Bone Marrow Aplasia

7 Bone Marrow Aplasia B one marrow aplasia refers to those hematologic conditions that are caused by a marked reduction and/or defect

Box 7.1  Classification of Bone Marrow Aplasia

in the pluripotent or committed stem cells, or the failure of the bone marrow microenvironment to support hematopoiesis. The clinical outcome is anemia, leukopenia, and/or thrombocytopenia. The term “aplastic anemia” (AA) is a misnomer, because the patients, in addition to anemia, also suffer from leukopenia and thrombocytopenia. In this chapter, constitutional and acquired AA, dyskeratosis congenita, Shwachman–Diamond syndrome, Diamond–Blackfan anemia (DBA), amegakaryocytosis, and paroxysmal nocturnal hemoglobinuria are discussed (Box 7.1). Bone marrow failure due to myelodysplastic syndromes (MDS), leukemias, myelofibrosis, and other disorders are discussed in the following chapters.

Constitutional Fanconi anemia Dyskeratosis congenita Schwachman–Diamond syndrome Diamond–Blackfan anemia Amegakaryocytosis

Acquired Idiopathic aplastic anemia Secondary aplastic anemia l Chemical and physical agents l Drugs and other chemicals l Radiation l Infection l Viral: hepatitis, EBV, HIV l Others: tuberculosis, dengue fever l Immunologic (humoral and/or cellular) l Metabolic (pancreatitis, pregnancy)

Fanconi Anemia Fanconi anemia (FA) is the most common form of congenital bone marrow aplasia. It is an autosomal recessive or X-linked disorder with a prevalence of about 1 in 300,000 in most populations, but with much higher frequencies in the Afrikaner population of South Africa and Ashkenazi Jews. Characteristic congenital malformations associated with FA include generalized skin hyperpigmentation (café au lait spots), microcephaly, hypogonadism, abnormality of fingers (Figure 7.1), and short stature, which are found in 60–70% of the affected children. FA affects males more than females with a ratio of about 2:1. The congenital AA without physical abnormalities is known as Eastern–Dameshek anemia. FA patients have an increased risk of developing clonal bone marrow cytogenetic abnormalities, such as myelodysplastic syndrome (MDS) and/or acute myeloid leukemia (AML). The actuarial risk of MDS and AML is over 50% by the age of 40. This risk is higher in patients with cytogenetic abnormalities. There is also an elevated risk of Atlas of Hematopathology. DOI: http://dx.doi.org/10.1016/B978-0-12-385183-3.00007-3 © 2013 Elsevier Inc. All rights reserved.

Paroxysmal nocturnal hemoglobinuria Others Hypoplastic myelodysplastic syndromes Bone marrow replacement l Malignant neoplasms l Fibrosis l Others

l l

squamous carcinoma of head and neck, gynecologic neoplasms, and various other solid tumors, in patients with FA.

MORPHOLOGY l

Bone marrow biopsy sections in early stages of the disease may appear hyper- or normocellular with some megaloblastic changes but eventually become hypoplastic and depict marked hypocellularity with scattered foci of hematopoietic cells, predominantly erythroid (Figure 7.2). l Often, there are increased proportions of plasma cells and lymphocytes.

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Bone Marrow Aplasia l Blood

examination is usually normal at birth. Usually, microcytosis is the first detected abnormality, followed by elevated levels of fetal hemoglobin, thrombocytopenia, and neutropenia between the ages of 5 and 10 years.

MOLECULAR AND CYTOGENETIC STUDIES l

FIGURE 7.1  Fanconi anemia. The hands of this child show symmetric abnormalities of the thumbs, resulting in their resemblance to fingers. From Hoffbrand AV, Pettit JE, Vyas P. Color Atlas of Clinical Hematology, 4th edn. Mosby/Elsevier, Philadelphia, 2010, with permission.

