Applied Cytology HEMATOLOGY

Peer Reviewed Case 1: Lameness & Pyrexia

What Your Hematology Analyzer Can’t Tell You

Heather L. Wamsley, DVM, PhD, Diplomate ACVP (Clinical Pathology) A 10-month-old, female bloodhound presented for acute onset University of Florida shifting leg lameness and pyrexia (104° F/40° C). Table 1 (page 32) provides blood analysis results; Figures 1 through 3 show the blood films. ollection of a peripheral blood sample allows a practi - tioner the opportunity to perform a complete blood Ccount (CBC), which includes automated analysis of ASK YOURSELF… the blood and microscopic evaluation of the peripheral G Is the patient truly thrombocytopenic as blood smear. suggested by the automated CBC? G Is the cause of clinical signs evident in The peripheral blood smear (blood film) not only confirms the blood film? and refines numeric data reported by the hematology ana - lyzer, but also adds useful information when evaluated by a Intragranulocytic, dark trained microscopist: blue, stippled, irregularly G Red blood cell (RBC) quantity (RBC count, hematocrit, 1 shaped morula adjacent to segmented hemoglobin) nucleus; platelets are not G RBC indices (mean cell volume, mean cell hemoglobin visible. ( Wright-Giemsa , concentration, RBC distribution width) 100×) G White blood cell (WBC) count G WBC differential G Platelet count G Platelet indices (mean platelet volume) 2 Each of the following cases highlight different reasons Intragranulocytic, dark blue, stippled, crescent- why it remains important for trained personnel to shaped morula adjacent examine blood films even when an advanced, to the segmented neu - automated hematology analyzer is used to trophil nucleus. ( Wright- Giemsa , 100×) obtain data.

3 Intragranulocytic, dark blue, stippled, round morula surrounded by the segmented neu - trophil nucleus. ( Wright- CBC = ; RBC = red blood cell; WBC = white Giemsa , 100×) blood cell

30 ...... NAVC Clinician’s Brief / January 2011 / Applied Cytology Case 2: Acute Onset of Congestive Case 3: Prior History of Leukemia & Heart Failure Chemotherapy

A 3.5-year-old, castrated male French bulldog was diagnosed An 8-year-old, castrated male mixed-breed dog with a prior with acute onset congestive heart failure. Table 1 provides history of leukemia and chemotherapy was presented for a fol - blood analysis results; Figures 4 and 5 show the blood films. low-up CBC. Table 1 provides blood analysis results; Figures 6 and 7 show the blood films.

ASK YOURSELF… G Are the erythrocyte and neutrophil ASK YOURSELF… morphologies abnormal? G Although the total leukocyte count is G Are the automated leukocyte count and normal, is the patient out of remission? leukocyte differential accurate? G Is the automated leukocyte differential G Is the patient truly thrombocytopenic? accurate? G Is the cause of clinical signs suggested G What are the intraerythrocytic inclusions? by the blood films?

In dogs, the predominant 4 Low-power scanning leukocyte is the neu - reveals 2 microfilariae. 6 trophil. However, in this Leukocytes are moder - case, large lymphoblasts ately increased with predominate. Pale stain - numerous band neu - ing, oval nucleoli are trophils present along visible in 2 of the lym - with a lesser number of phoblasts ( lower left segmented , cell, lower right cell ). 2 , and 1 Erythrocyte density is . Erythrocyte moderately reduced and density is moderately numerous intraerythro - decreased consistent with cytic piroplasms are pres - numeric RBC data; 1+ ent. ( Wright-Giemsa , anisocytosis and 1+ poly - 100×) chromasia are also pres - ent. Platelets are mildly decreased, which is con - sistent with thrombocy - topenia. ( Wright-Giemsa , 20×) 7

