Rodak's Hematology: Clinical Principles and Applications, 5th Ed.

CHAPTER 1. An overview of clinical laboratory hematology

George A. Fritsma

OUTLINE

History

Red Blood Cells

Hemoglobin, Hematocrit, and Red Blood Cell Indices

Reticulocytes

White Blood Cells

Platelets

Complete Blood Count

Blood Film Examination

Endothelial Cells

Coagulation

Advanced Hematology Procedures

Additional Hematology Procedures

Hematology Quality Assurance and Quality Control

The average human possesses 5 liters of blood. Blood transports oxygen from lungs to tissues; clears tissues of carbon dioxide; transports glucose, proteins, and fats; and moves wastes to the liver and kidneys. The liquid portion is plasma, which, among many components, provides coagulation enzymes that protect vessels from trauma and maintain the circulation.

Plasma transports and nourishes blood cells. There are three categories of blood cells: red blood cells (RBCs), or erythrocytes; white blood cells (WBCs), or leukocytes; and platelets (PLTs), or thrombocytes.1Hematology is the study of these blood cells. By expertly staining, counting, analyzing, and recording the appearance, phenotype, and genotype of all three types of cells, the medical laboratory professional (technician or scientist) is able to predict, detect, and diagnose blood diseases and many systemic diseases that affect blood cells. Physicians rely on hematology laboratory test results to select and monitor therapy for these disorders; consequently, a complete blood count (CBC) is ordered on nearly everyone who visits a physician or is admitted to a hospital.

History

The first scientists such as Athanasius Kircher in 1657 described “worms” in the blood, and Anton van Leeuwenhoek in 1674 gave an account of RBCs,2 but it was not until the late 1800s that Giulio Bizzozero described platelets as “petites plaques.”3 The development of Wright stain by James Homer Wright in 1902 opened a new world of visual blood film examination through the microscope. Although automated instruments now differentiate and enumerate blood cells, Wright’s Romanowsky-type stain (polychromatic, a mixture of acidic and basic dyes), and refinements thereof, remains the foundation of blood cell identification.4

In the present-day hematology laboratory, RBC, WBC, and platelet appearance is analyzed through automation or visually using 500× to 1000× light microscopy examination of cells fixed to a glass microscope slide and stained with Wright or Wright-Giemsa stain (Chapters 15 and 16). The scientific term for cell appearance is morphology, which encompasses cell color, size, shape, cytoplasmic inclusions, and nuclear condensation.

Red blood cells

RBCs are anucleate, biconcave, discoid cells filled with a reddish protein, hemoglobin (HGB), which transports oxygen and carbon dioxide (Chapter 10). RBCs appear pink to red and measure 6 to 8 μm in diameter with a zone of pallor that occupies one third of their center (Figure 1-1A), reflecting their biconcavity (Chapters 8 and 9).

Image 

FIGURE 1-1 Normal cells in peripheral blood: A, Erythrocyte (red blood cell, RBC); B, Neutrophil (segmented neutrophil, NEUT, SEG, polymorphonuclear neutrophil, PMN); C, Band (band neutrophil, BAND); D, Eosinophil (EO); E, Basophil (BASO); F,Lymphocyte (LYMPH); G, Monocyte (MONO); H, Platelet (PLT).

Since before 1900, physicians and medical laboratory professionals counted RBCs in measured volumes to detect anemia or polycythemia. Anemia means loss of oxygen-carrying capacity and is often reflected in a reduced RBC count or decreased RBC hemoglobin concentration (Chapter 19). Polycythemia means an increased RBC count reflecting increased circulating RBC mass, a condition that leads to hyperviscosity (Chapter 33). Historically, microscopists counted RBCs by carefully pipetting a tiny aliquot of whole blood and mixing it with 0.85% (normal) saline. Normal saline matches the osmolality of blood; consequently, the suspended RBCs retained their intrinsic morphology, neither swelling nor shrinking. A 1:200 dilution was typical for RBC counts, and a glass pipette designed to provide this dilution, the Thoma pipette, was used routinely until the advent of automation.

The diluted blood was transferred to a glass counting chamber called a hemacytometer (Figure 14-1). The microscopist observed and counted RBCs in selected areas of the hemacytometer, applied a mathematical formula based on the dilution and on the area of the hemacytometer counted (Chapter 14), and reported the RBC count in cells per microliter (μL, mcL, also called cubic millimeter, mm3), milliliter (mL, also called cubic centimeter, or cc), or liter (L).

