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The New Hematology Analyzer Sysmex XE-2100

Performance Evaluation of a Novel White Blood Cell Differential Technology


Katharina Ruzicka, MD; Mario Veitl, MD; Renate Thalhammer-Scherrer, MD; Ilse Schwarzinger, MD

Context.The new hematology analyzer Sysmex XE2100 (TOA Medical Electronics, Kobe, Japan) has a novel, combined, white blood cell differential technology and a special reagent system to enumerate nucleated red blood cells. Design.Performance evaluation of both technologies of the Sysmex XE-2100 according to the H20-A protocol of the National Committee for Clinical and Laboratory Standards and comparison of the results with those for the hematology analyzer Sysmex NE-8000 (TOA Medical Electronics). Specimens.Five hundred forty-four blood samples randomly chosen from various inpatient and outpatient departments of the Vienna University hospital. Results.Five-part white blood cell differential counts on the XE-2100 revealed excellent correlation with the he white blood cell differential (WBC-diff) technologies of hematology analyzers can be roughly divided into electric and optical methods.1 In the electric impedance method, cells are classied on the basis of a combination of cell size data from direct-current resistance information and intracellular data from alternating-current capacitance information. The optical method discriminates between cells on the basis of forward- and side-scattered light. To optimize the WBC-diff capacities, these technical methods may be combined with chemical alterations of either blood cells and/or reagents. Flow-cytochemical differential instruments classify cells using a combination of the optical method and enzyme cytochemistry.2 The Abbott Cell Dyn 4000 combines the optical method with a special reagent that allows for discrimination of nucleated red blood cells (NRBCs) from WBCs by staining NRBC nuclei with a specic uorochrome dye.3 The Sysmex hematology analyzer (SE-9000) combines the electric impedance method with special reagents that disrupt the mature WBCs but x immature WBCs, which are then identied in a special immature cell (IMI) channel.4 The new hematology analyzer Sysmex XE-2100 performs the WBC-diff count by combining 3 kinds of optical information (forward-scattered light, side-scattered light, and side uorescence) with the preestablished impedAccepted for publication September 25, 2000. From the Department of Laboratory Medicine, University of Vienna, Vienna, Austria. Reprints: Ilse Schwarzinger, MD, Department of Laboratory Medicine, University of Vienna, Wahringer Gurtel 18-20, A-1090, Vienna, Austria (e-mail: ilse.schwarzinger@univie.ac.at). Arch Pathol Lab MedVol 125, March 2001

