Chapter 3: Examination of the Marrow

The first recorded examinations of marrow in living patients occurred in the first decades of the 20th century, first using the tibia as the source of marrow and then surgical bone biopsies. Neither technique led to routine examination of the marrow, because in the former case the tibia was usually hypocellular in adults, and in the latter case, because of the invasiveness of an open procedure and the discomfort and risk of infection and bleeding. In 1923, Arinkin devised the marrow aspiration technique,¹ which was the prototype for our current aspiration procedure. Thirty years passed before the suggestion that the pelvis might be preferable to the sternum gained hold, and another 10 years passed before a practical marrow biopsy instrument was put to use. Regular use of the posterior iliac crest for aspiration and biopsy and regular use of biopsy to complement aspiration did not occur until the 1970s, when staging of lymphoma made biopsy a frequent procedure and new simpler biopsy instruments became readily available.

The International Council for Standardization in Hematology has published guidelines for marrow aspirate and biopsy to promote consistency in performance and reporting.² Although marrow aspiration and biopsy techniques are safe, they should be performed with a clear idea as to how the results will help distinguish the differential diagnoses under consideration or provide followup of treatment.^3,4,5 In many hematologic disorders, such as most cases of iron-deficiency anemia, thalassemia, and acquired and inherited hemolytic anemia, examination of the blood and specialized laboratory tests usually suffice to make the diagnosis without the need for a marrow examination.

When examination of the marrow is indicated, the decision as to whether an aspirate or an aspirate plus biopsy is desired should be made. Aspiration is always attempted because of the superior morphology offered by examination of the aspirate smear. However, a marrow biopsy is superior to the aspirate in quantifying marrow cellularity and diagnosing infiltrative diseases of the marrow and should be performed when these conditions are part of the differential diagnosis.^6,7 Marrow biopsy is useful for diagnosing and following the course of disorders that are commonly associated with reticulin fibrosis, such as megakaryoblastic leukemia, hairy cell leukemia, and the chronic myeloproliferative neoplasms.⁸ In myelodysplastic syndromes, marrow biopsy is useful for evaluating abnormal localization of immature precursor cells and abnormal megakaryocytes. Marrow necrosis and gelatinous transformation are more readily detected in marrow sections than in aspirate films. Marrow aspirate alone may be appropriate in some clinical settings where the diagnostic question is very targeted, such as diagnosis of childhood immune thrombocytopenia purpura or surveillance followup of leukemia patients in apparent remission.

Depending on the diagnostic question, availability of material, and expected frequency of the abnormal cells, an appropriate selection of specialized diagnostic methods may be needed to support the clinical diagnosis. Morphology of marrow cells is still the gold standard for diagnosis of hematologic malignancy and allows construction of a good differential diagnosis for nonmalignant disorders. Immunocytochemistry provides excellent phenotype–morphology correlation on an individual cell basis, but is limited to epitopes that resist destruction by fixation, decalcification, and paraffin embedding. Flow cytometry allows study of almost any surface or intracellular protein, with the added ability to detect important quantitative changes in cellular proteins and simultaneous determination of multiple proteins within the same cell. However, flow cytometry requires that cells be viable and dissociated from tissue. Gene expression arrays allow analysis of complex patterns of RNA expression by sophisticated mathematical algorithms to discover diagnostic patterns based on gene expression. These studies may point the way to a smaller more practical set of proteins that can be studied by immunocytochemistry or immunofluorescence. Molecular assays target oncogenic DNA sequence alterations from the chromosome to the nucleotide, and include classic metaphase cytogenetics, fluorescence in situ hybridization (FISH), reverse transcriptase polymerase chain reaction (PCR), and targeted or whole-genome sequencing.

At birth, all bones contain hematopoietic marrow. Fat cells begin to replace hemopoietic marrow in the extremities in the fifth to seventh year. By adulthood, the hemopoietic marrow is limited to the axial skeleton and the proximal portions of the extremities (Chaps. 5 and 9). Fatty marrow appears yellow, whereas hematopoietic marrow is red. Red marrow contains fat, however, and fat droplets are visible grossly in aspirated marrow specimens. Histologically, yellow marrow consists almost entirely of fat cells and supporting connective tissue. Red marrow contains an abundance of hematopoietic cells, fat cells, and connective tissue. The marrow fills the spaces between the trabeculae of bone in the marrow cavity. Marrow is soft and friable and can be readily aspirated or biopsied with a needle.

The posterior iliac crest (Fig. 3–1) is the preferred site for marrow aspiration and biopsy. In adults, the anterior iliac crest and rarely the sternum have been used (Fig. 3–2). The sternum should be used for aspiration only. The anterior iliac crest is less preferred than the posterior crest in adults because of its thick cortical bone. The anteromedial surface of the tibia is an option for infants younger than 1 year old (particularly newborns), but the posterior iliac crest is still the preferred site. Serious adverse outcomes after marrow aspiration or biopsy are rare, occurring in less than 0.05 percent. One direct fatality and three episodes of prolonged but not permanent disability were reported in nearly 55,000 marrow biopsies.⁹ Morbidity most frequently involved hemorrhage, which was associated more with platelet function impairment than thrombocytopenia or coagulation factor defects.⁹ Infection and reactions to anesthetic agents are other infrequent complications. Penetration of the bone with damage to the underlying structures is possible with all marrow aspirations, but the hazard is greatest in sternal aspirations because the sternum at the second interspace is only approximately 1 cm thick in adults, and the distance from posterior sternal cortex to the ascending aorta varies greatly and may be as little as 4 to 5 mm,¹⁰ giving rise to the rare but dramatic consequence of aortal wall tear. To prevent this, a guard should be in place on the needle if a sternal aspirate needs to be done.

