURSOR B-CELLALL (EARLY PRE-B AND PRE-B)
ALL WITH t(12;21): TEL–AML1
ALL WITH t(1;19): PBX1–E2A
ACUTE LYMPHOID LEUKEMIA (ALL) WITH t(9;22): BCR–ABL
ALL WITH t(4;11): AF4–MLL
PRECURSOR T-CELLALL
Burkitt’s Leukemia/Lymphoma (Mature B-Cell ALL)
Childhood versus Adult ALL
Treatment of Acute Leukemia
Treatment of ALL
Treatment of AML
APL
AML WITH INV(16) OR t(8;21)
AML IN THE ELDERLY
Measurement of Response to Therapy for Acute Leukemia
Table 16–16 Some Targeted Drugs Currently in Development for AML
Case Study 1
Figure 16-36 Case study—bone marrow (AML).
Case Study 2
Case Study 3
Questions
SUMMARY CHART
REFERENCES
Chapter 17 Chronic Myeloproliferative Disorders I: Chronic Myelogenous Leukemia
OBJECTIVES
Introduction to Chronic Myeloproliferative Disorders
Historical Perspective
Definition and Classification
Table 17–1 Characteristics of the Chronic Myeloproliferative Disorders
Figure 17-1 Relationship between the various myeloproliferative disorders. Frequencies of transitions between each of these bone marrow stem cell disorders are shown. AML = acute myeloblastic leukemia; PV = polycythemia vera; IMF = idiopathic myelofibrosis; CML = chronic lymphoblastic leukemia; CNL = chronic neutrophilic leukemia.
Table 17–2 WHO Cassification of Chronic Myeloproliferative Diseases
Chronic Myelogenous Leukemia
Historical Perspective
Definition
Incidence and Etiology
Pathogenesis
Figure 17-2 A metaphase spread from a patient with CML showing t(9;22). The abnormal chromosomes 9 and 22 are marked with arrows. The Philadelphia chromosome is the abnormal chromosome 22 on the right; the normal chromosome 22 is on the left.
Figure 17-3 Molecular basis of the Philadelphia (Ph) chromosome. (A) Sequence of molecular and biochemical events involved in generating the Ph chromosome and its phenotypic consequences. (B) Southern blot analysis of DNA from CML cells analyzed with a bcr probe to show clonal rearrangements in the bcr region. Lane 1: Ph-positive CML DNA showing one rearranged band; lane 2: Ph-positive CML as in lane 1 but with a different break point in the bcr region; lane 3: Ph-negative leukemic cell DNA showing no rearranged bcr; and lane 4: molecular weight markers.
Clinical Features
Laboratory Findings
Figure 17-4 Peripheral blood from a patient with CML shows numerous mature neutrophils along with bands, metamyelocytes, and myelocytes. Note the two basophils at the upper right and bottom center.
Figure 17-5 Pergeroid (pseudo-Pelger–Huët) neutrophil in CML. Note that the cytoplasm is mature and the nucleus is small and round with condensed chromatin. This cell can be mistaken for a myelocyte. A basophil is in the upper left corner.
Figure 17-6 A micromegakaryocyte with giant platelets, from the peripheral blood of a patient with CML.
Table 17–3 Laboratory Features at Presentation of Chronic Myelogenous Leukemia
Table 17–4 Chronologic Sequence of Appearance of Findings in CML
Figure 17-7 A bone marrow aspirate from a patient with CML in chronic phase shows granulocytic hyperplasia with orderly maturation through segmented neutrophils. Blasts are not significantly increased. A basophil is at upper left.
Figure 17-8 Pseudo-Gaucher cells (sea-blue histiocytes) in the marrow of a patient with CML.
Figure 17-9 This bone marrow biopsy from a patient with CML is stained with a reticulin (silver) stain to show reticulin fibrosis, which in this case is quite extensive.
Figure 17-10 Leukocyte alkaline phosphatase (LAP) is decreased in peripheral blood neutrophils in this patient with CML. Compare with Figure 17–11.
Figure 17-11 Leukocyte alkaline phosphatase (LAP) is increased in peripheral blood neutrophils in this patient with a leukemoid reaction related to infection.
Differential Diagnosis
Table 17–5 Diagnostic Criteria for Accelerated Phase of Chronic Myelogenous Leukemia
Table 17–6 Diagnostic Criteria for Blast Phase of Chronic Myelogenous Leukemia
Table 17–7 Differential Diagnosis Between Leukemoid Reaction and Chronic Myelogenous Leukemia
CHRONIC NEUTROPHILIC LEUKEMIA
CHRONIC EOSINOPHILIC LEUKEMIA
ATYPICAL CHRONIC MYELOID LEUKEMIA
Prognosis
Treatment
Case Study
QUESTIONS
ANSWERS
DISCUSSION
Questions
SUMMARY CHART
REFERENCES
Chapter 18 Chronic Myeloproliferative Disorders II: Polycythemia Vera, Essential Thrombocythemia, and Idiopathic Myelofibrosis
OBJECTIVES
Introduction to Myeloproliferative Disorders
Historical Perspective
Definition and Classification
Table 18–1 Differential Characteristics of the Chronic Myeloproliferative Disorders
Polycythemia Vera
Historical Perspective
Definition
Table 18–2 World Health Organization Classifications
Table 18–3 Features of Polycythemia Vera, Secondary Hypoxic Polycythemia, and Relative Erythrocytosis
Incidence
Table 18–4 PVSG Criteria for Diagnosis of Polycythemia Vera
Pathogenesis
Table 18–5 WHO Criteria for Diagnosis of Polycythemia Vera
Clinical Features
Laboratory Findings
Figure 18-1 Peripheral blood smear seen in polycythemia vera. Note hypochromia and increased cellularity. (magnification ×400)
Figure 18-2 Bone marrow showing panhyperplasia in polycythemia vera. Note increased number of megakaryocytes (arrows). H&E stain (low power)
Figure 18-3 Leukocyte alkaline phosphatase (LAP) stain of peripheral blood showing increased activity in polycythemia vera (red staining). LAP negative stain in CML (bottom).
Differential Diagnosis
Treatment
Essential Thrombocythemia
Historical Perspective
Definition
Table 18–6 Updated PVSG Criteria for the Diagnosis of Essential Thrombocythemia
Incidence
Pathogenesis
Table 18–7 WHO Criteria for Diagnosis of Essential Thombocythemia
Clinical Features
Laboratory Findings
Figure 18-4 Essential thrombocythemia, peripheral blood megakaryocyte and numerous platelets.
Figure 18-5 Essential thrombocythemia, bone marrow. Note increased megakaryocytes.
Differential Diagnosis
Table 18–8 Causes of Reactive Thrombocytosis
Table 18–9 Differentiating Features Between Essential Thrombocytosis and Reactive Thrombocytosis
Treatment
Idiopathic Myelofibrosis
Historical Perspective
Definition
Table 18–10 The American Polycythemia Vera Study Group’s Definition of Chronic Idiopathic Myelofibrosis (CIMF)
Table 18–11 Updated Cologne Criteria for IMF Diagnosis and Staging
Incidence and Etiology
Clinical Features
Figure 18-6 Hepatosplenomegaly, a characteristic finding in patients with idiopathic myelofibrosis with myeloid metaplasia (IMF/MM).
Figure 18-7 Extramedullary hematopoiesis in the liver of a patient with IMF.
Laboratory Findings
Table 18–12 Lille System of Prognostic Factors Predicting Survival for IMF Patients
Figure 18-8 Leukoerythroblastosis, teardrop poikilocytosis, and abnormal platelet morphology associated with idiopathic myelofibrosis. A. Leukoerythroblastosis. Note the myeloblast at the large arrow and the numerous nucleated red blood cells at the small arrows. B. Teardrop poikilocytosis. C. Dwarf megakaryocyte (or micromegakaryocyte). This pathologic alteration of a megakaryocyte may be found in any of the myeloproliferative disorders. Although often difficult to distinguish from cells of other lineages, observation of the marked cytoplasmic granularity and further comparison of this cytoplasm to that of other platelets present on the peripheral smear will aid in identification. D. Dwarf megakaryocyte. The cell at the pointer displays cytoplasmic blebs or budding, which is another characteristic of a micromegakaryocyte. Also note the giant platelets present on this peripheral blood smear.
Figure 18-9 Teardrop-shaped cells (arrows): peripheral blood in a patient with myelofibrosis.
Figure 18-10 Micromegakaryocyte found on the peripheral blood smear of a patient with essential thrombocythemia.
Figure 18-11 Histopathology of the bone marrow in idiopathic myelofibrosis. A. Early hyperplastic state without fibrosis. B. Advanced stage with a conspicuous increase in reticulin fibers, a still hypercellular marrow, and a lymphoid infiltrate (arrows). C. Late osteosclerotic state with endophytic bone formation, a residual cluster of hematopoiesis, and large areas of fatty tissue. D. Moderate degree of reticulin fibers surrounding atypical megakaryocytes in early IMF. E. Coarse bundles of obvious collagen fibers encompassing a few hematopoietic elements in terminal stages of IMF. F. Clusters of pleomorphic megakaryocytes displaying an abnormal maturation and mitosis (arrowhead). A-C, magnification ×140; D-F, ×350. A–C and F, periodic acid-Schiff (PAS) stain; D and E, Gomori’s silver impregnation.
Differential Diagnosis
Table 18–13 Differential Diagnosis of Myelofibrosis
Treatment
Table 18–14 The 2008 World Health Organization Diagnostic Criteria for Primary Myelofibrosis*
Case Study 1
COMMENT
QUESTIONS
ANSWERS
Case Study 2
COMMENT
QUESTIONS
ANSWERS
Case Study 3
COMMENT
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 19 Myelodysplastic Syndromes
OBJECTIVES
Introduction
Epidemiology—Etiology—Pathogenesis
MDS: Clonal Proliferative Diseases
CELL OF ORIGIN OF THE NEOPLASTIC CLONE
Figure 19-1 Schematic representation of marrow stem cell hierarchy showing the stem cell theoretically involved in the pathogenesis of myelodysplastic syndromes (MDS), according to cytogenetic and fluorescence in situ hybridization (FISH) analyses (see text). CFU-GEMM = colony-forming unit–granulocyte–erythrocyte–megakaryocyte-macrophage; BFU-E = burst-forming unit-erythroid; CFU-Meg = colony-forming unit-megakaryocyte; CFU-GM = colony-forming unit-granulocyte-macrophage.
Ineffective Hematopoiesis
Figure 19-2 Apoptosis of progenitor cells in bone marrow of patient with MDS. There is an increased production of inhibitory cytokines such as TNF-α and TGF-β in MDS patients. This may contribute to upregulation of Fas and co-expression of Fas-ligand (FL). This is one of the several pathways that could activate programmed cell death. Cysteine proteases (caspases) will be activated and will execute the cell.
Genetic Anomalies
Biologic Characteristics of Disease Progression
Morphological Characteristics of Blood and Bone Marrow
Definitions of Specific Morphological Characteristics
BLASTS
Table 19–1 Hallmark of Morphologic Findings in MDS
Figure 19-3 RAEB-t (bone marrow): blast cells (A), immature granulocytes with some degree of hypogranularity and vacuolization (B), and Pelgeroid hypogranular neutrophil (C).
SIDEROBLASTS
Lineage Dysplasias
DYSERYTHROPOIESIS
PERIPHERAL BLOOD
Figure 19-4 RAEB-t (bone marrow): two blasts with azurophilic rods, blast with an Auer rod (arrow).
Figure 19-5 5q– syndrome (peripheral blood): anisomacrocytosis and thrombocytosis.
Figure 19-6 RAEB (peripheral blood): anisomacrocytosis, neutrophil and macrothrombocyte (arrow).
Figure 19-7 RARS (peripheral blood): dimorphic macrocytosis. Note the small lymphocyte in the center which can be used as a micrometer.
Figure 19-8 An erythrocyte with basophilic stippling (A), one with a Howell-Jolly body (B), and two erythroblasts connected by internuclear bridging (peripheral blood) (C).
BONE MARROW
DYSGRANULOCYTOPOIESIS
PERIPHERAL BLOOD
Figure 19-9 RAEB-t (bone marrow): dysplastic multinucleated erythroblasts with asynchronous maturation.
Figure 19-10 An erythroblast with basophilic stippling (A), and two erythroblasts connected through internuclear bridging (bone marrow) (B).
Figure 19-11 RARS (bone marrow): vacuolization of erythroblasts.
Figure 19-12 RAEB (bone marrow). Note the pronormoblast (arrow) and the dyserythropoiesis and dysgranulopoiesis with nuclear hyposegmentation and hypogranulation.
Figure 19-13 RARS (bone marrow, Prussian blue stain): three-ringed sideroblasts (sideroblasts type III).
Figure 19-14 RARS (bone marrow, Prussian blue stain). Note the ringed sideroblasts.
Figure 19-15 RAEB (peripheral blood): pseudo-Pelger–Huët anomaly, mononuclear Stodtmeister type.
Figure 19-16 Bilobulated and monolobulated neutrophil (peripheral blood).
Figure 19-17 RAEB (peripheral blood): anisomacrocytosis and Pelgeroid neutrophil.
Figure 19-18 RAEB-t (peripheral blood): pseudo–Pelger–Huët and Stodtmeister neutrophils.
BONE MARROW
DYSMEGAKARYOCYTOPOIESIS
PERIPHERAL BLOOD
BONE MARROW
Classification of MDS Subtypes
Figure 19-19 RAEB (bone marrow): monolobular (dwarf) megakaryocyte.
Figure 19-20 RAEB (bone marrow): large megakaryocyte with multilobulated nuclei, some of them distinctly detached and small in size (botryoid, “pawn-ball” shape).
Figure 19-21 5q– syndrome (bone marrow): monolobulated micromegakaryocytes.
Figure 19-22 5q– syndrome (bone marrow): monolobulated micromegakaryocytes.
Figure 19-23 RA (bone marrow): hypogranular megakaryocyte.
Figure 19-24 RA (bone marrow): detached nuclei megakaryocyte.
Figure 19-25 RA (bone marrow): vacuolated immature megakaryocyte.
FAB Classification
Refractory Anemia
Table 19–2 FAB Classification and Criteria of MDS
Refractory Anemia with Ringed Sideroblasts
Refractory Anemia with Excess Blasts
Figure 19-26 RAEB (bone marrow): myeloblast (A) and Pelgeroid neutrophils (B).
Figure 19-27 RAEB (bone marrow): myeloblasts, mitosis, and Pelgeroid neutrophil.
Refractory Anemia with Excess Blasts in Transformation
Chronic Myelomonocytic Leukemia
Figure 19-28 RAEB-t (bone marrow): myeloblast with Auer rods.
Figure 19-29 RAEB-t (bone marrow): myeloblasts, megaloblastoid erythroblast with Howell-Jolly body (arrow).
Figure 19-30 CMML (peripheral blood): promonocytes–monocytes and Pelgeroid neutrophil (arrow).
Figure 19-31 CMML (bone marrow): myeloblasts, monocytes, and promonocytes.
WHO Classification3,38
Table 19–3 WHO Classification and Criteria of MDS
Table 19–4 WHO Classification of the Myelodysplastic Myeloproliferative Diseases
Laboratory Features
Cytochemistry
Table 19–5 Diagnostic Criteria for Chronic Myelomonocytic Leukemia (CMML)
Figure 19-32 RA (peripheral blood, peroxidase stain): neutrophil, positive reaction.
Figure 19-33 RA (bone marrow, peroxidase stain): pseudo-Pelger–Huët-negative cells.
Figure 19-34 RAEB (bone marrow, PAS stain): erythroblasts, positive granules.
Bone Marrow Histology
Cytogenetics and Molecular Abnormalities
Figure 19-35 RA (peripheral blood, Kleihauer–Betke staining): positive RBCs, HbF.
Figure 19-36 RAEB (bone marrow, reticulin stain, magnification ×400): severe fibrosis.
Table 19–6 Abbreviations and Terminology Used in Describing Chromosomes and Their Abnormalities
Figure 19-37 Karyotype GTG banding, resolution 450 bands, showing complex clonal abnormalities involving at least 8 chromosomes within the same cell, including del(5)(q13q33), −7, del(20)(q11.2).
THE 5q– SYNDROME
Figure 19-38 Karyotype (GTG banding) showing the t(5;12)(q33;p13) translocation and a loss of chromosome 7 in a case of CMML.
Figure 19-39 The 5q– syndrome. Several genes related to hematopoiesis are localized on 5q. A. The interstitial deletion may comprise any region located between band 5q13 and 5q33. B. The band 5q31 is consistently deleted in most patients.
Figure 19-40 Karyotype with GTG banding, 450 bands resolution, showing the interstitial deletion of chromosome 5 → 46,XY,del(5) (q13q33) characteristically found in the 5q– syndrome.
THE FUTURE OF CYTOGENETICS
Immunology
Figure 19-41 Fluorescence in situ hybridization (FISH), +8 in neutrophil.
Figure 19-42 RAEB-2, spectral karyotyping (SKY): complex karyotype with numerous chromosomal translocations and trisomy 22.
Evaluation of Progenitor Cell Growth in Semisolid Media
Cellular Dysfunction
RED BLOOD CELLS
WHITE BLOOD CELLS
PLATELETS
Secondary Myelodysplastic Syndromes
Myelodysplastic Syndromes in Children
Clinical Features
Evolution and Prognosis
Diagnostic Problems in MDS
MDS with Hypoplastic Marrow
MDS with Severe Myelofibrosis
MDS with Thrombocytosis
Figure 19-43 Survival and leukemic progression for each subgroup of MDS.
Table 19–7 International Prognostic Scoring System (IPSS) for MDS Based on Percentage of Bone Marrow Blasts, Karyotype, and Blood Cytopenias
MDS with Features of Chronic Myelogenous Leukemia
Figure 19-44 Overlap between MDS and other hematologic disorders. AA = aplastic anemia; MPD = myeloproliferative disorder; PNH = paroxysmal nocturnal hemoglobinuria; AML = acute myeloid leukemia.
MDS versus Acute Erythroid Leukemia
Treatment
Figure 19-45 Algorithm to distinguish MDS from different types of acute myeloid leukemia (AML) according to FAB criteria.
