GENERAL PRINCIPLES
Approach to the Hematology Patient
Hematologic diseases are a heterogeneous group of diseases that can have multiple clinical and laboratory manifestations that mimic nonhematologic diseases. History, physical exam, labs, peripheral smear, and bone marrow biopsy are critical in making the correct diagnosis. The diseases can be approached by identifying the primary hematologic component that is affected: RBCs, WBCs, platelets, or the coagulation system. The major abnormalities in hematology are quantitative in nature, with either excessive or deficient production of one of the hematopoietic constituents (e.g., leukemias, anemias). Qualitative abnormalities that can be inherited (e.g., sickle cell disease) or acquired also occur.
DIAGNOSIS
Clinical Presentation
History
The medical history is, of course, the first step in hematology diagnostic assessment. Table 1-1 offers some general questions for evaluation of a hematologic disorder.
Physical Exam
The physical exam is also an important part of the diagnostic process. Along with the history, it can suggest a diagnosis, guide lab testing, and aid in the differential diagnosis. Table 1-2 offers some general physical exam findings that are useful in the hematology patient.
Diagnostic Testing
Laboratories
The clinician should be comfortable using the complete blood count (CBC) and peripheral smear to evaluate patients for possible hematologic disorders. Patients may be referred to a hematologist based on a lab abnormality that is drawn for a reason other than the diagnosis of a primary hematologic disorder. There are certain limiting values in hematology that can help exclude or confirm the need for further testing or warn us of the possibility of potential physiological consequences (see Table 1-3).
The Peripheral Smear. The visual study of peripheral blood is necessary to diagnose hematologic and nonhematologic diseases, for example, thrombotic thrombocytopenic purpura or malaria. In these cases, as in others, automated hematology analyzers are able to provide a large number of data regarding all the blood cells but will not be able to detect subtle anomalies critical in the diagnosis.
Slides for a peripheral smear are typically prepared either by automated methods or by qualified technicians in a specialized laboratory. This step is critical since poorly processed samples can lead to incorrect diagnoses. Smears may be prepared on glass slides or coverslips. Ideally, blood smears should be prepared from uncoagulated blood and from a sample collected from a finger stick. In practice, most slides are prepared from blood samples containing anticoagulants and are thus prone to the introduction of morphologic artifacts. Blood smears are normally stained using Wright or May–Grünwald–Giemsa stain.
Examination of the Peripheral Smear. Examination of the smear should proceed systematically and begin under low power to identify a portion of the slide with optimal cellular distribution and staining, which normally corresponds to the thinner edge of the sample. As a general rule, the analysis starts with RBCs, continues with leukocytes, and finishes with platelets. Under low power (×10 to ×20) it is possible to analyze general characteristics of RBCs to discover, for example, the presence of Rouleaux associated with multiple myeloma, estimate the WBC and platelet counts, and determine the presence of abnormal populations of cells, such as blasts, by scanning over the entire smear. Under high power (×100), each of the cell lineages is examined for any abnormalities in number or morphology.
Red Blood Cells. Quantitative analysis of RBCs is difficult on a peripheral smear. Automated analyzers are used to calculate:
MCHC, the mean cell Hgb concentration, expressed as grams per deciliter;
MCH, the mean corpuscular Hgb, expressed as picograms; and
MCV, the mean corpuscular volume, expressed as femtoliters (10−15 L).
Qualitative analysis of RBCs should demonstrate uniform round cells with smooth membranes and a pale central area with a round rim of red Hgb. Variations in size are called anisocytosis, and variations in shape, poikilocytosis. The following abnormalities may be observed:
Hypochromia corresponds to a very thin rim of Hgb and a larger central pale area. These red cells are often microcytic and are seen in iron deficiency, thalassemias, and sideroblastic anemia.
Microcytosis (<6 μm): Differential diagnosis includes iron-deficiency anemia, anemia in chronic disease, thalassemias, and sideroblastic anemia. These cells are usually hypochromic and have prominent central pallor.
Macrocytosis (>9 μm in diameter): Differential diagnosis includes liver disease, alcoholism, aplastic anemia, and myelodysplasia. Megaloblastic anemias (B12 and folate deficiencies) have macro-ovalocytes (large oval cells). Reticulocytes are large immature red cells with polychromatophilia.
Schistocytes (fragmented cells) are caused by mechanical disruption of cells in the microvasculature by fibrin strands or by mechanical prosthetic heart valves. Differential diagnosis includes thrombotic thrombocytopenic purpura/hemolytic uremic syndrome, disseminated intravascular coagulation, hemolysis/elevated liver enzymes/low platelet count (HELLP) syndrome, and malignant hypertension.
