017 the New Hematology Analyzer Sysmex XE-2100

Arch Pathol Lab Med—Vol 125, March 2001 Hematology Analyzer Sysmex XE-2100—Ruzicka et al 391

The New Hematology Analyzer Sysmex XE-2100Performance 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 XE-2100 (TOA Medical Electronics, Kobe, Japan) has a novel,combined, white blood cell differential technology and aspecial reagent system to enumerate nucleated red bloodcells.

Design.—Performance evaluation of both technologiesof the Sysmex XE-2100 according to the H20-A protocolof the National Committee for Clinical and LaboratoryStandards and comparison of the results with those for thehematology analyzer Sysmex NE-8000 (TOA Medical Elec-tronics).

Specimens.—Five hundred forty-four blood samples ran-domly chosen from various inpatient and outpatient de-partments of the Vienna University hospital.

Results.—Five-part white blood cell differential countson the XE-2100 revealed excellent correlation with the

manual reference method for neutrophils, lymphocytes,and eosinophils (r 5 .925, .922, and .877, respectively) andgood correlation for monocytes and basophils (r 5 .756and .763, respectively). The efficiency rates of flagging forthe presence of $1% abnormal white blood cells were83% (XE-2100) and 66% (NE-8000). The correlation ofautomated and microscopic nucleated red blood cellcounts was excellent (r 5 .97).

Conclusions.—From the present evaluation and our for-mer experience with other types of Sysmex analyzers, weconclude that the new white blood cell differential tech-nology of the XE-2100 represents a further developmenttoward more efficient flagging of abnormal white bloodcells.

(Arch Pathol Lab Med. 2001;125:391–396)

The white blood cell differential (WBC-diff) technologiesof hematology analyzers can be roughly divided into

electric and optical methods.1 In the electric impedancemethod, cells are classified on the basis of a combinationof cell size data from direct-current resistance informationand intracellular data from alternating-current capacitanceinformation. The optical method discriminates betweencells on the basis of forward- and side-scattered light. Tooptimize the WBC-diff capacities, these technical methodsmay be combined with chemical alterations of either bloodcells and/or reagents. Flow-cytochemical differential in-struments classify cells using a combination of the opticalmethod and enzyme cytochemistry.2 The Abbott Cell Dyn4000 combines the optical method with a special reagentthat allows for discrimination of nucleated red blood cells(NRBCs) from WBCs by staining NRBC nuclei with a spe-cific fluorochrome dye.3 The Sysmex hematology analyzer(SE-9000) combines the electric impedance method withspecial reagents that disrupt the mature WBCs but fix im-mature WBCs, which are then identified in a special im-mature cell (IMI) channel.4

The new hematology analyzer Sysmex XE-2100 per-forms the WBC-diff count by combining 3 kinds of opticalinformation (forward-scattered light, side-scattered light,and side fluorescence) with the preestablished imped-

Accepted for publication September 25, 2000.From the Department of Laboratory Medicine, University of Vienna,

Vienna, Austria.Reprints: Ilse Schwarzinger, MD, Department of Laboratory Medi-

cine, University of Vienna, Wahringer Gurtel 18-20, A-1090, Vienna,Austria (e-mail: [email protected]).

ance/IMI method.5 Furthermore, the XE-2100 has a specialchannel to enumerate NRBCs. The present evaluation fo-cuses on the flagging efficiency of this new combined tech-nology for abnormal WBCs and on the accuracy of theanalyzer’s NRBC enumeration. The results are presentedin comparison with those from our current routine he-matology analyzer, the Sysmex NE-8000.

