EVALUATION OF THE AUTOMATED IMMATURE GRANULOCYTE COUNT PRODUCED BY THE SYSMEX XE-2100 HAEMATOLOGY ANALYSER C. Briggs, D. Grant, J.

Staves and S.J. Machin. Department of Haematology, University College Hospital, Grafton Way, London, U.K.
INTRODUCTION
The XE 2100TM (Sysmex) (Figure 1) is a fully automated cell counter that uses new technology to improve the range of quantitative cell analysis to include an immature granulocyte (IG) count (metamyelocytes, myelocytes and promyelocytes) in addition to the standard five cell differential. The analyser also counts nucleated red blood cells, reticulocytes, the immature reticulcyte fraction and has the ability to measure platelets in two ways, by conventional impedance as well as an optical platelet count using a fluorescent dye. Linearity showed an r2 value of 0.991 The IG count was stable over 72 hours at 4 oC but began to fall after 24 hours at room temperature. (Figure 6) No carryover for the IG count was detectable. Reproducibility on 10 consecutive measurements on a sample with a mean IG count of 0.57 x 109/L gave a CV of 5.99%.

Figure 1 - The XE-2100

MANUAL IG VS XE 2100 IG COUNT
5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 y = 0.7401x + 0.0619 r = 0.8154

XE 2100 IG X 10^9/L

Figure 3 - Location of cells in an abnormal DIFF scattergram

Immature Granulocytes

METHODS
The anticoagulant used was K3EDTA in vacutainers; (BD Haemoguard) and all samples were processed within 4 hours of venesection. 465 samples were used in the study, 100 from apparently healthy normal adults, the rest pathological samples preselected to cover a wide range of haematological abnormalities. The XE-2100 IG count was compared to a manual 400 cell differential count performed The analyser has a maximum throughput of 150 samples per hour and uses 130 l of blood in closed mode and 200 l in open mode. The workstation has the capacity to store data on 10,000 samples, including graphics. The IG count is measured in the DIFF channel. The combination of side scatter, (inner complexity of the cell) forward scatter, (volume) and fluorescent intensity of nucleated cells gives a concise but precise image of each cell detected in the peripheral blood. A well defined physical description of the different leucocyte populations (clusters) is obtained. Abnormal and immature cells, with their larger nuclear volume show higher fluorescence intensity than normal cells, and are easily distinguishable in the DIFF channel. (Figures 2 and 3) The IG flag is derived from the IMI (immature information) channel. The XE-2100 IG flag was compared to the manual observation of the blood film and the sensitivity and specificity of the flag calculated using the Galen and Gambino formula, as recommended by the ICSH. Linearity, reproducibility, carryover and stability of the immature granulocytes were all assessed. according to the NCCLS H20 - A protocol. 2 x 200 cell counts by at least two observers. Immature granulocytes were defined as metamyelocytes, myelocytes and promyelocytes. Blast cells were not included in the comparison, nor were stab cells or band cells, which

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 MANUAL IG X 10^9/L

Figure 4 - Correlation of the manual and XE-2100 IG counts

Figure 5 - Scatterplot from a patient with Myelodysplastic Syndrome. XE-2100 reported 9% IG, the manual count was 12%.
XE 2100 IG % STABLIITY AT ROOM TEMP.

XE 2100 IG % STABLITY AT 4 DEG C

6 5 6 4 5 3 4 2 3 1 2 0 1
XE 2100 XE 2100 IG % IG %

5
XE 2100 IG %

were classified as neutrophils with film comment of left shift as is the standard practise in the U.K.

4 3 2 1 0 O 2 4 8 24 48 72 HRS HRS HRS HRS HRS HRS HRS TIME

O 2 4 8 24 48 72 0 HRS HRS HRS HRS HRS HRS HRS O 2 4 TIME 24 8 48 72 HRS HRS HRS HRS HRS HRS HRS TIME

Figure 6 - IG stability

RESULTS
The XE-2100 gave an IG count on 278 out of 465 samples. The total number of samples showing immature granulocytes by the manual differential was 129 out of 465. The XE-2100 has a lower limit of detection than the manual count and reports immature granulocytes as low as 0/1%. The lowest count seen on the manual film was 0.5%. The number of samples with an IG count of 0.5% or above on the XE-2100 and no immature cells seen on the manual film was 25, (5.3%) range of counts 0.5% - 4.4%. There were 21 (4.5%) samples which showed an IG count on the manual film but not on the XE-2100, range of counts 0.5% - 1.5%. There were no consistent features or diagnosis that would account for this discrepancy. The number of samples showing immature granulocytes on both the XE-2100 and manual differential was 108. The correlation for these samples can be seen in Figure 4. The IG flag was seen on 29% of samples. 19.7% true positives, 9.4% false positives and

CONCLUSIONS
Moderately good correlation was observed between the XE-2100 and manual IG counts considering the low number of cells being analysed and the subjectivity of the manual classification of immature granulocytes, especially at the marked left shift (band cell) or metamyelocyte stage and the limitation of a routine manual 100 cell differential to detect abnormal cells below the 1% level. Nearly all the outliers on the correlation graph are from samples with a high total white cell count, so a relatively small difference in the percentage IG count by the two methods translates into a significant absolute count difference. The performance of the IG flag was similar to that of the SE-9500, 9% false positives, so an IG count is potentially more useful. The introduction of fluorescent flow cytometric analysis allows extended quantitation of additional cell populations and allows the detection of immature granulocytes at an earlier stage of the laboratory process and so potentially improves screening and monitoring of various pathological states,

Figure 2 - Location of Leucocytes in the XE-2100 DIFF channel.

2.2% false negatives.