Acanthocytes (spiculated cells with irregular projections of varying length) are seen in liver disease.
Burr cells (cells with short, evenly spaced cytoplasmic projections) may be an artifact of slide preparation or found in renal failure and uremia.
Bite cells (cells with a smooth semicircle extracted) are due to spleen phagocytes that have removed Heinz bodies consisting of denatured Hgb. They are found in hemolytic anemia due to glucose-6-phosphate dehydrogenase deficiency.
Spherocytes (round, dense cells with absent central pallor) are seen in immune hemolytic anemia and hereditary spherocytosis.
Sickle cells (sickle-shaped cells) are due to polymerization of Hgb They are found in sickle cell disease but not in sickle cell trait.
Target cells (cells with extra Hgb in the center surrounded by a rim of pallor; bull’s-eye appearance) are due to an increase in the ratio of cell membrane surface area to Hgb volume within the cell. These have a central spot of Hgb surrounded by a ring of pallor from the redundancy in cell membrane. They are found in liver disease, postsplenectomy, in hemoglobinopathies, and in thalassemia.
Teardrop cells/dacryocytes (teardrop-shaped cells) are found in myelofibrosis and myelophthisic states of marrow infiltration.
Ovalocytes (elliptical cells) are due to the abnormal membrane cytoskeleton found in hereditary elliptocytosis.
Polychromatophilia (blue hue of cytoplasm) is due to the presence of RNA and ribosomes in reticulocytes.
Howell–Jolly bodies (small, single, purple cytoplasmic inclusions) represent nuclear remnant DNA and are found after splenectomy or with functional asplenism.
Basophilic stippling (dark-purple inclusions, usually multiple) arises from precipitated RNA found in lead poisoning and thalassemia.
Nucleated red cells are not normally found in peripheral blood. They appear in hypoxemia and myelofibrosis or other myelophthisic conditions, as well as with severe hemolysis.
Heinz bodies (inclusions seen only on staining with violet crystal) represent denatured Hgb and are found in glucose-6-phosphate dehydrogenase after oxidative stress.
Parasites, including malaria and babesiosis, may be seen within red cells.
Rouleaux (red cell aggregates resembling a stack of coins) is due to the loss of normal electrostatic charge–repelling red cells due to coating with abnormal paraprotein, such as in multiple myeloma.
Leukoerythroblastic smear (teardrop cells, nucleated red cells, and immature white cells) is found in marrow infiltration or fibrosis (myelophthisic conditions).
White Blood Cells. WBCs normally seen on the peripheral smear include mature granulocytes (neutrophils, eosinophils, and basophils) and mature agranulocytes (lymphocytes and monocytes). Under normal conditions, immature myeloid and lymphoid cells are not seen and their presence is related to conditions such as infections and hematologic neoplasias.
Neutrophils. Neutrophils comprise 55% to 60% of total WBCs (1.8 × 109 to 7.7 × 109/L). They have nuclei containing three or four lobes and granular cytoplasm. The normal size is 10 to 15 μm. Hypersegmented neutrophils contain more than five lobes and are found in megaloblastic anemias. The cytoplasmic granules correspond to enzymes that are used during the acute phase of inflammation. Increased prominence of cytoplasmic granules is indicative of systemic infection or therapy with growth factors and is known as toxic granulation. Neutrophils develop from myeloblasts through promyelocyte, myelocyte, metamyelocyte, and band forms and progress to mature neutrophils. Only mature neutrophils and bands are normally found in peripheral blood. Metamyelocytes and myelocytes may be found in pregnancy, infections, and leukemoid reactions. The presence of less mature forms in the peripheral blood is indicative of hematologic malignancy or myelophthisis.
Lymphocytes. Lymphocytes comprise 25% to 35% (1 × 109 to 4.8 × 109/L, or thousands per cubic millimeter) of total WBCs. They contain a dark, clumped nucleus and a scant rim of blue cytoplasm. The differentiation of T and B cells using light microscopy is very difficult. The normal size is 7 to 18 μm. Atypical (or reactive) lymphocytes seen in viral infections contain more extensive, malleable cytoplasm that may encompass surrounding red cells.
Eosinophils. Eosinophils comprise 0.5% to 4% of total WBCs (0.2 ×109/L, or thousands per cubic millimeter). These are large cells containing prominent red/orange granules and a bilobed nucleus. The normal size is 10 to 15 μm. Increased numbers are found in parasitic infections and allergic disorders.