For either a marrow biopsy or aspiration, sedation minimizes anxiety and pain,¹¹ particularly in children,¹² for whom propofol, with or without fentanyl, administered under carefully controlled conditions¹³ with monitoring of oxygen saturation, blood pressure, and vital signs, is frequently used. Midazolam (Versed) is a popular choice for conscious sedation of adult patients, although a variety of other premedications have been used. There is a relative lack of empirical research and consensus guidelines on the subject of pain reduction during adult marrow procedures.^14,15 The experience of marrow procedures from the patient’s point of view is worth reading.¹⁶ The only significant correlates with severe/unbearable pain (experienced by 4 percent of patients) during marrow examination were quality of the information about the procedure provided before the examination and previous painful experiences.¹⁷ Marrow biopsies and aspirations for lymphoma staging purposes often can be performed while the patient is under anesthesia for other procedures. Several different types of needles are available for marrow aspiration.³ For adults, a 16-gauge needle is sufficiently large to permit aspiration of adequate specimens; larger needles are unnecessary. The patient is prone or in the left or right lateral decubitus position. Sterile precautions must be observed. The skin over the puncture site is shaved if necessary and cleansed with a disinfectant solution. The skin, subcutaneous tissues, and periosteum are infiltrated with a local anesthetic solution, such as 1 percent lidocaine. Adequate infiltration of the anesthetic at the periosteal surface is important to minimize severe pain during the procedure, but no more than 20 mL of 1 percent lidocaine should be used in an adult.¹⁸ Adequate anesthesia can be achieved with much less lidocaine in virtually all cases. An air gun can be used to anesthetize the skin surface prior to application of anesthetic to the periosteal surface by injection. After the anesthesia has taken effect, usually in 3 to 5 minutes, the marrow needle is inserted through the skin, subcutaneous tissue, and cortex of the bone using a slight twisting motion. In obese patients, the length of the needle must be sufficient to reach the iliac crest. The stylet should be locked into place on the hub of the needle to prevent plugging of the needle with tissue prior to needle entry into the marrow cavity. Penetration of the cortex can be sensed by a slight, rapid forward movement accompanied by a sudden increase in the ease of advancing the needle. The stylet of the needle is removed promptly, the hub is attached to a 10- or 20-mL syringe, and approximately 0.5 to 1.5 mL of fluid is aspirated. The actual aspiration of the marrow causes a transient painful sensation for most patients. If additional specimen volume is required, another syringe is fitted on the marrow needle, the syringe and needle is rotated and an adjacent area is entered and marrow is aspirated. The stylet may be reinserted and the marrow needle slightly repositioned between aspirations. When aspiration is complete, the stylet is reinserted and the needle immediately removed from the bone. Pressure is applied to the skin over the aspiration site for at least 5 minutes to minimize bruising at the site. If platelet number or function is decreased, firm pressure should be applied for at least 10 to 15 minutes. The bloody fluid that is aspirated contains light-colored particles of marrow approximately 0.5 to 1 mm in diameter. They often are readily visible in the syringe, but may not be detected until the syringe contents are discharged on glass slides for film preparation.

If nothing enters the syringe when aspiration is performed, the needle may not be properly placed in the marrow cavity. The needle can be cautiously advanced 1 to 2 mm after reinsertion of the stylet and aspiration attempted again, or the needle can be removed from the bone and reinserted in a nearby site in the anesthetized area. The thickness of the bone must be considered when the needle is being adjusted in the bone. Occasionally the needle must be rotated on its longitudinal axis, or in a larger orbit, in order to loosen the marrow mechanically before the marrow can be aspirated. If a small amount of blood has been aspirated, a new needle should be used because of the probability of clotting the aspirate when it finally is obtained. Aspiration with a 50-mL syringe may succeed if use of a smaller syringe fails. Fibrotic or densely packed leukemic marrow may resist all attempts at aspiration, in which case a biopsy is necessary. The most common cause of failure to obtain marrow is faulty positioning of the needle, and a second attempt at aspiration usually succeeds. A specimen preparation checklist used at the time of procedure to verify presence of spicules, length of biopsy, and other protocol items has been found to increase biopsy specimen length and decrease frequency of non-diagnostic samples.¹⁹

Needle biopsy usually is performed with the Jamshidi needle, using the same preparation as described above. The Jamshidi instrument (see Fig. 3–1) consists of a cylindrical needle with constant bore, except for a concentrically tapered distal portion ending in a sharp, beveled cutting tip. The stylet fits precisely inside the opening at the tapered tip, interlocks at the hub of the needle, and extends 1 to 2 mm beyond the end of the needle. An 11-gauge needle is most commonly used in the United States. After the skin and the periosteum of the biopsy site are anesthetized, a 3-mm incision is made in the skin. The needle, with obturator in place, is inserted into the skin incision and through the subcutaneous tissue to the cortex of the bone. The needle is directed toward the posterior iliac spine and advanced with a twisting motion. Penetration of the cortex is sensed by a decreased resistance to forward movement of the needle. The obturator is removed, and the needle is slowly advanced with reciprocal clockwise–counterclockwise twisting motions around the long axis. After sufficient penetration of the bone (up to approximately 3 cm), the needle is rotated several times on its axis and withdrawn approximately 2 to 3 mm. Some needles now come with a “trap” that snares the biopsy so that the needle can be directly removed. The needle is reinserted to the original depth at a slightly different angle, taking care not to bend the needle, and rotated several times to free the specimen from attachments in the marrow cavity. The needle is slowly withdrawn, with the same twisting motion used during insertion. The core of marrow inside the needle is removed by inserting the probe through the cutting tip and extruding the specimen through the hub of the needle. The smaller size of the cutting aperture relative to the bore of the shaft of the Jamshidi instrument yields a specimen that fits loosely inside the needle and therefore is less subject to compression, distortion, or fragmentation. The technique reliably produces good quality biopsy specimens. Marrow biopsy should be performed before marrow aspiration is attempted (or in a slightly different site on the iliac crest) to avoid hemorrhage and distorted marrow architecture in the biopsy core. With the availability of the biopsy needles described in this section, open (surgical) biopsies rarely are necessary but may be performed, for example, for diagnosis of deeply situated bone lesions or at the time of a surgical procedure performed for a related indication (e.g., staging). An FDA-approved battery-powered drill that inserts a biopsy needle into the posterior iliac bone of adult patients provides more consistent and longer biopsy cores and shortens procedure time.²⁰

Several types of preparations can be made from the marrow aspirate to maximize use of the diagnostic material. Most important is the direct film, which is made immediately from a drop of marrow suspension from the unmanipulated aspirate. This preparation is the best for evaluating cellular morphology and differential counts of the marrow. The particle film is best for estimating marrow cellularity and megakaryocyte abundance, but morphology is obscured in the thicker parts of the film. A concentrate film, which is prepared from a concentrate of nucleated cells (marrow buffy coat) achieved by centrifugation of a small volume of anticoagulated marrow, is sometimes used for detecting low-abundance cells when the marrow is hypocellular. The relative proportions of cell lineages are not maintained in the concentrate film preparation (often erythroid precursors are relatively enriched). In addition, this preparation is subject to anticoagulant-induced changes in nuclear morphology or cytoplasmic vacuolization. The touch imprint from the biopsy is quite valuable and sometimes diagnostically necessary for evaluating cellular morphology when the aspirate is hypocellular.²¹

MARROW FILMS

After aspiration, approximately 0.5 mL of marrow is placed on a glass slide; the rest is mixed into a tube containing ethylenediaminetetraacetic acid (EDTA) solution. The marrow specimen is examined to ensure the presence of “spicules” or particles of marrow containing bony or fatty pieces, indicating successful aspiration of the marrow cavity. Direct marrow films are immediately prepared by transferring drops of the unanticoagulated marrow pool to fresh slides and making push films with coverslips. Sufficient films should be made for special stains. Heparinization of the aspirate is not necessary if the operator works rapidly and should be avoided because heparinization may introduce artifacts. Formalin vapor artifact that can distort morphology can be avoided by making sure formalin containers are not opened until aspirate smears are prepared and put away.