manual reference method for neutrophils, lymphocytes, and eosinophils (r .925, .922, and .877, respectively) and .756 good correlation for monocytes and basophils (r and .763, respectively). The efciency rates of agging for the presence of 1% abnormal white blood cells were 83% (XE-2100) and 66% (NE-8000). The correlation of automated and microscopic nucleated red blood cell counts was excellent (r .97). Conclusions.From the present evaluation and our former experience with other types of Sysmex analyzers, we conclude that the new white blood cell differential technology of the XE-2100 represents a further development toward more efcient agging of abnormal white blood cells. (Arch Pathol Lab Med. 2001;125:391396) ance/IMI method.5 Furthermore, the XE-2100 has a special channel to enumerate NRBCs. The present evaluation focuses on the agging efciency of this new combined technology for abnormal WBCs and on the accuracy of the analyzers NRBC enumeration. The results are presented in comparison with those from our current routine hematology analyzer, the Sysmex NE-8000. MATERIALS AND METHODS Hematology Analyzers
Sysmex XE-2100. The Sysmex XE-2100 (TOA Medical Electronics, Kobe, Japan) has a throughput of 150 samples per hour and provides 32 parameters, including reticulocyte and NRBC counts.5 Measurement of WBCs is performed by ow cytometry using a semiconductor laser to detect forward- and side-scattered light information. Red cell lysis is performed by a reagent that selectively suppresses the degranulation of basophils, resulting in their separation from other forms of WBCs (Figure 1, A). In the DIFF channel, WBCs are permeabilized to enable staining of their DNA and RNA with a uorescence dye. Cells are then categorized according to their side-scattered light and uorescence intensity characteristics. A 4-part WBC-diff is created from the WBC populations: lymphocytes, monocytes, eosinophils, and neutrophils plus basophils (Figure 1, B). In the IMI channel, a special reagent acts on the lipid pattern of the cell membrane to selectively protect immature WBC against disruption, whereas mature leukocytes are disrupted (Figure 1, C).4 After this reaction, cells are categorized by direct-current resistance and alternating-current capacitance information. The presence of abnormal WBCs is indicated by the suspect messages: Blasts?, Immature Gran?, Left Shift?, Atypical Lympho?, and Abn Lympho/ L Blasts?. The suspect messages are generated by combining pattern abnormalities in the 4-DIFF and IMI scattergrams; the
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areas of abnormal WBC locations in the respective scattergrams are shown in Figure 2, A and B. The XE-2100 quanties NRBCs by using a reagent that, after red cell lysis, simultaneously denucleates, shrinks, and slightly stains the nuclei of NRBCs. The reagent does not alter the shape of WBCs but stains their intracytoplasmic organelles and nuclei. The difference in size and staining intensity allows discrimination of NRBCs from WBCs and enumeration of NRBCs (Figure 3). The NRBCs are indicated as absolute numbers per 100 WBCs. The WBC count is automatically adapted according to the results of the NRBC channel, by subtracting the NRBC count from the WBC count measured in the WBC/BASO channel. The software version used in the present evaluation was version 12. Sysmex NE-8000. On the Sysmex NE-8000 (TOA Medical Electronics), the traditional direct-current technique is used for cell sizing, whereas information on the nuclear size and density are gained by radiofrequency detection. Complex algorithms are used to determine the optimum discriminator placement for separation of each cell population.6 The software version used for the present study was version 13. The NE-8000 creates the suspect WBC messages Blasts?, Left Shift?, Immature Gran?, and Atypical Lymph? The areas used to dene cells of the Left Shift and Immature Gran categories are separated in the 4-DIFF and IMI scattergrams of the XE-2100, whereas on analyzers of the NE-series, the Left Shift area is integrated in the Immature Gran area. Thus, the XE2100 might create both ags concomitantly, whereas the NE-8000 suppresses the Left Shift ag in the presence of the Immature Gran ag. The WBC suspect ag Abn Lympho/L Blasts? is only created by the XE-2100 and assists in the detection of lymphoid blasts.

Table 1. Regression Analyses for Comparison of White Blood Cell Differential Counts
Cell Type Comparisons*

Slope

Intercept

XE-2100 NE-8000 Lymphocytes XE-2100 NE-8000 Monocytes XE-2100 NE-8000 Eosinophils XE-2100 NE-8000 Basophils XE-2100 NE-8000 * Comparisons were made with reference method.

Neutrophils

.925 0.853 .907 0.826 .922 0.838 .916 0.864 .756 0.899 .470 0.414 .877 1.063 .857 1.173 .763 0.616 .626 0.489 counts determined by

8.114 12.10 4.440 4.241 2.574 2.813 0.019 0.068 0.039 0.149 the manual

tained any of the suspect WBC ags described previously. To account for the different strategies used by the analyzers to create a Left Shift ag, this ag was not analyzed separately, but was combined with the Immature Gran ag under the heading Myeloid Precursors. After the suspect ags were compared with the results of the microscopic reference method, they were classied as true positive (TP), true negative (TN), false positive (FP), and false negative (FN).