A

DNA testing of several of the FA genes is now available, consisting of either gene sequencing or targeted mutation analysis such as the predominant IVS4 1A → T mutation in the Ashkenazi-Jewish population. l Population-based carrier screening (i.e., for those couples with no family history of the disorder) is offered in some centers for Ashkenazi Jews of reproductive age, in whom the carrier frequency for mutations in the gene for FA type C is 1 in 90. l Conventional karyotyping is performed by stimulating and culturing lymphocytes from peripheral blood. Baseline breakage (with no DNA damaging agent) is recorded with age matched-controls, followed by analysis for chromosome breaks, gaps, and other aberrations for conditions supplemented with DNA-damaging agents Mitomycin C (MMC) and Diepoxybutane (DEB). l Individuals with FA will exhibit an increased rate of spontaneous chromatid/chromosome breaks, triradials, quadriradials, and are hypersensitive to the clastogenic effect of DNA cross-linking agents. The increased rates of spontaneous chromosomal breakage, and the radial forms distinguish FA from other chromosomal breakage syndromes. The increased sensitivity to DEB/MMC is present regardless of phenotype, congenital anomalies, or severity of the disease

Other Congenital Bone Marrow Aplasias DYSKERATOSIS CONGENITA

B FIGURE 7.2  Bone marrow biopsy section from a patient with Fanconi anemia demonstrating marked hypocellularity with only small foci of hematopoietic cells: (A) low power and (B) high power views.

Bone marrow smears may show increased mast cells. There is evidence of hemophagocytosis, particularly in early stages of the disease. l These bone marrow morphologic features are not pathognomonic for FA and are also observed in patients with acquired AA.

Dyskeratosis congenita (DC) is an X-linked recessive trait which is characterized by bone marrow hypoplasia and a triad of mucosal leukoplakia, nail dystrophy, and abnormal skin pigmentation. About 20% of the patients may also suffer pulmonary dysfunction characterized by reduced diffusion capacity. The approximate median ages for the demonstration of somatic abnormalities and bone marrow failure are 8 and 10 years, respectively. Over 90% of the affected patients are male. DC patients have a higher tendency to develop MDS, AML, and skin and oropharynx cancer.

Morphology Bone marrow becomes markedly hypoplastic with morphologic features similar to those of FA.

l

l

Molecular and Cytogenetic Studies l l

The dyskeratin gene (DKC1) at chromosome Xq28 is mutated. Sequence analysis of this gene is available in a small number of laboratories.

Other Congenital Bone Marrow Aplasias

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A

FIGURE 7.4  Diamond–Blackfan syndrome. Three-year-old boy showing sunken bridge of the nose. From Hoffbrand AV, Pettit JE, Vyas P. Color Atlas of Clinical Hematology, 4th edn. Mosby/Elsevier, Philadelphia, 2010, with permission.

l l

B FIGURE 7.3  Shwachman–Diamond syndrome. Bone marrow smears show reduced number of neutrophils and bands: (A) low power; (B) high power. l

Telomere length can also be assayed to rule out the diagnosis. This is especially important in the younger child presenting with macrocytosis who may not yet exhibit the characteristic cutaneous or oral manifestations of DC.

SHWACHMAN–DIAMOND SYNDROME Shwachman–Diamond syndrome or Shwachman– Diamond– Oski syndrome is a rare autosomal disorder characterized by skeletal anomalies, short stature, pancreatic insufficiency, and progressive bone marrow hypoplasia and neutropenia (Figure 7.3). Patients are prone to infection, particularly caused by gram-negative organisms, such as Haemophilus influenzae, or Staphylococcus aureus.

Molecular and Cytogenetic Studies l Mutations

of a gene referred to as Shwachman–Bodian– Diamond syndrome (SBDS) have been reported. Sequencing of the SBDS gene is available in several reference laboratories. l Cytogenetically, over 6% of the aberrations frequently involve chromosome 7, typically in the form of an isochromosome 7q [i(7)(q10)], followed by del(20q) often occurring as a secondary event to i(7)(q10). The chromosomal aberrations can be transient and are not necessarily indicative of an imminent transformation to MDS/AML.