A useful technique is preparing a thick film of marrow by discharging a drop or two of the aspirate on a slide, covering the aspirate with a second slide, gently pressing the slides together to express most of the blood into a gauze sponge, and then pulling the slides apart longitudinally. Such preparations may contain an increased number of broken cells if too much pressure is applied, but they provide a large number of particles from which marrow cellularity can be estimated and which are useful for estimating the amount of hemosiderin present. Broken cells may be minimized using a squash technique with coverslips instead of glass slides, which compared favorably to push (wedge) films in achieving representative distribution of intact cells derived from marrow particles.²²

The EDTA-anticoagulated sample may be centrifuged (1500 g for 10 minutes) in a Wintrobe tube to concentrate the cellular elements of the marrow. After centrifugation, the fatty layer and plasma are removed, and the “buffy coat” is mixed with an equal amount of plasma. Multiple films of this preparation are made. All smears should be thoroughly air-dried, which can take longer in a humid environment, before staining to avoid artifacts.

TOUCH PREPARATIONS

After a biopsy specimen is obtained using the Jamshidi needle, the specimen should be extruded through the hub of the needle and then gently rolled across a glass slide (using an applicator stick to move the specimen) before it is placed in fixative, taking care to avoid crushing. The touch preparations are allowed to dry and are stained in the same manner as films.

SPECIAL STUDIES

It is essential to formulate the diagnostic question before performing a marrow aspiration to ensure an adequate sample is obtained for all the special studies that may be needed to make the correct diagnosis, while avoiding aspiration of more marrow than is needed, which can lead to dilution of the sample.²³ A sterile anticoagulated sample containing viable unfixed cells in single-cell suspension is the best substrate for nearly all special studies. Specifically, flow cytometry is best performed on EDTA- or heparin-anticoagulated aspirate specimens, which are stable for at least 24 hours at room temperature. For cytogenetic or cell culture analysis, preservative-free heparin-anticoagulated marrow should be added to tissue culture medium and analyzed as soon as possible to maintain optimal cell viability. Cytogenetic samples are generally not adversely affected by overnight incubation.²⁴ In cases where the marrow aspirate is dry, a duplicate biopsy specimen can be disaggregated to produce a cell suspension for morphology, flow cytometry, and cytogenetic studies.²⁵ FISH for detection of chromosomal deletions, duplications, and translocations can be performed in marrow biopsies when EDTA-based decalcification protocols are used.²⁶

For molecular analysis of fresh specimens, sample storage should be minimized, and storage at 4°C is preferable. EDTA is the preferred anticoagulant because heparin can interfere with some molecular assays. DNA is relatively stable, but RNA has a variable half-life in an intact cell and is degraded rapidly (on the order of seconds to minutes) in a cell lysate by ubiquitous ribonucleases. Sample storage prior to RNA isolation should be minimized.²⁷ Collection tubes have been designed for stabilization of RNA, but for maximal RNA recovery, samples should be transported to the laboratory immediately, where cell suspensions (typically buffy coat or mononuclear cell preparations) can be prepared and nucleic acids extracted under conditions that inhibit ribonucleases. DNA and messenger RNA can be extracted and analyzed from paraffin-embedded tissue sections²⁸ and dried stained films,²⁹ with variable degree of degradation dependent on the length of sequence required.

Archival storage of marrow specimens is important in light of advances in molecular diagnosis that may necessitate validation studies using samples of known origin or retrospective testing of diagnostic material. Isolated DNA or RNA can be stored for long periods at −70°C, whereas viable, intact cells can be reliably preserved only by controlled rate freezing in dimethylsulfoxide (DMSO) and storage in liquid nitrogen.

HISTOLOGIC SECTIONS

A variety of techniques for preparing aspirated material for histologic study have been advocated. All of the techniques are designed to collect a sufficient number of marrow particles in a small volume so that adequate sections can be prepared. This goal can be accomplished by discharging the marrow aspirate onto a glass slide, allowing the particles to settle for a few seconds, and then gently tilting the slide so that the excess blood runs off. The particles then are pushed together with an applicator stick, and the remaining blood is allowed to clot. The clot is promptly fixed, typically in buffered formalin, for tissue processing and sectioning. An alternative method using filtration of the anticoagulated aspirate specimen has been described.³⁰

The core marrow biopsy specimen is processed for histologic examination typically by fixation in neutral buffered formalin, followed by decalcification and embedding in paraffin. Decalcification can be accomplished with acid reagents or EDTA, the latter of which is preferred as it provides better preservation of nucleic acid and protein antigens. Sections of high quality cut at 3 μm and stained with hematoxylin and eosin are satisfactory for routine work. Refinements in fixation and embedding techniques have enabled use of many immunologic markers in decalcified paraffin-embedded marrow biopsy specimens. Fixation in neutral-buffered formalin and embedding without decalcification in plastic resin has the advantage of superior morphology,³¹ but is less frequently used as the potential for immunostaining and molecular assays is more restricted.

OVERVIEW

The Wright-Giemsa–stained direct marrow aspirate film should be examined as quickly as possible to provide a preliminary assessment of the marrow morphology and allow setup of specialized testing based on this preliminary evaluation while the sample is fresh. Final interpretation of the marrow biopsy and aspirate should be integrated with results from the clinical history, blood film, cell counts, laboratory data, cell marker studies, and molecular or cytogenetic data. No other histologic specimen exists in which a state-of-the-art interpretation is dependent on such an array of supportive data. The challenge for the hematopathologist and hematologist is to understand the advantages and limitations of each diagnostic approach so that results can be reconciled and placed into perspective. Some common pitfalls in preparation and interpretation of marrow aspirates⁴ and biopsies⁵ have been reviewed.

ADEQUACY OF THE MARROW SAMPLE

The first question in interpreting the marrow is whether the sample is adequate for diagnosis. At the time of the procedure, the presence of marrow particles in the aspirate is the best indicator that the needle entered the medullary cavity and marrow was successfully withdrawn. Marrow particles are bony with a glistening appearance caused by fat in the particles. Specimens containing cortical bone, muscle, or other tissue with little or no medullary bone are inadequate for marrow interpretation. Samples with extensive crush artifact or hemorrhage are suboptimal, underscoring the importance of proper technique in obtaining a useful sample. An unspoken assumption is that the piece of marrow provided for diagnostic evaluation is representative of the marrow as a whole. Based on reproducibility of bilateral biopsies, this more likely is true in leukemia and myeloma than in lymphoma and metastatic tumor.³² A biopsy specimen should contain at least a 0.5-cm length of marrow cavity. However, for detection of lymphoma or metastatic tumor, current recommendations suggest a biopsy length of 1.6 to 2.0 cm.³³ A significant proportion of biopsies obtained in routine practice may fall short of this recommended length.³⁴

