Cell Identification

VPB

S-02

Participants Identification No. % Evaluation nRBC, norm/ABN morphology 833 85.5 Educational Lymphocyte 118 12.1 Educational Lymphocyte, reactive 19 2.0 Educational The image is that of a nucleated RBC, as correctly identified by 85.5% of participants. The designation of

nucleated RBC represents any stage of normoblast or nucleated red cell precursor found circulating in the blood regardless of the maturational stage. Nucleated RBCs are round with blue-gray to pink cytoplasm (dependent on the stage of maturation and degree of hemoglobinization).They have a centrally placed round nucleus and clumped chromatin. The chromatin imparts a cartwheel appearance and lacks nucleoli. In later stages of maturation, the cell nucleus may appear pyknotic with very dense chromatin that lacks distinguishable structure. The centrally placed nucleus often has a clearly defined nuclear membrane, helping to distinguish it from plasma cells.

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VPB

S-03

Participants Identification No. % Evaluation Monocyte 920 94.5 Educational Monocyte, immature 21 2.2 Educational Neutrophil, metamyelocyte 10 1.0 Educational The image is that of a monocyte, as correctly identified by 94.5% of participants. Monocytes are

leukocytes that are larger than a neutrophil and have abundant gray-blue cytoplasm. They may show cytoplasmic vacuoles and cytoplasmic pseudopods (present in this case). At high power, one can appreciate fine azurophilic granules. The nuclear contour show many indentations and folds. The chromatin is condensed and lacks a nucleolus.

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VPB

S-04

Participants Identification No. % Evaluation Platelet, giant 835 85.8 Educational Platelet, normal 96 9.9 Educational Megakaryocytic cell 15 1.5 Educational Platelet hypogranular 15 1.5 Educational The image is that of a giant platelet (macrothrombocyte), as correctly identified by 85.8% of participants.

Giant platelets are larger than the size of a normal red blood cell. They are distinguished from leukocytes due to their lack of a nucleus. They may be distinguished from red blood cells as their cytoplasm often contains variable numbers of fine azurophilic granules that are usually centrally located with a zone of clear cytoplasm surrounding them. Occasionally the granules fuse to form larger granules. Giant platelets may have a round shape with smooth borders or appear irregular with scalloped edges. Giant platelets usually represent immature platelets that are being released prematurely from the bone marrow.

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VPB

S-05

Participants Identification No. % Evaluation Fragmented red cell 953 97.8 Educational Acanthocyte (spur cell) 17 1.8 Educational The image is that of a fragmented red blood cell, as correctly identified by 97.8% of participants.

Fragmented red blood cells (schistocytes) show irregular cellular shapes and an absence of central pallor. These cells result from mechanical destruction or by mechanical trauma to red blood cells as they undergo rips and tears in the circulation from passing through fibrin or platelet clots. Fragmented red blood cells may have a variety of shapes and include helmet cells, keratocytes (horn cells) and triangulocytes.

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VPB

S-06

Participants Identification No. % Evaluation Neutrophil, myelocyte 762 78.2 Educational Neutrophil promyelocyte 107 11.0 Educational Neutrophil, metamyelocyte 38 3.9 Educational Lymphocyte, reactive 16 1.6 Educational Neutrophil promyelocyte abnormal 16 1.6 Educational The image is that of a myelocyte, as correctly identified by 78.2% of participants. Myelocytes are left

shifted (immature) myeloid cells characterized by moderate nuclear to cytoplasmic ratios, abundant granules in the cytoplasm, typically eccentric round to ovoid nuclear contours, early chromatin clumping, and the absence of a nucleolus. There is a mixture of both azurophilic (liliac) and specific (pale orange/pink) granules in the cytoplasm. Occasionally a perinculear hof is seen which represents the golgi apparatus. Myelocytes are not typically present in the peripheral blood. Their presence can be associated with both reactive and neoplastic conditions including infection or other reactive conditions and chronic myelogenous leukemia.