DIAMOND–BLACKFAN ANEMIA Diamond–Blackfan anemia (DBA) is a pure red cell aplasia predominantly demonstrated in infancy and early childhood. DBA is about 45% familial and is often associated with physical anomalies, such as thumb malformations, growth retardation, and craniofacial deformities (Figure 7.4).

Morphology and Laboratory Findings l

Morphology/Laboratory Findings l

Neutropenia is the major hematologic feature of this disorder, which is often intermittent or cyclic.

Elevated levels of fetal hemoglobin are detected in up to 80%. Marked bone marrow hypoplasia (AA) is reported in 20–25% of the cases.

Marked bone marrow erythroid hypoplasia (Figure 7.5) Macrocytic anemia and elevated fetal hemoglobin levels l Increased erythrocyte adenosine deaminase activity. l DBA patients may eventually develop pancytopenia and aplastic bone marrow. l

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Bone Marrow Aplasia

A

A

B

B

FIGURE 7.5  Bone marrow smears from a patient with pure red cell aplasia (Diamond–Blackfan syndrome) demonstrating lack of erythroid precursors: (A) low power; (B) high power.

Molecular and Cytogenetic Studies l

l l

l

l

At least 9 different genes encoding ribosomal proteins (6 of the small subunit (RPS7, RPS10, RPS17, RPS19, RPS24, and RPS26) and 3 of the large subunit (RPL5, RPL11, and RPL35a) have been associated with DBA. Currently it is possible to evaluate for 6 of these, and the presence of a gene mutation confirms the diagnosis of DBA. However, these mutations currently are found in only 50% of the patients. RPS19, the first one discovered, is the most frequently involved, with mutations found in approximately 25% of patients. The genetics of the disorder are complicated by the absence of family history in many cases, which could be due to either sporadic incidence or a dominant gene with low penetrance. Testing for parvovirus B19 by PCR on bone marrow samples may be performed as part of the differential diagnosis of red cell aplasia in an infant. Chromosomal abnormalities involving the DBA (ribosomal protein S19) region at 19q13, such as t(X;19), t(8;19), and 19q microdeletions have been reported. In addition, deletions of chromosome 3q involving the coding region for RPL35a has also been observed.

FIGURE 7.6  Amegakaryocytosis. (A) Bone marrow biopsy section and (B) bone marrow smear showing lack of megakaryocytes.

AMEGAKARYOCYTOSIS Congenital amegakaryocytosis (amegakaryocytic thrombocytopenia) is a rare disorder of infancy with markedly reduced or absent megakaryocytes in bone marrow and therefore isolated thrombocytopenia. The cause of this disorder in some children appears to be due to the mutations of the thrombopoietin receptor gene, MPL, on chromosome 1p34. Clinical symptoms include bleeding into the mucous membranes, gastrointestinal tract, and skin. Absence of radial bones is observed in some of the patients (thrombocytopenia with absent radius, TAR syndrome).

MORPHOLOGY AND LABORATORY FINDINGS l

Absent or rare megakaryocytes in bone marrow samples with marked thrombocytopenia (Figure 7.6). l Approximately 50% of these patients may eventually develop AA and pancytopenia. l The serum concentration of thrombopoietin is elevated.

Acquired Aplastic Anemia

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Molecular and Cytogenetic Studies l

The autosomal recessive form of amegakaryocytosis is caused by mutations in the MPL gene. l Complete sequencing of the gene is available in select reference laboratories.

