Atlas of Hematopathology. DOI:

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7Bone Marrow Aplasia

Bone marrow aplasia refers to those hematologic condi-tions that are caused by a marked reduction and/or defect in the pluripotent or committed stem cells, or the failure of the bone marrow microenvironment to support hema-topoiesis. 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 thrombocytope-nia. In this chapter, constitutional and acquired AA, dys-keratosis 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.

Fanconi Anemia

Fanconi anemia (FA) is the most common form of con-genital bone marrow aplasia. It is an autosomal reces-sive or X-linked disorder with a prevalence of about 1 in 300,000 in most populations, but with much higher fre-quencies in the Afrikaner population of South Africa and Ashkenazi Jews. Characteristic congenital malformations associated with FA include generalized skin hyperpig-mentation (café au lait spots), microcephaly, hypogonad-ism, 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 myelo-dysplastic syndrome (MDS) and/or acute myeloid leu-kemia (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

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

MORPHOLOGYl Bone marrow biopsy sections in early stages of the disease

may appear hyper- or normocellular with some megaloblas-tic 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.

Box 7.1 Classification of Bone Marrow Aplasia

ConstitutionalFanconi anemiaDyskeratosis congenitaSchwachman–Diamond syndromeDiamond–Blackfan anemiaAmegakaryocytosis

AcquiredIdiopathic aplastic anemiaSecondary 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)

Paroxysmal nocturnal hemoglobinuriaOthersl Hypoplastic myelodysplastic syndromes

l Bone marrow replacement

l Malignant neoplasms

l Fibrosis

l Others

100 BOnE MARROw AplASIA

AA

BB

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.

l Bone marrow smears may show increased mast cells.l There is evidence of hemophagocytosis, particularly in early

stages of the disease.l These bone marrow morphologic features are not pathog-

nomonic for FA and are also observed in patients with acquired AA.

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 STUDIESl DNA testing of several of the FA genes is now available, con-

sisting of either gene sequencing or targeted mutation analy-sis 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 cultur-ing 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 sponta-neous chromatid/chromosome breaks, triradials, quadrira-dials, 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

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 demonstra-tion 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 mor-phologic features similar to those of FA.

Molecular and Cytogenetic Studiesl The dyskeratin gene (DKC1) at chromosome Xq28 is mutated.l Sequence analysis of this gene is available in a small number

of laboratories.

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.

101OTHER COngEnITAl BOnE MARROw AplASIAS

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, pancre-atic insufficiency, and progressive bone marrow hypoplasia and neutropenia (Figure 7.3). Patients are prone to infec-tion, particularly caused by gram-negative organisms, such as Haemophilus influenzae, or Staphylococcus aureus.

Morphology/Laboratory Findingsl Neutropenia is the major hematologic feature of this disorder,

which is often intermittent or cyclic.

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

of the cases.

Molecular and Cytogenetic Studiesl 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 second-ary 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 child-hood. 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 Findingsl Marked bone marrow erythroid hypoplasia (Figure 7.5)l Macrocytic anemia and elevated fetal hemoglobin levelsl Increased erythrocyte adenosine deaminase activity.l DBA patients may eventually develop pancytopenia and aplas-

tic bone marrow.

AA

BB

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

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.

102 BOnE MARROw AplASIA

Molecular and Cytogenetic Studiesl 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 cur-rently are found in only 50% of the patients.

l RPS19, the first one discovered, is the most frequently involved, with mutations found in approximately 25% of patients.

l 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.

l 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.

l 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.

AMEGAKARYOCYTOSIS

Congenital amegakaryocytosis (amegakaryocytic throm-bocytopenia) 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 muta-tions of the thrombopoietin receptor gene, MPL, on chro-mosome 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 FINDINGSl 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.

AA

BB

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.

AA

BB

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

103ACquIRED AplASTIC AnEMIA

Molecular and Cytogenetic Studiesl The autosomal recessive form of amegakaryocytosis is caused

by mutations in the MPL gene.l Complete sequencing of the gene is available in select refer-

ence laboratories.

Acquired Aplastic Anemia

Acquired AA is characterized by severe bone marrow hypo-cellularity and pancytopenia (anemia, leukopenia, and thrombocytopenia). The term “acquired” refers to non-congenital causative mechanisms which could be immu-nologic, 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 infec-tions 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 dis-eases. 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 cat-egories: (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 age-dependent, 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) transplanta-tion. 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 FINDINGSl 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 ran-domly 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.

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.

AA

BB

CC

104 BOnE MARROw AplASIA

l Bone marrow smears consist predominantly of adipo-cytes and stromal tissue with scattered hematopoietic cells. Occasionally, some of the aspirated smears may show cellu-lar marrow particles, giving the wrong impression of a nor-mocellular 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, mac-rophages, and mast cells. These cells either appear as well-defined 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.

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.

l Sometimes these patients are evaluated for associated leu-kemia or MDS, such as by clonal gene rearrangement analy-sis, 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 7.8 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.

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

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

105pAROxySMAl nOCTuRnAl HEMOglOBInuRIA

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 character-ized by hemolytic anemia (often with hemoglobinuria), venous thrombosis, and bone marrow failure:

1. Acquired intravascular hemolysis demonstrated by hemo-globinemia, 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 cere-bral 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 sup-plementation, red blood cell transfusion, plus use of pred-nisone 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 monoclo-nal antibody against complement C5. This reagent is effec-tive in controlling hemolysis, and results in improvement in quality-of-life measures.