Visual RBC counting was developed before 1900 and, although inaccurate, was the only way to count RBCs until 1958, when automated particle counters became available in the clinical laboratory. The first electronic counter, patented in 1953 by Joseph and Wallace Coulter of Chicago, Illinois, was used so widely that today automated cell counters are often called Coulter counters, although many high-quality competitors exist (Chapter 15).5 The Coulter principle of direct current electrical impedance is still used to count RBCs in many automated hematology profiling instruments. Fortunately, the widespread availability of automated cell counters has replaced visual RBC counting, although visual counting skills remain useful where automated counters are unavailable.

Hemoglobin, hematocrit, and red blood cell indices

RBCs also are assayed for hemoglobin concentration (HGB) and hematocrit (HCT) (Chapter 14). Hemoglobin measurement relies on a weak solution of potassium cyanide and potassium ferricyanide, called Drabkin reagent. An aliquot of whole blood is mixed with a measured volume of Drabkin reagent, hemoglobin is converted to stable cyanmethemoglobin (hemiglobincyanide), and the absorbance or color intensity of the solution is measured in a spectrophotometer at 540 nm wavelength.6 The color intensity is compared with that of a known standard and is mathematically converted to hemoglobin concentration. Modifications of the cyanmethemoglobin method are used in most automated applications, although some automated hematology profiling instruments replace it with a formulation of the ionic surfactant (detergent) sodium lauryl sulfate to reduce environmental cyanide.

Hematocrit is the ratio of the volume of packed RBCs to the volume of whole blood and is manually determined by transferring blood to a graduated plastic tube with a uniform bore, centrifuging, measuring the column of RBCs, and dividing by the total length of the column of RBCs plus plasma.7 The normal ratio approaches 50% (refer to inside front cover for reference intervals). Hematocrit is also called packed cell volume (PCV), the packed cells referring to RBCs. Often one can see a light-colored layer between the RBCs and plasma. This is the buffy coat and contains WBCs and platelets, and it is excluded from the hematocrit determination. The medical laboratory professional may use the three numerical results—RBC count, HGB, and HCT—to compute the RBC indices mean cell volume (MCV), mean cell hemoglobin(MCH), and mean cell hemoglobin concentration (MCHC) (Chapter 14). The MCV, although a volume measurement recorded in femtoliters (fL), reflects RBC diameter on a Wright-stained blood film. The MCHC, expressed in g/dL, reflects RBC staining intensity and amount of central pallor. The MCH in picograms (pg) expresses the mass of hemoglobin and parallels the MCHC. A fourth RBC index, RBC distribution width (RDW), expresses the degree of variation in RBC volume. Extreme RBC volume variability is visible on the Wright-stained blood film as variation in diameter and is called anisocytosis. The RDW is based on the standard deviation of RBC volume and is routinely reported by automated cell counters. In addition to aiding in diagnosis of anemia, the RBC indices provide stable measurements for internal quality control of counting instruments (Chapter 5).

Medical laboratory professionals routinely use light microscopy at 500× or 1000× magnification (Chapters 4 and 16) to visually review RBC morphology, commenting on RBC diameter, color or hemoglobinization, shape, and the presence of cytoplasmic inclusions (Chapters 16 and 19). All these parameters—RBC count, HGB, HCT, indices, and RBC morphology—are employed to detect, diagnose, assess the severity of, and monitor the treatment of anemia, polycythemia, and the numerous systemic conditions that affect RBCs. Automated hematology profiling instruments are used in nearly all laboratories to generate these data, although visual examination of the Wright-stained blood film is still essential to verify abnormal results.8

Reticulocytes

In the Wright-stained blood film, 0.5% to 2% of RBCs exceed the 6- to 8-μm average diameter and stain slightly blue-gray. These are polychromatic (polychromatophilic) erythrocytes, newly released from the RBC production site: the bone marrow (Chapters 8 and 17). Polychromatic erythrocytes are closely observed because they indicate the ability of the bone marrow to increase RBC production in anemia due to blood loss or excessive RBC destruction (Chapters 23 to 26 [Chapter 23 Chapter 24 Chapter 25 Chapter 26]).