5 Closer magnification of Figure 4 reveals 2+ toxicity in many Figure 6 magnified to show intraerythrocytic piroplasms consistent band neutrophils; 1 band is 3+ toxic ( far right ). Erythrocyte with Babesia canis . Immunosuppressive chemotherapy likely density is moderately reduced with 1+ anisocytosis, 1+ poly - unmasked a latent B canis infection, which caused parasitemia and chromasia, and a single nucleated RBC ( upper left ). Platelets contributed to this dog’s anemia. ( Wright-Giemsa , 100×) are mildly decreased and there are 2 RBC fragments, including a schistocyte ( left ). ( Wright-Giemsa , 50×) CONTINUES

Applied Cytology / NAVC Clinician’s Brief / January 2011 ...... 31 Applied Cytology CONTINUED

PURPOSE OF BLOOD FILM EVALUATION AS PART OF A Table 1. Laboratory Results FULL CBC (Highlighted values are outside the reference interval) 1. Confirm & refine numeric data reported by hematology analyzer Parameter Case 1 Case 2 Case 3 Reference • RBC quantity: RBC count, hema - Interval tocrit, hemoglobin Manual • RBC indices: MCV, MCHC, RDW Spun packed cell volume (%) 41 23.4 Not done 37–54 • WBC count • WBC differential Total plasma protein (g/dL) 7.3 6.5 Not done 6–7.8 • Platelet count Automated Analyzer • Platelet indices: MPV Calculated hematocrit (%) 38.5 21.4 23.5 37–54 2. Add useful information that only a Red blood cells (10 6/mcL) 5.73 2.89 2.98 5.7–8.2 trained microscopist can provide • RBC morphology: Size, shape, Hemoglobin (g/dL) 14 7.2 7.6 14–20 color, & inclusions Mean cell volume (fL) 67.2 74.1 79 65–75 • WBC morphology: Detect changes consistent with inflam - MCHC (g/dL) 36.3 33.7 32.2 34–37 matory, neoplastic, or hereditary RDW (%) 11.2 15.8 Not available 11–14 diseases Platelets (10 3/mcL) 37 91 25 124–351 • Platelet morphology: Size & inclusions Mean platelet volume (fL) 19.4 17.8 Not available 10–16 • Detect infectious diseases White blood cells (/mcL) 10,280 32,170 11,800 5000–14,500 Neutrophils (/mcL) 5500 27,380 354 3000–11,500 CBC = complete blood count; MCHC = mean cell hemoglobin concentration; MCV = mean cell vol - Lymphocytes (/mcL) 4000 2230 9676 1000–4800 ume; MPV = mean platelet volume; nRBC = Monocytes (/mcL) 410 2000 1770 150–1350 nucleated red blood cell; RBC = red blood cell; RDW = red blood cell distribution width; WBC = (/mcL) 410 120 0 100–1250 white blood cell (/mcL) 0 30 0 100 <

Table 2. Case 2: Hematology Analyzer versus Blood Film Findings

Parameter Before Blood Film Evaluation After Blood Film Evaluation Reference Interval Nucleated RBCs Not available 8 nRBC/100 WBC Platelets (10 3/mcL) 91 Confirmed, 91 124–351 WBCs (/mcL) 32,170 Corrected, 29,780 5000–14,500 Neutrophils (/mcL) 27,380 22,000 3000–11,500 Bands (/mcL) Not available 4800 0–300 Metamyelocytes (/mcL) Not available 300 0 Lymphocytes (/mcL) 2230 1200 1000–4800 Monocytes (/mcL) 2000 1500 150–1350 Eosinophils (/mcL) 120 0 100–1250 Basophils (/mcL) 30 0 < 100 Morphology, parasites Not available 1+ anisocytosis, 1+ polychromasia, few schistocytes, toxicity (most 2+, few 3+), reactive lymphocytes, reactive monocytes, circulating microfilariae

32 ...... NAVC Clinician’s Brief / January 2011 / Applied Cytology Case 1: Lameness & Pyrexia Case 2: Acute Onset of Congestive Heart Failure