Acquired Aplastic Anemia Acquired AA is characterized by severe bone marrow hypocellularity and pancytopenia (anemia, leukopenia, and thrombocytopenia). The term “acquired” refers to noncongenital causative mechanisms which could be immunologic, environmental, or unknown. The incidence of AA is significantly higher (about fivefold) in the Far East than in the West. Clinical manifestations of AA are non-specific and are usually related to pancytopenia. Pallor, fatigue, purpura and mucosal hemorrhage, and recurrent infections are common findings. Several studies support the destruction or suppression of bone marrow stem cells by immune mechanisms as the major contributing factors. Clonal expansion of cytotoxic T-cells (T-large granular lymphocytic leukemia) may play a role. Some patients have a history of exposure to a wide spectrum of chemical and physical agents and various diseases. However, since there is daily exposure to unlimited and widespread chemicals, such as insecticides, fertilizers, food additives, and herbal medicine, the exact causative factor(s) is not detected in about 50–75% of AA patients. Therefore, acquired AA is divided into two major categories: (1) idiopathic AA (with no known etiology) and (2) secondary AA. The outcome of untreated severe AA is very poor, with over 70% death rate within 1 year. Prognosis is also agedependent, with better outcome in patients under 49 years than those over 60 years. The treatment of choice under the age of 45 is hematopoietic stem cell (HSC) transplantation. But, only 25–30% of AA patients find proper donors. Immunosuppressive therapy is recommended for patients over the age of 45. Immunosuppressive agents include ATG, corticosteroids, and cyclosporine. Hematopoietic growth factors, such as G-CSF, have been added to the immunosuppressive regimen with some beneficial effects.

MORPHOLOGY AND LABORATORY FINDINGS l Bone

marrow is markedly hypocellular with a very high proportion of fatty tissue and stromal cells (Figure 7.7). All hematopoietic elements are decreased but are morphologically normal. The bone marrow biopsy sections show scattered islands of hematopoietic cells randomly distributed throughout the fatty marrow. These islands are predominantly erythroid and contain very few megakaryocytes. There is no evidence of a malignant infiltrate or diffuse fibrosis.

A

B

C FIGURE 7.7  Aplastic anemia. Bone marrow biopsy sections (A, low power; B, high power) demonstrating marked hypocellularity. (C) The bone marrow smear shows fatty tissue and stromal cells.

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Bone Marrow Aplasia

l Bone

marrow smears consist predominantly of adipocytes and stromal tissue with scattered hematopoietic cells. Occasionally, some of the aspirated smears may show cellular marrow particles, giving the wrong impression of a normocellular or even hypercellular marrow. For this reason, bone marrow biopsies are preferred for the establishment of the diagnosis of AA. Some bone marrow smears may show increased proportion of lymphocytes, plasma cells, macrophages, and mast cells. These cells either appear as welldefined aggregates or are diffusely dispersed in the stroma. There may be evidence of hemophagocytosis, particularly in early stages of the disease. l Peripheral blood shows pancytopenia with reduced reticulocyte count. Anemia is usually normochromic and normocytic, but macrocytosis and anisocytosis may be present. Neutrophils may show toxic granulation. The lymphocyte count is normal or low. Occasionally, cytotoxic CD8+ T-lymphocytosis can be seen.

FIGURE 7.8  Capillary electrophoresis readout of immunoglobulin heavy chain gene rearrangement analysis in a patient with aplastic anemia, showing only background signal and a few nonspecific spurious signal peaks.

AA is defined as severe when: 1. bone marrow cellularity is <25% of normal cellularity for age in biopsy sections, or 2. bone marrow cellularity is <50% of normal cellularity for age, with <30% hematopoietic cells, plus at least two of the following: a. absolute erythrocyte count <40,000/μL. b. absolute neutrophil count <500/μL. c. platelet count <20,000/μL.

When the criteria for severe AA are met and the absolute neutrophil count is <200/μL, the patient is considered to have a very severe AA.

MOLECULAR AND CYTOGENETIC STUDIES Because of its heterogeneous and non-genetic etiology, there are no specific molecular tests for acquired AA. Mutation testing of genes associated with the hereditary disorders, and PCR-based detection of implicated viruses, may be performed as part of the differential diagnostic work-up. Sometimes these patients are evaluated for associated leukemia or MDS, such as by clonal gene rearrangement analysis, but the very low white blood cell counts can make this a challenge (Figure 7.8). l Approximately 4% of patients with AA show cytogenetic abnormalities, such as 5q−, monosomy 7, and trisomy 6 or 8 (Figures 7.9, and 7.10). l Patients with AA and abnormal cytogenetics have different clinical characteristics compared with AA patients with normal cytogenetics. Patients with abnormal cytogenetics are generally younger, and are associated with a higher cumulative leukemic transformation rate and lower leukemic transformation-free survival. Furthermore, abnormal cytogenetics is an independent predictor of a poor response to immunosuppressive therapy.