Statistics
Correlations of the 5 WBC-diff parameters were estimated by regression analyses. The predictive value of instrument agging for the presence of abnormal WBC after clinical review was expressed by the following parameters8: Sensitivity (%) Specificity (%) Efficiency (%) [TP/TP [TN/TN FN] FP] 100 100

Specimen Collection
Whole blood was collected in Vacutainer K3-EDTA tubes (Becton Dickinson, Mountain View, Calif) and analyzed within 4 hours after collection. Samples for evaluation were randomly chosen from various inpatient and outpatient departments of the Vienna University hospital, and included specimens from patients with normal hematologic proles, specimens from patients with reactive hematologic abnormalities, and specimens from patients with known hematologic disorders.

Percentage of Subjects Correctly Classified [TP TN/TP FP FN TN ] 100

Reference Method
Blood lms were prepared by the manual wedge technique and stained according to a modied Wright technique. Reference differential counts were performed in accordance with the National Committee for Clinical Laboratory Standards (NCCLS) H20-A protocol.7 Briey, 2 manual, 200-cell WBC-diff counts were performed from each lm by 2 independent, qualied medical technologists. Films were considered positive for pathologic WBCs if they showed more than 10% band forms and/or 1% metamyelocytes (corresponding to Left Shift), or 1% myelocytes and/or promyelocytes (corresponding to Immature Gran), or 1% blast cells, or 5% atypical lymphocytes. Lymphocytes were classied as atypical if they exhibited either nucleoli or nuclear shape abnormalities, reactive morphology, or plasmacytoid morphology. The NRBCs were counted outside the percentage count of WBCs and were indicated as absolute numbers per 100 WBCs.

RESULTS Correlation of Automated 5-Part WBC-Diff Parameters Correlations of the 5 WBC-diff parameters provided by the automated analyzers with the manual reference counts are shown in Table 1. Only samples with complete, automated, 5-part WBC-diff counts were included for regression analyses. Correlation coefcients for neutrophils, lymphocytes, and eosinophils were slightly better and correlation coefcients for monocytes and basophils were clearly better for the XE-2100. Flagging of Abnormal WBCs Four hundred eighty-six differential counts were compared to assess the sensitivity and specicity of the analyzers suspect WBC ags. The predictive values of instrument agging for detection of 1% pathologic WBCs (ie, either blasts and/or myeloid precursor cells and/or atypical lymphocytes) are shown in Table 2. One hundred forty-nine samples exhibited 1% abnormal WBCs on microscopic examination. The agging efciencies were 83% for the XE-2100 and 66% for the NE-8000. This difference

Automated WBC-Diff Counts


The 5 automated WBC-diff parameters performed by the 2 analyzers were compared with the manual reference counts. Automated WBC-diff counts were considered positive if they con

Figure 1. A, White blood cell/basophil (WBC/BASO) scattergram. B, 4-DIFF scattergram. C, Immature cell (IMI) scattergram. Figure 2. A, Abnormal cell locations on a 4-DIFF scattergram. B, Abnormal cell locations on an immature cell (IMI) scattergram. Figure 3. Nucleated red blood cell scattergram. Arch Pathol Lab MedVol 125, March 2001

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Table 2. Sensitivity, Specicity, and Efciency of Instrument Flagging for Detection of 1% Abnormal WBCs*
WBC Count 4 All Samples 109/L (n 486) (n 72) 410 10 109/L 109/L (n 275) (n 139)

Analyzer

XE-2100 True positive, n True negative, n False positive, n False negative, n Sensitivity, % Specicity, % Efciency, %

113 292 45 36 76 87 83

20 39 2 11 65 95 82 16 27 14 15 52 66 60

44 199 13 19 70 94 88 36 173 39 27 57 82 76

49 54 30 6 89 64 74 54 17 67 1 98 20 51

NE-8000 True positive, n 106 True negative, n 217 False positive, n 120 False negative, n 43 Sensitivity, % 71 Specicity, % 64 Efciency, % 66 * WBC indicates white blood cell.