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Case Discussion This case represents a patient with a microangiopathic hemolytic anemia, more specifically thrombotic thrombocytopenia purpura (TTP). Several features in the clinical history and peripheral blood smear help in recognition of this entity including the clinical presentation of lethargy with laboratory tests confirming a normocytic anemia and marked thrombocytopenia. The peripheral blood smear is also essential in the diagnosis of this entity as the smear demonstrates evidence of intravascular hemolysis with identification of shistocytes or fragmented red cells, small numbers of spherocytes, and thrombocytopenia. Microangiopathic hemolytic anemia (MAHA) encompasses various clinical entities that result in red blood cell destruction in the blood vessels which manifests as increased schistocytes in the peripheral blood smear. These schistocytes are subsequently removed from the circulation leading to an anemia. Depending upon the time frame, the body compensates for the anemia by releasing immature red blood cell precursors from the bone marrow. These immature red blood cells include nucleated red blood cells and polychromatic red blood cells (reticulocytes). Various clinical entities can result in MAHA including thrombotic thrombocytopenia purpura (TTP), hemolytic uremic syndrome (HUS), HELLP (Hemolysis, Elevated Liver enzymes, and Low Platelets) syndrome, and mechanical heart valves. Each of these clinical entities will be briefly discussed below. Thrombotic thrombocytopenia purpura (TTP) is a hematologic disorder due to deficient activity of a plasma enzyme von Willebrand Factor (vWF)-cleaving metalloproteinase (ADAMTS-13). This enzyme is responsible for cleaving vWF multimers into smaller molecules in the blood. vWF circulates in the plasma and is found on platelets where it acts as a natural component of the coagulation cascade. Deficient to absent activity of ADAMTS-13 can be idiopathic, inherited or secondary to various causes such as drugs, cancer, autoimmune disorders, and human immunodeficiency virus infection (HIV). The acquired form of TTP results from an autoantibody against ADAMTS-13 that acts to block the activity of the enzyme. Rarely, TTP results from an inherited deficiency of ADAMTS-13. Due to the decrease in ADAMTS-13 function, abnormally large vWF multimers circulate in the blood and bind platelets thus forming microthrombi in the microcirculation (small vessels or capillaries) that block normal blood flow. Red blood cells are forced through them causing disruption and fragmentation. This gives rise to the important diagnostic features seen in the blood smear of fragmented red blood cells and decreased platelets (which are consumed in the formation of the microthrombi). The fragmented red cells have a shorter than normal lifespan due to destruction in the spleen and vessels, leading to hemolytic anemia. In response to the red cell destruction and platelet utilization, the bone marrow may release immature platelets (including giant platelets) and nucleated red cells into the circulation. Often there is an increase in polychromatophilic red cells or reticulocytes, representing annucleated but immature red cells in response to red cell destruction. TTP presents with a pentad of clinical symptoms including hemolytic anemia, thrombocytopenia, renal failure, neurologic change, and low grade fever. However, not all symptoms may be present and commonly patients may present with only hemolytic anemia, thrombocytopenia, and elevated lactic dehydrogenase (LDH). Coomb’s test will be negative for antibodies to red blood cells. Blood findings include schistocytes along with thrombocytopenia. The diagnosis of TTP relies on the clinical presentation and the above laboratory findings including blood evidence of microangiopathic hemolysis and decreased ADAMTS-13 activity. Early diagnosis is critical as the mortality rate of untreated patients can be as high as 90%. While a special laboratory test for the detection of ADAMTS-13 level is available, treatment should not depend on the results of this test as it is often performed in a reference laboratory, and results may not be immediately available. Patients with TTP require plasma pheresis to remove the abnormally large vWF multimers that are causing clotting, with replacement by compatible fresh frozen plasma replacement to restore normal ADAMS-13 activity.

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MAHA from disrupted RBCs in the microvasculature and can occur in various other clinical settings including disseminated intravascular coagulation (DIC), hemolytic uremic syndrome (HUS), HELLP syndrome, and in patients with mechanical heart valves. The specific cause of the microangiopathic hemolytic anemia relies on additional clinical history and ancillary laboratory testing. A major diagnostic consideration when a diagnosis of TTP is being considered is DIC, which results from systemic fibrin clot formation in the vasculature due to activation of the coagulation cascade from various causes including malignancy, infection, and tissue injury. The fibrin clot formed in DIC consumes platelets and coagulation factors in the blood. Thus, patients with DIC (a consumptive coagulopathy) will have abnormal coagulation studies including prolonged prothrombin time (PT), prolonged activated thromboplastin time (aPTT), decreased fibrinogen levels, and increased D-dimers. These coagulation abnormalities are not observed in TTP since the clots of TTP (a non-consumptive coagulopathy) do not utilize coagulation factors. HUS is another MAHA that can mimic TTP but the clinical presentation of HUS in children with Escherichia coli associated diarrhea sets it clinically apart from TTP which is a disorder of predominately adults with no Escherichia coli association. Clotting in HUS occurs due to activation of clotting due to exposure to a toxin released from the bacteria. HELLP syndrome is also clinically distinct from TTP clinically as HELLP occurs exclusively in females as a complication of pregnancy. The clinical history of heart valve replacement with persistent schistocytes in the blood will suggest mechanical heart valve as the potential culprit for the MAHA.