Figure 19-46 Treatment options for MDS patients according to IPSS.
Supportive Care and Hematopoiesis-Improving Therapies
Therapies Oriented Toward Improving Survival
Case Study
Questions
SUMMARY CHART
REFERENCES
Chapter 20 Chronic Lymphocytic Leukemia and Related Lymphoproliferative Disorders
OBJECTIVES
Introduction
Chronic Lymphocytic Leukemia/Small Lymphocytic Lymphoma
Table 20–1 Lymphoproliferative Disorders
Figure 20-1 Photomicrograph of peripheral blood smear from a patient with chronic lymphocytic leukemia (CLL). Note the characteristic mature-appearing lymphocyte morphology with hypercondensed nuclear chromatin creating a “soccer-ball” pattern. Two smudge cells are also seen. Note the lack of platelets in this thrombocytopenic patient. A, denotes lymphoblasts; B, lymphocytes; and C, smudge cells.
Etiology and Pathophysiology
Figure 20-2 Photomicrograph of bone marrow aspirate smear from a patient with CLL. Note monotonous appearance of mature-appearing lymphocytes with condensed nuclear chromatin.
Figure 20-3 Severe generalized herpes zoster with a varicelli-form rash in a patient with CLL.
Immunologic Features and Methods for Studying Lymphocytes
Figure 20-4 The pathophysiology of CLL. The three major processes that typically interact are marrow replacement by long-lived lymphocytes, hypersplenism, and autoimmunity. ITP = immune thrombocytopenic purpura.
Table 20–2 Methods Used to Study Lymphocytes in Lymphoproliferative Disorders
Table 20–3 Cluster Differentiation (CD) Markers and Clinical Application
Figure 20-5 Hypothetical scheme of marker expression and gene rearrangement during normal B-cell ontogeny.
Figure 20-6 ▪ Postulated B-lymphocyte development scheme. Lymphoblasts undergo several maturation steps in the microenvironment of the bone marrow (BM), giving rise to mature (virgin or naive) lymphocytes. Naive B lymphocytes enter blood vessels (BV) to reach lymph nodes and populate the mantle zone of the lymphoid follicles. These cells differentiate into centroblasts (large, noncleaved follicular center cells) in the dark zone of the germinal center. Centroblasts undergo rearranged-immunoglobulin gene hypermutation and develop into centrocytes (cleaved follicular center cells). Centrocytes that bind antigen on the surface of the follicular dendritic cells survive, whereas those that fail to bind die by apoptosis. Surviving centrocytes in the light zone of the germinal center may differentiate into monocytoid B cells, which populate the marginal zone of the lymphoid follicle, and plasma cells, which reenter the bone marrow and memory cells. Memory cells actively recirculate between the blood, lymph, and lymphoid organs.
Table 20–4 Immunophenotypic Features and Genetic Abnormalities of B-Lymphoproliferative Disorders
Figure 20-7 Flow cytometric analysis of chronic lymphocytic leukemia. Leukemia bone marrow lymphocytes are gated by CD45-scattered analysis. The plot of CD5 versus CD19 is used to demonstrate dual-positive neoplastic lymphocytes with weak CD19 fluorescence intensity. The plot of CD23 versus FMC7 illustrates positive staining of the cells for CD23 but no staining of FMC7. The cells are then plotted for both CD19 versus κ and CD19 versus λ, showing κ light-chain clonality with a 15:1 ratio of κ to λ for the dual CD19+, CD5+ cells.
Clinical Features
Figure 20-8 Hypothetical scheme of marker expression and gene rearrangement during T-cell ontogeny. TCR = T-cell receptor.
Figure 20-9 Flow cytometric analysis of T-prolymphocytic leukemia (T-PLL). Leukemic peripheral blood lymphocytes are gated by CD45-side scattered analysis. Comparison of CD5 versus CD19 shows a predominance of CD5-positive cells (upper panel). Comparisons of CD3 versus CD4 and CD3 versus CD8 demonstrate a predominance of CD4+ T lymphocytes (middle panel). CD4+ T lymphocytes also express CD7 antigen (lower panel).
Laboratory Features
Figure 20-10 Photomicrograph of bone marrow biopsy section showing involvement by CLL, nodular pattern, characterized by distinct, randomly distributed aggregates of small lymphocytes.
Figure 20-11 A. Photomicrograph of bone marrow biopsy section showing involvement by CLL, diffuse pattern. The entire bone marrow space between bone trabeculae is replaced by small lymphocytes. B. Immunohistochemistry ZAP-70 expression in CLL cells indicates poor prognosis. Flow cytometry can also be used to detect ZAP-70 expression.
TUMOR AND MICROENVIRONMENT
Chromosomal Abnormalities
Table 20–5 Staging Systems for Chronic Lymphocytic Leukemia
Clinical Course, Prognostic Factors, and Staging
Table 20–6 Leukemia-Cell Parameters Associated with Aggressive Disease Independent of the Disease Stage
Treatment
Table 20–7 Treatment Options for Lymphocytic Leukemia
Differential Diagnosis
Figure 20-12 Peripheral blood smears of various lymphoproliferative disorders. A. Chronic lymphocytic leukemia (CLL). B. Acute lymphoblastic leukemia (ALL). C. Prolymphocytic leukemia (PLL). D. CLL with occasional prolymphocyte. E. Small lymphocytic lymphoma (SLL) in leukemic phase. F. Small cleaved-cell lymphoma (SCCL). G. Hairy-cell leukemia (HCL). H. Sézary syndrome. I. Adult T-cell leukemia/lymphoma. J. T-gamma lymphocytosis with large granular lymphocytes. K. Infectious mononucleosis with atypical lymphocytes. L. Plasma cell dyscrasia.
Table 20–8 Morphological and Immunological Characteristics of Lymphoproliferative Disorders
Figure 20-13 α-Naphthol acetate esterase (ANAE) stain showing localized “dotlike” positivity in two T lymphocytes.
Figure 20-14 Small lymphocytic lymphoma (SLL), lymph node.
Figure 20-15 Flow cytometric analysis of mantle cell lymphoma in leukemia phase. Leukemic marrow lymphocytes are gated by CD45-scattered analysis. The plot of CD5 versus CD19 demonstrates dual positive neoplastic lymphocytes. In contrast to CLL, the neoplastic lymphocytes in mantle cell lymphoma show positive staining for FMC7 but no staining of CD23. Cyclin D1 immunohistochemical stain was positive.
Figure 20-16 Small cleaved-cell lymphoma (SCCL), lymph node.
Figure 20-17 Hairy-cell leukemia (HCL), bone marrow aspirate.
Figure 20-18 Tartrate-resistant acid phosphatase (TRAP) stain of peripheral blood showing positivity in hairy cell and no staining in neutrophilic.
Figure 20-19 Flow cytometric analysis of hairy-cell leukemia. Large mononuclear cells in leukemic bone marrow are gated by CD45–side scattered analysis (left lower panel). The plots of CD22 versus CD11c and CD20 versus CD103 demonstrate predominance of dual positive B cells (upper panel). The plots for both CD20 and λ show λ light-chain clonality (middle panel). The B cells are also reactive with anti-CD25 (anti-interleukin-2 receptor) (lower right-sided histogram).
Figure 20-20 Infiltration of the epidermis and upper dermis by lymphocytes, many with convoluted (cerebriform) nuclei, histiocytes, and formation of Pautrier microabscesses, characteristic of the cutaneous T-cell lymphoma mycosis fungoides.
Table 20–9 Typical Features of Chronic Lymphocytic Leukemia
Case Study 1
Case Study 2
Questions
SUMMARY CHART
REFERENCES
Chapter 21 The Lymphomas
OBJECTIVES
Introduction
Hodgkin Lymphoma
Etiology and Pathogenesis
Figure 21-1 Reed–Sternberg cell (arrows). Prototypic cell of Hodgkin lymphoma (HL)is characterized by large size, abundant eosinophilic cytoplasm, multinucleation, and inclusion-like macronuclei.
Pathology
NODULAR LYMPHOCYTE-PREDOMINANT HODGKIN LYMPHOMA
Table 21–1 Diseases or Disorders in Which Reed–Sternberg-like Cells Have Been Reported
Figure 21-2 A. Mononuclear Hodgkin cell (arrows). Variant RS cell with single monolobed nucleus and inclusion-like macronucleolus. B. L&H (popcorn) cell (arrows). Characteristic cell of nodular lymphocytic-predominant Hodgkin lymphoma (NLPHL). C. Lacunar cell. Large cell with artifactual clear space (lacuna) due to formalin fixation that surrounds the nucleus. Wisps of cytoplasm are visible in the space. D. Pleomorphic Reed–Sternberg cell. Large cell with bizarre multinucleated/multilobated nucleus.
Table 21–2 WHO Classification of Hodgkin Lymphoma
CLASSIC HODGKIN LYMPHOMA
Figure 21-3 Nodular lymphocyte predominant Hodgkin lymphoma. Low-power microscopic view showing vaguely nodular pattern.
NODULAR SCLEROSIS HODGKIN LYMPHOMA
MIXED CELLULARITY HODGKIN LYMPHOMA
Table 21–3 HL versus NHL
Figure 21-4 Classic Hodgkin lymphoma, nodular sclerosis subtype. Low-power microscopic view demonstrating nodules of cells produced by orderly bands of collagen.
Figure 21-5 Grouped lacunar cells in nodular sclerosis Hodgkin lymphoma (NSHL) showing central area of necrosis.
Figure 21-6 Mixed cellularity Hodgkin lymphoma. Easily identified Reed–Sternberg cells and RS variants are seen admixed with small lymphocytes, plasma cells, eosinophils, and histiocytes.
LYMPHOCYTE-RICH HODGKIN LYMPHOMA
LYMPHOCYTE DEPLETION HODGKIN LYMPHOMA
UNCLASSIFIED HODGKIN LYMPHOMA
HISTOLOGIC PROGRESSION
Clinical Features
Diagnostic Evaluation and Staging
Treatment and Prognosis
Non-Hodgkin Lymphoma
Etiology and Pathogenesis
Table 21–4 Staging Workup for Hodgkin Lymphoma
Table 21–5 Cotswold Staging
Table 21–6 “B” Symptoms of Hodgkin Lymphoma
Table 21–7 Conditions Associated with Increased Risk of Developing Non-Hodgkin lymphoma (NHL)
Pathology
Table 21–8 Common Chromosomal Abnormalities Associated with Malignant Lymphomas
Figure 21-7 Reciprocal translocation t(8;14) (q24;q32) is seen in majority of cases of Burkitt’s lymphoma as well as some non-Burkitt’s high-grade lymphomas. A reciprocal translocation of genetic material occurs between chromosomes 8 and 14. A distal portion of the long arm of chromosome 8, containing the c-myc oncogene, is translocated to a site adjacent to the immunoglobulin heavy-chain locus on chromosome 14.
Figure 21-8 A. Follicular growth pattern of lymphoma. Neoplastic cells are organized into closely packed nodules (follicles). B. Diffuse growth pattern of lymphoma. Neoplastic cells are arranged in sheet-like fashion without follicular organization.
Table 21–9 World Health Organization (WHO) Classification
Figure 21-9 Anatomical compartments of the lymph nodes.
Figure 21-10 Maturation (ontogeny) of (A) B lymphocytes and (B) T lymphocytes in relation to the pertinent anatomical compartments.
B-CELL LYMPHOMAS
MATURE B-CELL LYMPHOMAS
Figure 21-11 A. Architecture of benign (reactive) follicle. Secondary follicle consisting of well developed mantle zone (1) and germinal center that is polarized into light (2) and dark (3) zones. Tingible body macrophages (arrows) are prominent in the dark zone. B. Malignant follicle of follicular lymphoma. Note poorly defined/absent mantle zone and lack of polarization of the germinal center into light and dark zones.
Figure 21-12 A. Follicular lymphoma, WHO grade 1. Malignant follicles are characterized by a predominance of centrocytes. B, Follicular lymphoma, WHO grade 3. Malignant follicles are characterized by a predominance of centroblasts.
Table 21–10 Indolent B-cell Lymphomas: Morphological Features
Table 21–11 Low-grade B-cell Lymphomas: Immunophenotypic Profile
Table 21–12 Low-grade B-cell Lymphomas: Clinical Features
Table 21–13 Low-grade B-cell Lymphomas: Large-cell Transformation
Figure 21-13 A. Mantle cell lymphoma. Neoplastic cells are “intermediate” between the small uniform lymphocytes of SLL and the centrocytes of FL. B. Majority of nuclei of MCL express BCL-1 protein (red-brown reaction product). Immunoperoxidase stain with monoclonal antibody to BCL-1/cyclin D1.
Figure 21-14 Endoscopic view of large polyp of the bowel in a case of lymphomatous polyposis (mantle cell lymphoma).
Figure 21-15 A. Extranodal marginal zone B cell (MALT) lymphoma of the stomach. MALT lymphoma (1) infiltrates the gastric mucosa and extends into gastric epithelium (2), Reactive follicles (3) are commonly present. B. Characteristic lymphoepithelial lesions (arrows) of gastric MALT lymphoma are composed of clusters of small, atypical lymphocytes (CD20 positive B cells) within the glandular epithelium.
Figure 21-16 A. Extranodal marginal zone B cell (MALT) lymphoma of the parotid gland. Neoplastic cells (red arrows) surround and infiltrate the metaplastic epithelium (yellow arrows) of the parotid gland. B. Detailed view of lymphoepithelial lesions (red arrows) involving metaplastic epithelium (yellow arrows) in a case of MALT lymphoma of the parotid gland.
Figure 21-17 Splenic marginal zone B-cell lymphoma. White pulp nodules (1) show expansion of the marginal zone (2) by a population of small monoclonal B cells which express the phenotype of splenic marginal zone cells. Neoplastic cells infiltrate the red pulp (3).
Figure 21-18 A. Small lymphocytic lymphoma. Diffuse effacement of lymph node by proliferation of small lymphocytes. Pale areas are growth centers (arrows). B. Cytological detail of small lymphocytes of SLL. Cells have scant cytoplasm with uniform nuclei, coarsely clumped chromatin and indistinct nucleoli. C. Growth center of SLL is composed of prolymphocytes and paraimmunoblasts, cells that are larger than the typical small lymphocyte of SLL
Figure 21-19 Diffuse large B-cell lymphoma. This common lymphoma is composed of a mixture of large B cells that exhibit centroblastic, immunoblastic, and pleomorphic morphology.
Figure 21-20 A. Burkitt lymphoma. Small uniform cells (yellow arrows) with moderate amount of cytoplasm, round nuclei and multiple small nuclei are admixed with cellular debris and tingible body macrophages (red arrow). B. High proliferation rate of Burkitt lymphoma detected by high percentage of nuclei showing positive staining for MIB-1/Ki-1 (red-brown reaction product). Immunoperoxidase stain with monoclonal antibody to the proliferation antigen MIB-1/Ki-1.
Figure 21-21 Burkitt’s lymphoma before (left) and after treatment (right).
Figure 21-22 Intravascular large cell lymphoma. Small vessels (capillaries) are occluded by atypical large cells (arrows) without extension into the parenchyma of the involved organ.
Figure 21-23 Primary effusion lymphoma (PEL). Cytocentrifuge preparation of PEL showing large atypical mononuclear cells that usually have immunoblastic features.
PRECURSOR B LYMPHOBLASTIC LYMPHOMA
T-CELL AND NATURAL KILLER (NK) CELL LYMPHOMAS
MATURE T AND NK CELL LYMPHOMAS
Figure 21-24 A. Precursor B lymphoblastic lymphoma. Cells have scant cytoplasm, finely dispersed (“blastic”) chromatin, and brisk mitotic activity. B. The neoplastic nuclei of lymphoblastic lymphoma express TdT (red-brown reaction product). Immunoperoxidase stain with polyclonal antibody to TdT.
Table 21–14 Mature T/NK Cell Lymphomas: Mode of Presentation
Figure 21-25 Peripheral T-cell lymphoma (PTCL), unspecified. PTCL usually consists of a mixture of atypical lymphoid cells which are variable in size, giving a polymorphous appearance.
Figure 21-26 A. Systemic anaplastic large cell lymphoma. Pleomorphic cells that vary in size and shape. “Hallmark” cells are readily apparent (arrows). B. Detailed view of several “hallmark” cells (arrows) showing kidney or horseshoe-shaped nuclei and juxtanuclear eosinophilic inclusion-like zone.
Figure 21-27 A. Angioimmunoblastic T-cell lymphoma. Mixture of lymphocytes, some of which are atypical, dendritic cells, and thick-walled high endothelial venules. B. Clusters of medium to large cells with clear cytoplasm (arrows) are characteristic of angioimmunoblastic T-cell lymphoma.
Figure 21-28 A. Myosis fungoides. Plaque lesion of MF showing band-like infiltrate (arrows) of dermis by atypical lymphocytes that extent into the overlying epidermis. B. Pautrier microabscess. Cluster of atypical lymphocytes within the epidermis.
Figure 21-29 Myosis fungoides, tumor stage. Tumor nodules are produced by massive local infiltrates of the skin by the characteristic cerebriform cells of mycosis fungoides.
Figure 21-30 Sézary cells in the peripheral blood from a patient with Sézary syndrome.
Figure 21-31 A. Primary cutaneous anaplastic large cell lymphoma. Large atypical cells with marked nuclear pleomorphism infiltrate the debris. B. Detail view of cytological features of primary cutaneous anaplastic large cell lymphoma.
PRECURSOR T-LYMPHOBLASTIC LYMPHOMA
HISTIOCYTIC AND DENDRITIC CELL TUMORS
HISTIOCYTIC TUMORS
DENDRITIC CELL TUMORS
DIAGNOSTIC EVALUATION
Table 21–15 T/NK with Primary Extranodal Presentation
Table 21–16 T/NK Lymphomas with Primary Leukemic/disseminated Presentation
Figure 21-32 Dendritic cell sarcoma. An atypical spindle cell proliferation characterizes this tumor which expresses a dendritic cell phenotype.
BENIGN VERSUS MALIGNANT
Figure 21-33 B-cell monoclonality detected by flow cytometry A. Overlay of two single-color (one-parameter) histograms demonstrating a monoclonal lambda population. B. Two-color (dualparameter) histogram with simultaneous analysis for κ (y-axis) and λ (x-axis) light chains. The dense cluster of events in the lower right quadrant indicates a monoclonal λ population.