The marrow cavity was entered if the aspirate contains marrow particles or hematopoietic precursors (e.g., megakaryocytes, nucleated red cells) not found in the blood film. However, this finding does not ensure the specimen is adequate for diagnosis, because the amount of marrow actually aspirated can vary significantly in disease states. Also, some cell types, notably fibroblasts and metastatic tumor cells, are not as readily removed from the marrow space by aspiration as are normal precursors. Lack of particles or precursor cells does not prove the marrow cavity was not entered, because marrow packed with leukemic cells or infiltrated with fibroblasts may yield few cells (“dry tap”).³⁵ Marrow aspirations resulting in a dry tap usually are a consequence of significant pathology (only 7 percent show normal histology on biopsy³⁵) and indicate the need to examine a biopsy specimen, which should include a touch imprint.²¹

MARROW CELLULARITY

The “gold standard” for overall marrow cellularity is examination of an adequate marrow biopsy specimen.³⁶ The normal cellularity percentage of marrow space occupied by hematopoietic cells as opposed to fatty and nonhematopoietic tissue of iliac crest marrow decreases from a mean of 80 percent in early childhood to 50 percent by age 30 years, with further decreases after age 70 years.³⁷ Consequently, marrow cellularity should be evaluated with reference to normal individuals of the same age as the patient.³⁸ When evaluating cellularity, consider that the marrow spaces directly adjacent to cortical bone frequently are fatty and are not representative of the cellularity of the deeper marrow spaces. A grid can be used to estimate marrow cellularity of a biopsy.³⁹

Cellularity assessment by examination of the direct marrow aspirate film is more difficult because of loss of histologic structure and mixture with blood. The aspirate may suggest the marrow is more hypocellular than indicated by the biopsy.⁴⁰ Marrow particles (seen in the direct film or a particle preparation) are the best indicators of cellularity. These particles are like “mini-biopsies” and contain sufficient hematopoietic and fatty elements to give some idea of marrow cellularity. Cellularity estimates based on careful examination of particles in the aspirate preparation agree well with cellularity estimated from the marrow biopsy.³⁸

The degree of dilution of marrow aspirate specimens with blood during the aspiration is variable and may affect interpretation of marrow cellularity. Adult marrows with greater than 30 percent lymphocytes plus monocytes likely are substantially admixed with blood, as shown by cytokinetic studies of paired marrow aspirate and biopsy preparations.⁴¹ A higher-than-expected proportion of mature neutrophils in the marrow differential is another clue to a hemodilute marrow aspirate. In patients with hematologic disease, from 6 to 93 percent of the nucleated cells were derived from the blood.⁴² The greatest admixture was observed in patients with leukemia. Substantial dilution with blood may occur in difficult aspirates or when multiple draws were taken from the same puncture site. For instance, contamination of marrow aspirates with blood cells was only 8 percent in the first 1 mL, but 20 percent in subsequent draws.⁴³

Cellularity of individual lineages is best assessed by examination of the biopsy specimen. Erythroid cells typically are arranged in clusters, whereas megakaryocytes are scattered throughout the biopsy. Erythroid and megakaryocytic cellularity is best appreciated at low power. In the aspirate, a myeloid-to-erythroid (M:E) ratio frequently is calculated to give some impression of the relative cellularity of these two major lineages. As a rule of thumb, the M:E ratio normally should be between 2:1 and 4:1 (Table 3–1 lists the normal ranges in men and women). The relative proportions of cell types should be assessed only on the direct marrow film, biopsy imprint, or particle preparation, not a concentrate film, which has been manipulated by centrifugation. A decreased M:E ratio can be interpreted as either myeloid hypocellularity or erythroid hyperplasia, depending on the overall marrow cellularity. Megakaryocyte numbers can be assessed from the direct marrow aspirate film, where at least five megakaryocytes should be present in the optimal portion of the film. In the particle preparation, most large particles should contain one or more megakaryocytes. Megakaryocyte number varies markedly in direct marrow aspirate films of normal subjects and depends on the degree of admixture of the specimen with blood. Megakaryocytes are enriched at the feathered edge of concentrate films.

INFILTRATIVE DISEASES OF THE MARROW

Malignant Neoplasms

Metastatic nonhematopoietic tumor in the marrow biopsy is characterized by disruption of the marrow architecture with groups of cytologically abnormal cells. Assessment of the tissue of origin is primarily based on morphology, clinical history, and immunocytochemical staining. The tendency of carcinoma cells to form tightly adherent clusters frequently is helpful in recognizing these neoplasms (Chap. 45). The clumps can appear on the marrow aspirate, but the aspirate is less sensitive than the biopsy for detecting metastatic tumor. Tumor clumps may occur only on side or feathered edges of the film, or only in the concentrate preparation. These tumor clumps must be distinguished from clumps of damaged hematopoietic cells, which commonly appear in aspirate preparations, especially the concentrate film. The distinction is best accomplished by examining cells at the periphery of the clumps to determine if the cells show the morphology of hematopoietic precursors or are cytologically atypical cells. Isolated nonhematopoietic tumor cells are seen infrequently in aspirate preparations, even when tumor is obvious in the biopsy, because of the adherent nature of most nonhematopoietic tumors. Examination of multiple films may be necessary to find isolated tumor cell clumps.⁴⁴ Methods for identifying rare micrometastatic tumor cells (disseminated tumor cells) in marrow aspirates and blood have continued to evolve, but have not yet found an established role in guiding clinical prognosis or therapy.^45,46

Myeloma⁴⁷ and lymphomas⁴⁸ are nonhomogeneously distributed and more reliably detected on the biopsy preparation. Abnormal lymphoid aggregates should be distinguished from lymphoid aggregates found in reactive conditions or in older patients. Neoplastic aggregates show cytologic atypia and a monomorphous cellular population, and they often are adjacent to bony trabeculae, but the distinction can be difficult in some cases. The cellular morphology often can be better appreciated on the marrow aspirate, but the key histologic features are lost. Lymphoma cells do not form the tight clusters seen in nonhematopoietic tumors on the marrow aspirate film. In hairy cell leukemia (Chap. 93), the hematopoietic cells are sufficiently adherent to each other and the marrow matrix with variably increased collagen matrix that the aspirate specimen is often markedly hypocellular (dry tap), whereas biopsy specimens show extensive infiltration with hairy cells. Immunohistochemistry is useful in the differential diagnosis of plasma cell myeloma and other lymphoproliferative disorders, as is flow cytometry when the abnormal cells are sufficiently represented in the aspirated material. Demonstration of immunoglobulin light-chain restriction in B-cell lymphoma is not possible by immunohistochemistry, but in situ hybridization for κ versus λ light-chain messenger RNA may be successful. Detection of clonal immunoglobulin gene rearrangements by PCR amplification of messenger RNA transcripts may be used for this purpose, but clinical interpretation of the results can be problematic, and morphology remains the standard in evaluating marrow involvement by lymphoma.⁴⁹ Positron emission tomography using [¹⁸F] fluorodeoxyglucose (FDG-PET) has been recommended to replace staging marrow biopsy in Hodgkin lymphoma,^50,51 and in many patients with diffuse large B-cell lymphoma.⁵²

Fibrosis

Marrow fibrosis typically is recognizable only on a marrow biopsy specimen; the aspirate merely shows reduced or absent recovery of hematopoietic cells. Early stages of fibrosis are characterized by increased stainable marrow reticulin fibers (Chap. 86). Fibrosis may accompany either primary hematopoietic disorders (e.g., myelofibrosis) or infiltrative diseases such as metastatic tumor.