DIAGNOSIS: DIAGNOSIS: Doxycycline-responsive tick-borne disease Caval syndrome associated with severe (Ehrlichia ewingii or Anaplasma phagocytophilum ) inflammation and fragmentation hemolysis

Interpretation & Discussion Interpretation & Discussion The blood film not only verified the thrombocytopenia shown Examination of the blood film in this case revealed several by the hematology analyzer, but also revealed numerous moru - significant findings that cannot be detected by hematology lae within neutrophils ( Figure 8 ), which cannot be detected by analyzers ( Table 2 ): analyzers. This important diagnostic finding, combined with G Data from the hematology analyzer was, depending on the the intragranulocytic morulae found in the patient’s synovial cell line, verified, refined, or corrected. fluid ( Figure 9 ), explained the patient’s clinical signs and pro - G The underlying etiology of the patient’s congestive heart vided a diagnosis. On a side note, at the time of this case pres - failure was indicated ( Figure 10 ). entation, only the SNAP 3Dx test (idexx.com) was available, G Abnormalities suggestive of disseminated intravascular which was negative. coagulation were found.

DID YOU ANSWER… Toxic Change. Hematology analyzers are not able to detect G The number of platelets in the blood film toxic change but it can be seen in the blood film ( Figure 11) . is reduced and consistent with the The toxic change occurred from severe inflammation with thrombocytopenia reported by the hema - accelerated neutrophil production by the marrow or the direct tology analyzer. effect of circulating bacterial endotoxins on differentiating G Intragranulocytic morulae are likely the neutrophils. cause of the patient’s acute pyrexia and CONTINUES lameness.

Closer magnification of 10 Figures 4 and 5 reveals a microfilaria sur - rounded by band neu - trophils with 1+ to 2+ Figure 1 magnified to toxic change. 8 show the intragranulo - cytic morula consistent with Ehrlichia ewingii or Anaplasma phago - cytophilum. A reactive with increased cytoplasmic 11 basophilia and punctate vacuola - Synovial fluid with neu - 9 tion is present between two 2+ to trophilic inflammation and 3+ toxic band neutrophils. The band intragranulocytic morula; 2 neutrophil on the left has a U-shaped segmented neutrophils are nucleus; the band neutrophil on the present on a granular right has an S-shaped nucleus and a eosinophilic background con - few small Döhle bodies. The erythro - sistent with mucin from syn - cyte density is moderately decreased; ovial fluid. One neutrophil there is 1+ anisocytosis. There is a (left ) contains a navy blue, single small platelet ( above & right stippled, circular morula. of center ).

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Left Shift. Hematology analyzers do not distinguish mature, This correction will also adjust inaccurately elevated absolute segmented neutrophils from more immature (left-shifted) cells, counts of each leukocyte type in the leukocyte differential. In such as band neutrophils and metamyelocytes ( Figure 11 ). This addition to the nRBCs, the dog also had other findings consis - dog had a significant regenerative left shift, suggesting inflam - tent with a regenerative anemia, including polychromasia and mation of sufficient severity that the marrow must release anisocytosis. immature, slightly less functional neutrophils (ie, bands and metamyelocytes) in order to satisfy the requirements of inflamed Microfilariae. Numerous microfilariae, which cannot be tissues. Evaluation of the peripheral blood film refined the neu - detected by hematology analyzers, were observed on the blood trophil data obtained from the hematology analyzer, allowing film and suggested the cause of the dog’s congestive heart fail - identification of the left shift. ure. Subsequent echocardiography revealed caval syndrome, a condition where a mass of worms in the right heart disrupts Platelets. The hematology analyzer’s platelet count was below normal blood flow and can cause fragmentation hemolysis, the reference interval. The blood film revealed a reduced num - which can, in turn, incite disseminated intravascular coagula - ber of platelets and absence of significant platelet clumping, tion (DIC). The few schistocytes observed in the blood film confirming thrombocytopenia. Falsely decreased platelet counts indicated mild fragmentation hemolysis. Schistocytes found in due to clumping occur so commonly that it is essential to tandem with anemia and confirmed thrombocytopenia also always verify a low automated platelet count by examining a indicated development of DIC. blood film ( Figure 12 ).