Figure 21-34 T-cell monoclonality demonstrated by Southern blot analysis: Abnormal bands designated by small bars indicate clonal rearrangement of the T-cell receptor gene. M = molecular weight markers; C = control DNA to locate position of germ-ling (nonrearranged) bands; P = patient DNA; 1 = BamHI digest; 2 = fcoRI digest; 3 = HindIII digest.
Figure 21-35 Fluorescent in Situ hybridization (FISH) analysis of B-cell lymphoma using two color break-apart probe. Arrowed cells show 1 normal fusion signal and 2 abnormal break-apart signals indicating breakage within the IgH (heavy chain) gene switch region.
LYMPHOMA VERSUS NONLYMPHOMA
T-CELL VERSUS B-CELL LYMPHOMA
HODGKIN LYMPHOMA (HL) VERSUS NON-HODGKIN LYMPHOMA (NHL)
Table 21–17 Antibody Panel for Distinguishing Among the Four Major Categories of Cancer
Figure 21-36 Expression of CD30 antigen by Reed–Sternberg cells and variants in CHL (red-brown reaction product). Three patterns of staining are present—cytoplasmic membrane, diffuse cytoplasmic, and juxtanuclear dot (“Golgi”). Immunoperoxidase staining using monoclonal antibody to CD30 antigen.
Staging
Figure 21-37 Expression of ALK-NPM protein in cytoplasm of neoplastic cells of ALCL (red-brown reaction product). Immunoperoxidase staining using monoclonal antibody to ALK-NPM protein (ALK-1).
Figure 21-38 Rosette of CD3+ T cells (red-brown reaction product) surrounding L&H (popcorn) cell of NLPHD. Immunoperoxidase stain using monoclonal antibody to CD3 antigen.
Treatment and Prognosis
Table 21–18 Staging Classification of Burkitt Lymphoma
Case Study
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 22 Multiple Myeloma and Related Plasma Cell Disorders
OBJECTIVES
Overview
Plasma Cell Development
Table 22–1 Classification of Monoclonal Gammopathies
Immunoglobulin
Structure and Function
Figure 22-1 Development of plasma cells; on encounter with an antigen (indicated by week 1), naive marginal-zone B cells differentiate into plasma cells. CSR = class-switch recombination.
Figure 22-2 Structure of the basic immunoglobulin unit. VH = variable region heavy chain; VL = variable region light chain; CH = constant region heavy chain; CL = constant region light chain.
Laboratory Recognition and Measurement
ELECTROPHORESIS
IMMUNOFIXATION
QUANTITATIVE IMMUNOGLOBULINS
FREE SERUM LIGHT CHAINS
BENCE-JONES PROTEINURIA
Clinical Consequences of Increased Monoclonal Immunoglobulin
HYPERVISCOSITY SYNDROME
Figure 22-3 Patterns of serum protein electrophoresis showing characteristic patterns of normal serum, monoclonal M-spike, polyclonal antibody production, IgM M-spike, and the absence of antibody production seen in hypogammaglobulinemia.
DECREASED PRODUCTION OF NORMAL IMMUNOGLOBULINS
CRYOGLOBULINS
Figure 22-4 Concentrations of κ and λ free-light-chains in sera from healthy individuals and those with myeloma.
Monoclonal Gammopathy of Undetermined Significance
Figure 22-5 Actuarial risk of full progression by serum monoclonal protein (M-protein) value at diagnosis of monoclonal gammopathy of undetermined significance (MGUS) in persons from southeastern Minnesota.
Multiple Myeloma
Figure 22-6 Progression from monoclonal gammopathy of undetermined significance to smoldering myeloma to malignant myeloma.
Epidemiology
Etiology
Pathophysiology
PLASMA CELL EXPANSION
Figure 22-7 SEER incidence and U.S. death rates from myeloma, by race and sex.
BONE MARROW STROMA
BONE DISEASE IN MULTIPLE MYELOMA
Figure 22-8 Mechanisms of disease in multiple myeloma. Skeletal destruction, abnormal immunoglobulin production, marrow failure, and decreased production of normal immunoglobulin all play a role.
HYPERCALCEMIA
Clinical Features
Diagnostic Workup
Figure 22-9 Bone marrow microenvironment. Effects of cytokines and transforming growth factors on the bone marrow (BM) microenvironment in patients with multiple myeloma (MM). Bone marrow stromal cells (BMSC) secrete: IL-6, VEGF, SDF-1α, and RANKL. This IL-6 secreted in the BM microenvironment promotes the binding of MM cells to the BMSCs augmenting more secretion of IL-6 and other cytokines from the BMSCs and MM cells. For example, TGFβ, TNFα, and VEGF from malignant myeloma cells enhance IL-6 secretion from BMSCs. The MM cells secrete IL-6, VEGF, bFGF, TNFα, TGFβ, and MIP-1α. The IL-6, VEGF, bFGF, secreted by the MM cells and the VEGF from BMSCs initiate angiogenesis in bone marrow endothelial cells (BMECs). The cytokines MIP-1α secreted by the MM cells and RANKL secreted by BMSCs initiate or activate osteoclast formation, leading to bone reabsorption and bone destruction in MM patients. Osteoclasts also secrete IL-6, inducing the growth of the malignant clone of MM cells. IL-6 = Interleukin 6; VEGF = vascular endothelial growth factor; SDF-1α = stromal cell-derived factor α RANKL = receptor activator of nuclear factor α–β ligand; bFGF = basic fibroblast growth factor; TNFβ = tumor necrosis factor; TGFα = transforming growth factor β MIP-1α = macrophage inflammatory protein alpha.
Figure 22-10 Bone destruction in multiple myeloma.
Table 22–2 Signs and Symptoms of Multiple Myeloma
Laboratory Studies
COMPLETE BLOOD COUNT AND PERIPHERAL BLOOD SMEAR
Table 22–3 Evaluation of Newly Diagnosed Multiple Myeloma Patients
CHEMISTRY STUDIES
BONE MARROW EXAMINATION
Figure 22-11 Peripheral blood showing marked rouleaux formation. Note the “stacked-coin” appearance of the red cells.
Figure 22-12 Bone marrow aspirate showing atypical and binucleated plasma cells and Russell bodies (arrow).
Figure 22-13 Bone marrow biopsy sample showing replacement of marrow by plasma cells.
Figure 22-14 Flame cell, sometimes associated with IgA myeloma.
CYTOGENETICS IN MULTIPLE MYELOMA
Figure 22-15 PCs with both the normal and abnormal pattern of hybridization. The depicted PCs show (A) a cell with the normal configuration of 2 pairs of signals for the probes localizing to the centromere 17 (CEP17; aqua) and the 17p13.1 (LSI p53) (red) probe. (B) A cell with deletion of 17p13.1. There are two green signals arising from the centromeric probe but only one red signal from the p53 locus probe. (C) A normal configuration of probes used to detect the t(14;16)(q32;q23). The locus-specific 14q32 probes are labeled in green, and the 16q23 probes are labeled in red. (D) A cell with fusion of probes for 14q32 (green) and 16q23 (red).
Figure 22-16 Overall survival of patients stratified by the hierarchic classification model. The survival curves show clear separation of patients into the good, intermediate, and poor prognosis category. The poor prognosis group includes patients with −17p13.1, t(4;14)(p13;q32), and/or t(14;16)(q32;q23); the intermediate prognosis group includes those patients with δ13; and the good prognosis group includes remaining patients, including those with the t(11;14)(q13;q32) and those with normal karyotype.
Table 22–4 Common Chromosomal Translocations in Multiple Myeloma
RADIOLOGICAL INVESTIGATIONS
PLAIN RADIOGRAPH—X-RAY EXAMINATION
Figure 22-17 Extensive lytic skull lesions in a patient with multiple myeloma.
Figure 22-18 Radiographic film of the left humerus of a patient with multiple myeloma. Areas with severe cortical bone destruction may be fractured by everyday activities such as lifting or walking (pathologic fractures).
COMPUTED TOMOGRAPHY SCANNING—CT SCAN
NUCLEAR MEDICINE—SCINTIGRAPHY
MAGNETIC RESONANCE IMAGING
POSITRON EMISSION TOMOGRAPHY
DUAL ENERGY X-RAY ABSORPTIOMETRY SCANNING
Figure 22-19 MRI showing compression fracture at L-2 (red arrow) and focal plasmacytoma (white arrows and circles).
Figure 22-20 PET-scan showing metabolic activity in multiple plasmacytomas throughout the skeleton.
Diagnostic Criteria
Table 22–5 Diagnostic Criteria for Multiple Myeloma
Staging
Treatment
Table 22–6 Durie Salmon Clinical Myeloma Staging System
Table 22–7 New International Staging System
Atypical Variants of Plasma Cell Syndromes
Smoldering Myeloma
Solitary Plasmacytoma of the Bone
Extramedullary Plasmacytoma
Plasma Cell Leukemia
Nonsecretory Myeloma
Figure 22-21 Peripheral blood in plasma cell leukemia showing presence of circulating plasma cells.
POEMS Syndrome
Supportive Care in Myeloma Patients
Waldenström’s Macroglobulinemia
Figure 22-22 Plasmacytoid lymphocytes in marrow aspirate from a patient with Waldenström’s macroglobulinemia.
Figure 22-23 Arm of patient with cryoglobulinemic purpura. Note the skin manifestations.
Amyloidosis
Figure 22-24 Amorphous amyloid deposits replacing normal liver architecture.
Light Chain Deposition and Heavy Chain Diseases
Case Study 1 Multiple Myeloma
QUESTIONS
ANSWERS
Case Study 2: Monoclonal Gammopathy of Undetermined Significance (MGUS)
QUESTIONS
ANSWERS
Case Study 3: Waldenström’s Macroglobulinemia
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 23 Lipid (Lysosomal) Storage Diseases and Histiocytosis
OBJECTIVES
Lipid (Lysosomal) Storage Diseases
Figure 23-1 Schematic structure of globoside and ganglioside to show site of action of the several catabolic enzymes, which, when defective, result in one of the storage diseases.
Table 23–1 Lipid Storage Diseases
Table 23–2 General Characteristics of Lipid Storage Diseases
Gaucher’s Disease
HISTORICAL PERSPECTIVES
Figure 23-2 Gaucher’s cell, bone marrow aspirate.
CLASSIFICATION AND CLINICAL FEATURES
Table 23–3 Gaucher’s Disease: Chronology
Table 23–4 The Common Clinical Triad of Gaucher’s Disease
TYPE I: ADULT GAUCHER’S DISEASE (CHRONIC NON-NEURONOPATHIC)
Table 23–5 Gaucher’s Disease: Clinical Subtypes
TYPE II: INFANTILE GAUCHER’S DISEASE (ACUTE OR MALIGNANT NEURONOPATHIC)
Table 23–6 Radiological Classification of Gaucher Bone Pathology
Figure 23-3 Anteroposterior radiograph of the knee shows diffuse mottled increased density of the distal femur and proximal tibia, characteristic of widespread bone infarction in Gaucher’s disease. The metaphyseal regions are broader than normal (arrow), resembling an Erlenmeyer flask deformity.
TYPE III: JUVENILE GAUCHER’S DISEASE (SUBACUTE NEURONOPATHIC)
LABORATORY DIAGNOSIS
Table 23–7 Clinical Presentations of Type I Gaucher’s Disease
Table 23–8 Comparison of Type IIIa and IlIb Gaucher’s Disease
PROGNOSIS
Table 23–9 Gaucher’s Disease: Laboratory Findings
Table 23–10 Disorders in Which “Pseudo-Gaucher’s Cells” Have Been Described
Table 23–11 Most Frequently Reported Hematologic Malignancies in Gaucher’s Disease
TREATMENT
Niemann–Pick Disease
CLASSIFICATION AND CLINICAL FEATURES
TYPE A
Table 23–12 Immunologic Abnormalities Described in Gaucher’s Disease
Table 23–13 General Characteristics of Niemann–Pick Disease
Figure 23-4 Niemann–Pick cell, bone marrow aspirate.
TYPE B
TYPE C
Table 23–14 Clinical Manifestations of Niemann–Pick Disease
LABORATORY DIAGNOSIS
Table 23–15 Comparison of Types A, B, and C Niemann–Pick Disease
PROGNOSIS AND TREATMENT
Tay–Sachs Disease
Figure 23-5 Tay–Sachs disease, vacuolated lymphocytes (also characteristic of Niemann–Pick disease).
CLINICAL FEATURES
LABORATORY DIAGNOSIS
Table 23–16 General Characteristics of Tay–Sachs Disease
PROGNOSIS AND TREATMENT
Mucopolysaccharidoses
Figure 23-6 Hurler’s anomaly.
CLASSIFICATION
CLINICAL FEATURES
Table 23–17 General Characteristics of Mucopolysaccharidoses
Table 23–18 Mucopolysaccharidoses (MPSs)
LABORATORY DIAGNOSIS
PROGNOSIS
TREATMENT
Histiocytosis
Sea-Blue Histiocyte Syndrome
Figure 23-7 Sea-blue histiocytes. Note the abnormally coarse azurophilic granules present in neutrophils, lymphocytes, and monocytes.
Other Histiocytic Disorders (Eosinophilic Granuloma, Hand–Schüller–Christian Disease, Letterer–Siwe Disease)
Table 23–19 General Characteristics of Sea-Blue Histiocytosis
Case Study
QUESTIONS
Table 23–20 Characteristics of Histiocytic Disorders
Questions
SUMMARY CHART
REFERENCES
PART 4 HEMOSTASIS AND INTRODUCTION TO THROMBOSIS
Chapter 24 Introduction to Hemostasis
OBJECTIVES
Platelets and Hemostatic Mechanisms
Overview
Figure 24-1 Hemostasis: A system in balance.
Table 24–1 Systems Involved in Maintaining Hemostasis
Vascular System
ADVANCED CONCEPTS
Table 24–2 Vessels and General Breach-Sealing Requirements
Table 24–3 Some Sources and Types of Bleeding
Table 24–4 Vessel Layer Composition and Function
Table 24–5 Actions of the Vascular System to Prevent Bleeding
Primary Hemostasis
Platelet Structure (Basic Information)
Table 24–6 Major Hemostasis-Related Substances Present in the Endothelium and/or Subendothelium and Their Function in Hemostasis
Table 24–7 Other Substances that Bind to Endothelial Cells and Their Role in Coagulation
Figure 24-2 Normal platelets: Wright-stained blood smear (peripheral blood).
Platelet Structure (Advanced)
Figure 24-3 Discoid platelets; (top) summary diagram of the platelet organelles; (bottom) transmission electron micrograph (TEM) of cross-sectioned platelets illustrating basic ultrastructure.
Figure 24-4 Internal anatomy of a stimulated platelet. Circumferential band of microtubules (MT) leads to reorganization of the internal structure of the platelet into three zones. The peripheral zone (PZ) is the region external to a circumferential band of microtubules (MT). The intermediate zone (IZ) (encircling arrows) includes the microtubules and the closely adjacent cytoplasmic material. The central zone (CZ) is internal to the microtubule band and contains many organelles such as granules (G), dense bodies (DB), dense tubular system (DTS), lysosomes (Ly), mitochondria (M), and many profiles of the open canalicular system (OCS). Magnification ×49,700.
PERIPHERAL ZONE
BASIC CONCEPTS
Table 24–8 Platelet Ultrastructural Zones
ADVANCED CONCEPTS
SOL–GEL ZONE
BASIC CONCEPTS
Table 24–9 Platelet Factors (PFs) 1 to 7
ADVANCED CONCEPTS
ORGANELLE ZONE
BASIC CONCEPTS
Table 24–10 Chemical Contents of Platelet Granules and Their Major Function in Hemostasis
Platelet Function
Overview
Table 24–11 Events that Occur in Platelets After Vessel Injury
Maintenance of Vascular Integrity
BASIC CONCEPTS
ADVANCED CONCEPTS
Platelet Plug Formation
Figure 24-5 SEM of platelet adherence at the site of endothelial loss. Short arrow points to a discoid intact platelet with a single pseudopod; long arrow points to an elongated adherent platelet; top double arrow marks densely adherent platelets appearing as elongated humps fused to the subendothelial layer.
ADHESION
SHAPE CHANGE
Figure 24-6 Pictorial representation of platelet adhesion to subendothelium through von Willebrand’s factor (vWF) bridge.
Figure 24-7 TEM of platelet adherence to subendothelial connective tissue at the focus of endothelial loss. (1) Intact platelet with pseudopod (thin arrow indicates a granule; thick arrow indicates dense body), (2) partially degranulated platelet, (3) degranulated platelet “ghost”, (4) internal elastic lamina.
AGGREGATION
Figure 24-8 TEM showing disk-to-sphere transformation of an activated platelet. Note progression from (1) disk shape to (2) pseudopod formation to (3) degranulated ballooned sphere.
Table 24–12 Some in Vitro Platelet Aggregators
Figure 24-9 TEM of an activated and a degranulated platelet. A. Early aggregation of activated platelet (the primary wave of aggregation, a reversible process). B. Degranulated platelet (the secondary wave of aggregation, an irreversible process).
Figure 24-10 A typical biphasic response of in vitro platelet aggregation to ADP, as recorded via an aggregometer.
SECRETION (THE RELEASE REACTION)
STABILIZATION OF THE HEMOSTATIC PLUG
Figure 24-11 TEM of viscous metamorphosis.
Figure 24-12 Sequence of events in hemostatic plug formation. (1) Platelet adhesion to exposed subendothelial connective tissue structures. (2) Platelet aggregation by ADP, thromboxane A2, and thrombin recruitment through transformation of discoid platelets into reactive spiny spheres that interact with one another through calcium-dependent fibrinogen bridges. (3) Contribution of platelet coagulant activity to the coagulation process, which stabilizes the plug with a fibrin mesh. (4) Consolidation of the platelet mass to provide a dense thrombus. (5) Fibrin polymerization and fibrin stabilization by factor XIII.
Figure 24-13 Synthesis of prostaglandins in platelets and endothelial cells during platelet plug formation.