Storage Diseases

Storage disorders, such as Gaucher and Niemann-Pick diseases (Chap. 72), are characterized by abnormal macrophages containing stored material in various forms seen in aspirate or biopsy.⁵³ Reactive cells, such as the histiocytes with “sea-blue” inclusion granules (Chap. 72) or pseudo-Gaucher cells associated with chronic myelogenous leukemia (CML) (Chap. 89),⁵³ can resemble the cells seen in storage disorders.

Amyloidosis

Amyloid refers to extracellular proteins that become insoluble as a result of alteration in secondary structure to form beta pleated sheets. Amyloid light-chain amyloidosis may result from a plasma cell neoplasm, and is associated with nephrotic syndrome, restrictive cardiomyopathy, neuropathy and other tissue involvement. Amyloid deposits can be identified in the marrow by characteristic birefringence or fluorescence of deposits when stained with Congo Red.⁵⁴

INFECTIONS

Infectious organisms with an intracellular location, such as Leishmania, Histoplasma, and Toxoplasma,⁵⁵ can be visualized in monocytic cells by morphologic examination of the marrow (Fig. 3–3). Identification of mycobacterial organisms in the marrow by acid-fast staining lacks sensitivity but allows early diagnosis in one-third of cases with HIV-related Mycobacterium avium complex infection.⁵⁶ Microscopic examination and culture of the marrow are the most sensitive diagnostic tests for disseminated leishmaniasis.⁵⁷ Mycobacteria, also, may be cultured from marrow. Marrow morphology also is a sensitive diagnostic tool for detecting disseminated histoplasmosis.⁵⁸ However, marrow culture has a low diagnostic yield in the workup of fever of unknown origin in nonimmunosuppressed patients.⁵⁹ Definitive diagnoses arising from marrow examination in this setting are usually hematologic malignancies.^59,60 The presence of marrow granulomas, recognizable only on biopsy specimens, necessitates examination by special stains for fungal and mycobacterial organisms, but the differential diagnosis is extensive.^53,61

NECROSIS AND GELATINOUS TRANSFORMATION

Marrow necrosis may occur in a variety of disorders, particularly sickle cell disease and neoplastic processes involving the marrow.⁶² Aspirates of necrotic marrow stained with polychrome stains contain cells with indistinct margins and smudged basophilic nuclei surrounded by acidophilic material. Marrow sections stained with hematoxylin and eosin show loss of normal marrow architecture, indistinct cellular margins, and a background of amorphous eosinophilic material. Patients with severe weight loss may develop gelatinous transformation of the marrow, characterized by amorphous extracellular material (proteoglycans), fat atrophy, and marrow hypoplasia.⁶³

OVERVIEW

Marrow aspirate films should be examined under low-power magnification to assess the cellularity of particles and estimate the number of megakaryocytes, plasma cells, and mast cells. Low-power examination may also permit detection of malignancy or abnormal storage cells. The entire film should be examined, including the particles, and higher magnification should be used to study any abnormalities discovered. Similarly, biopsy sections are examined at low power to assess adequacy, overall cellularity, presence of infiltrative disease, and cellularity of the major hematopoietic lineages.

After the low-power survey, the films should be examined at higher power and under oil-immersion magnification to determine the various hemopoietic cell types present and assess adequacy of differentiation in each hematopoietic lineage. For most diagnostic questions, careful and systematic visual examination of the marrow is sufficient to assess differentiation, but a marrow differential cell count can be performed to quantify blasts or other abnormal cells. Based on the diagnostic question at issue, the marrow differential count may require examination of 300 to 500 nucleated cells. Table 3–1 lists the normal values for these determinations, including data for infants from birth to age 18 months.⁶⁴ Between birth and age 1 month, lymphocytes increase and erythroid and granulocytic precursors decrease. After 1 month, the marrow differential count varies little to age 18 months, the duration of the study.⁶⁴ The proportion of segmented neutrophils increases with large volumes of aspirate, probably because of dilution of marrow cells by mature granulocytes in the blood.⁶⁵ The range of normal for all cell types is broad, and differential counts and M:E ratios should be considered rough guides to the character of the marrow as a whole.

Progenitors of all lineages typically are unremarkable cells without distinctive morphologic attributes. Precursors and mature cells of the hematopoietic lineages show characteristic diagnostic morphologic changes as described below. Further details of the morphology of these cells are discussed in the relevant specific chapters of this book as referenced below.

GRANULOCYTES

Granulocytes are precursors or mature forms of leukocytes characterized by neutrophilic, eosinophilic, or basophilic granules in their cytoplasm in the more mature stages of development. This series sometimes is referred to as the myeloid series (Chap. 60). The overall trend is a gradual decrease in nuclear size and enhanced clumping of nuclear chromatin as cells lose proliferative capacity, while granules of varying types progressively appear in the cytoplasm.

The myeloblast is round and large, with a nucleus occupying most of the cell. The nuclear chromatin is very fine, and two to five nucleoli are present. The cytoplasm is basophilic, but less so than the cytoplasm of the erythroid series. Few azurophilic granules may be present.⁶⁶ The promyelocyte is larger than the myeloblast, with a coarser chromatin, but still containing nucleoli. The cytoplasm is basophilic with a clear Golgi area and a small number of prominent, large red granules—the primary, nonspecific, or azurophilic granules. The myelocyte is slightly smaller than the promyelocyte, and is the most mature mitotic cell in the myeloid lineage. Its nucleus is round or oval and often eccentrically located. The chromatin pattern is coarser than that of the promyelocyte, and nucleoli usually are not visible. The defining feature is the presence of specific granules in the perinuclear cytoplasm, which identify the cell lineage. The granules may be neutrophilic (fine, variable size, lilac color), eosinophilic (larger, round, orange–red), or basophilic (larger still, irregular in size, deep blue). The metamyelocyte is about the same size as the myelocyte and resembles it closely, except that the nucleus is indented, the chromatin is more coarse, and the cytoplasm is less basophilic. The band cell is characterized by a nucleus that is horseshoe shaped or lobular but is not narrowly segmented. The cytoplasm is yellowish–pink or nearly colorless with abundant lineage specific granules. Segmented (polymorphonuclear) granulocytes differ from band cells by the multilobed character of the nucleus. At least two separate lobes are defined by a complete rounded shape, whether or not the thin filament joining them is seen. Nuclear chromatin is very dense. The mature eosinophil typically has only two lobes, whereas the nuclei of most neutrophils have two to four lobes. Basophil nuclei may be obscured by the abundant basophilic granules.

