Dita Gratzinger, MD, PhD Hematogones are maturing B cell precursors with a characteristic immunophenotypic pattern. The earliest forms have a B lymphoblast immunophenotype with low-moderate sidescatter, dim CD45, CD34, TdT, CD10bright, CD19dim, CD38bright (stage 1/early); later forms are usually more prominent and show low sidescatter, dim to moderate CD45, CD10moderate, CD19moderate, CD38bright, with variable CD20 and variable surface light chain expression (stages 2-3/intermediate-late). Stage 3 hematogones can also show coexpression of CD5. Mature B cells show low-moderate sidescatter, bright CD45, CD19, CD20, and surface light chain with CD38 ranging from negative to moderate. In cases of left-shifted hematogone hyperplasia, as in recovering or regenerating marrow, stage 1 hematogones may be more prominent and stage 3 hematogones and mature B cells can be markedly decreased. The immunophenotype of hematogones, particularly stage 1 hematogones, overlaps with that of B lymphoblastic leukemia; however review of the scatter plots with attention to the overall pattern of the stage 1, 2, and 3 hematogones however is highly characteristic and helps distinguish hematogones from B lymphoblastic leukemia. B lymphoblastic leukemia in contrast to hematogones generally has uniform CD19 and CD10 intensity, lacks CD20, and has somewhat higher sidescatter than stage 2-3 hematogones. B lymphoblastic leukemia also frequently shows uniform CD34 and TdT expression and shows immunophenotypic aberrancies such as myeloid antigen expression.
Morphologic features of hematogones reflect their maturation pattern, with slightly larger early hematogones showing and immature chromatin, and admixed more numerous intermediate-late stage hematogones smaller with more mature chromatin similar to mature lymphocytes. Hematogones have a characteristic high N:C ratio and round to oval or egg shape. Morphologic differentiation from blasts can be difficult; clues include the admixed more mature forms, and overall very small size, often smaller than admixed red blood cells. Hematogones are quite difficult to detect morphologically in bone marrow core biopsies unless there is quite pronounced hematogone hyperplasia Late stage hematogones can also be detected in the peripheral blood, most prominently in infants but also in children and adults.
Various clinical circumstances can be associated with increased hematogones, including post-chemotherapy, post-stem cell transplant, immune thrombocytopenic purpura, copper deficiency, viral infections, and solid tumor. By contrast, myelodysplastic syndromes, pediatric aplastic anemia, and GATA2-deficiency syndromes are associated with decreased hematogones. The differential diagnosis of B lymphoblastic leukemia and hematogone hyperplasia can be challenging based on morphologic grounds alone and should include clinicopathologic correlation and immunophenotyping.
Morphologic features of lymphoid blasts versus hematogones:
Very small (
Small to medium, often dimorphic
Absent to moderate, rarely vacuolated or granular
Very high to high, range
Usually very high or high
Immature to mature, range
Flow immunophenotypic features of lymphoid blasts versus hematogones:
Stage I low,
Stages II-III very low
Uniform, low to moderate
Stage I dim,
Stages II-III dim-mod
Uniform, usually dim
Stage I bright, Stages II-III moderate
Uniform, often bright
Stage I dim, Stages II-III moderate
Stages II-III variable
Stage I+ (small subset)
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Case 2: Megaloblastic Anemia
Tracy I. George, MD The diagnosis in this case is megaloblastic anemia, specifically vitamin B12 deficiency. The case shows typical findings of cytopenias, including a macrocytic anemia with very high MCV of 128 fL, and an elevated LDH reflecting increased cellular turnover in the bone marrow due to ineffective hematopoiesis. Peripheral blood smear findings including hypersegmented neutrophil, oval macrocytes, dacrocytes, and nucleated RBC are classic, with an erythroid hyperplasia in the bone marrow characterized by marked megaloblastic change in the erythroid and granulocytic lineages, with the latter displaying giant bands and metamyelocytes. Dyspoiesis, manifested as irregular nuclear contours and ‘blebbing,’ in erythroid precursors is mild.
A megaloblastic anemia is defined as a macrocytic anemia with megaloblastic erythropoiesis. Causes of megaloblastic anemias are either vitamin B12 (cobalamin) related, folate related, or independent of vitamin B12 and folate. Vitamin B12, found in animal food and bacteria, is most commonly deficient due to malabsorption, pernicious anemia, and dietary deficiency. Folate, found in food of animal and vegetable origin, is most commonly deficient due to diet; this may be seen in chronic alcohol abusers, poor, elderly, and developmentally disabled individuals. Megaloblastic anemia that is independent of vitamin B12 and folate is typically due to drug-induced impairment of nucleic acid synthesis, such as purine and pyrimidine analogues and chemotherapeutic agents. Often, a detailed history including information regarding diet, neurologic symptoms, malabsorption, prior surgery, family history, medications, and chemical exposures can help suggest a possible diagnosis.