Secondary Hemostasis: Fibrin-Forming (Coagulation) System
Table 24–13 Nomenclature of Coagulation Factors
Classification of Coagulation Factors by Hemostatic Function
Table 24–14 Classifications of Coagulation Factors by Hemostatic Function
Classification of Coagulation Factors by Physical Properties
Table 24–15 Classification of Coagulation Proteins by Physical Properties
Blood Coagulation: The “Cascade” Theory
Table 24–16 Consequences of Factor Deficiency
Table 24–17 Physical Properties of the Coagulation Factors
Extrinsic Pathway (Factor VII)
Intrinsic Pathway (Factors XII, XI, IX, and VIII)
BASIC CONCEPTS
Figure 24-14 The extrinsic pathway and the role of factor Vila in activation of factor X and IX.
Figure 24-15 The intrinsic pathway and its role in activation of factor X. HMWK = high-molecular-weight kininogen; PF3 = platelet factor 3.
Figure 24-16 The common pathway and formation of fibrin clot. PL = phospholipid source.
Common Pathway (Factors X, V, II, and I)
Thrombin-Mediated Reactions in Hemostasis (Advanced Concepts)
Figure 24-17 Blood coagulation: The “cascade” theory of coagulation. The extrinsic system, the intrinsic system, and the common pathway and the appropriate laboratory tests for evaluation of each. HMWK = high-molecular-weight kininogen; PF3 = platelet factor 3; PK = prekallikrein; PT = prothrombin time; APTT = activated partial thromboplastin time.
Thrombin-Mediated Platelet Aggregation
Thrombin Formation: Role of Extrinsic Pathway
Thrombin Formation: Role of Common Pathway
Figure 24-18 An overview of coagulation cascade, the intrinsic and extrinsic pathways, and the interaction between the two. The fibrinolytic pathway and its action on fibrinogen and fibrin. HMWK = high-molecular-weight kininogen; PK = prekallikrein; FpA = fibrinopeptide A.
Thrombin-Mediated Anticoagulant Activity
Figure 24-19 Platelet membrane phospholipid provides a surface for the interaction of coagulation factors and the formation of “tenase complex” and “prothrombinase complex.”
Figure 24-20 Activation of factor X at the beginning of the common pathway and the “tenase” complex.
Table 24–18 Factor VIII Complex
Figure 24-21 Conversion of prothrombin to thrombin by prothrombinase complex.
Table 24–19 Actions of Thrombin
Figure 24-22 Thrombin activity on fibrinogen.
Table 24–20 Interpretation of Coagulation Test Results
Fibrinogen Conversion and Fibrin Stabilization via Thrombin
Thrombin-Mediated Tissue Repair
Antithrombin (AT) and Protein C Pathways
Figure 24-23 The inhibitor pathway of coagulation. AT = antithrombin; PC = protein C; APC = activated protein C; PS = protein S; C4b-BP = C4b-bound protein; TM = thrombomodulin.
Table 24–21 Thrombin-Mediated Reactions in Hemostasis
Blood Coagulation: A Cell-Based Model of Hemostasis
Fibrin-Lysing (Fibrinolytic) System
Table 24–22 Some Inhibitors of Plasmin
Figure 24-24 Cell-based model of hemostasis. Phase 1 = initiation; phase 2 = amplification; phase 3 = propagation.
Action of Plasmin
Figure 24-25 Plasmin activity on fibrinogen. FDP = fibrin degradation product.
Kinin System
Table 24–23 The Actions of Plasmin
Figure 24-26 Interrelationship of coagulation, fibrinolytic, kinin, and complement systems. HMW = high molecular weight; LMW = low molecular weight.
Protease Inhibitors
Complement System
Table 24–24 Some Important Serine Protease Inhibitors
Laboratory Evaluation of Hemostasis
Table 24–25 Some Common Laboratory Screening Tests for Hemostatic Disorders
Case Study
Table 24–26 Classification of Bleeding Disorders by Screening Tests
Questions
SUMMARY CHART
REFERENCES
Chapter 25 Disorders of Primary Hemostasis: Quantitative and Qualitative Platelet Disorders and Vascular Disorders
OBJECTIVES
Introduction
Laboratory Evaluation of Disorders of Primary Hemostasis
Table 25–1 Clinical Manifestations of Platelet and Vascular Disorders (Primary Hemostasis)
Table 25–2 Classification of Bleeding Disorders by Screening Tests
Table 25–3 Laboratory Tests to Assess Disorders of Primary Hemostasis
Quantitative Platelet Disorders: Thrombocytopenia
Table 25–4 Classification of Disorders Causing Thrombocytopenia
Deficient Platelet Production
INEFFECTIVE THROMBOPOIESIS
CONGENITAL DISORDERS
Abnormal Distribution of Platelets
Table 25–5 Congenital Disorders Associated with Decreased Platelet Production
Increased Destruction of Platelets
PRIMARY IMMUNE-MEDIATED THROMBOCYTOPENIAS
IDIOPATHIC THROMBOCYTOPENIC PURPURA
Figure 25-1 Oral cavity of a patient with idiopathic thrombocytopenic purpura (ITP).
Figure 25-2 Petechial bleeding of the lower extremities in a patient with ITP.
Figure 25-3 ITP, bone marrow aspirate. Note the increased number of megakaryocytes with normal cellularity (M/E 3:1).
POSTTRANSFUSION PURPURA (PTP)
Figure 25-4 Posttransfusion purpura (PTP).
ISOIMMUNE NEONATAL THROMBOCYTOPENIA
DRUG-INDUCED IMMUNE THROMBOCYTOPENIA
HEPARIN-INDUCED THROMBOCYTOPENIA AND THROMBOSIS
ACQUIRED SECONDARY IMMUNE-MEDIATED THROMBOCYTOPENIA
LYMPHOPROLIFERATIVE DISORDERS/COLLAGEN VASCULAR DISORDERS
VIRAL INFECTIONS
HIV-RELATED THROMBOCYTOPENIA
MICROANGIOPATHIC THROMBOCYTOPENIA
THROMBOTIC THROMBOCYTOPENIC PURPURA
Table 25–6 Comparison of Acute and Chronic ITP
Figure 25-5 Microangiopathic hemolytic anemia. Note the presence of schistocytes (arrows) and nucleated red cell (top border).
Figure 25-6 Renal biopsy from a patient with thrombotic thrombocytopenic purpura (TTP) showing glomerular deposits of platelet-fibrin microvascular occlusion.
NONIMMUNE THROMBOCYTOPENIA
HEMOLYTIC UREMIC SYNDROME
Table 25–7 Comparison of TTP and HUS
DISSEMINATED INTRAVASCULAR COAGULATION
PREGNANCY-ASSOCIATED THROMBOCYTOPENIA
GESTATIONAL THROMBOCYTOPENIA
PREECLAMPSIA–ECLAMPSIA AND HELLP SYNDROME
Quantitative Platelet Disorders: Thrombocytosis
Primary Thrombocytosis
Reactive Thrombocytosis
Qualitative Platelet Disorders
Congenital Disorders of Platelet Function
PLATELET MEMBRANE DEFECTS
GLANZMANN’S THROMBASTHENIA
Table 25–8 Congenital Disorders of Platelet Function
Table 25–9 Comparison of Glanzmann’s Thrombasthenia and Bernard–Soulier Syndrome
Figure 25-7 This graph depicts the lack of platelet aggregation to epinephrine, 10 µm (blue); ADP, 5 µm (black); collagen, 2 µg/mL (red); and arachidonic acid, 0.5 µg/mL (green)—typical of a patient with Glanzmann’s thrombasthenia.
BERNARD–SOULIER SYNDROME
PLATELET RELEASE (SECRETION) DEFECTS
Table 25–10 Comparison of Platelet Aggregation Results
STORAGE POOL DEFICIENCIES (GRANULE DEFECTS)
PRIMARY SECRETION DEFECTS (ENZYMATIC PATHWAY DEFECTS)
DEFECTS IN PLATELET COAGULANT ACTIVITY
von Willebrand Disease
Figure 25-8 Factor VIII/von Willebrand factor (vWF) complex in plasma.
Figure 25-9 Schematic representation of the interactions between vWF, platelets, and collagen of the subendothelium. vWF synthesized by endothelial cells is released in plasma and is stored in the α granules of platelets, and can be released after stimulation. vWF mediates platelet adhesion through binding to collagen and to platelet glycoprotein Ib (GPIb) in the presence of ristocetin, as well as the platelet GPIIb/IIIa in the presence of physiologic agonists (thrombin, collagen, ADP). vWF also binds to factor VIII (FVIII) and heparin.
CLASSIFICATION
LABORATORY EVALUATION
TREATMENT
Acquired Qualitative Platelet Disorders
UREMIA
Table 25–11 Acquired Qualitative Platelet Disorders
LIVER DISEASE
PARAPROTEINEMIAS
MYELOPROLIFERATIVE DISORDERS
ACQUIRED VON WILLEBRAND DISEASE
CARDIOPULMONARY BYPASS
ACQUIRED STORAGE POOL DEFICIENCIES
DRUG THERAPY
Table 25–12 Drugs that can Inhibit Platelet Function
Vascular Disorders
Table 25–13 Vascular Purpuras
Primary Purpura
Secondary Purpura
INFECTIOUS PURPURA
ALLERGIC PURPURA
METABOLIC PURPURA
Figure 25-10 Anaphylactoid (Schönlein–Henoch) purpura. Purpuric lesions of the foot.
Figure 25-11 Steroid purpura (skin manifestations).
PURPURA SECONDARY TO DYSPROTEINEMIA
Figure 25-12 Typical lower extremity vascular lesions seen in patients with paraproteinemia.
Figure 25-13 Amyloid purpura. Note characteristic periorbital distribution.
Vascular and Connective Tissue Disorders
HEREDITARY HEMORRHAGIC TELANGIECTASIA
Figure 25-14 Tongue of a patient with hereditary hemorrhagic telangiectasia. Note the multiple vascular telangiectases, which can occur in the nares, the oral mucous membranes, and throughout the gastrointestinal tract. Recurrent bleeding requiring transfusions is a common manifestation.
ANGIODYSPLASIA
GIANT HEMANGIOMAS (KASABACH–MERRITT SYNDROME)
CONGENITAL CONNECTIVE TISSUE DISORDERS
Case Study 1
QUESTIONS
ANSWERS
Case Study 2
QUESTIONS
ANSWERS
Case Study 3
QUESTIONS
ANSWERS
Case Study 4
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 26 Disorders of Plasma Clotting Factors
OBJECTIVES
INTRODUCTION
Table 26–1 Factor Deficiencies
Table 26–2 Factor Deficiencies and Test Results
The Plasma Clotting Factors, Associated Disorders, and Laboratory Evaluation
Factor I (Fibrinogen)
AFIBRINOGENEMIA
HYPOFIBRINOGENEMIA
Table 26–3 Differential Diagnosis of Fibrinogen Disorders and Heparin
DYSFIBRINOGENEMIA
HYPERFIBRINOGENEMIA
Factor II (Prothrombin)
HYPOPROTHROMBINEMIA
DYSPROTHROMBINEMIA
PROTHROMBIN G20210A MUTATION
Factor V (Proaccelerin; Labile Factor)
FACTOR V LEIDEN MUTATION (R506Q)
Factor VII (Proconvertin; Stable Factor; Plasma Thromboplastin Component)
Figure 26-1 Activated protein C (APC) resistance of factor V Leiden. (Top) Normal inactivation of factors V and VIII:C by APC. (Bottom) The binding site for APC on the factor V Leiden molecule is altered, thereby permitting activated factor V (factor Va Leiden) to continue thrombin generation and subsequent fibrin formation.
Table 26–4 Laboratory Findings for Factor VII Deficiency
Factor VIII (Antihemophilic Factor) and von Willebrand Factor
Table 26–5 Abbreviations for factor VIII and von Willebrand Factor*
Table 26–6 Selected Properties of Factor VIII:C and von Willebrand Factor*
F VIII:C
VON WILLEBRAND FACTOR
VON WILLEBRAND FACTOR MULTIMERIC STRUCTURE
BLEEDING TIME
Figure 26-2 Ristocetin-induced platelet aggregation. A. (1) Normal response to ristocetin 1.2 U/mL. (2) Normal response to ristocetin 0.6 U/mL (low-dose). B. Abnormal response to ristocetin 0.6 U/mL characteristic of type 2B von Willebrand’s disease. Each mark on the x axis represents 1-minute intervals.
Figure 26-3 Quantitative immunoelectrophoresis of vWF:Ag; Laurell rockets in agarose gel. The first four peaks are the standard curve dilutions, followed by various vWF:Ag levels. Peak height is proportional to concentration.
Figure 26-4 vWF multimers. Samples of (1) normal plasma, (2) von Willebrand’s plasma, and (3) cryoprecipitate, underwent electrophoresis in sodium dodecyl sulfate (SDS)-agarose gel. A Western blotting technique was performed. The gel on nitrocellulose paper was incubated first with a rabbit anti-human vWF: Ag antibody and then with goat anti-rabbit IgG, after which it was stained.
HEMOPHILIA A
VON WILLEBRAND DISEASE
Table 26–7 Comparison of Hemophilia and Classic von Willebrand Disease
VON WILLEBRAND DISEASE TYPES AND SUBTYPES
TYPE 2 vWD
Type 2A
Type 2B
Table 26–8 Laboratory Diagnosis of Classic von Willebrand Disease (Type 1) and Variants
Type 2M
Type 2N
Type 3 vWD
Platelet-Type/Pseudo-vWD
ACQUIRED VON WILLEBRAND DISEASE SYNDROME
Factor IX (Christmas Factor; Plasma Thromboplastin Component [PTC])
HEMOPHILIA B
Factor X (Stuart–Prower Factor)
Table 26–9 Laboratory Findings for Factor X Deficiency
Factor XI (Plasma Thromboplastin Antecedent [PTA])
HEMOPHILIA C
Factor XII (Hageman Factor)
Factor XIII (Fibrin-Stabilizing Factor)
Prekallikrein (Fletcher Factor)
High-Molecular-Weight Kininogen (Fitzgerald Factor; Williams–Fitzgerald–Flaujeac Factor)
Circulating Anticoagulants/Acquired Inhibitors
Specific Inhibitors
Nonspecific Inhibitors: Lupus Anticoagulants
Table 26–10 Screening Tests for Lupus Anticoagulants: Decreased Phospholipid
Table 26–11 Confirmatory Tests for Lupus Anticoagulants: Increased Phospholipid*
Case Study 1
QUESTIONS
ANSWERS
Case Study 2
QUESTIONS
ANSWERS
Case Study 3
QUESTIONS
ANSWERS
Case Study 4
QUESTIONS
ANSWERS
Case Study 5
QUESTIONS
ANSWERS
Case Study 6
QUESTIONS
ANSWERS
Case Study 7
QUESTIONS
ANSWERS
Case Study 8
QUESTIONS
ANSWERS
Case Study 9
QUESTIONS
ANSWERS
Case Study 10
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
Acknowledgment
REFERENCES
Chapter 27 Interaction of the Fibrinolytic, Coagulation, and Kinin Systems; Disseminated Intravascular Coagulation; and Related Pathology
OBJECTIVES
Systems and Related Pathology
Molecular Components: Physicochemical and Functional Properties
Plasminogen
Figure 27-1 Summary of the interaction of the coagulation, fibrinolytic, and kinin systems. High-molecular-weight kininogen (HMWK) and prekallekrein catalyze the activation of factor XII to factor XIIa. Factor XIIa then promotes the conversion of prekallekrein to kallekrein. The latter liberates the kinins from HMWK, thus completing the positive feedback loops of the contact phase of coagulation. These components stimulate endothelial cells to release plasminogen activator. Thrombin generated through the extrinsic and intrinsic coagulation cascades then converts fibrinogen to fibrin, induces platelet activation, activates protein C, and stimulates both tissue plasminogen activator (tPA) and urokinase-like plasminogen activator (uPA) from the endothelium, which then can convert plasminogen to plasmin. Thrombin exerts a negative feedback by simultaneously stimulating plasminogen activator inhibitor 1 (PAI-1) release from the endothelial cells. PAI-1 will serve to bind tPA and uPA to dampen plasmin generation. Plasmin, once formed, will initiate clot lysis by proteolytically cleaving fibrin and fibrinogen into fibrin/fibrinogen degradation products (FDPs). In addition, plasmin will inactivate a number of coagulation factors including factors V and VIII and convert activated factor XII into XIIa fragments. Excess plasmin in circulation is prevented by the presence of α2-antiplasmin which forms a complex with plasmin, thus inactivating it.
Table 27–1 Components of the Fibrinolytic System
Plasminogen Activators
Plasminogen Activator Inhibitor-1
Plasmin
Plasmin Inhibitors
Thrombomodulin
Thrombin-Activatable Fibrinolysis Inhibitor
Congenital Abnormalities
Figure 27-2 Control of fibrinolysis. Plasminogen activators tissue plasminogen activator (t-PA) and, to a lesser degree, urokinase (u-PA) are synthesized in the endothelium. Both t-PA and u-PA are rapidly inhibited by activated plasminogen activator inhibitor-1 (PAI-1), also synthesized in the endothelium. The interaction of t-PA with PAI-I regulates fibrinolysis, and under basal conditions most t-PA released is bound to PAI-1. Plasminogen-tPA binds to fibrin in clots generating plasmin at the LYSYL (lysine) residue site and clot lysis occurs resulting in the formation of fibrin degradation products (FDPs) including D-dimers. The plasmin inhibitor α2-antiplasmin binds to free circulating plasmin at the same lysine binding site, thus limiting plasmin activity to the area of fibrin deposition. Excess levels of thrombin activate the plasma protein thrombin activatable fibrinolysis inhibitor (TAFI), which binds with fibrin at the plasminogen binding site, thus preventing plasmin formation and fibrinolysis.