MONOCYTES

Monocytes in normal marrow are identical morphologically to those in the blood. Promonocytes (Chap. 67) have delicate lace-like chromatin similar to a monoblast, but with indented or convoluted nuclear outline.⁶⁷ These are important cells to identify as they are considered blast equivalents in the evaluation of myelodysplastic syndrome (MDS) and acute leukemia.

MACROPHAGES (HISTIOCYTES)

These cells are derived from monocytes but are larger, reaching 20 to 30 μm in the longest dimension (Chap. 67). The nucleus is oval with delicate reticular chromatin and one or two small nucleoli. The cytoplasm ranges from blue-gray to pale and colorless, and often contains phagocytosed cells, degenerating cell debris, and vacuoles. Normally, intact red cells are rarely visible inside marrow macrophages. Erythrophagocytic macrophages are a feature of autoimmune hemolytic anemia, hemophagocytic lymphohistiocytosis (HLH), a severe uncontrolled hyperinflammatory reaction that can occur in a variety of clinical settings, such as infection, neoplasia, and autoimmune disorders (where it is termed macrophage activation syndrome), in addition to certain rare genetic disorders of cytotoxic granule function or immunodeficiency states⁶⁸ (Chap. 71).

ERYTHROID CELLS

During erythroid differentiation, the nucleus progressively becomes smaller and nuclear chromatin more condensed, as the cell’s proliferative capacity decreases. The cytoplasm gradually loses the bluish color imparted by RNA, which is replaced by the pink-staining hemoglobin. Cells in the erythroid series are termed erythroblasts (previously the term “normoblast” was used to distinguish the normal sequence from the sequence observed in megaloblastic anemia). These stages are arbitrary divisions within a continuum of differentiation. Chapter 31 provides more detailed descriptions of normal red cell precursors.

The proerythroblast is a large, round cell measuring from 15 to 20 μm in diameter. The nucleus occupies most of the cell and contains nucleoli. The chromatin is present in a fine reticular or stippled pattern but is usually more densely stained than the chromatin of the myeloblast. The cytoplasm typically is more basophilic than the myeloblast. The basophilic, polychromatophilic, and orthochromatophilic erythroblasts are characterized by cytoplasm gradually changing from blue to gray to pink in color as hemoglobin is produced and RNA reduced. The erythrocyte is the mature anucleate red cell. Polychromatophilic erythrocytes are mature anucleate red cells that are just released from the marrow (corresponding to early reticulocytes) and still have sufficient residual RNA to impart a slight grayish tinge to the cytoplasm (Chap. 32).

EVALUATION OF IRON STORES

Marrow examination often should include evaluation of the iron stores, especially if the patient is anemic. The examination is accomplished by staining a marrow film or section by the Prussian blue technique. Because decalcification of marrow biopsy specimens results in decreased recovery of stainable iron,⁶⁹ a nondecalcified specimen or aspirate should be stained when evaluating iron stores in the differential diagnosis of anemia. Marrow macrophages (seen best in the aspirate particle preparation) are evaluated for storage iron (see Fig. 3–3), and erythroblasts (best evaluated in the direct film or concentrate) are examined for the presence of iron granules in the cytoplasm (sideroblasts). Late erythroblasts are readily identified by their small size and the size, shape, and chromatin pattern of the nucleus. The proportion of normal late erythroblasts that contain one to four small Prussian blue granules is extremely variable (3–69 percent) in normal subjects.⁷⁰ Pathologic ring sideroblasts are characterized by an increased number of iron granules arranged in a ring encircling at least 1/3 of the nucleus, reflecting accumulation of iron in mitochondria (Chap. 87).

MEGAKARYOCYTES

Chapter 111 discusses the megakaryocyte in detail. Megakaryocytes are large cells (30–150 μm) with darkly stained, irregularly lobed nuclei. The cytoplasm is blue “cotton candy” textured, and the more mature cells contain many azurophilic granules.

LYMPHOCYTES

In normal marrow, lymphocytes similar to those found in the blood occur in variable numbers, depending on the degree of blood contamination of the marrow (Chap. 73). Immature lymphoid cells with a high nuclear-to-cytoplasmic ratio and moderately dense, but finely distributed, chromatin (“hematogones”) often are seen in marrow aspirates of children, and mostly represent B-cell precursors.⁷¹ These cells may cause diagnostic difficulty in some clinical settings, such as the “rebound” lymphocytosis that occurs after cessation of maintenance chemotherapy for acute lymphoblastic leukemia.

PLASMA CELLS

Normal plasma cells vary in size but usually are 12 to 16 μm in diameter when spread on a slide. They are round or oval. The nucleus is small, round, eccentrically placed, and stained densely purple. The chromatin is coarse and clumped. Nucleoli are not visible. The cytoplasm is deep blue, often with a paranuclear clear zone (Chap. 73). Binucleate forms may be found in normal marrow.

OTHER CELL TYPES

Mast cells are readily recognized by their content of dark-blue granules, which usually completely fill the cytoplasm and may obscure the nucleus (Chap. 63). The cells are round or spindle-shaped and often are located deep in the particles, frequently lying along blood vessels. The nucleus often is not visible but when seen is round or oval with a vesicular chromatin pattern.

Osteoclasts and osteoblasts are uncommon, and are more likely seen in hypocellular marrow or marrow obtained from children and from adults with hyperparathyroidism or osteoblastic reactions to tumors. Osteoclasts are large cells and may be larger than 100 μm in diameter (see Fig. 3–3). They superficially resemble megakaryocytes but contain multiple separated nuclei that have a moderately fine chromatin pattern with nucleoli. The cytoplasm varies from slightly basophilic to intensely acidophilic because of the content of acidophilic granules. Osteoclasts may contain coarse basophilic debris. Osteoblasts usually are oval cells up to 30 μm in the longest diameter (see Fig. 3–3). They often occur in groups. The nucleus usually is quite eccentric and may seem to be spilling out of the cell. The chromatin pattern is uniform, and one to three nucleoli are present. The cytoplasm is light blue and may contain a few red granules. Osteoblasts may be mistaken for plasma cells. In osteoblasts, the pale centrosomal region of the cytoplasm is separated from the nucleus, in contrast to that of the plasma cell, in which the centrosomal region directly abuts the nucleus.

Immunophenotyping is complementary to morphology in the contemporary practice of marrow cell identification. Flow cytometers use similar principles to the automated hematology analyzers discussed in Chap. 2, with the notable difference that fluorescence-labeled monoclonal antibodies directed toward cluster of differentiation (CD) antigens are the primary diagnostic tool. As described in the World Health Organization classification of hematologic malignancies,⁷² immunophenotypic data (expression of cell surface, intracytoplasmic, and nuclear antigens) are key determinants of diagnosis and classification of hematopoietic malignancies. The principle of immunophenotyping is to diagnose and follow neoplastic cell populations by virtue of differential patterns of protein expression. Only the basic principles of flow cytometry analysis are described in this chapter, so that the reader has the basis for understanding the phenotypic characteristics associated with the hematopoietic disorders described in greater detail in other chapters of this book.