Methylene blue dyes, called nucleic acid stains or vital stains, are used to differentiate and count these young RBCs. Vital (or “supravital”) stains are dyes absorbed by live cells.9 Young RBCs contain ribonucleic acid (RNA) and are called reticulocytes when the RNA is visualized using vital stains. Counting reticulocytes visually by microscopy was (and remains) a tedious and imprecise procedure until the development of automated reticulocyte counting by the TOA Corporation (presently Sysmex Corporation, Kobe, Japan) in 1990. Now all fully automated profiling instruments provide an absolute reticulocyte count and, in addition, an especially sensitive measure of RBC production, the immature reticulocyte count or immature reticulocyte fraction (Chapter 15). However, it is still necessary to confirm instrument counts visually from time to time, so medical laboratory professionals must retain this skill.

White blood cells

WBCs, or leukocytes, are a loosely related category of cell types dedicated to protecting their host from infection and injury (Chapter 12). WBCs are transported in the blood from their source, usually bone marrow or lymphoid tissue, to their tissue or body cavity destination. WBCs are so named because they are nearly colorless in an unstained cell suspension.

WBCs may be counted visually using a microscope and hemacytometer. The technique is the same as RBC counting, but the typical dilution is 1:20, and the diluent is a dilute acid solution. The acid causes RBCs to lyse or rupture; without it, RBCs, which are 500 to 1000 times more numerous than WBCs, would obscure the WBCs. The WBC count ranges from 4500 to 11,500/μL. Visual WBC counting has been largely replaced by automated hematology profiling instruments, but it is accurate and useful in situations in which no automation is available. Medical laboratory professionals who analyze body fluids such as cerebrospinal fluid or pleural fluid may employ visual WBC counting.

A decreased WBC count (fewer than 4500/μL) is called leukopenia, and an increased WBC count (more than 11,500/μL) is called leukocytosis, but the WBC count alone has modest clinical value. The microscopist must differentiate the categories of WBCs in the blood by using a Wright-stained blood film and light microscopy (Chapter 16). The types of WBCs are as follows:

• Neutrophils (NEUTs, segmented neutrophils, SEGs, polymorphonuclear neutrophils, PMNs; Figure 1-1B). Neutrophils are phagocytic cells whose major purpose is to engulf and destroy microorganisms and foreign material, either directly or after they have been labeled for destruction by the immune system. The term segmented refers to their multilobed nuclei. An increase in neutrophils is called neutrophilia and often signals bacterial infection. A decrease is called neutropenia and has many causes, but it is often caused by certain medications or viral infections.

• Bands (band neutrophils, BANDs; Figure 1-1C). Bands are less differentiated or less mature neutrophils. An increase in bands also signals bacterial infection and is customarily called a left shift. The cytoplasm of neutrophils and bands contains submicroscopic, pink- or lavender-staining granules filled with bactericidal secretions.

• Eosinophils (EOs; Figure 1-1D). Eosinophils are cells with bright orange-red, regular cytoplasmic granules filled with proteins involved in immune system regulation. An elevated eosinophil count is called eosinophilia and often signals a response to allergy or parasitic infection.

• Basophils (BASOs; Figure 1-1E). Basophils are cells with dark purple, irregular cytoplasmic granules that obscure the nucleus. The basophil granules contain histamines and various other proteins. An elevated basophil count is called basophilia. Basophilia is rare and often signals a hematologic disease.

• The distribution of basophils and eosinophils in blood is so small compared with that of neutrophils that the terms eosinopenia and basopenia are theoretical and not used. Neutrophils, bands, eosinophils, and basophils are collectively called granulocytes because of their prominent cytoplasmic granules, although their functions differ.

• Leukemia is an uncontrolled proliferation of WBCs. Leukemia may be chronic—for example, chronic myelogenous (granulocytic) leukemia—or acute—for example, acute myeloid leukemia. There are several forms of granulocytic leukemias that involve any one of or all three cell lines, categorized by their respective genetic aberrations (Chapters 30, 33 to 35 [Chapter 33 Chapter 34 Chapter 35]). Medical laboratory scientists are responsible for their identification using Wright-stained bone marrow smears, cytogenetics, flow cytometric immunophenotyping, molecular diagnostic technology, and occasionally, cytochemical staining (Chapter 17 and Chapters 30 to 32 [Chapter 30 Chapter 31 Chapter 32]).