Disseminated Intravascular Coagulation
Triggering Mechanisms: Associated Clinical Disorders
Figure 27-3 Integrated system of hemostasis. 1. Disruption of endothelial continuity releases tissue factor (TF). 2. In the presence of ionized calcium, TF forms a complex with factor VII or VIIa leading to the conversion of factor X to Xa and rapid generation of small amounts of thrombin and fibrin formation via the extrinsic pathway. In addition, the TF–factor VIIa-Xa complex is rapidly inhibited by the presence of tissue factor pathway inhibitor (TFPI). 3. The VIIa–TF complex also activates factor IX to IXa, initiating the intrinsic pathway and the formation of larger amounts of thrombin on the surface of activated platelets. 4. Enhanced expression of TF by monocytes also occurs in response to endotoxin or cytokines secondary to sepsis. 5. Thrombin stimulates platelet activation and the release of platelet agonists. 6. Activated platelets undergo shape change, resulting in the exposure of phospholipids and cofactors V and VIII on the surface of the platelet membrane. 7. Secondarily, and simultaneously, thrombin complexes with thrombomodulin (TM) on the endothelial surface. 8. Protein C, once bound to this complex, is rapidly converted to its activated state. 9. Activated protein C indirectly causes release of tissue plasminogen activator in cells from the subendothelium, in addition to direct stimulation and release of this glycoprotein by thrombin. 10. Plasmin-induced proteolysis of the fibrin clot results in the formation of fibrin degradation products.
Figure 27-4 Diffuse hemorrhage, a clinical manifestation in a patient with disseminated intravascular coagulation (DIC). Note the multiple cutaneous ecchymoses.
Table 27–2 Clinical Conditions Associated with Disseminated Intravascular Coagulation
Clinical Presentation
Laboratory Diagnosis
Table 27–3 Laboratory Tests to Detect Excess Thrombin and/or Plasmin Activity
Figure 27-5 DIC (peripheral blood). Note presence of schistocytes (arrows) and nucleated red cell (top border).
Figure 27-6 DIC (skin biopsy). Note both partial (small arrow) and complete (large arrow) occlusion of blood vessels by RBC/fibrin clot.
Table 27–4 Laboratory Tests to Diagnose DIC
Therapy
Table 27–5 Profile of DIC
Related Disorders
Table 27–6 Laboratory Differentiation Between DIC and Primary Fibrinolysis
Table 27–7 Other Causes of Fibrinolytic Activation
Case Study
Questions
SUMMARY CHART
REFERENCES
Chapter 28 Introduction to Thrombosis and Anticoagulant Therapy
OBJECTIVES
Introduction
Regulation of Coagulation and Fibrinolysis
The Role of Endothelium
ANTICOAGULANT ROLE
PROTHROMBOTIC ROLE
Platelets
Procoagulant Factors and Generation of Thrombin
Table 28–1 Role of Thrombin
Natural Inhibitors of Coagulation Factors (Plasma Components)
ANTITHROMBIN (ANTITHROMBIN-III)
Figure 28-1 Physiologic inhibitors of coagulation. AT = antithrombin; APC = activated protein C; Xlla = activated factor XII; HC-II = heparin cofactor II; PS = protein S; TFPI = tissue factor pathway inhibitor
HEPARIN COFACTOR II (HC-II)
Figure 28-2 Thrombin binds to thrombomodulin on the surface of endothelial cell. Thrombin/thrombomodulin complex acts on protein C, resulting in the formation of activated protein C (APC). APC cleaves factors Va and VIIIa in the presence of its cofactor, protein S. APC also inactivates plasminogen activator inhibitor-1 (PAI-1). Inhibition of APC occurs by PCI-1 and PCI-2. TR = thrombin; TM = thrombomodulin; PC = protein C; PS = protein S; PCI = protein C inhibitor.
THROMBIN/THROMBOMODULIN INTERACTION
THE PROTEIN C AND S SYSTEM
TISSUE FACTOR PATHWAY INHIBITOR
Fibrinolytic System
Figure 28-3 Shown here is the interaction of tissue factor pathway inhibitor (TFPI) with factors VII, IX, and X.
Table 28–2 Inhibitory Effect of the Serine-Protease Inhibitors
Table 28–3 TPA Inhibitors
Inherited Thrombophilia
Activated Protein C Resistance
Figure 28-4 Data on the functional assay for activated protein C are shown here. This is an APTT-based assay and the APC-R ratio can be calculated by measuring the patient clot time with and without activated protein C (APC) in the presence of CaCl2. In a normal person, the ratio is more than 0.9. In case 1, the final ratio is 1.2 and thus the test is negative for activated protein C resistance (APC-R). In case 2, the ratio is less than 0.9 and the test for APC-R is positive. In case 2, therefore, the patient should be tested for factor V Leiden mutation by polymerase chain reaction (PCR). APC-V = activated protein C-factor V ratio.
Protein C Deficiency
Table 28–4 Classification of Hereditary PC Deficiencies
Table 28–5 Causes of Acquired Deficiency of Coagulation Inhibitors
Protein S Deficiency
Table 28–6 Classification of Hereditary PS Deficiencies
Antithrombin Deficiency
Prothrombin Nucleotide G20210A Mutation
Table 28–7 Classification of Hereditary AT Deficiencies Activity
Hyperhomocysteinemia
Figure 28-5 Homocysteine can be metabolized to methionine (via remethylation) and to cysteine (via transulfuration). The numbers represent the enzymes involved in the reactions: (1) Betaine homocysteine methyltransferase; (2) methionine synthase in the presence of cofactor vitamin B12; (3) cystathione β-synthetase in the presence of cofactor vitamin B6; (4) methylene tetrahydrofolate reductase (MTHFR).
Tissue Factor Pathway Inhibitor Deficiency
Factor XII Deficiency
Heparin Cofactor II Deficiency
Dysfibrinogenemia
Elevated Plasma Factor VIII Coagulant Activity
Lipoprotein a and Thrombosis
Other Coagulant Factors Associated with Thrombosis
Acquired Thrombotic Disorders
Lupus Anticoagulant/Antiphospholipid Syndrome
HISTORY OF ANTIPHOSPHOLIPID SYNDROME
Table 28–8 Revised Classification Criteria for the Antiphospholipid Antibody Syndrome
IMMUNOLOGY
MECHANISM OF THROMBOSIS
THE LABORATORY’S CONTRIBUTION TO THE DIAGNOSIS OF aPL SYNDROME
Table 28–9 Screening and Confirmatory Assays That Can Be Used in LA Testing
Table 28–10 Criteria for Lupus Anticoagulants
A WALK THROUGH THE ALGORITHM
Figure 28-6 In the hematology laboratory, detection of LA usually begins with an elevated APTT. This figure is a flowchart that illustrates an approach to the diagnosis of LA. The shaded boxes show the typical abnormalities that can be seen when LA is present in the sample.
THERAPY AND MONITORING
Figure 28-7 Incubated mixing study can be performed as shown in this figure. For details, refer to the accompanying text.
Heparin-Induced Thrombocytopenia
CLINICAL MANIFESTATIONS
MECHANISM
Figure 28-8 Heparin complexed with PF4 is the antigen usually responsible for initiating HIT (1). An antibody is formed against heparin-PF4 complexes (2). The antibody complexes with heparin-PF4 and the Fc portion of the antibody binds to the platelet FcRIIa receptor (3), resulting in platelet activation and release of more PF4 (4), which then binds to more heparin.
LABORATORY DIAGNOSIS
THERAPY AND MONITORING
Other Acquired Conditions Associated with Thrombosis
Thrombosis with Pregnancy and Use of Oral Contraceptives
Thrombosis and Nephrotic Syndrome
Thrombosis and Medications
Thrombosis in Cancers and Other Conditions
Thrombosis and Major Trauma
Diagnostic Approach and Issues in Laboratory Testing in Patients with Thrombosis
Table 28–11 Differential Diagnosis of Hypercoagulable States
Table 28–12 Risk Factors for Venous Thromboembolism
Table 28–13 Evaluation of a Patient with Suspected Thrombophilia (Hereditary/Acquired)
Table 28–14 Suggested Evaluation Criteria for Inherited Thrombophilia
Table 28–15 Testing for Inherited Thrombophilia
Table 28–16 Coagulation Defects and Sites of Thrombosis
Complete History and Physical Examination
Table 28–17 Issues in Laboratory Testing in Patients with Thrombosis
USE OF D-DIMER ASSAY IN THE DIAGNOSIS OF THROMBOEMBOLISM
TESTING DURING THE ACUTE EVENT
Conditions That Can Interfere with Test Results
Testing in the Appropriate Clinical Setting
Functional Assays
In Arterial Thrombosis, Consider the Additional Evaluation of Hyperhomocysteinemia and Lipoprotein a
Repeat Testing Prior to Diagnosis
Performing Testing and Making Evaluation Worthwhile
Anticoagulant Therapy
Unfractionated Heparin Therapy
Administration and Monitoring
Low-Molecular-Weight Heparin
Oral Anticoagulation
Alternative Anticoagulants
DIRECT THROMBIN INHIBITORS
FACTOR Xa INHIBITORS
OTHER ANTICOAGULANTS
Antiplatelet Agents
Thrombolytic Therapy
Case Study 1
PERTINENT HISTORY
PERTINENT PHYSICAL FINDINGS
LABORATORY FINDINGS
QUESTIONS
ANSWERS
Case Study 2
PERTINENT HISTORY
PERTINENT PHYSICAL FINDINGS
LABORATORY FINDINGS
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
Acknowledgment
REFERENCES
PART 5 LABORATORY METHODS
Chapter 29 Quality Management, Quality Assurance, and Quality Control
OBJECTIVES
Introduction
Figure 29-1 The history of quality.
Quality Management
Definition of Quality Management and Quality Testing
Legal Implications
Figure 29-2 The elements of a quality laboratory result.
“Quality Management Improves Bottom Lines”
Quality Management Plans
Figure 29-3 Quality award-winning companies outperform control firms.
Quality Approaches
BENCHMARKING
ROOT CAUSE ANALYSIS
INTERNATIONAL STANDARDS ORGANIZATION (ISO)
SIX SIGMA
RISK MANAGEMENT AND FAILURE MODE AND EFFECTS ANALYSIS
LEAN
TOTAL QUALITY MANAGEMENT
MALCOLM BALDRIGE NATIONAL QUALITY AWARD
Divisions of Quality Management
ORGANIZATION
FACILITIES AND SAFETY
INFORMATION MANAGEMENT
OCCURRENCE MANAGEMENT
CUSTOMER SERVICE
Figure 29-4 The 12 divisions of Quality Management (QM), also called Quality Systems Essentials (QSE).
PERSONNEL
EQUIPMENT
DOCUMENTS AND RECORDS
ASSESSMENTS
PURCHASING AND INVENTORY
PROCESS CONTROL
PROCESS IMPROVEMENT
Figure 29-5 Flowchart of a sedimentation rate procedure.
Quality Software
Quality Assurance and Quality Control
Definitions of Quality Assurance, Quality Control, and Quality Assessment
General Quality Assurance Control Activity Guidelines
Preanalytical, Analytical, Postanalytical, and Nonanalytical Factors in Testing
PREANALYTICAL FACTORS
ANALYTICAL FACTORS
POSTANALYTICAL FACTORS
NONANALYTICAL FACTORS
Accuracy, Precision, and Error
HOW TO MEASURE ACCURACY
Table 29–1 Errors in the Testing Process
Figure 29-6 Accuracy and precision can be compared to a target. A. Example of accuracy and precision (the shooter hit the “target” value, i.e., the bulls-eye, and also reproduced the shot several times. B. Precise, but not accurate.
HOW TO MEASURE PRECISION
WHAT IS THE EQUATION FOR THE STANDARD DEVIATION (SD)?
Figure 29-7 Accuracy: constant and proportional systematic error in relation to the mean.
Table 29–2 Differences Between Accuracy and Precision
Figure 29-8 Example of Gaussian distribution curve for data of normal populations.
WHAT IS A CV?
WHY IS A CV USEFUL?
TOTAL ERROR
Box 29–1 Coefficient of Variation
Table 29–3 CLIA Definitions for Total Error (TE)
Method Validation
WHAT STUDIES NEED TO BE RUN ON A NEW METHOD OR INSTRUMENT?
Box 29–2 Method Validation Plan
INTERFERENCE EXPERIMENT
Box 29–3 Method Validation Experiments
REPLICATION EXPERIMENT
REPORTABLE RANGE (LINEARITY) EXPERIMENT
REFERENCE RANGE (NORMAL RANGE) EXPERIMENT
COMPARISON OF METHODS EXPERIMENT
Figure 29-9 Correlation study of hematology analyzers (hemoglobin).
RECOVERY EXPERIMENT
Quality Control Definitions
QUALITY CONTROL MATERIAL
QUALITY CONTROL STATISTICS
MATRIX
ASSAYED CONTROL MATERIAL
UNASSAYED CONTROL MATERIAL
QUALITY CONTROL LEVELS
Box 29–4 Judging a Method’s Performance
STATISTICAL PROCESS CONTROL
RUN
CONTROL LIMITS
CONTROL RULE
MOVING AVERAGES
CONTROL CHART
SHIFT
TREND
ELECTRONIC QC
Minimum Quality Control Requirements, CLIA-88 CFR
Figure 29-10 Different types of errors and QC actions.
Box 29–5 Minimum QC Requirements (CLIA-88)
ESTABLISHING IN-HOUSE QC MEAN AND SD
Levy–Jennings Graphs
Figure 29-11 A typical Levy–Jennings graph. Each data point is plotted so an overall view of the data is possible.
Figure 29-12 Example of a shift in QC values on a Levy–Jennings graph. Notice that between day 8 and day 20 the data has abruptly shifted above the mean.
SHIFTS AND TRENDS
Figure 29-13 Example of a trend in QC values on a Levy-Jennings graph. Notice that between day 8 and day 20 there is a slow trend above the mean.
Westgard MultiRule Quality Control
Figure 29-14 A Levy-Jennings graph showing a violation of the Westgard Rule R4s.
CHOOSING WESTGARD RULES
Sigma-metrics
Table 29–4 Westgard MultiRules: Probability of Detecting Errors, False Rejections
Box 29–6 Six Sigma Details
Figure 29-15 Imprecision and the Six Sigma scale.
CALCULATING QUALITY
Troubleshooting Quality Control Problems
SYSTEMATIC ERRORS
RANDOM ERRORS
Peer Group Quality Control
STATISTICS
REVIEW OF PEER GROUP DATA
Box 29–7 SDI and CVI
Hematology Laboratory Applications
Quality Plan Example
Method Validation Studies
EXAMPLE OF METHOD VALIDATION STUDIES
Quality Control
Laboratory Quality Updates
Quality Meeting Launches New Organization
Equivalent Quality Control Option 4
Case Study 1
QUESTIONS
ANSWERS
Case Study 2
QUESTIONS
ANSWERS
Questions
SUMMARY CHART
REFERENCES
Chapter 30 Body Fluid Examination: The Qualitative, Quantitative, and Morphologic Analysis of Serous, Cerebrospinal, and Synovial Fluids
OBJECTIVES
Introduction
Types of Body Fluids and Anatomy
Pericardial, Pleural, and Peritoneal (Serous) Fluids
Cerebrospinal Fluid
Synovial Fluid
Specimen Collection and Preparation
Collection
Preparation
Table 30–1 Advantages of the Cytocentrifugation Method for Body Fluids
Table 30–2 Disadvantages of the Cytocentrifugation Method for Body Fluids
Figure 30-1 Artifact of cytocentrifugation. Peripheralization, vacuolization, and hyperlobulation of neutrophils. Wright stain, ×1000 magnification.
Figure 30-2 Artifact of cytocentrifugation. Prominence of nucleoli, vacuolization in lymphocytes. Wright stain, ×1000 magnification.
Figure 30-3 Artifact of cytocentrifugation. Cytoplasmic projections of lymphocytes. Wright stain, ×1000 magnification.
Cellular Components of Body Fluids
Neutrophils
Lymphocytes
Monocytes
Figure 30-4 Histiocytes/macrophages (center). Also monocytes and neutrophils. Wright stain, ×1000 magnification.
Figure 30-5 Macrophages. Also a lymphocyte (A) and a plasma cell (B). Wright stain, ×1000 magnification.
Figure 30-6 Signet-ring type macrophages. Wright stain, ×1000 magnification.
Tissue Cells
Figure 30-7 Mesothelial cells. Normal, quiescent. Wright stain, ×1000 magnification.
Figure 30-8 Mesothelial cells; degenerated with peripheral vacuoles, resembling macrophages. Wright stain, ×1000 magnification.
Eosinophils, Basophils, Mast Cells
Figure 30-9 Macrophages (right) and mesothelial cells (left). Wright stain, ×1000 magnification.
Pleural, Pericardial, and Peritoneal (Serous) Fluids
Effusions: Transudates and Exudates
Table 30–3 Effusions: Causative Factors
Table 30–4 Comparison of Transudates and Exudates
Cellular Responses, Microorganisms, and Malignant Cells in Serous Fluids
Table 30–5 Comparison of Chylous and Pseudochylous Effusions
Table 30–6 Reactive Processes and Cell Types Present in Serous Effusions
Figure 30-10 Reactive mesothelial cell; binucleated. Also macrophages, neutrophils, and lymphocytes. Wright stain, ×1000 magnification.
Figure 30-11 Mesothelial cell; multinucleated. Also macrophages (A) and lymphocytes (B). Wright stain, ×1000 magnification.
Table 30–7 Mesothelial Cell Morphological Variation
Figure 30-12 Hyperplastic mesothelial cells. Wright stain, ×400 magnification.
Figure 30-13 Reactive mesothelial cells in sheets (epithelioid) with prominent nucleoli. Wright stain, ×1000 magnification.
Figure 30-14 Phagocytic macrophages/mesothelial cell. Note the ingested RBCs (left). Wright stain, ×1000 magnification.
Figure 30-15 Senescent mesothelial cell (right); becoming signet-ring configuration. Wright stain, ×1000 magnification.
Figure 30-16 Bacteria in body fluid: Staphylococcus species; note the intra- and extracellular distribution. Wright stain, ×1000 magnification.
Figure 30-17 Bacteria in body fluid: Neisseria species; note the intracellular diplococci in neutrophils. Wright stain, ×1000 magnification.
Figure 30-18 Clusters of malignant cells. Wright stain, ×1000 magnification.
Figure 30-19 Cell-ball formation; malignant cell cluster. Wright stain, ×500 magnification.
Figure 30-20 Indian file cellular arrangements: Breast carcinoma. Wright stain, ×500 magnification.
Figure 30-21 Nuclear molding. Wright stain, ×1000 magnification.