The diagnosis of megaloblastic anemias includes a complete blood count (CBC), peripheral blood smear examination, and laboratory testing for vitamin B12 and folate. A serum vitamin B12 level is recommended, with a few caveats. Patients with antibodies to intrinsic factor antibodies may show spuriously high measured B12 levels when actually they are B12 deficient with some analyzers; autoantibodies have the potential to bind intrinsic factor reagents in competitive-binding luminescence assays, with this binding blocking the binding site for vitamin B12, preventing the formation of luminescent complexes, which ultimately results in an overestimate of the concentration of serum B12. However, other analyzers do not show such results and appear reliable, even in the presence of intrinsic factor antibodies. Hence, additional testing such as homocysteine and methylmalonic acid (MMA) levels can be useful. To explain homocysteine and MMA levels in B12 deficiency, we must remember that vitamin B12 is required in two reactions in humans as follows:
Methylmalonyl CoA mutase Hence, deficiency of B12 results in increased homocysteine and increased methylmalonic acid, as well as folate deficiency. Whereas, folate deficiency results in increased homocysteine, but not methylmalonic acid. This is summarized in the table below. With respect to folate testing, RBC folate levels reflect folate intake over 3 months and is the preferred test, with serum folate reflecting recent folate intake. Many authors recommend folate testing only in patients at risk for folate deficiency, given the folate supplementation of grains in the US since 1998. In my own practice, while these conditions are rare, I have seen far more B12 deficiency than folate deficiency in the southwestern US, with folate deficiency found only in malnourished chronic alcoholics.
Serum vit B12
↔ or ↑
↔ or ↑
Deficiency in B12 and folate results in impaired DNA synthesis with megaloblastic hematopoiesis resulting in a hypercellular marrow of all 3 cell lines, but especially the erythroid lineage, a decreased myeloid-to-erythroid ratio typically, and megaloblastic change in multiple lineages. This nuclear to cytoplasmic dyssynchrony results in enlarged erythroblasts with enlarged nuclei and sieve-like chromatin, where the nucleus is less mature than the cell’s cytoplasm. Granulocytic cells are also megaloblastic with giant bands and metamyelocytes, but myeloblasts are not significantly increased. Actual dysplasia in the erythroids, such as nuclear budding and binucleate forms, tends to be mild. Storage and sideroblastic iron are often increased.
The differential diagnosis for megaloblastic anemia can be approached in a number of ways. Often, the pathologist is first confronted with an abnormal CBC showing a macrocytic anemia. The differential diagnosis for a macrocytic anemia includes megaloblastic anemia, myelodysplasia, chronic alcohol abuse or liver disease, drugs, hypothyroidism, chronic lung disease, heavy smoking, chronic hemolytic anemia with reticulocytosis, myeloma, aplastic anemia, and physiologic etiologies (e.g. normal neonates, subset of normal pregnant women). In this differential diagnosis, the pathologist is typically examining a blood smear for signs of a possible megaloblastic anemia including oval macrocytes, hypersegmented neutrophils, dacrocytes, fragmented cells, and nucleated RBCs. Left shifted granulocytes are not uncommon. It’s also important to remember that patients that are B12 deficient can be macrocytic without anemia, and lack macrocytosis and anemia, presenting primarily with neurologic symptoms.
A paradoxical inverse correlation between hemoglobin and severity of neurologic symptoms has been well described. Clinical history and laboratory results can help narrow the differential diagnosis, but in some cases a bone marrow may be performed. In these cases, megaloblastic erythropoiesis (in contrast to normal erythropoiesis) can narrow the diagnosis to megaloblastic anemia, myelodysplasia, chronic alcohol or liver disease occasionally, and drug effect. While drugs/medications, alcohol, and liver disease is typically known, distinguishing myelodysplastic syndrome (MDS) from megaloblastic anemia can be more challenging. Indeed, some cases of megaloblastic anemia are so frightening that a diagnosis of acute erythroid leukemia has been contemplated. Lab testing results should readily distinguish the two conditions, but the dysplasia seen in MDS is typically of a wider variety, if present. Hence, neutrophils may be hypogranular, hypolobated, and rarely, hypersegmented, or show abnormal granulation. Significant dyspoiesis can be observed and megakaryocytes can be small and hypolobulated, hyperlobulated, show separated nuclear lobes, and hyperchromatic forms, and blasts may be increased, as well as ring sideroblasts are present. Bone marrows referred in consultation for either MDS or acute erythroid leukemia that ultimately resulted in diagnoses of megaloblastic anemia showed marked megaloblastic change, a striking erythroid hyperplasia, no increased myeloblasts, but showed a normal karyotype that seemed unusual if this were MDS or acute erythroid leukemia. Or, the patient was of a young age without a compelling history to support a hematologic malignancy. In such cases, additional lab testing documented a B12 deficiency.