Nucleated Red Blood Cells. Nucleated red blood cells (nRBCs) are counted by hematology analyzers as nucleated DID YOU ANSWER… G The erythrocytes show increased poly - cells that are not distinguished from leukocytes, so nRBCs are chromasia and anisocytosis, and there lumped into the total leukocyte count. When there are more are some nRBCs and schistocytes. The than 5 nRBCs per 100 WBCs, a correction formula can be neutrophils are 2+ to 3+ toxic and left- used to adjust the automated leukocyte count down to a more shifted, with many band neutrophils in accurate value: circulation. G nRBCs are counted with other nucleated (Uncorrected WBC count per mcL × 100 / nRBCs per 100 WBCs + 100) cells by hematology analyzers and = Corrected WBC count per mcL included in the total leukocyte count, causing a falsely increased total leuko - cyte count if there are 5 nRBCs/100 WBCs. Band neutrophi>ls and further left- shifted neutrophils are usually counted as mature, segmented neutrophils by 12 hematology analyzers. G The number of platelets in the blood film In this blood film, is reduced, consistent with the thrombo - a few platelet cytopenia reported by the hematology clumps are pres - analyzer. Concurrent thrombocytopenia, ent, but the anemia, and poikilocytosis due to schis - platelets trapped within the clumps tocytes raise concern for possible under - are not counted by lying DIC. the hematology G Circulating microfilariae suggest heart - analyzer. worm infection, which is likely related to this patient’s congestive heart failure.

DIC = disseminated intravascular coagulation; nRBC = nucleated red blood cell; WBC = white blood cell

34 ...... NAVC Clinician’s Brief / January 2011 / Applied Cytology Case 3: Prior History of Leukemia & Chemotherapy

DIAGNOSIS: Acute lymphocytic leukemia (no longer in remis - DID YOU ANSWER… sion) with Babesia canis infection unmasked by G The patient is out of remission since the predominant nucleated cell is the lym - immunosuppressive chemotherapy phoblast. G The automated leukocyte differential is Interpretation & Discussion not accurate since the blast cells were Although the total leukocyte count was within the reference not reported by the analyzer. interval, evaluation of leukocyte morphology in the blood film G The intraerythrocytic inclusions are revealed that abnormal leukocytes, specifically large lym - presumably Babesia canis . phoblasts, were the predominant cell type ( Table 3 ). Once the blasts were included in the manual leukocyte differential as a separate category, the absolute counts of the other leukocyte types decreased.

Erythrocyte morphology was also abnormal. There were numerous intraerythrocytic piroplasms consistent with Babesia canis , which cannot be detected by hematology analyzers ( Fig - ure 13 ). In this case, the blood film contained important addi - tional information that only a trained microscopist could discern during evaluation. 13 Figure 6 magnified to show intraerythro - cytic piroplasms consistent with Babesia canis and a lymphoblast with a pale stain - ing, oval nucleolus at the nuclear margin (right ).

Table 3. Case 3: Hematology Analyzer versus Blood Film Findings

Parameter Before Blood Film Evaluation After Blood Film Evaluation Reference Interval Platelets (10 3/mcL) 25 Confirmed, 25 124–351 Neutrophils (/mcL) 354 110 3000–11,500 Lymphocytes (/mcL) 9676 1180 1000–4800 Monocytes (/mcL) 1770 0 150–1350 Eosinophils (/mcL) 00100–1250 Blasts (/mcL) Not counted separately 10,500 0 Morphology, parasites Not available Large lymphoblasts present, many intraerythrocytic piroplasms consistent with Babesia canis seen

Applied Cytology / NAVC Clinician’s Brief / January 2011 ...... 35