FIGURE 7.9  A G-banded karyotype showing monosomy 7 (arrow) in a patient with AA.

l

FIGURE 7.10  FISH studies for chromosome 8 in interphase. Cells demonstrating trisomy 8 (arrows).

Paroxysmal Nocturnal Hemoglobinuria

Paroxysmal Nocturnal Hemoglobinuria Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired stem cell disorder associated with a defect in cell membrane glycosyl phosphatidylinositol (GPI) anchor due to mutation of the PIG-A gene. This defect leads to partial or complete loss of GPI-linked membrane proteins, such as CD14, CD16, CD24, CD48, CD52, CD55, CD58, CD59, CD66, and CD73 (Table 7.1). Some of these proteins, such as CD55 and CD59, play an inhibitory role in the activation of the complement system, and therefore their absence leads to complement-induced lysis and hemolytic anemia. CD55, also known as decay accelerating factor (DAF), is expressed by all hematopoietic cells and is an inhibitor of C3 and C5 convertase. Similarly, CD59 is expressed by all hematopoietic cells. It is referred to as membrane inhibitor of reactive lysis (MIRL) and inhibits complement membrane attack complex by binding to the C8 component and prevents C9 from binding and polymerizing. The International PNH Interest Group (I-PIG) has divided PNH into three main categories: (1) classical PNH with hemolysis and thrombosis; (2) PNH in the setting of other specified bone marrow disorders such as AA and MDS; Table 7.1

Some of the GPI-Linked Proteins Deficient in Paroxysmal Nocturnal Hemoglobinuria1 Molecule

CD

Complement Regulatory Molecules DAF CD55 MIRL CD59

1

Comments Decay accelerating factor Membrane inhibitor of reactive lysis

Enzymes Ecto-5′-nucleotidase ADP-ribosyl transferase

CD73 CD157

Lymphocytes T cells and neutrophils

Adhesion Molecules Blast-1 LFA-3 Adhesion molecule 1 NCA-95 NCA-50/90 Carcinoembryonic antigen

CD48 CD58 CD66a CD66b CD66c CD66e

Leukocytes; binds CD24 All hematopoietic cells Granulocytes, epithelium Granulocytes Granulocytes, epithelium Epithelium

Others NA1/NA2 Campath-1

CD16 CD52

BA-1 Thy-1

CD24 CD90

Neutrophils and NK cells Lymphocytes and monocytes B cells and granulocytes Stem cell subset, T cell subset

Adapted from Hall C, Richards SJ, Hillmen P. The glycosyl phosphatidylinositol anchor and paroxysmal nocturnal haemoglobinuria/aplasia model. Acta Haematol 2002; 108: 219–230.

105

and (3) subclinical PNH, in which patients have small PNH clones but no evidence of hemolysis or thrombosis. Classical PNH may affect patients at any age, but the peak incidence is between 20 and 35 years. It is characterized by hemolytic anemia (often with hemoglobinuria), venous thrombosis, and bone marrow failure: 1. Acquired intravascular hemolysis demonstrated by hemoglobinemia, hemoglobinuria, hemosiderinuria, and negative direct antiglobulin (Coombs) test. 2. Thrombosis of the relatively large veins in odd places, such as hepatic (Budd–Chiari syndrome), mesenteric, portal, or cerebral veins. Venous thrombosis is the major cause of death in PNH patients. Arterial thrombosis is rare. 3. Bone marrow hypoplasia leading to pancytopenia.

