Blood Cell Identification Graded Case History An 11-year-old girl presented to the emergency room with wheezing and was diagnosed as having asthma. Her CBC showed WBC = 12.5 x 10 9 /L; Hgb = 12.4g/dL; and PLT = 211 x 10 9 /L. /-01 Erythrocyte, normal 17 100.0 1323 95.4 3701 99.1 Good The arrowed cells are normal erythrocytes that were correctly identified by 100.0% of referees and 98.1% of participants. Erythrocytes are mature non-nucleated red cells which are 6.7-7.8 μm in diameter. They have a biconcave disk shape, which appears round to ovoid on smear. This patient is not anemic, has a normal MCV and normal erythrocyte morphology. /-02 Monocyte 17 100.0 1356 97.8 3658 97.9 Good The arrowed monocyte was correctly identified by 100.0% of referees and 97.9% of participants. Monocytes are slightly larger than neutrophils at 12-20 μm, have abundant gray-blue cytoplasm and an indented nucleus with condensed nuclear chromatin. 2
Blood Cell Identification Graded /-03 Eosinophil, any stage 17 100.0 1383 99.7 3726 99.7 Good The arrowed cell is an eosinophil that was correctly identified by 100.0% of referees and 99.7% of participants. Eosinophils have numerous characteristic cytoplasmic coarse, orange-red granules. The majority of eosinophils have a bilobed nucleus which is connected by a thin filament. Occasional trilobed eosinophils can be seen in patients with peripheral eosinophilia. /-04 Basophil, any stage 16 94.1 1330 95.9 3628 97.1 Good Alder anomaly/inclusion 1 5.9 - - - - Unacceptable This arrowed basophil was correctly identified by 94.1% of referees and 96.8% of participants. Basophils are myeloid cells which have moderate to many coarse densely stained granules of varying sizes and shapes. These granules are typically blue-black, but may be purple to red and may overlay the nucleus. Basophils are increased in the peripheral blood in patients with hypersensitivity reactions, chronic renal disease, iron deficiency, inflammation, and myeloproliferative disorders. 3
Blood Cell Identification Graded /-05 Platelet, normal 17 100.0 1382 99.6 3729 99.8 Good The arrowed object is a normal platelet that was correctly identified by 100.0% of referees and 99.8% of participants. Platelets are fragments of megakaryocytic cytoplasm which are single or in small aggregates. The cytoplasm contains fine, purple-red granules which are aggregated at the center or dispersed throughout the cytoplasm. 4
Discussion This 11-year-old young lady with a known history of asthma presents to the Emergency Department with wheezing. Clinical evaluation reveals findings consistent with an exacerbation of asthma. As a part of the evaluation, a CBC was performed. The CBC reveals a mild leukocytosis, with a WBC = 12.5 X 10 9 /L (reference range 4.7-10.30 X 10 9 /L) and 12% eosinophils, for an absolute eosinophil count of 1500/μL (1.5 x10 9 /L). Eosinophilia is defined as a peripheral blood absolute eosinophil count greater than 500/μL (0.5 x10 9 /L). Absolute eosinophilia can be seen in both benign and malignant disorders, with secondary (reactive) eosinophilia occurring most commonly. Causes of reactive eosinophilia include allergic/atopic disorders, drug hypersensitivity, parasitic infections, connective tissue and pulmonary disorders, cutaneous disorders, and in association with hematopoietic and solid tumors. In the United States, allergic/atopic disorders are the number one cause of eosinophilia. Patients with asthma, allergic rhinitis, eosinophilic enteritis and atopic dermatitis may have a mild peripheral eosinophilia which fluctuates with the clinical status of the primary disease process. Worldwide, parasitic infections are the primary cause of eosinophilia due to the high prevalence of ascariasis, schistosomiasis, trichinosis, visceral larva migrans, strongyloidiasis and paragonimiasis infections. Eosinophils are myeloid elements derived from a hybrid eosinophil/basophil precursor cell in the bone marrow. After maturation, eosinophils are released into the peripheral blood and tissues in response to cytokines. While many cytokines influence eosinophil production, interleukin (IL)-5, IL-3 and granulocytemacrophage colony-stimulating factor (GM-CSF) are the primary stimuli for eosinophil production. In addition to increasing the number of eosinophils produced, these cytokines also enhance eosinophil longevity and eosinophil activity. Chemotactic cytokines (chemokines) are responsible for recruiting eosinophils from the bone marrow into the peripheral blood and to solid tissue. These chemokines include eotaxin-1, eotaxin-2 and RANTES (regulated on activation normal T cell expressed and secreted). The main functions of eosinophils are to modulate immediate hypersensitivity reactions and to destroy parasites. When eosinophils are activated, their secondary granules release cationic proteins that contribute to the immunologic response against infections and can contribute to the