• Lymphocytes (LYMPHs; Figure 1-1F). Lymphocytes comprise a complex system of cells that provide for host immunity. Lymphocytes recognize foreign antigens and mount humoral (antibodies) and cell-mediated antagonistic responses. On a Wright-stained blood film, most lymphocytes are nearly round, are slightly larger than RBCs, and have round featureless nuclei and a thin rim of nongranular cytoplasm. An increase in the lymphocyte count is called lymphocytosis and often is associated with viral infections. Accompanying lymphocytosis are often variant or reactive lymphocytes with characteristic morphology (Chapter 29). An abnormally low lymphocyte count is called lymphopenia or lymphocytopenia and is often associated with drug therapy or immunodeficiency. Lymphocytes are also implicated in leukemia; chronic lymphocytic leukemia is more prevalent in people older than 65 years, whereas acute lymphoblastic leukemia is the most common form of childhood leukemia (Chapters 35 and 36). Medical laboratory scientists and hematopathologists classify lymphocytic leukemias largely based on Wright-stained blood films, flow cytometric immunophenotyping, and molecular diagnostic techniques (Chapters 31 to 32 [Chapter 31 Chapter 32]).

• Monocytes (MONOs; Figure 1-1G). The monocyte is an immature macrophage passing through the blood from its point of origin, usually the bone marrow, to a targeted tissue location. Macrophages are the most abundant cell type in the body, more abundant than RBCs or skin cells, although monocytes comprise a minor component of peripheral blood WBCs. Macrophages occupy every body cavity; some are motile and some are immobilized. Their tasks are to identify and phagocytose (engulf and consume) foreign particles and assist the lymphocytes in mounting an immune response through the assembly and presentation of immunogenic epitopes. On a Wright-stained blood film, monocytes have a slightly larger diameter than other WBCs, blue-gray cytoplasm with fine azure granules, and a nucleus that is usually indented or folded. An increase in the number of monocytes is called monocytosis. Monocytosis may be found in certain infections, collagen-vascular diseases, or in acute and chronic leukemias (Chapters 29,33, and 35). Medical laboratory professionals seldom document a decreased monocyte count, so the theoretical term monocytopenia is seldom used.

Platelets

Platelets, or thrombocytes, are true blood cells that maintain blood vessel integrity by initiating vessel wall repairs (Chapter 13). Platelets rapidly adhere to the surfaces of damaged blood vessels, form aggregates with neighboring platelets to plug the vessels, and secrete proteins and small molecules that trigger thrombosis, or clot formation. Platelets are the major cells that control hemostasis, a series of cellular and plasma-based mechanisms that seal wounds, repair vessel walls, and maintain vascular patency (unimpeded blood flow). Platelets are only 2 to 4 μm in diameter, round or oval, anucleate (for this reason some hematologists prefer to call platelets “cell fragments”), and slightly granular (Figure 1-1H). Their small size makes them appear insignificant, but they are essential to life and are extensively studied for their complex physiology. Uncontrolled platelet and hemostatic activation is responsible for deep vein thrombosis, pulmonary emboli, acute myocardial infarctions (heart attacks), cerebrovascular accidents (strokes), peripheral artery disease, and repeated spontaneous abortions (miscarriages).

The microscopist counts platelets using the same technique used in counting WBCs on a hemacytometer, although a different counting area and dilution is usually used (Chapter 14). Owing to their small volume, platelets are hard to distinguish visually in a hemacytometer, and phase microscopy provides for easier identification (Chapter 4). Automated profiling instruments have largely replaced visual platelet counting and provide greater accuracy (see Chapter 15).

One advantage of automated profiling instruments is their ability to generate a mean platelet volume (MPV), which is unavailable through visual methods. The presence of predominantly larger platelets generates an elevated MPV value, which sometimes signals a regenerative bone marrow response to platelet consumption (Chapters 13 and 40).

Elevated platelet counts, called thrombocytosis, signal inflammation or trauma but convey modest intrinsic significance. Essential thrombocythemia is a rare malignant condition characterized by extremely high platelet counts and uncontrolled platelet production. Essential thrombocythemia is a life-threatening hematologic disorder (Chapter 33).