MORPHOLOGY AND LABORATORY FINDINGSl 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 prepon-derance. Stainable iron is usually absent, primarily due to loss of iron secondary to hemoglobinuria and hemosiderinuria.

l Blood examination commonly reveals severe anemia with ele-vated 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

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 activa-tion 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 inhibi-tor of reactive lysis (MIRL) and inhibits complement mem-brane 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 Comments

Complement Regulatory Molecules

DAF CD55 Decay accelerating factor

MIRl CD59 Membrane inhibitor of reactive lysis

Enzymes

Ecto-5′-nucleotidase CD73 lymphocytes

ADp-ribosyl transferase CD157 T cells and neutrophils

Adhesion Molecules

Blast-1 CD48 leukocytes; binds CD24

lFA-3 CD58 All hematopoietic cells

Adhesion molecule 1 CD66a granulocytes, epithelium

nCA-95 CD66b granulocytes

nCA-50/90 CD66c granulocytes, epithelium

Carcinoembryonic antigen CD66e Epithelium

Others

nA1/nA2 CD16 neutrophils and nK cells

Campath-1 CD52 lymphocytes and monocytes

BA-1 CD24 B cells and granulocytes

Thy-1 CD90 Stem cell subset, T cell subset

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

106 BOnE MARROw AplASIA

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.

FLOW CYTOMETRY

Detection of GPI-anchor-deficient cells by flow cytome-try is the established method of choice for diagnosis and monitoring of PNH. The International Clinical Cytometry Society (ICCS) has recently published consensus guide-lines 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 clini-cal 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 accept-able 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 monitor-ing classical PNH in hemolytic and thrombotic patients, since the size of PNH clones in those patients is usually greater than 10%.

l 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 underesti-mated 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 mono-

cytes. Lymphocytes are not suitable targets.

FIGURE 7.11 paroxySmal nocturnal hemogloBinuria (pnh). Bone marrow biopsy section from a patient with pnH, demonstrating marked hypocellularity.

Box 7.2 Clinical Indications for PNH Testing1

Intravascular hemolysis as evidenced by hemoglobinuria or ele-vated plasma hemoglobin.

Evidence of unexplained hemolysis with accompanying:

l Iron-deficiency, or

l Abdominal pain or esophageal spasm, or

l Thrombosis, or

l granulocytopenia and/or thrombocytopenia.

Other acquired Coombs-negative, non-schistocytic, non-infectious hemolytic anemia.

Thrombosis with unusual features:

l 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.

Evidence of bone marrow failure:

l Suspected or proven aplastic or hypoplastic anemia

l MDS/Refractory cytopenia with unilineage dysplasia

l Other cytopenias of unknown etiology after adequate work-up

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

107pAROxySMAl nOCTuRnAl HEMOglOBInuRIA

l FLAER (fluorescent aerolysin) is considered the single most useful reagent in detecting WBC PNH clones. It binds spe-cifically to the GPI anchor, and is reliably absent from GPI-anchor-deficient neutrophils and monocytes.

l It is recommended that at least two GPI-anchored markers including FLAER are used to assess PNH clones, and addi-tional antibodies like CD45, CD33, and CD15 are helpful in gating the target populations.

High-Sensitivity Assays (Figure 7.14)l High-sensitivity assays are not required in diagnosis of clas-

sical PNH. Instead, they are useful in identifying small PNH clones that are commonly associated with bone marrow fail-ure 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 CYTOGENETICSl 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 hemato-poietic cells that are glycosyl phosphatidylinositol-anchored protein (GPI-AP)-positive. Various chromosomal aberrations have been reported in up to 24% of patients with PNH, includ-ing monosomy 5, trisomy 6, trisomy 8, and monosomy 7.

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 7.13 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.

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.

108 BOnE MARROw AplASIA

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 impera-tive for accurate diagnosis. It is important to remem-ber that a proportion of patients with constitutional or acquired AA may eventually develop MDS or AML.

Table 7.2

Differential Diagnoses in Bone Marrow Aplasia

Disorder Bone Marrow Morphology Immunophenotype Cytogenetics and Molecular

Constitutional Aplasias normo- to hypercellular at early stages, hypocellular marrow at later stages

non-contributory Frequent chromosomal breakage, sometimes −7, mutations in causative genes

Acquired AA Hypocellular marrow Often increased cytotoxic T cells, strong association with HlA-DR2

Sometimes 5q−, −7, +6, +8, viral pCR

pnH Hypocellular marrow loss of gpI-linked proteins, such as CD55 and CD59

Mutations in pig-A gene

Hypocellular MDS Hypocelluar marrow with significant dysplastic changes, and sometimes increased blasts

Abnormal phenotypic patterns, sometimes increased CD34+ and/or CD117+ cells

−7, +8, 5q−, 20q−, and other chromosomal aberrations

Hypoplastic AMl Hypocellular marrow with ≥20% blasts

Increased CD45dim+ cells expressing myeloid markers, often CD34 and/or CD117

Frequent chromosomal aberrations involving 11q, 16q, or t(15;17), t(8;11), t(9;22), and others

Hypocellular hairy cell leukemia

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

TRAp+, CD103+, CD25+, CD22+, CD11c+

not known

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

109ADDITIOnAl RESOuRCES

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