tissue damage seen in allergic and autoimmune diseases. Some of the cationic proteins include eosinophil cationic protein (ECP), eosinophil peroxidase (EPO), Charcot-Leyden crystal lysophospholipase, major basic protein (MBP) and eosinophilderived neurotoxin (EDN). Secondary eosinophilia associated with a neoplastic disorder is likely due to dysregulated production of cytokines. Classical Hodgkin lymphoma, non-hodgkin lymphoma, Langerhans cell histiocytosis, transitional cell bladder carcinoma, adenocarcinoma of the stomach, colon and uterus and large cell lung carcinoma are some of the malignancies in which eosinophilia may be seen. Primary eosinophilia is a persistent eosinophilia of 1.5 x10 9 /L for greater than 6 months, without evidence of a reactive cause and with signs and symptoms of organ involvement. Primary eosinophilia is subdivided into clonal eosinophilia and idiopathic hypereosinophilic syndrome. If the eosinophils are found to be clonal based on abnormal molecular or cytogenetic studies, the case is further categorized based on criteria outlined in the recently revised World Health Organization (WHO) classification. These disorders include chronic eosinophilic leukemia and myeloid or lymphoid neoplasms associated with eosinophilia that have specific genetic abnormalities (i.e. PDGFRA, PDGFRB, FGFR1). Idiopathic hypereosinophilic syndrome 5
is diagnosed in patients with similar clinical findings but no identifiable cytogenetic or molecular abnormalities and no increase in blasts. The diagnostic evaluation for a new patient presenting with peripheral eosinophilia should include a complete history and physical examination, to include a detailed drug history and travel history. In many patients, such as the one in this case example, the clinical history of asthma is sufficient and no further workup is warranted. In other patients, stool and serologic studies to exclude a parasitic infection, follow up blood counts to determine if the eosinophilia persists, cytogenetic and/or molecular analysis on peripheral blood or bone marrow and possibly a bone marrow examination may be indicated. References: 1. Swerdlow SH, Campo E, Harris N, et al. (Eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC: Lyon. 2008. 2. Foucar, K. Eosinophilia in Practical Diagnosis of Hematologic Disorders, 4 th edition. 2006;203-211. 3. Glassy EF, ed. Color Atlas of Hematology. An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists: 1998. 4. Dinauer, M. The Phagocyte System in Hematology of Infancy and Childhood, 6 th edition. 2003. 5. Rosenberg, H. Eosinophil trafficking in allergy and asthma. J Allergy Clin Immunol. 2007; 119:1303-10. Deborah A. Perry, MD Hematology and Clinical Microscopy Resource Committee 6
Blood Cell Identification Ungraded Case History The patient is a 74-year-old man with a myelodysplastic syndrome (refractory anemia with excess blasts). The CBC showed WBC = 5.2 x 10 9 /L; RBC = 2.63 x 10 12 /L; Hgb = 8.8 g/dl; Hct = 27%; MCV = 101.8 fl; MCH = 33.5 pg; MCHC = 32.9 g/dl; PLT = 39 x 10 9 /L /-06 Neutrophil w/dysplastic nucleus and/or agranular cytoplasm Neutrophil w/pelger Huët nucleus (acquired or congenital) Neutrophil, segmented or band Neutrophil necrobiosis (degenerated neutrophil) 8 57.1 438 32.4 557 15.2 Educational 6 42.9 735 54.3 2798 76.5 Educational - - 26 1.9 259 7.1 Educational - - 134 9.9 21 0.6 Educational The arrowed object is a dysplastic neutrophil, or neutrophil with Pelger-Huët nucleus, and was correctly identified by 100.0% of referees and 90.3% of participants. The two nuclear lobes in this bilobed neutrophil are round and connected by a thin strand of chromatin. These cells are termed Pelger-Huët cells and may be seen in a variety of conditions. In the inherited autosomal dominant form (Pelger-Huët anomaly), virtually all neutrophils will demonstrate bilobed nuclei. Neutrophil function remains normal. The chromatin is mature and denser than in normal neutrophils. Monolobated forms may also be seen in patients with the rare homozygous form of the anomaly. The nuclear anomaly appears to be due to abnormalities in the laminin beta receptor gene which maps to chromosome 1q41-q43. Other conditions in which similar bilobed nuclei can be seen include reactive conditions associated with severe infections, in association with medications such as valproate, or in cases of myelodysplastic syndromes. When these cells are observed under these conditions, they are referred to as pseudo- Pelger-Huët cells or pelgeroid cells. When they are seen in association with myelodysplastic syndromes or other myeloid neoplasms, cytoplasmic hypogranulation may also be seen, as is demonstrated in the arrowed neutrophil in this image. 7