METHODOLOGY

Flow cytometers are automated hematology analyzers that use principles of light scatter and fluorescence to define cellular populations in which to analyze expression of proteins typically identified by fluorescent tagged antibodies. A single-cell suspension is aspirated into a laminar flow of isotonic diluent that passes in front of one or more laser beams. Light scatter and fluorescence data are collected using specific photomultiplier tubes with appropriate filtration to collect scattered light (same wavelength as the incident laser light) or fluorescence emitted light (at a longer wavelength determined by the dye used). Multiple detectors with different filtration coupled with single or multiple lasers are used to collect highly multiplexed data. As with automated hematology analyzers, light scatter information is collected at a low angle (correlates with cell size) and 90-degree angle (correlates with cellular granularity and nuclear complexity; Chap. 2, Fig. 2–1). The latter measurement is especially useful in separating developing myeloid progenitors, monocytes, and mature granulocytes from lymphoid cells and blasts.

Immunophenotyping can be achieved by using monoclonal antibodies specific to certain cell surface proteins, most of which have CD designations as defined by international workshops. A primary requirement for flow cytometry analysis is that cells must be viable and in single-cell suspension prior to staining, which is why this method is used largely for hematopoietic malignancies and immunologic disorders, and not for analysis of solid tumors. This consideration also explains differences in results between flow cytometry and morphologic or immunohistochemical observations when samples with highly adherent neoplastic cells are analyzed. For instance, in multiple myeloma or large cell lymphoma the proportion of malignant cells is typically lower (or absent) by flow cytometry compared with marrow biopsy. In a well-equipped and appropriately staffed clinical laboratory, preliminary information often can be provided within 3 to 4 hours after the initial sample collection, thereby facilitating institution of appropriate therapy (e.g., in the case of newly diagnosed acute leukemias).

Clinical laboratories typically use four- to six-color analysis, plus side and forward light scatter, for routine diagnostic panels. For research studies, simultaneous analysis of up to 20 simultaneous fluorochromes is possible, by excitation with up to 5 lasers and separate collection of the emitted light produced by interaction with each laser. At present, routine use of that many simultaneously measured markers is not necessary for clinical diagnosis. An important consideration is that analysis with more simultaneous colors places greater demand on resources for development, maintenance and ongoing quality assurance. Most clinically important phenotypic markers are analyzed as cell surface proteins by directly adding conjugated antibodies to cell suspension, followed by washing and lysis of red cells.⁷³ Assessment of intracytoplasmic and nuclear-associated proteins is accomplished after staining for surface makers by then fixing cells in suspension and adding the relevant antibodies in conjunction with a membrane-permeabilizing agent. Some lineage-specific markers (CD3 in precursor T cells; CD79a and CD22 in B cells; myeloperoxidase in granulocyte lineage; cyclin D1 in mantle cell lymphoma) are expressed only in the cytoplasm at certain stages of development. Fluorescence and light scatter data are stored electronically as list mode data files that can be archived and later reanalyzed using appropriate software. As the number of parameters collected on individual cells increases, standard ways of looking at multiple two-parameter histograms of gated cell populations become more difficult. Data analysis techniques and automation appropriate to discovery and interpretation of multidimensional data sets such as those generated by various “-omics” analyses may become part of the multiparameter flow cytometry workflow.^74,75 Computational methods for identifying cell populations in highly multidimensional data sets have been shown to be more effective in reliable and consistent identification of clinically relevant cell populations in multicolor flow cytometry than manual gating and analysis,⁷⁶ particularly in the context of a consensus approach using an ensemble of algorithms, as is commonly done today in weather forecasting.

GATING STRATEGIES

In heterogeneous specimens such as marrow, in which the relevant clinical population (such as blasts) may be a minor population overall, a strategy for specifically identifying the population(s) of interest is necessary. As discussed in Chap. 2, this is accomplished for blood cells by very complex cluster analysis using multiple physical parameters. Because the flow cytometer has a much more sophisticated analytical capability at the back end with the fluorescent markers, the front-end selection of cells is not intended to be definitive, but should include the cells of interest and exclude nonrelevant cells, particularly those that may create an interpretive problem if included in the analysis. This process, referred to as gating, is typically accomplished by a combination of CD45 (common leukocyte antigen) and 90-degree light scatter (side scatter). As shown in Figure 3–4, lymphocytes, monocytes, myeloid precursors, and blast cells can be reasonably distinguished in marrow using this method. It is important to exclude monocytes, if they are not the cells one wishes to phenotype, as they express high-affinity Fc receptors that nonspecifically bind antibodies and may cause false-positive fluorescence signals. Individual lineages, such as eosinophils, basophils, and neutrophils, or stages of neutrophilic maturation, are not distinguished as automated hematology analyzers do for blood, but this is not necessary for the diagnostic questions usually asked by flow cytometry. The “blast gate,” defined by dim CD45 expression and low to intermediate side scatter, is a helpful region within which to identify and phenotype blast cells using more specific markers (only a minority of cell in this gate may be blasts,⁷⁷ but many cells with confounding immunophenotypes are excluded). Care must be taken to look for cells with unusual light scatter patterns not fitting in the usual “gates” to make sure the abnormal cells are not “hiding” in these regions. In particularly complex clinical circumstances, several fluorescent markers can be used just to identify a rare or subtly defined neoplastic subset, which can then be more definitively phenotyped in additional tubes containing those “backbone” markers to define the cells of interest plus additional markers to phenotype them. This strategy benefits from the ability to simultaneously measure up to 8 fluorescent markers in currently available clinical systems.⁷⁵ For samples with low cell viability, gating strategies based on light scatter and/or vital exclusion dyes, such as 7α-actinomycin-D, to limit analysis to the viable cell population only, may be used.⁷³ Strategies are commonly used to exclude cell doublets, for instance, based on the relationship of the pulse width (duration of signal) to pulse height of the forward light scatter signal. Immunocytochemistry of a marrow biopsy specimen is the preferred method for phenotyping solid tumors and is highly complementary to flow cytometry in diagnosis of lymphoid infiltrates.