A low platelet count, called thrombocytopenia, is a common consequence of drug treatment and may be life-threatening. Because the platelet is responsible for normal blood vessel maintenance and repair, thrombocytopenia is usually accompanied by easy bruising and uncontrolled hemorrhage (Chapter 40). Thrombocytopenia accounts for many hemorrhage-related emergency department visits. Accurate platelet counting contributes to patient safety because it provides for the diagnosis of thrombocytopenia in many disorders or therapeutic regimens.

Complete blood count

A complete blood count (CBC) is performed on automated hematology profiling instruments and includes the RBC, WBC, and platelet measurements indicated in . The medical laboratory professional may collect a blood specimen for the CBC, but often a phlebotomist, nurse, physician assistant, physician, or patient care technician may also collect the specimen (Box 1-1Chapters 3 and 42). No matter who collects, the medical laboratory professional is responsible for the integrity of the specimen and ensures that it is submitted in the appropriate anticoagulant and tube and is free of clots and hemolysis (red-tinted plasma indicating RBC damage). The specimen must be of sufficient volume, as “short draws” result in incorrect anticoagulant-to-specimen ratios. The specimen must be tested or prepared for storage within the appropriate time frame to ensure accurate analysis (Chapter 5) and must be accurately registered in the work list, a process known as specimen accession. Accession may be automated, relying on bar code or radio-frequency identification technology, thus reducing instances of identification error.

BOX 1-1

Complete Blood Count Measurements Generated by Automated Hematology Profiling Instruments

RBC parameters

RBC count

HGB

HCT

MCV

MCH

MCHC

RDW

RETIC

Platelet parameters

PLT count

MPV

WBC parameters

WBC count

NEUT count: % and absolute

LYMPH count: % and absolute

MONO count: % and absolute

EO and BASO counts: % and absolute

BASO, Basophil; EO, eosinophil; HGB, hemoglobin; HCT, hematocrit; LYMPH, lymphocyte; MCH, mean cell hemoglobin; MCHC, mean cell hemoglobin concentration; MCV, mean cell volume; MONO,monocyte; MPV, mean platelet volume; NEUT, segmented neutrophil; PLT, platelet; RBC, red blood cell; RDW, RBC distribution width; RETIC, reticulocyte; WBC, white blood cell.

Although all laboratory scientists and technicians are equipped to perform visual RBC, WBC, and platelet counts using dilution pipettes, hemacytometers, and microscopes, most laboratories employ automated profiling instruments to generate the CBC. Many profiling instruments also provide comments on RBC, WBC, and platelet morphology (Chapter 15). When one of the results from the profiling instrument is abnormal, the instrument provides an indication of this, sometimes called a flag. In this case, a “reflex” blood film examination is performed (Chapter 16).

The blood film examination (described next) is a specialized, demanding, and fundamental CBC activity. Nevertheless, if all profiling instrument results are normal, the blood film examination is usually omitted from the CBC. However, physicians may request a blood film examination on the basis of clinical suspicion even when the profiling instrument results fall within their respective reference intervals.

Blood film examination

To accomplish a blood film examination, the microscopist prepares a “wedge-prep” blood film on a glass microscope slide, allows it to dry, and fixes and stains it using Wright or Wright-Giemsa stain (Chapter 16). The microscopist examines the RBCs and platelets by light microscopy for abnormalities of shape, diameter, color, or inclusions using the 50× or 100× oil immersion lens to generate 500× or 1000× magnification (Chapter 4). The microscopist then visually estimates the WBC count and platelet count for comparison with their respective instrument counts and investigates discrepancies. Next, the microscopist systematically reviews, identifies, and tabulates 100 (or more) WBCs to determine their percent distribution. This process is referred to as determining the WBC differential (“diff”). The WBC differential relies on the microscopist’s skill, visual acuity, and integrity, and it provides extensive diagnostic information. Medical laboratory professionals pride themselves on their technical and analytical skills in performing the blood film examination and differential count. Visual recognition systems such as the Cellavision® DM96 or the Bloodhound automate the RBC and platelet morphology and WBC differential processes, but the medical laboratory professional or the hematopathologist is the final arbiter for all cell identification. The results of the CBC, including all profiling and blood film examination parameters and interpretive comments, are provided in paper or digital formats for physician review with abnormal results highlighted.