Blood Cell Identification Ungraded /-07 Monocyte 14 100.0 723 53.3 2905 79.4 Educational Neutrophil, metamyelocyte - - 287 21.2 206 5.6 Educational Monocyte, immature (promonocyte, monoblast) Neutrophil, toxic (to include toxic granulation and/or Dohle bodies, and/or toxic vacuolization) - - 100 7.4 287 7.8 Educational - - 84 6.2 41 1.1 Educational The arrowed cell is a mature monocyte that was correctly identified by 100.0% of referees and 72.3% of participants. Adjacent to the monocyte is a band neutrophil. Monocytes are slightly larger than neutrophils and measure 12-20 μm in diameter. The cytoplasm is abundant, gray to gray blue in color, and may contain fine pink azurophilic granules and/or vacuoles, as in the monocyte depicted here. The nucleus is usually indented, folded or band-like as seen in the monocyte in the image. Nuclear chromatin is condensed, but less than that of a neutrophil. Nucleoli are generally absent or indistinct. The edges of the monocyte are smooth but may also demonstrate pseudopod like extensions. Metamyelocytes are approximately10-18 μm in diameter, with condensed nuclear chromatin. The nucleus is indented to less than half of the potential round nucleus. The cytoplasm is amphophilic and contains rare primary and many fine specific granules. While the nucleus of the depicted monocyte demonstrates an indentation less than half of the nucleus, granules are not identified in the cytoplasm, which is not amphophilic; the nuclear chromatin is also not condensed, as seen in the adjacent band neutrophil. A monoblast is a large cell, 15-25 μm in diameter with finely dispersed chromatin and distinct nucleoli. Nucleoli are not apparent in the arrowed monocyte. A promonocyte demonstrates nuclear and cytoplasmic characteristics that are between those of monoblasts and the mature monocyte as described above. Promonocytes are usually larger than mature monocytes and have similar appearing gray blue cytoplasm.cytoplasmic vacuoles are not typically seen in promonocytes, unlike the monocyte in the image which contains cytoplasmic vacuoles. Nucleoli are present in promonocytes and absent in mature monocytes such as the one depicted by the arrow. Monocytes may be seen as normal constituents of peripheral blood and can be increased in chronic infections, inflammatory bowel disease and hematologic malignancies, which can include chronic myelomonocytic leukemia. 8
Blood Cell Identification Ungraded /-08 Platelet, giant (macrothrombocyte) 14 100.0 1327 97.9 3630 99.1 Educational The arrowed object is a giant platelet that was correctly identified by 100.0% of referees and 98.7% of participants. Normal platelets are usually 1-4 μm in diameter, while giant platelets are usually 10-20 μm in diameter. For purposes of proficiency testing, the term giant platelet is used when the platelet is larger than the size of the average red blood cell in the field, assuming that there is a normal MCV. The arrowed platelet in the field appears to be larger than the majority of surrounding erythrocytes. The periphery of the platelet may be rounded, scalloped or stellate. The cytoplasm may lack granules or contain a normal amount of centrally located fine azurophilic granules, as seen in the image. Giant platelets may be seen in a variety of conditions. They may be increased in diseases associated with increased platelet loss or destruction with compensatory platelet production. These can include leukemoid reactions, following severe blood loss, autoimmune thrombocytopenia and inherited conditions, such as May-Hegglin anomaly. Giant platelets may be seen in hematologic neoplasia such as in myeloproliferative neoplasms (for example, essential thrombocythemia) or myelodysplastic syndromes, as in this patient s case. 9
Blood Cell Identification Ungraded /-09 Target cell (codocyte) 13 92.9 1346 99.2 3647 99.5 Educational Neutrophil with dysplastic nucleus and/or agranular cytoplasm 1 7.1 1 0.1 1 0.1 Educational The arrowed target cell was correctly identified by 92.9% of referees and 99.4% of participants. Adjacent to the target cell is a monolobated neutrophil (pseudo-pelger Huët cell). Target cells may be seen in a wide variety of disease states, but can also be an artifact of smear preparation. Target cells are formed as a result of an increased surface to volume ratio with the excess membrane sinking or falling into the relatively less dense interior of the cell and then being pushed up at the cell s central portion forming a bell shape. These cells may be normocytic or slightly macrocytic. Conditions where the membrane to interior volume ratio is increased include: thalassemias, iron deficiency anemia, as well as liver disease. Air drying can also produce target cell formation. The target cell in this case may represent an artifact of drying or possibly indicate the presence of a hemoglobinopathy or liver disease. 10