The possible association between the PNH clone and other primary bone marrow failure disorders such as AA, MDS, and hypocellular acute myeloid leukemia has been the subject of study for some time. Recent published results of large study series suggest that PNH clones can be found in as many as 60–70% AA and 20–55% of MDS using high sensitivity flow cytometric studies. Identification of PNH clones may predict a good response to immunosuppressant in these patients, and therefore a better clinical outcome. Management of PNH includes treatment of hemolytic and non-hemolytic anemia with iron and folic acid supplementation, red blood cell transfusion, plus use of prednisone and androgen derivatives. Anticoagulation therapy is used for episodes of thrombosis. Hematopoietic stem cell transplantation has been used with success in recent studies. Eculizumab is a therapeutic agent newly approved by the FDA for classical PNH. It is a humanized monoclonal antibody against complement C5. This reagent is effective in controlling hemolysis, and results in improvement in quality-of-life measures.

MORPHOLOGY AND LABORATORY FINDINGS l

Bone marrow in most instances is markedly hypocellular and presents morphologic features similar to those of AA (Figure 7.11). However, some patients may show a normocellular or even a hypercellular marrow. There is often erythroid preponderance. Stainable iron is usually absent, primarily due to loss of iron secondary to hemoglobinuria and hemosiderinuria. l Blood examination commonly reveals severe anemia with elevated reticulocyte count and some degree of granulocytopenia and thrombocytopenia. The leukocyte alkaline phosphatase (LAP) score is reduced. l There is evidence of intravascular hemolysis by the presence of hemoglobinuria, hemosiderinuria. Plasma haptoglobulin levels are reduced and plasma lactate dehydrogenase (LDH) levels are elevated. l For years, the diagnosis of PNH was based on the sensitivity of the red cells to lysis by complement. This was determined by the sucrose lysis screening test and the confirmatory Ham acid hemolysis test. In the sucrose lysis test, the patient’s red cells are incubated with serially diluted isotonic sucrose solutions. Under these conditions the complement system is

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Box 7.2  Clinical Indications for PNH Testing1 Intravascular hemolysis as evidenced by hemoglobinuria or elevated plasma hemoglobin. Evidence of unexplained hemolysis with accompanying: Iron-deficiency, or Abdominal pain or esophageal spasm, or l Thrombosis, or l Granulocytopenia and/or thrombocytopenia. l l

Other acquired Coombs-negative, non-schistocytic, non-infectious hemolytic anemia. Thrombosis with unusual features: Unusual sites l Hepatic veins (Budd–Chiari syndrome) l Other intra-abdominal veins (portal, splenic, and splanchnic) l Cerebral sinuses l Dermal veins l With signs of accompanying hemolytic anemia (see above) l With unexplained cytopenia. l

FIGURE 7.11  Paroxysmal nocturnal hemoglobinuria (PNH). Bone marrow biopsy section from a patient with PNH, demonstrating marked hypocellularity.

Evidence of bone marrow failure:

activated and the test is considered positive if there is evidence of hemolysis. In the Ham test, the pH of serum is reduced to activate the complement system and to induce hemolysis in the PNH red cells. However, nowadays, immunophenotyping by flow cytometry (see the following section) is considered the standard procedure.

Suspected or proven aplastic or hypoplastic anemia MDS/Refractory cytopenia with unilineage dysplasia l Other cytopenias of unknown etiology after adequate work-up l l

1 

Adopted and modified from the ICCS Guidelines for the Diagnosis and Monitoring of Paroxysmal Nocturnal Hemoglobinuria and Related Disorders by Flow Cytometry.

FLOW CYTOMETRY Detection of GPI-anchor-deficient cells by flow cytometry is the established method of choice for diagnosis and monitoring of PNH. The International Clinical Cytometry Society (ICCS) has recently published consensus guidelines on PNH testing by flow cytometry, addressing both routine and high sensitivity assays.

Clinical Indications for PNH Testing According to the ICCS guidelines, there are several clinical indications for PNH testing (Box 7.2). The category of “evidence of bone marrow failure” is of particular interest since it is more frequently encountered in clinical practice than is classical PNH.

Sample Requirements Peripheral blood is the preferred specimen, and the acceptable anticoagulants include EDTA, heparin, and ACD. Bone marrow should not be used other than in a research setting. It is recommended that blood samples are tested within 24–48 hours after collection.