Figure 30-22 Abnormal mitotic activity; acinar (glandular) arrangement of malignant cells. Wright stain, ×1000 magnification.
Figure 30-23 Foamy, bizarre vacuolization in cytoplasm of malignant cells. Wright stain, ×1000 magnification.
Figure 30-24 Nuclear molding; cannibalism of malignant cell (right). Wright stain, ×1000 magnification.
Table 30–8 Features of Nonhematologic Malignant Cells in Serous and Cerebrospinal Fluid
Types of Effusions, Laboratory Analysis, and Clinical Correlations
Pleural and Pericardial Effusions
SPECIMEN COLLECTION AND PROCESSING
Table 30–9 Body Fluid and Collection Procedure Nomenclature
LABORATORY ANALYSIS AND CLINICAL CORRELATIONS
QUALITATIVE ANALYSIS
QUANTITATIVE MICROSCOPIC AND MORPHOLOGIC ANALYSIS
Figure 30-25 Neutrophils with vacuolization, fragmentation, decreased granulation; one pyknotic neutrophil (center). Wright stain, ×1000 magnification.
Figure 30-26 One plasma cell (A), one plasmacytoid lymphocyte, reactive and normal lymphocytes in a chronic effusion; note the monocyte/macrophages (B). Wright stain, ×1000 magnification.
Figure 30-27 Reactive mesothelial cells: Pleural fluid; note the prominent nucleoli and multinucleation. Wright stain, ×1000 magnification.
Peritoneal Effusions
SPECIMEN COLLECTION AND PROCESSING
LABORATORY ANALYSIS AND CLINICAL CORRELATIONS
QUALITATIVE ANALYSIS
QUANTITATIVE MICROSCOPIC AND MORPHOLOGIC ANALYSIS
Cerebrospinal Fluid
Specimen Collection and Processing
Laboratory Analysis and Clinical Correlations
QUALITATIVE ANALYSIS
BIOCHEMICAL ANALYSIS
QUANTITATIVE MICROSCOPIC AND MORPHOLOGIC ANALYSIS
Table 30–10 Normal Values for Cerebrospinal Fluid
Cellular Responses, Malignant Cells, and Microorganisms in CSF
Figure 30-28 Normal CSF; two monocytes and one lymphocyte (A). Wright stain, ×1000 magnification.
Figure 30-29 Reactive lymphocytosis in CSF. Wright stain, ×1000 magnification.
Figure 30-30 Lymphocytic pleocytosis, CSF. Wright stain, ×400 magnification.
Figure 30-31 Reactive (plasmacytoid) lymphocytes in CSF in multiple sclerosis. Wright stain, ×1000 magnification.
Figure 30-32 Siderophages in CSF: hemosiderin pigment in macrophages (A). Wright stain, ×400 magnification.
Figure 30-33 Siderophage with hematoidin (hematin) pigment. Wright stain, ×1000 magnification.
Figure 30-34 Eosinophils (A) and reactive lymphocytes (B) in CSF; ventricular shunt. Wright stain, ×1000 magnification.
Figure 30-35 Ependymal or choroid plexus cells (neuroepithelial tissue) in sheet formation. Wright stain, ×400 magnification.
Figure 30-36 Papillary cluster of neuroepithelial lining cells. Wright stain, ×1000 magnification.
Figure 30-37 Acute lymphoblastic leukemia in CSF; note the prominent nucleoli. Wright stain, ×1000 magnification.
Figure 30-38 Burkitt’s lymphoma in CSF. Wright stain, ×1000 magnification.
Figure 30-39 Cleaved lymphoma cells in CSF. Wright stain, ×1000 magnification.
Figure 30-40 Lymphoma cells in CSF. Wright stain, ×1000 magnification.
Synovial Fluid
Specimen Collection and Processing
Table 30–11 Normal Values for Synovial Fluid
Table 30–12 Synovial Fluid Characteristics by Disease Category
Laboratory Analysis and Clinical Correlations
QUALITATIVE ANALYSIS
Quantitative Analysis: Biochemical Analysis and Microscopic Examination
BIOCHEMICAL ANALYSIS
MICROSCOPIC EXAMINATION
Crystal Examination
TYPES OF CRYSTALS, ANALYSIS, AND CLINICAL CORRELATION
MONOSODIUM URATE CRYSTALS
CALCIUM PYROPHOSPHATE DIHYDRATE CRYSTALS
BASIC CALCIUM PHOSPHATE CRYSTALS
OXALATE CRYSTALS
CHOLESTEROL AND LIPID CRYSTALS
CORTICOSTEROID CRYSTALS
HEMATIN OR HEMATOIDIN CRYSTALS
ARTIFACTS
Summary
Table 30–13 Body Fluid Collection, Testing, and Storage Requirements
Case Study 1
QUESTIONS
ANSWERS
Case Study 2
QUESTIONS
ANSWERS
Case Study 3
QUESTIONS
ANSWERS
Case Study 4
QUESTIONS
ANSWERS
Acknowledgments
Questions
SUMMARY CHART
REFERENCES
Chapter 31 Hematology Methods
OBJECTIVES
Introduction
Specimen Collection
Patient Identification
HOSPITALIZED PATIENTS
OUTPATIENTS
Safety
Verification of Laboratory Requisitions
Labeling the Blood Specimen
Table 31–1 Information Present on Laboratory Requisition
Specimen Accessioning
Figure 31-1 Example of a specimen label with bar code.
Figure 31-2 Argox A-50 Barcode printer.
Method 1: Venipuncture
Figure 31-3 Argox AS-8250 CCD Barcode Scanner.
Figure 31-4 Evacuated tube system consisting of a doublepointed needle, a plastic holder, and a vacuum tube with a rubber stopper.
Figure 31-5 Application of the rubber tourniquet.
Figure 31-6 The path of veins from the arm.
Method 2: Capillary Blood Collection
Figure 31-7 Acceptable heel puncture sites for a dermal puncture.
Figure 31-8 Acceptable finger puncture sites and correct puncture angle.
Figure 31-9 Microtainer tube containing EDTA.
Unopette Technology—Manual Counts
Figure 31-10 Spencer Bright-Line double counting system with improved Neubauer ruling. This represents an enlarged view of one of the two ruled squares of the hemacytometer. The four corner primary squares are used for counting white blood cells. The arrows in the upper left corner square represent the suggested counting pathway of cells. Five secondary squares (labeled RBC) of the center primary square are used for counting red blood cells. In platelet enumeration, all 25 squares of the center primary square are counted.
Figure 31-11 Hemacytometer, side view.
Method 3: Red Blood Cell Counts
Figure 31-12 InCyto C-Chip disposable hemacytometer.
Figure 31-13 Unopette system, consisting of a reservoir containing a diluent, a pipet used to deliver the specimen, a reservoir, and a pipet shield used to puncture the plastic seal on reservoir, and serve as a cap for the pipet to prevent evaporation.
Figure 31-14 Schematic for using Unopette system. A. Using shield of capillary pipet, puncture diaphragm of reservoir. B. Fill capillary with sample from fingerstick or venous blood specimen. C. Transfer sample to reservoir by squeezing reservoir slightly to force out some air. Do not expel any liquid. Cover opening of over flow chamber with index finger. Maintain pressure until pipet is secured in reservoir neck. Squeeze reservoir several times to rinse capillary bore without expelling any liquid. D. Place index finger over upper opening and gently invert several times to thoroughly mix sample with diluent.
Figure 31-15 Unopette conversion to dropper assembly by withdrawing pipet from reservoir and securing it in reverse position
Figure 31-16 Charging of hemacytometer by Unopette inversion of reservoir dropper assembly. The first three or four drops are discarded before hemacytometer is loaded.
Figure 31-17 Red blood cells are illustrated at ×400 using Unopette methodology.
Method 4: White Blood Cell Counts
Method 5: Platelet Counts
Evaluation of the Peripheral Blood Smear
Method 6. Slide Preparation and Wright Stain
Figure 31-18 Photograph of platelets loaded onto a hemacytometer using a ×40 objective (phase contrast). The platelets appear as dense, refractile, dark bodies that can be round, oval, or rod shaped with a diameter of approximately 2 to 4μm.
Figure 31-19 A. A drop of blood is placed on glass slide. B. A spreader slide is situated at a 30- to 45-degree angle. C. Draw back the slide against the drop of blood and spread the blood smoothly. D. Adequate blood smear with feathered edge. E. Unacceptable blood smear. F. Acceptable and unacceptable stained smears.
Method 7. The White Blood Cell Differential
Table 31–2 Romanowsky Staining Pattern of Hematologic Cells
Figure 31-20 The Hema-Tek Slide Stainer and Stain-Pak.
Methods Used in Detection and Monitoring of Anemia
Method 8: Hemoglobin Determination
Method 9: Microhematocrit Determination
Figure 31-21 Microhematocrit centrifuge.
Figure 31-22 Securing the capillary tube with sealing clay.
Method 10. Red Blood Cell Indices
Figure 31-23 Microhematocrit rotor.
Figure 31-24 Diagram of a centrifuged microhematocrit tube of whole blood.
Figure 31-25 Microhematocrit reader.
RBC DISTRIBUTION WIDTH
MEAN PLATELET VOLUME
Table 31–3 Classification of Anemia
Method 11. Reticulocyte Counts
Figure 31-26 Photograph of a reticulocyte using new methylene blue stain.
Table 31–4 Maturation Time for Reticulocytes
Method 11A. Reticulocyte Counts Using the Miller Disc
Standard Methods for Specific Anemias
Method 12. Sickle Screen
Figure 31-27 Positive (left) and Negative (right) results of sickle solubility test.
Method 13. Helena SPIFE® Alkaline Hemoglobin Electrophoresis
Table 31–5 Hemoglobins that Produce Positive/Negative Solubility Tests
Method 14. Helena SPIFE® Acid Hemoglobin Electrophoresis
Figure 31-28 Comparative hemoglobin electrophoresis. Hemoglobin electrophoresis on cellulose acetate and citrate agar, indicating patterns of mobility. The width of the band is not indicative of hemoglobin concentration.
Method 15. Hemoglobin A2 Determination
Method 16. Isoelectric Focusing
Figure 31-29 Schematic of isoelectric focusing pattern.
Method 17. Hemoglobin F Acid Stain
Figure 31-30 Kleihauer–Betke stain of blood from a newborn. Red-staining cells contain hemoglobin F; clear-staining cells contain hemoglobin A.
Method 18. Screening Test for Glucose-6-Phosphate Dehydrogenase Deficiency
Method 19. Staining for Heinz Bodies
Figure 31-31 Heinz bodies.
Method 20. Screening Method for Detection of Red Cell Pyruvate Kinase
Methods to Detect Red Cell Membrane Disorders
Method 21. Sugar Water Test
Method 22. Ham’s Test
Figure 31-32 A positive (right) and control (left) sugar water test.
Method 23. Osmotic Fragility
Figure 31-33 Results of a Ham’s test.
Method 24. Autohemolysis Test
Figure 31-34 Comparative osmotic fragility curve (blue line, sickle cell anemia; purple line, hereditary spherocytosis). Normal range is shaded. 1, normal biconcave disc; 2, disc-to-sphere transformation; 3, disc-to-sphere transformation; 4, lysis.
Figure 31-35 Incubation hemolysis test. This test provides a further measure of cell resistance to hemolysis. Pyruvate kinase–deficient blood demonstrates an abnormal rate of hemolysis that is independent of the presence or absence of glucose in the incubation media. In contrast, the blood from a patient with hereditary spherocytosis shows more marked hemolysis when glucose is absent.
Nonspecific Tests of Inflammation
Method 25. The Westergren Erythrocyte Sedimentation Rate
Figure 31-36 Westergren methodology for ESR.
Table 31–6 Diseases Associated with an Elevated ESR
Method 26. The Automated Streck ESR-100
Table 31–7 Factors Affecting the ESR
Figure 31-37 Streck ESR-100. A high-capacity erythrocyte sedimentation rate (ESR) analyzer designed for laboratories that run over 100 samples daily.
Case Study
QUESTIONS
Questions
SUMMARY CHART
REFERENCES
Chapter 32 Principles of Automated Differential Analysis
OBJECTIVES
Introduction
Evaluation of Blood Specimens by AccuCount Cell Volume and VCS Technology: The Beckman Coulter® LH Series
Red Cell Histogram Analysis
Figure 32-1 Coulter RBC Histogram.
Platelet Histogram Analysis
Table 32–1 Calculation of the RDW (CV) and RDW – SD as Determined by Beckman Coulter
Figure 32-2 Coulter platelet count determination. A. The platelet histogram has a lower limit of 2 fL and an upper limit of 20 fL. B. Using the least squares regression formula, the formula defines a log normal curve. The log normal curve is plotted from 0 to 70 fL to include giant platelets. C. Integral calculus is used to find the area under the curve. The area under the curve is calibrated to a reference platelet count. D. RBC pulses between 0 and 70 fL are excluded from the PLT count by the electronic curve. The extrapolated area under the PLT curve includes even giant platelets.
Leukocyte Differential Analysis—VCS Technology
Table 32–2 Measurements Associated with Coulter’s VCS Technology
Figure 32-3 The five-part differential is determined using three energy probes (Volume, Conductivity and Laser Light Scatter) and plotting individual cells in three dimensions. The VCS cube displays the WBC populations as separate and distinct populations in the colors indicated in the Figure.
Reticulocyte Analysis
Figure 32-4 Using the same energy probes as the WBC differential and the supravital stain new methylene blue, reticulocytes are plotted in a three-dimensional scatterplot. The colors of the scatterplot indicate red blood cells (red), platelets (green), and reticulocytes (blue). Additional reticulocyte parameters are derived from a variety of information provided by the VCS technology.
Nucleated Red Blood Cell Analysis
White Cell Interference Algorithms
Abnormal Cell Flagging
Figure 32-5 The LH 700 Series counts nucleated red blood cells (NRBCs) when it finds cells present in a signature position for NRBCs in the VCS scatterplot and detects interference at the lower discriminator of the WBC histogram. If there are enough cells present to significantly interfere with the WBC count, the count is corrected for the presence of the interference. Both the corrected WBC count and the uncorrected WBC count are available.
Table 32–3 Summary of Coulter LH Series Analyzer Flagging Messages
Decision Rules
Research Population Data
Figure 32-6 For each of the neutrophil, lymphocyte, monocyte, eosinophil, reticulocyte, and non-reticulocyte (RBCs in the reticulocyte count) populations, a mean and standard deviation is displayed for the volume, conductivity, and light scatter measurements. Those values appear as Research Population Data (RPD) on the WBC and Retic display tabs.
Body Fluid Cell Counting
Beckman Coulter LH Series Hematology Systems
Figure 32-7 Beckman Coulter LH 1502 Hematology System.
Evaluation of Blood Specimens by Light Scattering and Cytochemical Analysis: The ADVIA 120/2120 Hematology Systems, Siemens Healthcare Diagnostics
ADVIA 120/2120 Hematology Systems
Unifluidics Technology
Figure 32-8 ADVIA 2120 System.
Figure 32-9 The Unifluidics Block of the ADVIA 2120 Hematology System.
Red Cell Analysis
Platelet Analysis
Figure 32-10 Cytograms derived from the various channels of the ADVIA 2120 Hematology System. A. RBC scatter cytogram. B. Platelet scatter cytogram. C. Peroxidase cytogram. D. Lobularity/Nuclear density (basophil channel) cytogram. E. Reticulocyte cytogram.
Reticulocyte Analysis
Leukocyte Analysis
Figure 32-11 Siemens ADVIA 120/2120 red blood cell cytograms. A. RBC cytogram and the position of hypochromic, normochromic, hyperchromic, microcytic, normocytic, and macrocytic cells. B. An increase of certain clusters of RBCs is consistent in various disease states. (Harris, N, et al: The ADVIA 2120 Hematology System: A flow cytometry-based analysis of blood and body fluids in the routine hematology laboratory).
Morphology Flagging
Cerebrospinal Fluid Analysis
Table 32–4 Examples of Morphology Flagging by the ADVIA 120/2120 Hematology Analyzer
Figure 32-12 ADVIA 2120 CSF assay cytograms. A. CSF scatter cytogram and the position of red cells (RBCs), lymphocytes (Lymphs), monocytes (Monos), neutrophils (Neuts). B. Scatter/absorption cytogram and relevant position of various cells.
Evaluation of Blood Specimens by Direct Current with Hydrodynamic Focusing, RF/DC Technology, and Fluorescent Flow Cytometry: The Sysmex XE, XT, and XS Series Hematology Analyzers
Table 32–5 Specifications of the XT, XE and XS Series Analyzers
Red Cell and Platelet Histogram Analysis
Figure 32-13 Normal RBC histogram on a Sysmex X-Series analyzer.
Hemoglobin and Hematocrit Analysis
Red Cell Distribution Width
Figure 32-14 Normal platelet histogram on a Sysmex X-Series analyzer.
Leukocyte Differential Analysis
Figure 32-15 Sysmex determination of RDW-SD and RDW-CV
Figure 32-16 Location of normal and abnormal cell populations on a WBC differential scattergram from a Sysmex XE-2100 analyzer.
Figure 32-17 Normal NRBC scattergram on a Sysmex XE-2100 analyzer.
Reticulocytes and Optical Platelets
Additional Parameters
Figure 32-18 Reticulocyte and optical platelet (PLT-O) scattergrams from a Sysmex XE-2100. Notice that the scattergrams are the same but the x-axis is moved to allow differentiation of either the Retics into HFR, MFR, and LFR on the RET scattergram or analysis of the optical platelet population in the PLT-O scattergram.
Figure 32-19 Sysmex reticulocyte scattergram showing differentiation of immature reticulocyte, mature RBC, and optical platelet populations.
Figure 32-20 PLT-O scattergram from a Sysmex XE-2100 showing the derivation of the immature platelet fraction (IPF).
Flagging
Body Fluid Analysis
Figure 32-21 Immature myeloid information (IMI) scattergram from a Sysmex XE-2100. Details show the source of specific IP (Interpretative) messages.
Table 32–6 Examples of Flagging Messages Generated by Sysmex Analyzers
Quality Control on the Sysmex X-Series Analyzers
Figure 32-22 Example of Sysmex Radar Chart. QC results are easily reviewed in this format. The inner line represents the lower acceptable limits and the outer most line represents the upper acceptable limits for the parameters represented on the chart. The green line is where the results of the most recent run of QC falls in relation to the limits.