COMMON FLOW CYTOMETRY APPLICATIONS IN HEMATOLOGY

Often the diagnostic question involves characterizing an expanded blast population or detection and analysis of a clonal lymphoid population (see Fig. 3–4). These determinations can be achieved by examining lineage-specific or maturation stage-specific markers. For instance, in marrow, immature cell populations can be identified by expression of antigens such as CD34 and CD117. In some instances, the stage of differentiation can be determined using combinations of markers that are expressed only during certain phases of differentiation (e.g., dual expression of CD4 and CD8 in an immature T-precursor population). Stage-specific phenotypes are sometimes valuable clues to clinically relevant diagnoses, such as the characteristic lack of human leukocyte antigen-D related (HLA-DR) expression in promyelocytic leukemia, a phenotype that mimics the normal loss of this antigen in promyelocytes. Among lymphoid leukemia/lymphomas, chronic lymphocytic leukemia (CLL)/small cell lymphoma, mantle cell lymphoma, hairy cell leukemia, and B- or T-precursor lymphoblastic leukemia, among others, have distinctive immunophenotypes. Methodologic advances such as multicolor analysis (6 or more simultaneous fluorescence colors) have allowed flow cytometers to detect and analyze diagnostically important rare subpopulations such as Reed-Sternberg cells in classical Hodgkin lymphoma.⁷⁸ Aberrant phenotype combinations suggestive of malignancy, such as coexpression of high levels of CD56 or CD117 on plasma cells in plasma cell myeloma, or loss of the T-cell markers CD7 or CD26 in a mature phenotype T cell in T-cell lymphoma, are diagnostically useful. Expression intensity is a frequent clue to diagnosis; for example, the weak surface immunoglobulin and weak CD20 expression in CLL. In reporting flow cytometry immunophenotyping results, the summary immunophenotype of the relevant population(s) should be described, rather than simply a listing of the percentage of cells positive for each marker, with subpopulations noted as observed.

Immunophenotyping of marrow by flow cytometry is a useful adjunct to established morphologic and cytogenetic criteria in diagnosing MDS,⁷⁹ and is predictive of later development of overt MDS in the diagnostically challenging group of cytopenic patients with initially morphologically equivocal marrow findings.⁸⁰ Abnormalities in MDS marrow include an increase in the percentages of CD34+ cells even when blasts are not morphologically increased; decreased CD34+ B progenitors; aberrant antigen expression by myeloid progenitors, mature granulocytic cells, or monocytes; and decreased side scatter of mature granulocytic cells. A simplified scoring system (so-called Ogata score) has been validated for use in an interlaboratory study,⁸¹ and international guidelines for a more extensive scoring panel have been published by the International/European Leukemia Net Working Group for Flow Cytometry in MDS.⁸² Immunophenotyping may provide therapeutically relevant prognostic information independent of existing risk factors incorporated in the commonly used (and recently revised) International Prognosis Scoring System (IPSS) score.⁸³

Clonality of immunoglobulin-expressing B-cell malignancies involving marrow (e.g., CLL and lymphoplasmacytic lymphoma) can be determined by simultaneous assessment of surface κ and λ immunoglobulin light-chain expression on the surface of B cells, often in combination with a characteristic neoplastic immunophenotype, such as CD5 expression in CLL B cells. Technical considerations are important to minimize nonspecific binding of serum monoclonal immunoglobulins to the surface of lymphocytes. Cytoplasmic κ and λ identification, in conjunction with aberrant surface immunophenotype, can also be useful in establishing the clonality of plasma cell neoplasms in marrow. T-cell clonality is not as easily demonstrated, because there are dozens of Vβ specificities expressed by T cells, and antibodies exist only to identify approximately 70 percent of these. Analysis of Vβ repertoire of the αβ T-cell antigen receptor in cells with an atypical immunophenotype, can identify clonal populations of T cells at diagnosis and posttherapy,⁸⁴ but this methodology is less-routinely used in clinical laboratories.

Presence of minimal residual disease (MRD) measured by either detection of a molecular target using PCR or aberrant immunophenotype using multicolor flow cytometry is increasingly used as a prognostic marker, often in the setting of clinical trials, in a variety of hematopoietic neoplasms, including acute leukemia, plasma cell myeloma, and CLL. In some settings, such as chronic phase CML, MRD monitoring by molecular assay is standard practice. In others, both flow cytometry and molecular methods for MRD detection are used successfully, each having their own advantages and limitations.⁸⁵ Flow cytometry assays for MRD are considerably more complex than standard diagnostic phenotyping and need to be designed for this specific purpose,⁸⁶ with collection of large numbers of events, multicolor strategies for detecting different types of subtle aberrant phenotypes, consistent data interpretation protocols, and confirmation of absence of “background” cells in noninvolved (but otherwise comparable; e.g., posttherapy) marrows expressing each aberrant phenotype to be tested. Immunophenotypic evidence of MRD can be sought by detection of specific leukemia associated immunophenotype, or by observation of a population in multidimensional space shifted significantly from any corresponding normal population (“different from normal” approach). Immunophenotypic shifts can occur during treatment, so identification of more than one aberrant phenotype to be tested is advisable when possible. Interpretation of MRD assays is dependent on type of neoplasia, timing of testing, treatment regimen, and standardization of assay methods. Standardization of flow cytometry MRD assays is a challenge that will need to be resolved as these assays move into more routine clinical practice. In acute leukemia, where MRD testing by flow cytometry has been most intensively studied, the assays provide posttreatment prognostic information, but the translation of this information into therapeutic decision making based on MRD risk stratification is further advanced in pediatric leukemia and acute promyelocytic than in adult acute myelogenous leukemia (AML).⁸⁷ MRD detection by either flow cytometry or PCR is standard clinical practice in pediatric acute lymphocytic leukemia (ALL),^85,86 where nearly all patients have leukemia associated targets suitable for either molecular or flow cytometry MRD assays. Detection of MRD in AML is more challenging because half of patients lack a molecular target suitable for MRD testing, but the majority of AML patients have a leukemia-associated phenotype that can be detected at the 0.1 percent level or below by multicolor flow cytometry. MRD detection in chronic lymphocytic leukemia (CLL)^88,89 and plasma cell myeloma⁹⁰ is also possible by flow cytometry and molecular techniques, and is becoming a relevant clinical issue now that therapeutic options in these malignancies are rapidly improving.

Flow cytometry is used to enumerate CD34+ progenitors when evaluating the adequacy of blood stem cell collections (Chaps. 23 and 28), with several routine assay kits available.⁹¹ Flow cytometry offers the potential to incorporate other markers defining clinically relevant progenitor and stem cell subpopulations.⁹² Lymphocyte subset quantitation is diagnostically important in acquired and congenital immunodeficiency states. Flow cytometry analysis of glycosylphosphatidylinositol (GPI) linked proteins in multiple blood cell types is the gold standard for diagnosis of paroxysmal nocturnal hemoglobinuria (Chap. 40).⁹³ Flow cytometry detection of paroxysmal nocturnal hemoglobinuria (PNH) clones is facilitated by using FLAER (fluorescently labeled inactive variant of the bacterial protein aerolysin), which binds to all (GPI) linked structures thus sensitively detecting GPI-linked protein expression in multiple cell lineages. Guidelines for standardized PNH flow assays have been published⁹⁴ and these assays are readily set up in clinical laboratories.