Table 3. Sensitivity, Specicity, and Efciency of Instrument Flagging for Detection of 1% Blasts and Myeloid Precursor Cells and 5% Atypical Lymphocytes
Analyzer Blasts Myeloid Immature Precursors Gran Atypical Lymph

XE-2100 True positive, n True negative, n False positive, n False negative, n Sensitivity, % Specicity, % Efciency, % NE-8000 True positive, n True negative, n False positive, n False negative, n Sensitivity, % Specicity, % Efciency, %

18 438 25 5 78 95 94 15 411 52 8 65 89 88

104 303 53 26 80 85 84 61 241 115 69 47 68 62

77 327 75 7 92 81 83 27 356 46 57 32 89 79

8 441 23 14 36 95 92 9 433 31 13 41 93 91

the XE-2100, 2 were agged by the Immature Gran ag, and 2 were agged by the Atypical Lymph ag; the fth sample did not produce a suspect WBC ag. All FN samples exhibited WBC counts below 2.5 109/L. The NE8000 gave twice as many FP results as the XE-2100 (11% vs 5%, respectively). Among the 25 samples with an FP result determined by the XE-2100, 13 contained 1% cells of the Immature Gran category, 8 contained singular ( 1%) myeloid precursor cells, 1 exhibited singular ( 1%) blasts, 1 contained 5% atypical lymphocytes, and 2 did not show any pathologic WBC on microscopic examination; 16 samples exhibited WBC counts that exceeded 10 109/L, 5 had normal WBC counts, and 4 were leukocytopenic. All FP samples were additionally agged by the Immature Gran ag. Immature Gran/Left Shift.One hundred thirty of 486 (27%) samples showed 1% myeloid precursor cells on microscopic examination. Eighty-four blood lms contained 1% cells of the Immature Gran category. The efciency rates for detection of myeloid precursor cells were 84% for the XE-2100 and 62% for the NE-8000 (Table 3). The XE-2100 was highly sensitive in detecting cells of the Immature Gran category: the rate of FN results was only 1%, compared with 12% on the NE-8000. Among the 7 samples with an FN result determined by the XE-2100, 4 were leukocytopenic, and 3 exhibited normal WBC counts. The rates of FP results were 16% on the XE-2100 and 9% on the NE-8000. Twelve of 75 FP results of the XE-2100 were truly negative, without any pathologic WBCs on microscopic examination; 37 samples contained singular ( 1%) cells of the Immature Gran category, with or without additional cells of the Left Shift category; 17 samples contained increased numbers of bands and/or metamyelocytes but no cells of the Immature Gran category; 4 samples exhibited blasts; and 5 samples contained atypical lymphocytes. Atypical Lymph. Twenty-two of 486 (5%) samples contained 5% atypical lymphocytes. The rates of FP and FN results were identical for both analyzers (Table 3). Abn Lympho/L Blasts?. The Abn Lympho/L Blasts? ag occurred in 31 of 486 (6%) samples. None of these samples contained blast cells. Thirteen samples contained atypical lymphocytes; 7 of those were also agged by the Atypical Lymph ag. Enumeration of NRBCs NRBC counts were determined for 544 samples, including the 486 samples that were analyzed for agging of abnormal WBC. One hundred six samples contained NRBCs on microscopic examination (range, 0.5428 /100 WBCs). The analyzer gave a positive NRBC result in 91 samples (77 TP and 14 FP). In 453 samples, the XE-2100 counted zero NRBC (424 TN and 29 FN). Among the 29 FN samples, 23 showed 1 NRBCs /100 WBCs, and 6 showed 2 NRBCs /100 WBCs on microscopic examination. The correlation between microscopic NRBC counts and NRBC counts provided by the XE-2100 was excellent (r .97) for 540 samples containing fewer than 100 NRBCs/100 WBCs (Figure 4). There was a trend toward lower NRBC counts on the XE-2100 for samples containing more than 15 NRBCs /100 WBCs. Four samples exhibited 428, 340, 214, and 213 NRBCs/100 WBCs; the respective counts on the XE-2100 were 312, 258, 96, and 60. The analyzer agged all NRBC counts above 100/100
Hematology Analyzer Sysmex XE-2100Ruzicka et al