Monocytes. Monocytes comprise 4% to 8% of total WBCs (0 to 0.3 × 109/L, or thousands per cubic millimeter). These are the bigger circulating cells with an eccentric U-shaped nucleus. They contain blue cytoplasm and are the precursors of the mononuclear phagocyte system (macrophages, osteoclasts, alveolar macrophages, Kupfer cells, and microglia). The usual size is 12 to 20 μm.
Basophils. Basophils comprise 0.01% to 0.3% of total WBCs (0 to 0.1 ×109/L, or thousands per cubic millimeter). Their cytoplasm contains large dark-blue granules and a bilobed nucleus. They are involved in inflammatory reactions and increased numbers are also seen in chronic myeloid leukemia. As for eosinophils, the normal size is 10 to 15 μm.
WBC Abnormalities. Quantitative anomalies result in leukopenia and leukocytosis. Main causes of leukopenia include bone marrow failure (aplastic anemia), myelophthisis (acute leukemia), drugs (immunosuppressive drugs, propylthiouracil), and hypersplenism (portal hypertension). Main causes of leukocytosis are infection, inflammation, malignancies, and allergic reactions.
Pelger–Huet anomaly (neutrophils have a bilobed nucleus connected by a thin strand and decreased granulation) is seen in myelodysplastic syndromes.
Hypersegmented neutrophils (more than four nuclear lobes) are found in megaloblastic anemias (vitamin B12 and folate deficiency).
Blast cells (myeloblasts or lymphoblasts; large cells with large nuclei and prominent nucleoli) are seen in acute leukemia.
Auer rods (rodlike granules in blast cytoplasm) are pathognomonic for acute myelogenous leukemia, especially acute promyelocytic leukemia (M3).
Hairy cells (lymphoid cells with ragged cytoplasm) are seen in hairy cell leukemia.
Sézary cells (atypical lymphoid cells with cerebriform nuclei) are seen in cutaneous T cell lymphoma.
Platelets. Platelets appear as small (1- to 2-μm-diameter), purplish cytoplasmic fragments without a nucleus, containing red/blue granules. Derived from bone marrow giant cells called megakaryocytes, they are involved in the cellular mechanisms of primary hemostasis leading to the formation of blood clots. Normal counts are 150,000 to 400,000 per cubic millimeter of peripheral blood. The number of platelets per high-power field multiplied by 20,000 usually estimates the platelet count per microliter. Alternatively, one should find 1 platelet for every 10 to 20 red cells.
Numbers of platelets can decrease due to bone marrow disease (myelophthisic bone marrow), consumption (disseminated intravascular coagulation), or drugs. An increase in numbers can be seen in bone marrow overproduction (myeloproliferative syndromes) or in a normal response to massive bleeding. Pseudo-thrombocytopenia represents clumping of platelets in blood samples collected in EDTA, resulting in spuriously low platelet counts. This phenomenon can be avoided by using citrate to anticoagulate blood samples sent for blood counts.
Diagnostic Procedures
Bone Marrow Evaluation. For many hematologic diseases that affect the bone marrow, evaluation of the peripheral blood smear does not provide sufficient information, and a direct examination of the bone marrow is required to establish the diagnosis. The bone marrow biopsy can be done at the bedside under local anesthesia alone or in combination with low doses of anxiolytics or opioids. Despite advances in the bone marrow biopsy and aspiration techniques, they are still commonly considered painful procedures, but with expertise, they can be performed safely and with minimal discomfort to the patient.
Indications and Contraindications. The most common indications for bone marrow evaluation are workup of bone marrow malignancies; staging of marrow involvement by metastatic tumors; assessment of infectious diseases that may involve the bone marrow (i.e., HIV, tuberculosis); determination of marrow damage in patients exposed to radiation, drugs, and chemicals; and workup of metabolic storage diseases. There are a few absolute contraindications for the procedure, including infection, previous radiation therapy at the site of biopsy, and poor patient cooperation. Thrombocytopenia is not a contraindication to bone marrow biopsy, although it may be associated with more procedure-related bleeding. Patients who have a coagulopathy require factor replacement or withholding of anticoagulation to minimize bleeding complications.