Endothelial cells

Because they are structural and do not flow in the bloodstream, endothelial cells—the endodermal cells that form the inner surface of the blood vessel—are seldom studied in the hematology laboratory. Nevertheless, endothelial cells are important in maintaining normal blood flow, in tethering (decelerating) platelets during times of injury, and in enabling WBCs to escape from the vessel to the surrounding tissue when needed. Increasingly refined laboratory methods are becoming available to assay and characterize the secretions (cytokines) of these important cells.

Coagulation

Most hematology laboratories include a blood coagulation–testing department (Chapters 42 and 44). Platelets are a key component of hemostasis, as previously described; plasma coagulation is the second component. The coagulation system employs a complex sequence of plasma proteins, some enzymes, and some enzyme cofactors to produce clot formation after blood vessel injury. Another 6 to 8 enzymes exert control over the coagulation mechanism, and a third system of enzymes and cofactors digests clots to restore vessel patency, a process called fibrinolysis. Bleeding and clotting disorders are numerous and complex, and the coagulation section of the hematology laboratory provides a series of plasma-based laboratory assays that assess the interactions of hematologic cells with plasma proteins (Chapters 42 and 44).

The medical laboratory professional focuses especially on blood specimen integrity for the coagulation laboratory, because minor blood specimen defects, including clots, hemolysis, lipemia, plasma bilirubin, and short draws, render the specimen useless (Chapters 3 and 42). High-volume coagulation tests suited to the acute care facility include the platelet count and MPV as described earlier, prothrombin time andpartial thromboplastin time (or activated partial thromboplastin time), thrombin time (or thrombin clotting time), fibrinogen assay, and D-dimer assay (Chapter 42). The prothrombin time and partial thromboplastin time are particularly high-volume assays used in screening profiles. These tests assess each portion of the coagulation pathway for deficiencies and are used to monitor anticoagulant therapy. Another 30 to 40 moderate-volume assays, mostly clot-based, are available in specialized or tertiary care facilities. The specialized or tertiary care coagulation laboratory with its interpretive complexities attracts advanced medical laboratory scientists with specialized knowledge and communication skills.

Advanced hematology procedures

Besides performing the CBC, the hematology laboratory provides bone marrow examinations, flow cytometry immunophenotyping, cytogenetic analysis, and molecular diagnosis assays. Performing these tests may require advanced preparation or particular dedication by medical laboratory scientists with a desire to specialize.

Medical laboratory scientists assist physicians with bedside bone marrow collection, then prepare, stain, and microscopically review bone marrow smears (Chapter 17). Bone marrow aspirates and biopsy specimensare collected and stained to analyze nucleated cells that are the immature precursors to blood cells. Cells of the erythroid series are precursors to RBCs (Chapter 8); myeloid series cells mature to form bands and neutrophils, eosinophils, and basophils (Chapter 12); and megakaryocytes produce platelets (Chapter 13). Medical laboratory scientists, clinical pathologists, and hematologists review Wright-stained aspirate smears for morphologic abnormalities, high or low bone marrow cell concentration, and inappropriate cell line distributions. For instance, an increase in the erythroid cell line may indicate bone marrow compensation for excessive RBC destruction or blood loss (Chapter 19 and Chapters 23 to 26[Chapter 23 Chapter 24 Chapter 25 Chapter 26]). The biopsy specimen, enhanced by hematoxylin and eosin (H& E) staining, may reveal abnormalities in bone marrow architecture indicating leukemia, bone marrow failure, or one of a host of additional hematologic disorders. Results of examination of bone marrow aspirates and biopsy specimens are compared with CBC results generated from the peripheral blood to correlate findings and develop pattern-based diagnoses.

In the bone marrow laboratory, cytochemical stains may occasionally be employed to differentiate abnormal myeloid, erythroid, and lymphoid cells. These stains include myeloperoxidase, Sudan black B, nonspecific and specific esterase, periodic acid–Schiff, tartrate-resistant acid phosphatase, and alkaline phosphatase. The cytochemical stains are time-honored stains that in most laboratories have been replaced by flow cytometry immunophenotyping, cytogenetics, and molecular diagnostic techniques (Chapters 30 to 32 [Chapter 30 Chapter 31 Chapter 32]). Since 1980, however, immunostaining methods have enabled identification of cell lines by detecting lineage-specific antigens on the surface or in the cytoplasm of leukemia and lymphoma cells. An example of immunostaining is a visible dye that is bound to antibodies to CD42b, a membrane protein that is present in the megakaryocytic lineage and may be diagnostic for megakaryoblastic leukemia (Chapter 35).