References 1. Glassy EF. Color Atlas of Hematology: an Illustrated Field Guide Based on Proficiency Testing. CAP. 1998. 2. Hillman RS et al. Hematology in Clinical Practice (5th). McGraw-Hill. 2011. 3. McPherson RA et al. Henry’s Clinical Diagnosis and Management by Laboratory Methods (21st). Saunders

Elsevier. 2007. 4. Kjeldsberg CR and Perkins SL. Practical Diagnosis of Hematologic Disorders (5th). ASCP Press. 2010.

Anna Ka-Yun Wong, MD and Sherrie Perkins, MD, Hematology and Clinical Microscopy Resource Committee

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Cell Identification

VPB

S-08

Participants Identification No. % Evaluation Spherocyte 822 84.4 Educational Erythrocyte, normal 84 8.6 Educational Micro w/central pallor 60 6.2 Educational The cells identified are spherocytes, as identified by 84.4% of the participants. The committee recognizes

that there is a hint of central pallor in these cells which may have led to some laboratories identifying these cells as microcytes instead. It can be difficult to distinguish spherocytes from microcytes on some peripheral blood smears. A spherocyte is formed when a portion of the red blood cell membrane is lost and the cell assumes the shape of a sphere as the result of the decreased cell surface to volume ratio. The increase in cell thickness makes the cell appear more densely stained than the surrounding red blood cells, with loss of central pallor. Spherocytes are commonly seen in hereditary spherocytosis, immune hemolytic anemias, thermal injury and in much fewer numbers in cases of microangiopathic hemolytic anemias.

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VPB

S-09

Participants Identification No. % Evaluation Polychromatophilic RBC 952 97.7 Evaluation Macrocyte oval/round 17 1.8 Evaluation The cells identified are polychromatophilic red cells, as correctly identified by 97.7% of the participants. A

polychromatophilic red cell is a non-nucleated, round or ovoid red cell that represents the final stage of red cell maturation after exiting the bone marrow. The polychromatophilic red cells are larger than a mature erythrocyte and are distinguishable by their more basophilic cytoplasmic coloration. It primarily contains hemoglobin with a small amount of RNA, and thereby stains homogeneously pink-gray or pale purple with Romanowsky stain.

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VPB

S-10

Participants Identification No. % Evaluation Neutrophil, seg/band 658 67.6 Educational Neutrophil, toxic 261 26.8 Educational Neutrophil, w/hypersegmented nucleus 24 2.5 Educational Alder anomaly inclusion 15 1.5 Educational The cells identified are neutrophils, as correctly identified by 67.6% of the participants. These cells show

the typical lobated nucleus containing clumped chromatin with no nucleoli present. The cells contain abundant pink cytoplasm with fine granules consistent with specific or secondary neutrophilic granules. These neutrophils lack toxic myeloid granulation, Döhle bodies and cytoplasmic vacuolization.

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VPB

S-11

Participants Identification No. % Evaluation Platelet, giant 937 96.2 Educational Megakaryocytic cell 19 2.0 Educational The blood component identified is a giant platelet, as correctly identified by 96.2% of the participants.

Giant platelets are identified based on their size (10-20um) which is larger than a normal sized red blood cell. The shape typically varies and the color is either clear or mildly blue-gray, either lacking granules (hypogranular giant platelet) or may contain purple-red granules. Giant platelets are formed from fragments of megakaryocyte cytoplasm. In non-myeloid disorders, large and giant platelets are young platelets and may be seen in disorders with increased platelet loss or destruction. The megakaryocytes which produce these platelets are typically smaller in size with less mature nuclear lobulation.

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VPB

S-12

Participants Identification No. % Evaluation Erythrocyte w/platelet 935 96.0 Educational Platelet, normal 32 3.3 Educational The cell identified is an erythrocyte with an overlying platelet, as correctly identified by 96.0% of the

participants. This is an artifact which is important to differentiate from red blood cell inclusions, abnormal cytoplasmic granules as seen in the lymphocytes of this case, or parasites such as plasmodium. Typically, platelets overlying red blood cells are surrounded by a thin clear zone or halo which is not seen in most red cell inclusions. In our case, the abnormal granules seen in the lymphocyte population are smaller and typically multiple in number.

