Discrepancy in differential WBC counts in a cat

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Martina Chase
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1 Discrepancy in differential WBC counts in a cat Harold Tvedten Clinical Chemistry Laboratory, University Animal Hospital (UDS), Swedish University of the Agricultural Sciences (SLU), Box 7038, SE Uppsala, Sweden Correspondence: harold.tvedten@slu.se, Phone Fax History A blood sample was sent to our lab because the 5-year-old cat had a severe anemia (Hct 15 %) and severe leukocytosis. It had lost weight, would not eat and was tired. The blood was analyzed with both an Advia 2120 and ProCyte Dx. Questions 1. Why are the automated differential WBC counts so different between the Advia 2120 and ProCyte Dx? 2. Which is correct? 3. How does one identify what is correct cell count? 4. What is a likely cause?

2 Table 1 Advia automated results Figure 1 Advia WBC Graphics

3 Table 2 Procyte automated results Figure 3 Procyte WBC graphical results Figure 3A Patient Flags: WBC abnormal distribution, band neutrophils suspected, nrbc suspected

4 Figure 3B Normal Cat Diagnosis Neither the Advia nor ProCyte had a correct automated differential WBC count! The cat had neither lymphoid leukemia (Advia 55 x 10 9 /L lymphocytes) nor a leukemoid neutrophil response(procyte 53.8 x 10 9 /L neutrophils). The cat had mainly metarubricytes (Figures 4 + 5). Manual NRBC count was 54 x 10 9 /L. To have 905 nrbc/100 WBC and 54 x 10 9 /L is exceptional. The NRBC confused both instruments in different ways. The manual differential WBC count had normal numbers of lymphocytes and almost normal numbers of neutrophils (Table 3).

5 Figure 4 Blood Smear with many metarubricytes. Figure 5 Blood Smear with many metarubricytes.

6 Discussion The Advia 2120 determines two total WBC counts. The peroxidase channel total WBC count (Perox) was x 10 9 /L and the basophil (Baso) total WBC was x 10 9 /L. This great discrepancy should flag for a serious error. The Baso count is the usual default total WBC and the Advia reported the Baso total WBC of x 10 9 /L in this case. The Perox total WBC count excluded a large number of the nrbc. The operator should look at hidden screens to find Advia s peroxidase total WBC result as a quality control check. Ulrika Falkenö presented case 8 in the ESVCP case session in That dog had 340 nrbc/100 WBC and there was a similar discrepancy in the two Advia total WBC counts. The Advia PEROX total WBC count was 7.7 x 10 9 /L and the Baso total WBC was 22.5 x 10 9 /L in Falkenö s case. ProCyte Dx used one channel (optical channel) to determine the total WBC of 65.5 x 10 9 /L. The Sysmex XT2000iV, precursor to the ProCyte Dx, uses both an optical and basophil channel to determine the total WBC count. The Sysmex basophil channel has a similar principle to the Advia basophil channel. But the smaller, in-clinic ProCyte Dx has only the optical total WBC count and not an impedance count. The total nucleated cell counts of the two instruments were similar and included nrbc for a total nucleated cell count (TCC). It was not apparent from the automated results that most cells were not leukocytes and not a true total WBC count. The ProCyte Dx did flag for WBC abnormal distribution, band neutrophils suspected, nrbc suspected. The large total nucleated cell count in this case was mainly metarubricytes and thus a falsely elevated total WBC count (Table 3). Table 3 Manual Differential WBC count

7 Advia relies mainly on the Peroxidase channel to identify leukocyte types by peroxidase staining (y-axis) and size (x-axis: forward scatter).the many NRBC filled the usual separation between lymphocytes and debris/platelets in Advia s Perox cytogram and caused a severe error in the automated differential WBC count. The nrbc lacked peroxidase staining and were similar in size to lymphocytes. This precluded correct identification of lymphocytes and thus a correct differential WBC count. Advia uses both the PEROX and BASO channels for its differential WBC counts. Advia s basophil reagent removes cytoplasm from the cells and thus mainly nuclei are counted in the Baso channel. The dense chromatin pattern of the metarubricyte nuclei has great optical density and metarubricytes (nrbc) are located more to the right on the basophil cytogram. The NRBC were in the area of neutrophils (red dots in the body of the worm ) in the basophil cytogram. The Advia basophil cytogram is used to separate granulocytes (found in the body of the worm shaped clusters) from mononuclear cells (lymphocytes and monocytes: blue dots) which are located in the head of the worm shaped cytogram. The Perox differential WBC count apparently is the default method for the Advia differential count. If the Baso channel was the main criteria,the nrbc would have been classified as neutrophils. The ProCyte Dx uses side fluorescence (SFL: RNA content) on its WBC Y-axis and side scatter (SSC: complexity) on WBC dot plot X-axis to separate leukocytes into separate clusters (Figure 3B). The WBC dot plot should show separation of leukocytes into clearly separate cell clusters in appropriate locations in order to trust the automated differential WBC count. The ProCyte Dx WBC cytogram of the patient cat shows no separation of neutrophils from lymphocytes. The nrbc filled that area and the instrument arbitrarily drew lines through the large cluster of cells to make an automated differential WBC count. Seeing a line through a cluster of cells should flag the operator to suspect an error in the automated WBC differential count. The main purpose of the case is to illustrate how a large number of nrbc can cause severe errors in laboratory results with two excellent hematology instruments. My diagnosis on the mailed in sample was the cat had myelodysplastic syndrome. This was based on a severe non-regenerative anemia with an inappropriate and extreme proliferation of metarubricytes. There were also 7 % blast cells and dysplastic changes in eosinophils, neutrophils and occasional megaloblastic rubicytes (Figures 6 + 7). It was a mailed in sample and there is no other information or final diagnosis available.

8 Figure 6 Blood Smear with atypical, binucleated immature cell. Figure 7 Blood Smear with atypical immature cell and metarubricyte.

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