Flow cytometers may be quantitative, such as clinical flow cytometers that have grown from the original Coulter principle, or qualitative, including laser-based instruments that have migrated from research applications to the clinical laboratory (Chapters 15 and 32). The former devices are automated clinical profiling instruments that generate the quantitative parameters of the CBC through application of electrical impedance and laser or light beam interruption. Qualitative laser-based flow cytometers are mechanically simpler but technically more demanding. Both qualitative and quantitative flow cytometers are employed to analyze cell populations by measuring the effects of individual cells on laser light, such as forward-angle fluorescent light scatter and right-angle fluorescent light scatter, and by immunophenotyping for cell membrane epitopes using monoclonal antibodies labeled with fluorescent dyes. The qualitative flow cytometry laboratory is indispensable to leukemia and lymphoma diagnosis.

Cytogenetics, a time-honored form of molecular technology, is employed in bone marrow aspirate examination to find gross genetic errors such as the Philadelphia chromosome, a reciprocal translocation between chromosomes 9 and 22 that is associated with chronic myelogenous leukemia, and t(15; 17), a translocation between chromosomes 15 and 17 associated with acute promyelocytic leukemia (Chapter 30). Cytogenetic analysis remains essential to the diagnosis and treatment of leukemia.

Molecular diagnostic techniques, the fastest-growing area of laboratory medicine, enhance and even replace some of the advanced hematologic methods. Real-time polymerase chain reaction, microarray analysis, fluorescence in situ hybridization, and DNA sequencing systems are sensitive and specific methods that enable medical laboratory scientists to detect various chromosome translocations and gene mutations that confirm specific types of leukemia, establish their therapeutic profile and prognosis, and monitor the effectiveness of treatment (Chapter 31).

Additional hematology procedures

Medical laboratory professionals provide several time-honored manual whole-blood methods to support hematologic diagnosis. The osmotic fragility test uses graduated concentrations of saline solutions to detect spherocytes (RBCs with proportionally reduced surface membrane area) in hereditary spherocytosis or warm autoimmune hemolytic anemia (Chapters 24 and 26). Likewise, the glucose-6-phosphate dehydrogenase assayphenotypically detects an inherited RBC enzyme deficiency causing severe episodic hemolytic anemia (Chapter 24). The sickle cell solubility screening assay and its follow-up tests, hemoglobin electrophoresis and high performance liquid chromatography, are used to detect and diagnose sickle cell anemia and other inherited qualitative hemoglobin abnormalities and thalassemias (Chapters 27 and 28). One of the oldest hematology tests, the erythrocyte sedimentation rate, detects inflammation and roughly estimates its intensity (Chapter 14).

Finally, the medical laboratory professional reviews the cellular counts, distribution, and morphology in body fluids other than blood (Chapter 18). These include cerebrospinal fluid, synovial (joint) fluid, pericardial fluid, pleural fluid, and peritoneal fluid, in which RBCs and WBCs may be present in disease and in which additional malignant cells may be present that require specialized detection skills. Analysis of nonblood body fluids is always performed with a rapid turnaround, because cells in these hostile environments rapidly lose their integrity. The conditions leading to a need for body fluid analysis are invariably acute.

Hematology quality assurance and quality control

Medical laboratory professionals employ particularly complex quality control systems in the hematology laboratory (Chapter 5). Owing to the unavailability of weighed standards, the measurement of cells and biological systems defies chemical standardization and requires elaborate calibration, validation, matrix effect examination, linearity, and reference interval determinations. An internal standard methodology known as the moving average also supports hematology laboratory applications.10 Medical laboratory professionals in all disciplines compare methods through clinical efficacy calculations that produce clinical sensitivity, specificity, and positive and negative predictive values for each assay. They must monitor specimen integrity and test ordering patterns and ensure the integrity and delivery of reports, including numerical and narrative statements and reference interval comparisons. As in most branches of laboratory science, the hematology laboratory places an enormous responsibility for accuracy, integrity, judgment, and timeliness on the medical laboratory professional.

References

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