Blood Cell Identification Ungraded /-10 Echinocyte (burr cell, crenated 14 100.0 1314 96.8 3547 96.8 Educational cell) Acanthocyte (spur cell) - - 30 2.2 95 2.6 Educational The arrowed burr cell, also termed an echinocyte, was correctly identified by 100.0% of referees and 96.8% of participants. These cells are erythrocytes that retain their central pallor and have 10-30 short, blunt projections evenly distributed over their surfaces. Their presence may be the result of slow drying on the glass slide or due to the use of aged blood. They may also be seen in disease states such as uremia or pyruvate kinase deficiency. 11
Discussion Myelodysplastic syndromes (MDS) are clonal hematopoietic stem cell disorders characterized by cytopenias in the peripheral blood and typically hypercellular bone marrows showing ineffective hematopoiesis and dysplasia in one or more of the myeloid cell lines. Blasts are variably increased in the peripheral blood and bone marrow, and chromosomal abnormalities are found in approximately 50% of patients. As the disease progresses, patients develop either bone marrow failure or an acute myeloid leukemia (AML). MDS is a disease of older adults with the median age at diagnosis being 70 years. While primary (de novo) MDS arises spontaneously, MDS can also arise secondary to previous chemotherapy, particularly alkylating agents or topoisomerase inhibitors, radiation therapy, cigarette smoking, or exposure to toxic agents, such as benzene. Refractory anemia with excess blasts (RAEB) is a MDS characterized by the presence of 2-19% blasts in the peripheral blood or 5-19% blasts in the bone marrow. Because of differences in survival and incidence of progression to an acute myeloid leukemia, there are two categories of RAEB recognized. RAEB-1 is defined by 5-9% blasts in the bone marrow or 2-4% blasts in the peripheral blood. RAEB-2 is characterized by 10-19% blasts in the bone marrow or 5-19% blasts in the peripheral blood. The peripheral blood smear in RAEB frequently demonstrates abnormalities in all three cell lines, as seen in this patient. Peripheral blood manifestations of RAEB can include the presence of anisocytosis and poikilocytosis, macrocytosis, as well as circulating nucleated red blood cells with abnormal nuclear lobation, such as budding or multinuclearity. Neutrophils can also manifest dysplastic changes including cytoplasmic hypogranulation, pseudo-chediak Higashi granules, abnormal nuclear hypolobation of neutrophils, as with the pseudo-pelger Huët anomaly seen here, hypersegmentation or small size. Giant platelets may also be a manifestation of myelodysplasia and abnormalities of granulation may be identified, such as hypogranulation. Cytogenetic abnormalities in MDS are important in determining prognosis and therapy. Approximately 30-50% of patients with RAEB demonstrate clonal cytogenetic abnormalities, including trisomy 8, -5, del(5q), -7,del(7q) and del(20q). Patients with abnormalities involving chromosome 7 and patients with 3 chromosomal abnormalities typically have a poor prognosis. MDS patients have a propensity to develop AML, particularly in patients with the more advanced subtypes, as in this case of RAEB. Approximately 25% of cases with RAEB-1 and 33% of cases with RAEB-2 will progress to AML. The median survival for RAEB-1 is approximately 16 months and 9 months for RAEB-2. Numerous treatment options are currently available. Therapy can consist of supportive transfusions, hematopoietic growth factor therapy, such as erythropoietin, lenalidomide in patients with deletions of chromosome 5, the hypomethylating agents azacytidine and decitabine and intensive chemotherapy. These therapies may improve cytopenias, reduce transfusion requirements and delay the time to leukemic transformation. Allogeneic stem cell transplantation is curative in 30-50% of patients with MDS. The best results are with transplantation in patients who have 5% blasts in the bone marrow. The patient in this case demonstrates a macrocytic anemia with thrombocytopenia. The presence of giant platelets and pseudo-pelger Huët anomaly are consistent with the patient s history of RAEB. Determination of the subtype of RAEB would require a bone marrow examination to evaluate the percentage of blasts present, along with evaluation of the percentage of blasts in the peripheral blood. Cytogenetic studies on the bone marrow could also offer prognostic information. 12
References: 1. Glassy EF, ed. Color Atlas of Hematology. An Illustrated Field Guide Based on Proficiency Testing. Northfield, IL: College of American Pathologists: 1998. 2. Hsi, E. Hematopathology. Elsevier: 2007. 3. Kantarjian H, O Brien S, Cortes J et al. Therapeutic advances in leukemia and myelodysplastic syndrome over the past 40 years. Cancer.2008;113:1933-1952. 4. Sekeres M, Schoonen W, Kantarjian H et al. Characteristics of US patients with myelodysplastic syndromes: results of six cross sectional physician surveys. JNCI. 2008; 100:1542-1551. 5. Swerdlow SH, Campo E, Harris N, et al. (Eds). WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. IARC: Lyon. 2008. Lydia C. Contis, MD Hematology and Clinical Microscopy Resource Committee 13