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Case Discussion

Mucopolysaccharidosis (MPS) is a rare group of inherited lysosomal storage disorders that are caused by the deficiency or absence of specific lysosomal enzymes necessary for the breakdown of complex carbohydrates. Mucopolysaccharidosis include 7 metabolic disorders, known as mucopolysaccharidosis types I-VII. The absence of one of these enzymes causes an accumulation of complex carbohydrates in cellular organelles such as lysosomes. These complex carbohydrates are known as mucopolysaccharides or glycosaminoglycans (GAGs) and serve as the building blocks for connective tissues in the body. Clinical manifestations of mucopolysaccharidosis occur due to impairment of the production of connective tissues in the body, as well as abnormal accumulation of GAGs. Clinical signs and symptoms, such as neurological and mental status developmental disorders, auditory and visual defects, cardiovascular functional impairments and hepatosplenomegaly often occur. Sanfilippo syndrome, also known as mucopolysaccharidosis type III or MPSIII , is a rare genetic disorder identified in 1963 by pediatrician Sylvester Sanfilippo, after whom the disorder was named. MPS III is the most common type of mucopolysaccharidosis and is characterized by the inability to breakdown the GAG known as heparin sulfate. Four different enzymatic deficiencies may cause accumulation of heparin sulfate, thus Sanfilippo syndrome is further subclassified into type IIIA, type IIIB, type IIIC and type IIID. (TABLE 1) All four types of MPS III are transmitted through autosomal recessive genes. Unlike other forms of MPS, symptoms in Sanfilippo syndrome usually appear later, often after the first year of life. A decline in learning ability typically occurs between ages 2 and 6. Delayed development is followed by deteriorating mental status. Other symptoms include behavioral problems, coarse facial features, diarrhea, sleep difficulties, stiff joints that may not extend fully, and walking problems. TABLE 1.

Genetics of MPS-III

MPS-III type Enzyme Deficiency gene location

MPS-III A heparan N-sulfatase 17q25.3

MPS-III B N-acetyl-alpha-D-glucosaminidase 17q21

MPS-III C acetyl-CoA:alpha-glucosaminide acetyltransferase 8p11-q13

MPS-III D N-acetylglucosamine-G-sulfate sulfatase 12q14 The diagnosis may be confirmed by evaluation of the enzyme level within tissue samples, as well as gene sequencing. Other testing modalities include slit lamp eye exam, skin fibroblast culture, and bone X-rays. Current treatment options include enzyme replacement therapy, substrate reduction therapy, gene therapy and stem cell transplantation. The accumulation of heparin sulfate may be manifested by inclusions in a variety of cell types, including leukocytes. Our current case is a 13 year old patient with a history of Sanfilippo syndrome A (MPSIIIA). The patient has a history of delayed speech and gait abnormality starting at 1-1/2 years of age, and currently suffers from developmental delays and behavior problems. Diagnostic testing including urine screening, genetic analysis and skin biopsy were performed and showed a deficiency of the heparin N-sulfatase enzyme consistent with Sanfilippo syndrome A (MPSIIIA).

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MPS cases may have certain morphological features evident on peripheral blood smear review. Lymphocyte abnormalities such as metachromatic inclusions, cytoplasmic vacuoles, and prominent azurophilic granules may be seen. Additional histological findings include prominent azurophilic granules in white blood cells and metachromatic inclusions in neutrophils. Our current case had a moderate normocytic anemia, and leukopenia, probably caused by impairment of bone marrow function. Evaluation of the peripheral blood smear showed lymphocytes with cytoplasmic azurophilic granules surrounded by halos. (Figure 1 A-D) The morphological findings in MPS cases may be subtle and difficult to distinguish from the normal granules seen in large granular lymphocytes. (Figure 1 E,F) Usually a majority of the lymphocytes in mucopolysaccharidosis patients will contain inclusions or granules, and the granules will be seen in both smaller and larger lymphocytes. This helps to distinguish them from large granular lymphocytes.

FIGURE 1. A-D. The lymphocytes seen in the peripheral blood smear contain dark, slightly larger cytoplasmic granules, many of which are surrounded by halos. Many of the lymphocytes have a higher N:C ratio when compared to a typical large granular lymphocyte. E, F. In contrast, large granular lymphocytes seen in the same patient show more abundant cytoplasm and larger size.

References

1. Foucar K. Non-Neoplastic Disorders of Lymphoid Cells. In: Foucar K, Reichard K, Czuchlewski D. Bone Marrow Pathology, 3rd Edition, volume 2. ASCP press, Chicago IL, 2010.

2. Valstar MJ, et al. Mucopolysaccharidosis type IIIA: clinical spectrum and genotype-phenotype correlations. Ann. Neurol. 2010; 68(6):876-87.

3. De Ruijter J. et al. Mucopolysaccharidosis type III (Sanfilippo Syndrome): Emerging treatment strategies. Curr Pharm Biotechnol. 2011; 12(6);923-30.

4. Cowan T, et al. Inborn Metabolic Errors. In: McClatchey K. Clinical Laboratory Medicine, 2nd Edition. Lippincott Williams & Wilkins, Philadelphia PA, 2002.

George Girgis and Kathryn Rizzo DO, PhD Hematology and Clinical Microscopy Resource Committee

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