Routine Assays The purpose of the routine assays is to detect and quantify cells lacking GPI-anchored proteins at a sensitivity level of

1%. Routine assays are critical in diagnosing and monitoring classical PNH in hemolytic and thrombotic patients, since the size of PNH clones in those patients is usually greater than 10%. RBCs (Figure 7.12) l The RBC analysis is to distinguish and quantify the following: – Type III cells—complete absence of GPI-anchored proteins; – Type II cells—partial loss of GPI-anchored proteins; – Type I cells—normal expression of GPI-anchored proteins. l Glycophorin A is used for gating RBCs, while CD59 or a combination of CD55/CD59 is used for GPI-anchored marker. The combined use of CD55 and CD59 is preferred, since rare cases of congenital CD55 or CD59 deficiency have been reported that have no association with PNH. l Testing RBC alone is inadequate in evaluation of PNH, since the size of PNH clones can be significantly underestimated as a result of hemolysis and/or transfusion. l WBCs (Figure 7.13). l It is the best method for assessing the true size of PNH clones. l Target populations include both neutrophils and monocytes. Lymphocytes are not suitable targets. l

Paroxysmal Nocturnal Hemoglobinuria

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FIGURE 7.12  Detection of PNH clones by routine RBC assay using multiparametric flow cytometry. Combined gating of light scatters (in log display) and Glycophorin A allows distinct separation of RBCs from debris and background. Dual parameter display (e.g., CD59 and Glycophorin A) is more sensitive than single parameter display in distinguishing type I, II, and III cells. Washing in sample preparation plus use of density plots can enhance the separation of those RBCs. FIGURE 7.14  Detection of small PNH clones by high-sensitivity assay using multiparametric flow cytometry. High sensitivity assay is performed on WBCs by collecting 500,000 to 1 million granulocytes using live-gate. Small PNH clones form discrete clusters revealing negative staining for both FLAER and CD24.

High-Sensitivity Assays (Figure 7.14) l

FIGURE 7.13  Detection of PNH clones by routine WBC assay using multiparametric flow cytometry. Combined CD45 gating plus CD15 vs. side scatter (R1 + R2) separates granulocytes from debris and background, whereas CD45 gating plus CD33 vs. side scatter (R1 + R3) distinguishes monocytes. At least two GPI-anchored proteins are needed for evaluating each target population. A PNH clone in granulocytes demonstrates double negativity for FLAER and CD24, while it is negative for both FLAER and CD14 in monocytes.

l

FLAER (fluorescent aerolysin) is considered the single most useful reagent in detecting WBC PNH clones. It binds specifically to the GPI anchor, and is reliably absent from GPIanchor-deficient neutrophils and monocytes. l It is recommended that at least two GPI-anchored markers including FLAER are used to assess PNH clones, and additional antibodies like CD45, CD33, and CD15 are helpful in gating the target populations.

High-sensitivity assays are not required in diagnosis of classical PNH. Instead, they are useful in identifying small PNH clones that are commonly associated with bone marrow failure disorders like AA and MDS. l A desired sensitivity level for high-sensitivity PNH assays is 0.01% according to the new PNH consensus. l Technical challenges are similar to those seen in other rare-event analysis, i.e., detection of minimal residual disease (MRD). In order to achieve this level of sensitivity, more events (e.g., up to 1 million) are required and live-gate may be necessary. l Multiparametric analysis is important, and several markers are needed for gating target cell populations in addition to evaluation of at least two GPI-anchored markers for each target.

MOLECULAR GENETICS AND CYTOGENETICS l

Reported mutations in the PIG-A gene on chromosome Xp22 are numerous and heterogeneous, and are further complicated by the presence of a pseudogene on chromosome 12; DNA sequencing is not generally available for clinical testing. l The gene mutations found in PNH are acquired, not inherited, so they will only be found in the abnormal cells. l Cytogenetic abnormalities in PNH usually occur in hematopoietic cells that are glycosyl phosphatidylinositol-anchored protein (GPI-AP)-positive. Various chromosomal aberrations have been reported in up to 24% of patients with PNH, including monosomy 5, trisomy 6, trisomy 8, and monosomy 7.