Figure 32-23 Example of Sysmex QC data displayed on a Levy–Jennings chart.
Evaluation of Blood Specimens by Multi-Angle Polarized Scatter Separation (MAPSS) Technology: Abbott CELL-DYN Sapphire® and CELL-DYN Ruby™
Multi-Angle Polarized Scatter Separation (MAPSS)
MAPSS Plus Fluorescent Analysis: The CELL-DYN Sapphire
Figure 32-24 The optics assembly of the CELL-DYN Sapphire. Laser light is reflected and focused onto a narrow stream of cells passing through the instrument flow cell. The forward light scatter detectors are able to detect axial light loss (AxLL or 0°) and intermediate angle scatter (IAS or 7°), which are measures of cellular size and complexity respectively. Polarized side scatter (PSS or 90°) measures cellular lobularity and depolarized side scatter (DSS or 90°D) is used to separate neutrophils from eosinophils. The filters FL1, FL2, and FL3 are used to detect green fluorescence (reticulocyte analysis and FITC labeled monoclonal antibody detection), orange fluorescence (phycoerythrin labeled monoclonal antibody detection), and red fluorescence (propidium iodide staining of nonviable WBCs and NRBCs).
Figure 32-25 Hydrodynamic focusing in the CELL-DYN Sapphire optical flow cell. Hydrodynamic focusing is the process where a suspension of cells is injected into a rapidly moving “sheath” of diluent. This has the effect of ensuring that the cells are focused in a path through the center of the laser light beam.
Figure 32-26 The list mode or raw data file of the CELL-DYN Sapphire contains digitized information on the signal strength of each of the detectors. As used in leukocyte analysis of the CBC the instrument collects five pieces of optical/fluorescence data equating to measurements of cellular size, complexity, lobularity, granularity, and DNA content. Each row represents the information collected on each one of up to 20,000 cellular events. During sequential MAPSS analysis, the cells are classified into NRBCs and the individual WBC types.
CELL-DYN Sapphire NRBC Detection
Figure 32-27 The sequential separation of nucleated cells using MAPSS analysis. NRBCs are identified using their size and fluorescence (FL3) characteristics (A). The remaining leukocytes are first separated into mononuclear and polymorphonuclear cells (B) using complexity and lobularity characteristics (7° and 90° light scatter). Neutrophils and eosinophils are separated based on the 90°/90°D characteristics (C). Lymphocytes and monocytes are separated based on cellular size (D) and basophil separation is achieved by consideration of both size and internal complexity.
Hemoglobin Measurement
Red Cell Analysis
Figure 32-28 An illustration of the multidimensional nature of MAPSS analysis. The Figure shows just three dimensions (AxLL, IAS, and FL3) of the potential five dimensions of data. The PSS and DSS data are not shown.
Figure 32-29 The DNA of nonviable (membrane-permeable) leukocytes are rapidly stained by the fluorochrome dye propidium iodide. Viable cells remain unstained and show low fluorescence.
Figure 32-30 A cluster of NRBCs on the CELL-DYN Sapphire AxLL/FL3 scatterplot. The number of NRBCs is expressed in both absolute terms and as the number per 100 WBCs.
Figure 32-31 Impedance transducer of the CELL-DYN Sapphire. The transducer uses hydrodynamic focusing of the cells under analysis to minimize RBC shape distortion effects and to ensure passage of cells through the optimal detection zone of the transducer.
Reticulocyte Analysis
CELL-DYN Ruby Reticulocyte Method
Figure 32-32 The CELL-DYN Sapphire proprietary fluorescent reticulocyte dye (CD4K530) brightly stains nucleic acids (RNA and DNA) (A), which permits clear separation of reticulocytes from nucleated cells. The amount of fluorescence produced by the reticulocyte stain shows a linear and progressive relationship to RNA concentration (B), thereby allowing assessment of the maturity of reticulocytes based on their RNA concentration.
Platelet Counting
Figure 32-33 Analysis of the quantitative reticulocyte information performed by CELL-DYN Sapphire. A two-dimensional gate is drawn around the mature red cells and reticulocytes. Platelets are excluded due to their lower 7° scatter. Nucleated cells (WBCs and NRBCs) are excluded owing to their higher fluorescence resulting from DNA (A). The fluorescence intensity of the events in the red cell gate is plotted as a histogram (B). Thresholds separate the mature red cells from the reticulocytes and define the immature reticulocyte fraction (IRF).
Figure 32-34 Reticulocyte analysis on the CELL-DYN Ruby. The 0° and 10° light scatter of RBCs stained with new methylene blue is used to differentiate mature RBCs and reticulocytes.
The Immunological Platelet Count
Figure 32-35 The CELL-DYN Sapphire has both impedance and two-dimensional optical platelet counts available on each CBC. The instrument reportable platelet count is the optical count. The impedance platelet count serves as a check on the optical count results and differences between them are alerted.
Figure 32-36 Platelet aggregate detection on the CELL-DYN Sapphire.
Automated Immunofluorescence Analysis
Quality Control Procedures
Quality Assurance and Quality Control Measures for Automated Complete Blood Count Instruments
Figure 32-37 Scatterplots produced in the immunoplatelet mode. A. Immunoplatelet analysis (FL1 and 7° scatter) used to separate platelets from non-platelets. B. Two-angle (90° and 7°) scatterplot for the platelet analysis, with platelet events recolored on the basis of their green fluorescence (FL1). C. Conventional two-dimensional optical platelet analysis. The position of platelet/RBC coincidence events is indicated.
Figure 32-38 T lymphocyte subset analysis on CELL-DYN Sapphire. A. Scatterplot from CD3/4 analysis indicating the position of the T-helper cells. B. CD3/8 analysis and the position of the T suppressor cells.
Case Studies: Leukocyte Histogram/Scattergram Analysis
Table 32–7 International Consensus Group for Hematology Review Consensus Rules
Case Study 1: Immature Granulocytes with Severe Dysplasia Seen in Myelodysplastic Syndrome
Figure 32-39 Coulter scatterplot depicting an increase in immature granulocytes in a patient with myelodysplastic syndrome.
Figure 32-40 Coulter scatterplot depicting immature granulocytes without dysplasia.
Case Study 2: Acute Myelocytic Leukemia
Figure 32-41 Siemens ADVIA 120/2120 cytograms depicting acute myeloid leukemia.
Case Study 3: Acute Myelocytic Leukemia (FAB M2)
Figure 32-42 CELL-DYN CBC results and scatterplots depicting acute myeloid leukemia FAB classification M2.
Case Study 4: Chronic Myelocytic Leukemia
Figure 32-43 Sysmex XT scatterplot showing a large population of cells in the neutrophil (blue) area, suggesting the presence of immature myeloid cells.
Figure 32-44 Sysmex XE-2100 IMI scatterplot. The large red population is indicative of a large amount of immature myeloid cells.
Case Study 5: Chronic Lymphocytic Leukemia
Figure 32-45 Siemens ADVIA 120/2120 cytograms depicting chronic lymphocytic leukemia.
Case Study 6: B-Cell Chronic Lymphocytic Leukemia
Figure 32-46 CELL-DYN CBC results and scatterplots depicting B-cell chronic lymphocytic leukemia.
Case Study 7: Multiple Myeloma
Figure 32-47 Coulter scatterplot depicting the presence of immature plasma cells.
Case Study 8: Infectious Mononucleosis
Figure 32-48 Sysmex XE-2100 Diff scatterplot shows a large, stretched out lymphocyte population (pink area), which is a characteristic appearance in cases of mononucleosis. The pink activity high on the y-axis suggests the presence of atypical lymphs.
Case Study 9: Eosinophilia
Figure 32-49 Coulter scatterplot and WBC histogram depicting eosinophilia.
Case Study 10: Myeloperoxidase Deficiency
Figure 32-50 Siemens ADVIA 120–2120 cytograms depicting myeloperoxidase deficiency.
Case Study 11: Premature Newborn with an Infection
Figure 32-51 Coulter scatterplot and WBC histogram depicting WBC interferences from an increase in NRBCs.
Case Studies: Red Cell and Platelet Analysis
Case Study 12: Leukoerythroblastosis
Figure 32-52 CELL-DYN CBC results and scatterplots depicting leukoerythroblastosis.
Case Study 13: Anemia with Eosinophilia
Figure 32-53 CELL-DYN CBC results and scatterplots depicting anemia with marked eosinophilia.
Case Study 14: Iron-Deficiency Anemia
Figure 32-54 Siemens ADVIA 120–2120 cytograms depicting iron-deficiency anemia.
Case Study 15: Vitamin B12 Deficiency
Figure 32-55 Coulter RBC histogram showing red cell abnormalities associated with vitamin B12 deficiency.
Case Study 16: Sickle Cell Anemia Case 1
Figure 32-56 Siemens ADVIA 120–2120 cytograms depicting sickle cell anemia.
Case Study 17: Sickle Cell Anemia Case 2
Figure 32-57 Sysmex XE-2100 Diff Scatterplot from a patient with sickle cell anemia. All three Sysmex analyzers showed similar scatterplots with an increase in the debris along the x-axis from the sickled red cells.
Figure 32-58 Sysmex XE-2100 NRBC scatterplot. The increased NRBCs are indicated by the pink cluster to the left of the WBCs (blue cluster).
Figure 32-59 Sysmex XE-2100 reticulocyte scatterplot in a sickle cell patient. The reticulocyte count is elevated at 9.88% reticulocytes
Case Study 18: Beta-Thalassemia Trait
Figure 32-60 Siemens ADVIA 120–2120 cytograms depicting beta-thalassemia trait.
Case Study 19: Hemolytic Anemia
Figure 32-61 Coulter RBC histogram and scatterplot depicting red cell abnormalities seen in a case of hemolytic anemia.
Case Study 20: Platelet Clumps
Figure 32-62 Coulter scatterplots, histograms, and photomicrographs depicting platelet clumps.
Case Study 21: May–Hegglin Anomaly
Figure 32-63 PLT histogram on a Sysmex XE-2100 from a patient with May–Hegglin anomaly. Note that the platelet curve does not return to the baseline of the histogram
Figure 32-64 A portion of the results screen from a Sysmex XE-2100. Note that the PLT parameter is flagged with an “&,” which indicates that the result came from the PLT-O scatterplot and not from the impedance count. The large platelets do not interfere with the flow cytometric counting in the optical platelet, so that result is reported
Figure 32-65 Sysmex XE-2100 PLT-O scatterplot. In the case of a patient with large platelets, the flow cytometric platelet count is reported to avoid interferences.
Case Study 22: Leukoagglutination with Platelet Satellitosis
Figure 32-66 Coulter VCS scatterplot depicting leukoagglutination with satellitosis.
Figure 32-67 Photomicrographs depicting platelet satellitosis and leukoagglutination.
Figure 32-68 Coulter VCS scatterplot of patient sample recollected in sodium citrate.
Case Study 23: Bacterial Meningitis
Figure 32-69 Siemens ADVIA 120–2120 cytogram and manual differential showing bacterial meningitis.
Questions
SUMMARY CHART
Acknowledgments
REFERENCES
Chapter 33 Coagulation Methods
OBJECTIVES
General Points Regarding Coagulation Procedures
Platelet Function Tests
Bleeding Time
Closure Time—PFA-100® (Siemens)
Platelet Aggregation
Table 33–1 Interpretation of PFA-100® Closure Time Results
Figure 33-1 Aggregation curves with various aggregating agents. A. Aggregation curve induced by collagen. Note the lag time before aggregation followed by a single wave of aggregation. B. Aggregation curve induced with epinephrine and thrombin. Note the biphasic wave of aggregation. C. Aggregation curve induced by ristocetin. A biphasic wave of aggregation as well as a single wave of aggregation may be seen. D. Aggregation curve induced by serotonin. Generally a single wave of aggregation followed by disaggregation is seen.
Figure 33-2 Aggregation curves induced with various concentrations of ADP. A. Very low concentrations of ADP induce a primary wave of aggregation followed by disaggregation. B. The optimal concentration of ADP induces a biphasic wave of aggregation. C. High concentrations of ADP induce a broad wave of aggregation.
Coagulation Screening Tests
Activated Partial Thromboplastin Time
Figure 33-3 Activated partial thromboplastin time (aPTT) assay.
One-Stage Prothrombin Time (Quick)11
Figure 33-4 Prothrombin time (PT) assay.
Thrombin Time
Mixing Studies—aPTT or PT 1:1 Mix
PROCEDURE: INCUBATED MIXING STUDIES
Coagulation Factor Assays
One-Stage Quantitative Assay Method for Factors II, V, VII, and X
Table 33–2 Preparation of Test Dilutions for Reference Plasma in the One-Stage Assay for Factors
One-Stage Quantitative Assay Method for Factors VIII, IX, XI, and XII
Figure 33-5 Factor V activity curve.
Factor XIII Screening Test
Coagulation Inhibitors
BETHESDA TITER
Tests to Monitor Anticoagulant Therapy
Monitoring Anticoagulant Therapy with Coagulation Screening Assays
HEPARIN THERAPY
ANTI-VITAMIN K (WARFARIN) THERAPY
Anti-FXa Assay (Heparin Activity)
Figure 33-6 Heparin anti-FXa assay.
Monitoring Direct Thrombin Inhibitors
Tests to Measure Fibrin Formation
Reptilase Time
Fibrinogen Activity
Table 33–3 Test Comparison
Tests for von Willebrand Disease
Figure 33-7 Fibrinogen calibration curve.
von Willebrand Factor Antigen
Table 33–4 Types of von Willebrand Disease
Figure 33-8 Latex immunoassay principle.
Figure 33-9 Principle of the sandwich enzyme-linked immunosorbent assay (ELISA) test for von Willebrand factor antigen (vWF: Ag).
von Willebrand Factor Activity (vWF:RCo, Ristocetin Cofactor)
von Willebrand Collagen Binding Activity
von Willebrand Factor Multimer Analysis
Figure 33-10 vWF multimer gel. Lane 11. Type 2A showing loss of high and moderate MW multimers. Lane 10. Type 1 showing a normal distribution with decreased quantity. Lane 9. Type 2B showing a loss of high MW multimers. Lane 8. Normal pattern.
Tests to Assess Hereditary Thrombotic Risk
Activated Protein C Resistance/Factor V Leiden
Antithrombin Assays
Antithrombin Functional Assay (Activity)
CHROMOGENIC SUBSTRATE ASSAY
Antithrombin Immunologic Assay (Antigen)
MICRO-LATEX PARTICLE IMMUNOLOGIC ASSAY
Protein C Assays
Protein C Immunologic Assay (Antigen)
Protein C Functional Assays (Activity)
CHROMOGENIC SUBSTRATE ASSAY
CLOT-BASED ASSAY
Protein S Assays
Protein S Functional Assay (Activity)–Clotting Assay
Protein S Immunologic Assay (Antigen)
Prothrombin G20210A (Factor II) Mutation
Tests for the Evaluation of Lupus Anticoagulants
Confirmatory Tests for Lupus Anticoagulants
Platelet Neutralization Procedure
Hexagonal Phospholipid Neutralization Assay
Anti-Phospholipid Antibody Assays
Tests for Fibrinolysis
D-Dimer Quantitative Test
Euglobulin Lysis Time
Fibrin Degradation Products: Latex Agglutination Method
Table 33–5 Dilutions
Markers of Coagulation Activation and Thrombin Generation
Coagulation Instrumentation
General Types of Coagulation Instrumentation
FULLY AUTOMATED METHOD
SEMIAUTOMATED METHOD
MANUAL (TILT-TUBE) METHOD
Methods of Endpoint Detection
MECHANICAL ENDPOINT DETECTION
PHOTO-OPTICAL ENDPOINT DETECTION
CHROMOGENIC ENDPOINT DETECTION
IMMUNOLOGIC ENDPOINT DETECTION
Case Study 1
DISCUSSION
Case Study 2
DISCUSSION
FOLLOW-UP:
Questions
SUMMARY CHART
REFERENCES
Chapter 34 Applications of Flow Cytometry to Hematopathology
OBJECTIVES
Basic Concepts of Flow Cytometry
Sample Preparation
SPECIMEN COLLECTION AND HANDLING
STAINING WITH FLUORESCENT DYES
STAINING WITH ANTIBODIES
Figure 34-1 General staining scheme using fluorescent dyes.
Table 34–1 Common Fluorescent Dyes and Fluorochromes Used in Clinical Flow Cytometry
Figure 34-2 General staining scheme for staining leukocytes with antibodies. RBCs = red blood cells.
Figure 34-3 Detection of five T-cell populations via three-color staining.
Cytometer Operation
STARTUP
QUALITY CONTROL
OPTIMIZATION
Table 34–2 Useful Antibodies for Hematology Applications
CYTOMETER OVERVIEW
Figure 34-4 Schematic of cell analysis using a two-laser flow cytometer.
THRESHOLD
PHOTODETECTORS
Table 34–3 Major Cytometer Models
AMPLIFICATION
FLUORESCENCE COMPENSATION
DATA COLLECTION
SHUTDOWN
Data Analysis
IDENTIFYING POPULATIONS
Figure 34-5 Leukocyte populations discernible in lysed whole blood. A. FSC/SSC display. B. CD45/SSC display. Note how in B, debris and basophils can be resolved from the lymphocyte population.
GATING
Figure 34-6 Populations discernible in whole blood.
QUADRANT STATISTICS
Figure 34-7 Effect of a lymphocyte gate (A) on fluorescence. Fluorescence events in B (ungated) and C (gated data) are color-coded to their respective populations in A using a gate provides a means for analyzing one population at a time. UL = upper left; UR = upper right; LL = lower left; LR = lower right.
Figure 34-8 Quadrant statistics from Figure 34–7C. UL = upper left; UR = upper right; LL = lower left; LR = lower right.
REGION STATISTICS
SINGLE-PARAMETER HISTOGRAM STATISTICS
Figure 34-9 Analysis using regions.
Figure 34-10 Analysis using a single-parameter histogram with markers.
ABSOLUTE CELL COUNTS
Figure 34-11 Analysis of DNA data using a single-parameter histogram and software modeling.