Technique. In adults, the most common places to do the procedure are the posterior and anterior iliac crests. Other potential biopsy sites are the sternum and tibia. The posterior iliac crest is the preferred site, as it allows collection of both aspirate and biopsy specimens and is associated with minimal morbidity or complications. Usually, a Jamshidi bone marrow aspiration and biopsy needle is used. Additional aspirate is often obtained for studies such as flow cytometry, cytogenetics, and cultures. In some instances, marrow cannot be aspirated and only a biopsy is obtained (a “dry tap”). This can be due to the technique or may signal myelofibrosis or previous local radiotherapy. In such cases, touch preparations of the biopsy can be made to allow for a cytologic exam. The biopsy specimen is embedded in a buffered formaldehyde-based fixation for further processing.
Complications. Bleeding at the site of puncture is the most common complication. It is easily controlled with compression, but some thrombocytopenic patients will require platelet transfusions. Other uncommon complications are infections, tumor seeding in the needle track, and needle breakage.
Bone Marrow Examination
The examination of the bone marrow aspirate begins under low power to obtain an impression of overall cellularity, an initial scan for any abnormal populations of cells or clumps of cells, and an evaluation of the presence or absence of bone marrow spicules. Megakaryocytes are normally seen under low power as large multinucleated cells. The overall cellularity of the marrow is difficult to estimate from the aspirate because of contamination with peripheral blood.
The myeloid-to-erythroid (M:E) ratio is also determined under low power and is normally 3:1 to 4:1. The ratio is increased in chronic myeloid leukemia due to an increase in granulocyte precursors and is increased in pure red cell aplasia due to a decrease in red cell precursors. The ratio is decreased in hemolytic disorders in which increased erythroid precursors are present or in agranulocytic conditions secondary to chemotherapeutic agents or other drugs.
Under high power, the aspirate should contain a variety of cells representative of various stages of myeloid and erythroid maturation. Myeloid cells progress from myeloblasts to promyelocytes, myelocytes, metamyelocytes, band forms, and then mature neutrophils. As these cells mature, their nuclear chromatin condenses, with a resultant decrease in the nuclear-tocytoplasmic ratio. Their cytoplasm gradually develops granules seen in mature neutrophils.
Erythroid precursors progress from proerythroblasts through varying stages of normoblasts known as basophilic, chromatophilic, and orthochromic. The nucleus gradually condenses, and the cytoplasm gradually takes on the pinkish hue of Hgb found in mature red cells.
Bone marrow core biopsies are fixed in a buffered formaldehyde-based solution and then embedded in paraffin or plastic. Biopsies are used to assess the cellularity of the bone marrow and the presence of neoplasias, infections, or fibrosis. Cellularity is estimated by observing the ratio of hematopoietic cells to fat cells. Cellularity is usually 30% to 60% but typically declines with advancing age.
Abnormalities in the Bone Marrow Evaluation. Listed below are some of the more common abnormal findings of the bone marrow.
Acute leukemia: The presence of >20% blasts in the bone marrow establishes the diagnosis of acute leukemia.
Myelodysplastic syndrome is a heterogeneous group of diseases characterized by the presence of immature erythroid precursors with loss of synchrony between nuclear and cytoplasmic maturation. Mature myeloid cells have decreased lobes (Pelger–Huet cells). Iron staining may reveal ring sideroblasts with iron granules surrounding the nucleus.
Chronic myeloid leukemia: Findings include a hypercellular marrow with an increased M:E ratio. Myeloblasts represent <5% of cells, with the marrow containing predominantly myelocytes, metamyelocytes, and mature neutrophils.
Chronic lymphocytic leukemia is marked by hypercellular marrow with small, round, mature lymphocytes with a thin rim of blue cytoplasm.
Myelofibrosis is often the cause of a “dry tap.” Bone marrow biopsy will reveal marrow infiltration with collagen and fibrous tissue.
Essential thrombocytosis: Megakaryocyte hyperplasia is a common finding.
Polycythemia vera is characterized by a hypercellular marrow.
Multiple myeloma: The marrow is replaced by large numbers of abnormal, often immature plasma cells with eccentric nuclei containing a cartwheel pattern of nuclear chromatin. Flame cells contain pink, flamelike cytoplasm and are associated with an IgA paraprotein.
Megaloblastic anemia: Findings include hypercellular marrow with abnormalities in myeloid and erythroid precursors. Megaloblasts are erythroid cells that are larger than normal, with more nuclear chromatin. There is loss of synchrony between nuclear and cytoplasmic maturation.
Storage diseases: Patients with Gaucher disease may have macrophages with striated cytoplasm due to accumulation of cerebrosides. Individuals with Niemann–Pick disease may have macrophages with a foamy cytoplasm secondary to contained sphingomyelin.