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Table 7.2

Differential Diagnoses in Bone Marrow Aplasia Disorder

Bone Marrow Morphology

Immunophenotype

Cytogenetics and Molecular

Constitutional Aplasias

Non-contributory

Acquired AA

Normo- to hypercellular at early stages, hypocellular marrow at later stages Hypocellular marrow

Frequent chromosomal breakage, sometimes −7, mutations in causative genes Sometimes 5q−, −7, +6, +8, viral PCR

PNH

Hypocellular marrow

Hypocellular MDS

Hypocelluar marrow with significant dysplastic changes, and sometimes increased blasts Hypocellular marrow with ≥20% blasts

Hypoplastic AML

Hypocellular hairy cell leukemia

Hypocellular marrow with the presence of hairy cells and often evidence of fibrosis

Often increased cytotoxic T cells, strong association with HLA-DR2 Loss of GPI-linked proteins, such as CD55 and CD59 Abnormal phenotypic patterns, sometimes increased CD34+ and/or CD117+ cells Increased CD45dim+ cells expressing myeloid markers, often CD34 and/or CD117 TRAP+, CD103+, CD25+, CD22+, CD11c+

Mutations in pig-A gene −7, +8, 5q−, 20q−, and other chromosomal aberrations Frequent chromosomal aberrations involving 11q, 16q, or t(15;17), t(8;11), t(9;22), and others Not known

AA, aplastic anemia; AML, acute myeloid leukemia; MDS, myelodysplastic syndromes; PNH, paroxysmal nocturnal hemoglobinuria.

Differential Diagnosis Morphologic features of bone marrow in advanced stages of constitutional marrow aplasias, acquired AA, and PNH are indistinguishable. Also, other bone marrow lesions, such as hypocellular MDS, hypoplastic AML, and

hypocellular hairy cell leukemia, may morphologically mimic AA (Table 7.2). Clinical history and information regarding other clinicopathologic parameters are imperative for accurate diagnosis. It is important to remember that a proportion of patients with constitutional or acquired AA may eventually develop MDS or AML.

ADDITIONAL RESOURCES

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Additional Resources Alter BP: Diagnosis, genetics, and management of inherited bone marrow failure syndromes, Hematology Am Soc Hematol Educ Program:29–39, 2007. Bagby GC, Alter BP: Fanconi anemia, Semin Hematol 43:147–156, 2006. Borowitz MJ, Craig FE, Digiuseppe JA, et al: Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry, Cytometry Part B 78B:211–230, 2010. Galili N, Ravandi F, Palermo G, et al: Prevalence of paroxysmal nocturnal hemoglobinuria (PNH) cells in patients with myelodysplastic syndromes (MDS), aplastic anemia (AA), or other bone marrow failure (BMF) syndromes: interim results from the explore trial, J Clin Oncol 27:15s, 2009. Hoffbrand AV, Pettit JE, Vyas P: Color atlas of clinical hematology, ed 4, Philadelphia, 2010, Mosby/Elsevier.

the management of bone marrow failure, Int J Hematol 84:118–122, 2006. Nishio N, Kojima S: Recent progress in dyskeratosis congenita, Int J Hematol 92:419–424, 2010. Richards SJ, Whitby L, Cullen MJ, et al: Development and evaluation of a stabilized whole-blood preparation as a process control material for screening of paroxysmal nocturnal hemoglobinuria by Flow Cytometry, Cytometry Part B 76:47–55, 2009. Shimamura A: Clinical approach to marrow failure, Hematology Am Soc Hematol Educ Program:329–337, 2009. Sutherland DR, Kuek N, Azcona-Olivera J, et  al: Use of a FLAERbased WBC assay in the primary screening of PNH clones, Am J Clin Pathol 132:564–572, 2009.

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