In summary, megaloblastic anemias must always be excluded when evaluating for MDS. Megaloblastic anemias typically present with a macrocytic anemia including a very high MCV, a hypercellular marrow involving all cell lineages, but with a decreased M:E ratio, and megaloblastic changes in multiple lineages. Serum vitamin B12 level (with RBC folate if clinically suspicious) is recommended, with reflex testing to include serum methylmalonic acid if serum vitamin B12 level is low/normal.
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E Ispir, MA Serdar, T Ozqurtas, et al. Comparison of four automated serum vitamin B12 assays. Clin Chem Lab Med 2015;53:1205-13.
DW Joelson, EW Fiebig, AH Wu. Diminished need for folate measurements among indigent populations in the post folic acid supplementation era. Arch Pathol Lab Med 2007; 131:477-80
SD Merrigan, DT Yang, JA Straseski. Intrinsic factor blocking antibody interference is not detected in five automated cobalamin immunoassays. Am J Clin Pathol 2014;141:701-5
E Scarpa, L Candiotto, R Sartori, et al. Undetected vitamin B12 deficiency due to false normal assay results. Blood Transfus 2013;11:627-9.
J Theisen-Toupal, G Horowitz, A Breu. Low yield of outpatient serum folate testing. Eleven years of experience. JAMA Intern Med 2014;174:1696-7.
JW Vardiman. Hematopathological concepts and controversies in the diagnosis and classification of myelodysplastic syndromes. Hematology Am Soc Hematol Educ Program. 2006:199-204.
SN Wickramasinghe. Diagnosis of megaloblastic anaemias. Blood Rev 2006;20:299-18.
DT Yang, RJ Cook. Spurious elevations of vitamin B12 with pernicious anemia. N Engl J Med 2012;366:1742-3.
Case 3: Fanconi Anemia
David R. Czuchlewski, MD The diagnosis in this case is Fanconi anemia (FA), a congenital syndrome characterized by bone marrow failure, variable congenital physical abnormalities, and increased cancer susceptibility. FA is caused by mutations in any of at least 16 genes that encode proteins responsible for cooperatively sensing and repairing DNA damage.
Patients with FA develop pancytopenia and multilineage bone marrow failure, generally coming to clinical attention within the first decade of life. However, age alone is an unreliable parameter for identification of FA, as some patients may present in adulthood. Congenital physical abnormalities are another possible clue to the presence of FA. The “classic” finding in FA is an absent or hypoplastic thumb and/or radius, thought other common features include short stature, abnormalities of skin pigmentation, microcephaly, microphthalmia, structural renal anomalies, and hypogonadism. However, up to 40% of patients with Fanconi anemia lack such physical findings, and absence of congenital abnormalities does not exclude the diagnosis.
In most cases, the bone marrow from FA patients at presentation is hypocellular, and the differential diagnosis includes the major classes of bone marrow failure. Extrinsic insults from toxins, medications, and infection should be ruled out, as well as hypocellular presentations of hematologic malignancies. If no apparent cause for the aplasia can be ascertained, the differential diagnosis would then chiefly include acquired aplastic anemia (AA) and inherited bone marrow failure such as FA. AA is essentially an immune-mediated attack on bone marrow progenitor cells, leading to markedly decreased cellularity and pancytopenia. Because there are no reliable morphologic clues to differentiate AA from FA, laboratory testing to identify the FA phenotype is critical. This involves culturing peripheral blood T cells in the presence of mitomycin C and/or diepoxybutane, toxic chemicals that induce breaks and crosslinks in the DNA of the cells. The cells are then harvested and stained as if to produce a routine karyotype. In normal patients, the intact DNA repair proteins are capable of repairing the damage induced by these “clastogenic agents” in culture. In contrast, cells from FA patients cannot repair the DNA breaks properly, and the chromosomes show numerous abnormal formations including chromosomal fragments, gaps, radials, and crosslinkages. DNA breakage studies should be performed to exclude FA whenever a diagnosis of AA is considered. If clinical suspicion for FA is high and DNA breakage studies are normal, repeat analysis may be indicated using cultured skin fibroblasts, as rare cases of FA feature subclones of peripheral blood cells that have corrected the causative mutation through subsequent “back mutation” to the wild-type state. Other inherited bone marrow failure syndromes, including dyskeratosis congenita and Shwachman-Diamond syndrome, should also be considered if DNA breakage studies show normal results.