was mainly due to the higher agging specicity of the XE-2100. Both analyzers produced comparable FN results, but the NE-8000 produced almost threefold the number of FP results produced by the XE-2100. Flagging efciencies were best for samples with normal WBC counts throughout all categories of abnormal WBC ags. On both analyzers, the agging specicity decreased with WBC counts of 10 109/L, an effect that was markedly less pronounced on the XE-2100. Conversely, the agging sensitivity was lowest among samples with WBC counts of less than 4 109/L, but again, this effect was less marked on the XE-2100 (Table 2). Blasts. Twenty-three of 486 (5%) blood lms showed 1% blasts on microscopic examination (Table 3). The rates of FN results were 1% for the XE-2100 and 2% for the NE-8000. Among the 5 samples that were missed by
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Figure 4. Correlation data of NRBC counts (XE-2100 vs manual). The correlation coefcient (r) is .97, the slope of the linear regression is 0.62, and the y-intercept is 0.09.

WBCs, as well as the corresponding WBC and WBC-diff counts. NRBC Counts and Abnormal WBC Flags Among the 91 samples with positive automated NRBC counts, 83 exhibited at least 1 additional abnormal WBC ag. Among the remaining 8 samples without additional abnormal WBC ags, 5 were TP and 3 were FP. Four of the TP samples were from severely anemic patients; the fth sample was obtained from a patient with known hairy cell leukemia. COMMENT The Sysmex XE-2100 is a new hematology analyzer that provides 32 parameters, including reticulocyte and NRBC counts.5 Data on precision, reproducibility, linearity, carry over, and stability have been provided.911 The present study focused on the performance of the novel WBC-diff technology that combines an optical laser/uorescence method with the impedance/IMI method, and on the accuracy of NRBC enumeration. The results were compared with those of standardized, microscopic 400-cell counts and with results obtained from the established hematology analyzer NE-8000. Correlation coefcients for neutrophils, lymphocytes, monocytes, eosinophils, and basophils were consistently better for the XE-2100 than for the NE-8000. The difference was marked for monocyte counts, which have been reported to be less accurate on the NE-8000,12,13 and for basophil counts, which have generally been reported to poorly correlate with manual reference methods.12,14,15 The sensitivity of agging 1% pathologic WBC was 76% on the XE-2100, which is similar to the agging sensitivities that we have previously observed for Sysmex analyzers of the NE, SF, and SE series.13,16 The agging specicity was markedly better for the XE-2100 when compared with the NE-8000 in the present study and also best
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when compared with previously established specicity rates for the SE-9000 and the SF-3000.12,16 Thus, among all hitherto evaluated Sysmex analyzers, we obtained the best agging efciency with the XE-2100. We have previously shown that the agging efciencies of hematology analyzers are associated with total WBC counts.12,16 The agging sensitivities were rather poor in leukocytopenic samples and highest in samples with more than 10 109 WBC/L. Conversely, the agging specicities markedly decreased with rising WBC counts. This trend was also recognizable on the XE-2100. However, for samples containing fewer than 4 109 WBC/L, the sensitivity rate of the XE-2100 was markedly better than the rates observed with the other analyzers.12,16 Thus, the XE2100 seems more suitable for screening for pathologic WBC in leukocytopenic samples. Furthermore, when compared with all other tested analyzers, the XE-2100 exhibited a better agging specicity in samples with leukocytosis.12,16 The agging sensitivity of the XE-2100 for blasts was excellent for samples with normal and elevated WBC counts. However, the presence of 1% blasts was missed in 5 samples with WBC counts of less than 2.5 109/L. This observation further stresses the importance of microscopic clarication of samples with unexplained leukocytopenia.16 Most of the FP samples exhibited elevated WBC counts, with myeloid precursor cells on microscopic examination, most probably representing reactive conditions. Combined evaluation of the Blast ag with the