FLUORESCENCE INTENSITY MEASUREMENTS
Applications of Flow Cytometry
Lymphocyte Subset Analysis and CD4 T-Cell Enumeration
SAMPLE PREPARATION
CYTOMETER OPERATION
DATAANALYSIS
Table 34–4 Panels for Lymphocyte Subsetting Analyses
Leukemia and Lymphoma Immunophenotyping
Figure 34-12 Automated four-color lymphocyte subset analysis.
SAMPLE PREPARATION
CYTOMETER OPERATION
DATAANALYSIS
Figure 34-13 Normal (A and C) and abnormal (B and D) scatter and staining patterns in bone marrow. The abnormal sample is from a patient with acute myelocytic leukemia. The normal lymphoid population is highlighted for reference. In B the abnormal population is apparent because of its low SSC and dim CD45 staining, and in D owing to the coexpression of CD15 and CD34. These two antigens are normally expressed in different maturation phases.
Leukemia and Lymphoma DNA Content Analysis
Figure 34-14 Normal (A and C) and abnormal (B and D) staining in peripheral blood. The abnormal sample is from a patient with chronic lymphocytic leukemia. In B, the abnormal population is apparent because of the coexpression of CD5 and CD19, two antigens normally found on different lineages. In D, where there are usually twice as many kappa cells as lambda cells, it is apparent that the abnormal clone has overgrown and replaced the normal population.
SAMPLE PREPARATION
CYTOMETER OPERATION
Figure 34-15 Quality control of the FL2-A amplifier using chicken erythrocyte nuclei stained with propidium iodide. Amplifier linearity is measured using the ratio of the doublet population mean to the single population mean. It should range between 1.95 and 2.05. Resolution is determined by measuring the coefficient of variation (CV) of the singlet peak. It should be less than 3%.
DATAANALYSIS
Hematopoietic Progenitor Cell Enumeration
Figure 34-16 DNA histogram of a leukemic T-cell line mixed with peripheral blood mononuclear cells (first peak). The statistics were calculated via software modeling.
SAMPLE PREPARATION
CYTOMETER OPERATION
DATAANALYSIS
Flow Crossmatching
SAMPLE PREPARATION
Figure 34-17 Enumeration of hematopoietic progenitor cells.
CYTOMETER OPERATION
DATAANALYSIS
Figure 34-18 Flow cytometry crossmatching. The negative (NHS) control (dotted) and positive control (solid) are shown.
Detection of Paroxysmal Nocturnal Hemoglobinuria
SAMPLE PREPARATION
CYTOMETER OPERATION
DATA ANALYSIS
Figure 34-19 Analysis of erythrocyte staining in paroxysmal nocturnal hemoglobinuria,. Normal control (A). Background staining is represented by the fluorescence from the patient’s subclass control sample and displayed as dotted lines. The controls were used for drawing regions to denote type I, II, and III populations (R1, R2, and R3, respectively). Patient sample (B) reveals the presence of type II and type III populations.
Residual White Blood Cell Enumeration
Figure 34-20 Analysis of granulocyte staining in paroxysmal nocturnal hemoglobinuria. Normal control (A) and patient sample (B). Note the abnormal population in Q4.
SAMPLE PREPARATION
CYTOMETER OPERATION
DATAANALYSIS
Figure 34-21 Detection of residual white blood cells (rWBC) in blood products. R1 = beads; R2 = rWBC.
Detection of Fetomaternal Hemorrhage
SAMPLE PREPARATION
CYTOMETER OPERATION
DATAANALYSIS
Platelet Studies
SAMPLE PREPARATION
Figure 34-22 Detection of fetomaternal hemorrhage using anti-hemoglobin F (HgbF) staining. Interference by autofluorescent leukocytes is removed by gating on the FL2 dim population (B). A control sample spiked with 1% cord blood is shown (C).
CYTOMETER OPERATION
DATAANALYSIS
Bead-Based Assays for Soluble Factors
Figure 34-23 Staining pattern of resting (A) and ADP-activated (B) platelets. Note the increase in size and CD62P staining. The size increase is a result of aggregation.
SAMPLE PREPARATION
CYTOMETER OPERATION
DATAANALYSIS
Figure 34-24 Qualitative bead-based assay for soluble factors. A. Negative control. B. Positive control.
Figure 34-25 Quantitative bead-based assay for soluble factors. A. Determination of the mean fluorescence intensity (MFI) for the IL-8. B. Conversion of the MFI into pg/ml of analyte.
Questions
SUMMARY CHART
Acknowledgments
REFERENCES
Chapter 35 Molecular Diagnostic Techniques in Hematopathology
OBJECTIVES
Structure of DNA
Figure 35-1 DNA is composed of two strands of nucleotides that are bound to each other through hydrogen bonds (depicted as diagonal bridges). Only four types of nucleotides are present: adenine (A), thymine (T), guanine (G), and cytosine (C). Because of the characteristic biochemical structure of each nucleotide, an A on one strand can bond only to a T on the other strand, and a G can bond only to a C. Therefore, the two strands of DNA are said to be “complementary” to each other. The strands are oriented in opposite directions with respect to the sugar and phosphate backbone, so each strand has a 5′ and a 3′ end. In the laboratory, the two strands of DNA may be dissociated from one another by heating them to near-boiling temperature (95°C) or treating them with an alkaline solution (high pH).
Applications of DNA Technology in Laboratory Medicine
Figure 35-2 A probe represents a short strand of nucleotides that can bind (or “hybridize”) to its complementary target nucleotide sequence. In the example depicted here, the probe was labeled with digoxigenin so that it could be subsequently detected via a colorimetric reaction involving an antibody to digoxigenin, alkaline phosphatase, and 4-nitroblue tetrazolium chloride (NBT).
Table 35–1 Hematologic Disease Amenable to Molecular Diagnosis
Sample Sources for Molecular Procedures
Nucleic Acid Extraction
Preparation of Samples
DNA Extraction from Fresh Cells or Frozen Tissue
Table 35–2 Genetic Abnormalities in Hematopoietic Cancers
DNA Extraction from Fixed, Paraffin-Embedded Tissue
RNA Extraction
Nucleic Acid Quantitation
Sequence-Specific Fragmentation by Restriction Endonucleases
Diagnostic Procedures for Analyzing DNA
Southern Blot Analysis
Figure 35-3 Southern blot analysis of DNA structure. In this procedure, DNA is extracted from cells and cut by restriction endonucleases at specific sequences. The resulting fragments are separated by size using gel electrophoresis, and then denatured into their single-stranded fragments and transferred to a nylon membrane. The membrane is soaked in a probe solution, permitting the labeled probe to hybridize to complementary nucleotide sequences on the membrane. In the particular example shown here, radiolabeled probe binds to only one target sequence in the patient sample, thus producing a single band on the autoradiograph.
Figure 35-4 A. Southern blot analysis can be used to identify B-cell tumors based on their characteristic clonal immunoglobulin gene rearrangements. Normally during B-cell differentiation, the immunoglobulin kappa light-chain gene (IGK) rearranges to produce a unique coding sequence that determines antibody specificity. This occurs through a process of splicing and deletion whereby 1 of 80 variable (V) regions is juxtaposed with 1 of 5 joining (J) regions. These rearrangements alter the size of the DNA fragments produced by the HindIII restriction endonuclease and recognized by hybridization to a probe (shown as a bar) spanning the J region. Whereas each benign B cell rearranges its IGK gene differently, malignant B cells contain exactly the same rearrangement that was present in the B cell from which the tumor arose. This clonal gene rearrangement alters the band pattern on a Southern blot. In addition to the 3.0-kb rearranged band that characterizes the particular rearrangement depicted here, there is also partial retention of the 5.4-kb unrearranged (germline) IGK gene originating from residual normal cells in the sample or from the other IGK allele in tumor cells. B. In the actual Southern blot autoradiograph shown here, a J region probe was used to detect clonal IGK gene rearrangement in a case of chronic lymphocytic leukemia (L). In each of three different restriction endonuclease digests, the presence of two extra bands suggests that both IGK alleles are clonally rearranged in the tumor cells or, less likely, that the tumor is biclonal. Normal control (C) tissue analyzed simultaneously identifies the position of the germline bands. A map of the J region depicts the expected size of the germline bands for each restriction endonuclease.
Polymerase Chain Reaction (PCR) and Real-Time PCR
REAL-TIME PCR
Figure 35-5 Polymerase chain reaction (PCR) is a method of copying a particular segment of DNA numerous times through a process of repeated cycles of heating, cooling, and DNA synthesis. To accomplish this, the target DNA is mixed with two short DNA probes called primers (shown as half-arrows) that are designed to span the segment of DNA to be amplified. Also added to the mixture is an enzyme called DNA polymerase, which converts single-stranded DNA into double-stranded DNA by incorporating nucleotides starting at the 3′ end of each primer. A thermocycler instrument is programmed to heat and cool the sample sequentially. In each heat/cool cycle, the sample is first heated to 95°C to dissociate the two strands of DNA, and then cooled to 55°C to permit binding of the primers, then warmed to 72°C for enzymatic DNA replication. After the first cycle, an exact copy of the original target DNA has been produced. Then, in subsequent cycles, the products of previous cycles serve as templates for DNA replication, permitting an exponential accumulation of DNA copies. After 30 cycles, which takes only a couple of hours, a billion copies of the target fDNA have been synthesized.
Figure 35-6 Quantitative real-time polymerase chain reaction (Q-PCR) allows estimation of the amount of DNA template in a patient sample. This is achieved in a thermocycler that has been modified to measure fluorescence at the end of each amplification cycle. The reaction contains a fluorochrome-labeled internal probe that hybridizes to the product and generates a signal in proportion to the level of the product. This signal, along with the signals generated by a series of standards, is visualized on the amplification plot shown above. The level of template in a given patient sample is estimated by extrapolation to the standards. Because the reaction vessels are never opened after amplification, the risk of amplicon contamination is minimized. Lower labor costs, faster turnaround time, and more precise measurement of the target DNA are added benefits of real-time compared with traditional gel-based detection of PCR products
Figure 35-7 Melt curve analysis can be used to detect a mutation in PCR-amplified DNA. A. Two fluorochrome-labeled, sequence-specific probes bind in tandem to the PCR product spanning the location of the putative mutation site. Gradual heating results in probe dissociation which is measured by the amount of fluorescence resonance energy transfer (FRET) between the two fluorochromes, F1 and F2. The temperature at which probe dissociation occurs depends on whether a mismatch is present, with a mismatch yielding a lower melting temperature (Tm). B. Based on the appearance of the melt curve, multiple patient samples are classified as normal or mutant (heterozygous or homozygous) for the HFE 845G:A mutation. This mutation encodes the HFE Cys282Tyr amino acid substitution in a protein that is responsible for regulating iron absorption from the diet.
Reverse Transcriptase Polymerase Chain Reaction
Figure 35-8 The reverse transcriptase polymerase chain reaction (RT-PCR) procedure is a method for detecting a particular RNA transcript. First, RNA serves as a template for construction of complementary DNA (cDNA) by the enzyme reverse transcriptase. Then routine PCR is done to convert the cDNA of interest to a double-stranded sequence and to amplify that sequence so that it may be readily detected or further analyzed. This procedure is a sensitive, specific, and rapid means of amplifying disease-associated RNA in a patient sample.
In Situ Hybridization to Cells or Tissues Immobilized on Glass Slides
Figure 35-9 The in situ hybridization technique permits visualization of nucleic acid in tissue sections on glass slides. In this example, hybridization to Epstein-Barr virus encoded RNA (EBER) transcripts reveals that the viral gene product is localized to the nucleus of a Reed-Sternberg cell in a case of Hodgkin lymphoma. In contrast, the background lymphocytes stain only with methyl green counterstain.
Fluorescence In Situ Hybridization
Figure 35-10 Fluorescence in situ hybridization (FISH) analysis helps in the diagnosis of DiGeorge syndrome, a congenital form of immunodeficiency caused by partial deletion of chromosome 22. In the example shown here, bright fluorescent signals identify the two number 22 chromosomes from among the 46 chromosomes in a cell. The DiGeorge probe and a control probe are visible on the normal chromosome 22 (center); however, only the control probe is seen on the other chromosome 22 (lower right), consistent with a deletion of DNA from the DiGeorge region.
DNA Sequencing
Array Technology for Gene Expression Profiling
Probe Production
Figure 35-11 Vectors are vehicles for capturing and replicating specific DNA sequences. The process of using a vector to capture and manipulate a particular DNA segment is called recombinant DNA technology. The recombined vector/insert DNA can be replicated inside bacterial hosts using natural cellular machinery. Specialized features built into the vector assist in tracking and controlling the vector and insert. Examples of vectors include plasmids, cosmids, and artificial chromosomes, the latter of which can hold extremely large segments of DNA (300 kb). These vectors facilitate production of abundant, homogeneous probes.
Probe Labels
Future Prospects of Molecular Assays
Figure 35-12 DNA probes are labeled by incorporating radioisotopes or non-isotopic markers into their nucleotide strands. A. The random primer method capitalizes on the ability of Klenow DNA polymerase to synthesize new complementary strands of DNA starting at the free 3′ ends where “hexamers” (6 base probes of random sequence) have bound to the template strand. Addition of labeled nucleotides to the reaction mixture results in incorporation of the label into the newly synthesized DNA. B. The nick translation method of probe labeling relies on the ability of the enzyme DNase 1 to randomly nick the backbone of DNA. Then the enzyme DNA polymerase 1 recognizes and repairs each nick, and subsequently proceeds to replace adjacent 3′ nucleotides with new ones. In this way, labeled nucleotides are incorporated into the newly synthesized DNA to form a DNA probe.
Case Study
Questions
SUMMARY CHART
Acknowledgments
REFERENCES
Chapter 36 Special Stains/Cytochemistry
OBJECTIVES
Definition and Historical Background of Cytochemistry
Technical Considerations
Figure 36-1 Cytochemical approach to the diagnosis of acute leukemias. AGL = acute granulocytic leukemia; NaF = sodium fluoride; AMonoL = acute monocytic leukemia; ALL = acute lymphoblastic leukemia; AEL = acute erythroleukemia; AmegL = acute megakaryoblastic leukemia; AUL = acute undifferentiated leukemia.
Cytochemical Enzyme Stains
Leukocyte Alkaline Phosphatase Stain
Figure 36-2 Positive LAP reaction. Increased LAP activity is seen in leukemoid reactions (infections) and in chronic myeloproliferative disorders such as polycythemia vera and idiopathic myelofibrosis.
Figure 36-3 Negative LAP reaction.
Table 36–1 Leukocyte Alkaline Phosphatase Scoring Scale and Characteristics
Table 36–2 Leukocyte Alkaline Phosphatase Score Calculation
Myeloperoxidase Stain
Table 36–3 Leukocyte Alkaline Phosphatase Reactivity in Various Disorders
Figure 36-4 Myeloperoxidase positivity in acute myeloid leukemia (M2). Note the bluish-black granules in the myeloid cells.
Alternative Method—Myeloperoxidase Stain
Cyanide-Resistant Peroxidase for Eosinophils
Figure 36-5 Cyanide-resistant peroxidase stain. Note that eosinophils and their precursors stain brown. Monocytes and granulocytes are negative for this stain.
Cytochemical Esterases
Figure 36-6 Nonspecific esterase reaction in monoblasts.
Figure 36-7 Nonspecific esterase stain with sodium fluoride inhibition in acute monocytic leukemia (M5b). Note the lack of staining in the monoblasts and immature monocytes.
Specific Esterase
NAPHTHOL-AS-D CHLOROACETATE ESTERASE
Table 36–4 Cytochemistry Markets for Subclassification of Nonlymphocytic Leukemia
Figure 36-8 Naphthol AS-D chloroacetate esterase stain in a patient with AML, M2. Note the bright red staining indicating that these two blasts are of myeloid origin.
Nonspecific Esterase (Fluoride Resistant)
α-NAPHTHYL BUTYRATE, α-NAPHTHYLACETATE
Figure 36-9 Nonspecific esterase α-naphthyl acetate positivity in acute monocytic leukemia (M5b). Note the granular positivity in the monoblasts and immature monocytes.
Figure 36-10 Positive α-naphthyl butyrate esterase stain in a patient with acute monocytic leukemia, M5b. Note the diffuse brick-red staining in the monoblasts, promonocytes, and monocytes.
Combined Esterase
Figure 36-11 Combined esterase positivity in acute myelomonocytic leukemia (M4). Granulocytes demonstrate a granular blue staining and monocytes demonstrate diffuse brown-red staining.
Other Stains
Acid Phosphatase Stain/Tartrate-Resistant Acid Phosphatase
Figure 36-12 Tartrate-resistant acid phosphatase (TRAP) stain of peripheral blood showing positivity in hairy cells and no staining in neutrophils. Note the presence of red precipitate in the hairy cells.
Figure 36-13 Acid phosphatase–positive T-cell lymphoblastic leukemia. Note: The enzyme activity is localized in the Golgi area and has a dotlike appearance.
Terminal Deoxynucleotidyl Transferase Test
Figure 36-14 Cytospin acid phosphatase positivity in lymphocytes.
Figure 36-15 TdT positivity in T-cell lymphoblasts in acute lymphocytic leukemia.
Nonenzyme Stains
Sudan Black B Stain
Figure 36-16 Sudan black B positivity in acute promyelocytic leukemia (M3). Note the many brownish-black staining granules in the promyelocytes.
Periodic Acid–Schiff Reaction
Figure 36-17 Positive Sudan black B (SBB) stain in a patient with AML, M2. Note the black staining cytoplasmic granules in the myeloblasts.
Figure 36-18 Positive PAS stain in acute megakaryocytic leukemia AML, M7.
Figure 36-19 Positive PAS stain in a child with ALL.
Figure 36-20 PAS positivity in erythroleukemia (M6). Note the intense staining of the large abnormal erythroblast.
Table 36–5 Summary of Cytochemical Stains
Questions
SUMMARY CHART
Back Matter
Answers to Chapter Review Questions
Glossary
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
R
S
T
U
V
W
X
Z
Bibliography
Chapter 5
Chapter 12
Chapter 26
Chapter 29
Chapter 30
Suggested Reading and Related Web Sites
Chapter 36
Index
Endsheets
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