Patients with FA are at significant risk of malignancy, including solid tumors (especially head and neck squamous carcinomas) and myeloid neoplasms (myelodysplastic syndrome or acute myeloid leukemia). Indeed, the cumulative incidence of MDS in FA patients by age 50 is at least 40%. Pathologists are often called upon to assess yearly follow-up bone marrows in FA patients to evaluate for the possibility of progression to MDS/AML. One potential complicating factor in these cases is the existence of cytogenetic clonal abnormalities in the bone marrow of some patients with FA who do not have MDS. These abnormal clones may fluctuate or even disappear over time, and should not be taken as de facto evidence of MDS. However, the presence of certain abnormalities such as amplification of chromosome 3q indicate high risk clones and are helpful markers for imminent disease progression. Dyserythropoiesis is a consistent finding in the bone marrow of all patients with FA, and is a less discriminatory criterion for progression to MDS in this setting. It should also be remembered that rare patients with FA come to initial clinical attention only after they have developed MDS or AML, in which case clinical clues are important in identifying the underlying etiology. Indeed, some experts recommend testing for FA, dyskeratosis congenita, and germline GATA2 mutations in all patients with MDS diagnosed younger than age 50 specifically to identify such cases.
Bone marrow transplant is the only cure for the hematologic complications of FA. If transplant is to be performed in this setting, special considerations must be made. First, all potential sibling donors must be carefully tested for evidence of FA. Because the phenotype of FA varies so dramatically, it is important to formally exclude clinically silent FA in these donors to avoid transplanting cells with the same pathogenic genotype into the affected patient. Second, FA patients require reduced intensity conditioning regimens prior to transplant. Due to their underlying inability to tolerate DNA damage, these patients experience inordinate toxicities with standard preparatory regimens. Other treatment options include androgens, G-CSF, and platelet transfusions to support peripheral counts.
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CASE 4: MonoMac syndrome
Dita Gratzinger, MD, PhD Monocytopenia is frequently seen as component of pancytopenia in patients receiving chemotherapy, or with marrow replacement or failure due to any number of etiologies. Much rarer is the finding of predominant or isolated monocytopenia, with a smaller or no decrease in neutrophils. This may be seen as an acute finding in a variety of clinical settings, such as acute infection/sepsis, corticosteroids or transfusion related acute lung injury (TRALI). In the relatively rare setting of a chronic monocytopenia pathologists are trained to assess for hairy cell leukemia. An important additional consideration is monocytopenia Mycobacterium Avium Complex infection syndrome (MonoMAC syndrome). MonoMAC is a rare heritable disorder with significant clinical implications that should be considered in both adults and children with chronic monocytopenia with or without chronic mild neutropenia and/or other evolving cytopenias.
MonoMac syndrome is a member of the GATA2 deficiency family of autosomal dominant and variably penetrant syndromes which include overlapping features of immunodeficiency with susceptibility to human papilloma virus (HPV) and atypical mycobacteria, bone marrow failure, myelodysplastic syndromes (MDS)/acute myeloid leukemia (AML), lymphedema, sensorineural deafness, and lung disease. GATA2 haploinsufficiency mutations vary from null alleles to regulatory mutations to mutations of the DNA-interacting zinc finger domain, and it is likely that the type of mutation in combination with genetic background contributes to the variable manifestations across kindreds and individuals within kindreds. MonoMac syndrome in particular refers to the combination of monocytopenia, and atypical mycobacterial infections; patients additionally have a deficiency of B and natural killer (NK) lymphocytes and increased risk of MDS/AML, and may also have pulmonary alveolar proteinosis. Other GATA2 deficiency syndromes include familial MDS/AML, Emberger syndrome (primary lymphedema and MDS/AML), and dendritic cell, monocyte, B, and NK cell deficiency.
What features of a diagnostic bone marrow biopsy in a patient with chronic profound monocytopenia should prompt the pathologist to suggest the possibility of MonoMAC syndrome and recommend GATA2 genetic testing? The most important component lies in the clinical history, including history of mycobacterial, HPV, or fungal infections, pulmonary alveolar proteinosis, and/or family members with early onset MDS/AML. Of course, such detailed clinical history is frequently unavailable or is only identified when the clinical presentation and bone marrow findings prompt the pathologist to seek it out. The CBC will frequently reveal more moderate cytopenias of other lineages, including thrombocytopenia, anemia, and/or neutropenia. Large granular lymphocytosis may be present, and this fairly frequent nonspecific finding should not be over-interpreted as large granular lymphocyte leukemia.
Flow cytometry findings in MonoMAC syndrome are quite distinctive, including marked monocytopenia in combination with markedly decreased B and NK cells (and, in the bone marrow, markedly decreased hematogones) but relatively preserved maturing granulocytes and T cells. Of note, there is no decrease in bone marrow histiocytes. The bone marrow findings, while less distinctive, frequently include hypocellularity, mild to moderate marrow fibrosis, and variable dyspoiesis, particularly of megakaryocytes. Cases that have progressed to MDS frequently show monosomy 7, but cytogenetics are otherwise normal.
Barbara Bain, Blood Cells: A Practical Guide, 2006