Abn Lympho/L Blasts ag on the XE-2100 did not increase the sensitivity of blast cell agging but resulted in an increase of FP results. The sensitivity of agging of Immature Gran was signicantly higher for the XE-2100 than for the NE-8000 in the present study and also signicantly higher than sensitivities we obtained on other hematology analyzers.12,16 The detection of Immature Gran was least sensitive in samples with low WBC counts. The better sensitivity of the XE-2100 was slightly outweighed by a decreased specicity at a positivity threshold of 1% pathologic cells. However, among the 75 FP samples, only 12 were truly negative, without any pathologic WBC on microscopic examination, whereas the majority of samples contained either few numbers of Immature Gran or pathologic WBCs of other categories. Thus, the Immature Gran ag on the XE-2100 seems to be highly sensitive for the presence of low numbers of myeloid precursor cells. The sensitivity of agging for the presence of 5% atypical lymphocytes was poor on both analyzers. This observation corresponds to our previous experiences with other hematology analyzers and might best be explained by the rather broad denition of an atypical lymphocyte.12,16 To screen for abnormalities of the peripheral blood lymphoid system, it might thus be more effective to check the absolute lymphocyte numbers. The new Abn Lympho/L Blasts ag detected few additional samples with atypical lymphocytes that had not been agged by the Atypical Lymph ag. The XE-2100 is the second hematology analyzer, after the Abbott Cell Dyn 4000 (Abbott Diagnostics Division, Mountain View, Calif), that provides numerical NRBC results.3 In the present evaluation, the correlation of microscopic and automated NRBC counts was excellent for samples with fewer than 100 NRBCs/100 WBCs. The observed trend toward lower NRBC counts in samples with
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more than 15 NRBCs/100 WBCs turned into a marked difference for the 4 samples with NRBC counts of more than 200/100 WBCs. A similar trend has been reported for NRBC counts on the Cell Dyn 4000.3 Thus, the value of automated NRBC enumeration in terms of concomitant correction of the WBC count is limited, since the necessity for correction of WBC counts increases with increasing NRBC counts. The clinical importance of automated NRBC enumeration has to be established. Beyond the neonatal period, the presence of NRBCs in the peripheral blood is highly indicative of a pathologic condition. The automated NRBC method offers a tool to screen for such pathologic samples. However, the additional request for an automated NRBC count on the XE-2100 would double the cost of each complete blood count. Thus, the automated enumeration of NRBCs cannot reasonably be considered for screening as long as the costs overshadow the possible benets of this method. Moreover, in the present study, 83 of the 91 samples with positive automated NRBC counts exhibited at least 1 additional abnormal WBC ag and would therefore have been further investigated anyway. The remaining 5 TP samples without an additional WBC ag exhibited numerical complete blood count abnormalities that would also have prompted further diagnostic workup. Automated NRBC enumeration might be benecial in neonatology, to facilitate the estimation of WBC counts on the one hand and to provide prognostic information on the other hand.1720 Monitoring the course of hemolytic disorders and the treatment of renal anemia with erythropoietin and iron in patients undergoing hemodialysis might be additional elds of application for automated NRBC counts.2123 In summary, our data suggest that when compared with other analyzers of the Sysmex series that we have tested, the new combined WBC-diff technology of the XE-2100 is superior in terms of predicting the presence of abnormal WBCs. We have already noted that, relative to the NE series, the SE and SF series have greater sensitivity for the detection of abnormal WBCs12,16; the XE-2100 represents further improvement in terms of specicity. The performance of automated NRBC counts on the XE-2100 compares very well with that reported for the Cell Dyn 4000.3
The authors wish to thank our laboratory staff for excellent technical assistance.
References
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