MATERIALS AND METHODS

Hematology Analyzers

Sysmex XE-2100. The Sysmex XE-2100 (TOA Medical Elec-tronics, Kobe, Japan) has a throughput of 150 samples per hourand provides 32 parameters, including reticulocyte and NRBCcounts.5 Measurement of WBCs is performed by flow cytometryusing a semiconductor laser to detect forward- and side-scatteredlight information. Red cell lysis is performed by a reagent thatselectively suppresses the degranulation of basophils, resultingin their separation from other forms of WBCs (Figure 1, A). Inthe DIFF channel, WBCs are permeabilized to enable staining oftheir DNA and RNA with a fluorescence dye. Cells are then cat-egorized according to their side-scattered light and fluorescenceintensity characteristics. A 4-part WBC-diff is created from theWBC populations: lymphocytes, monocytes, eosinophils, andneutrophils plus basophils (Figure 1, B). In the IMI channel, aspecial reagent acts on the lipid pattern of the cell membrane toselectively protect immature WBC against disruption, whereasmature leukocytes are disrupted (Figure 1, C).4 After this reac-tion, cells are categorized by direct-current resistance and alter-nating-current capacitance information. The presence of abnor-mal WBCs is indicated by the suspect messages: Blasts?, Imma-ture Gran?, Left Shift?, Atypical Lympho?, and Abn Lympho/LpBlasts?. The suspect messages are generated by combiningpattern abnormalities in the 4-DIFF and IMI scattergrams; the

392 Arch Pathol Lab Med—Vol 125, March 2001 Hematology Analyzer Sysmex XE-2100—Ruzicka et al


Table 1. Regression Analyses for Comparison ofWhite Blood Cell Differential Counts

Cell Type Comparisons* r Slope Intercept

Neutrophils

Lymphocytes

Monocytes

XE-2100NE-8000XE-2100NE-8000XE-2100NE-8000

.925

.907

.922

.916

.756

.470

0.8530.8260.8380.8640.8990.414

18.114112.1014.44014.24112.57412.813

Eosinophils

Basophils

XE-2100NE-8000XE-2100NE-8000

.877

.857

.763

.626

1.0631.1730.6160.489

10.01910.06810.03910.149

* Comparisons were made with counts determined by the manualreference method.

←

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.

areas of abnormal WBC locations in the respective scattergramsare shown in Figure 2, A and B.

The XE-2100 quantifies NRBCs by using a reagent that, afterred cell lysis, simultaneously denucleates, shrinks, and slightlystains the nuclei of NRBCs. The reagent does not alter the shapeof WBCs but stains their intracytoplasmic organelles and nuclei.The difference in size and staining intensity allows discrimina-tion of NRBCs from WBCs and enumeration of NRBCs (Figure3). The NRBCs are indicated as absolute numbers per 100 WBCs.The WBC count is automatically adapted according to the resultsof the NRBC channel, by subtracting the NRBC count from theWBC count measured in the WBC/BASO channel. The softwareversion used in the present evaluation was version 12.

Sysmex NE-8000. On the Sysmex NE-8000 (TOA MedicalElectronics), the traditional direct-current technique is used forcell sizing, whereas information on the nuclear size and densityare gained by radiofrequency detection. Complex algorithms areused to determine the optimum discriminator placement for sep-aration of each cell population.6 The software version used for thepresent study was version 13. The NE-8000 creates the suspectWBC messages Blasts?, Left Shift?, Immature Gran?, and Atyp-ical Lymph?

The areas used to define cells of the Left Shift and ImmatureGran categories are separated in the 4-DIFF and IMI scattergramsof the XE-2100, whereas on analyzers of the NE-series, the LeftShift area is integrated in the Immature Gran area. Thus, the XE-2100 might create both flags concomitantly, whereas the NE-8000suppresses the Left Shift flag in the presence of the ImmatureGran flag. The WBC suspect flag Abn Lympho/LpBlasts? is onlycreated by the XE-2100 and assists in the detection of lymphoidblasts.

Specimen CollectionWhole blood was collected in Vacutainer K3-EDTA tubes (Bec-

ton Dickinson, Mountain View, Calif) and analyzed within 4hours after collection. Samples for evaluation were randomly cho-sen from various inpatient and outpatient departments of the Vi-enna University hospital, and included specimens from patientswith normal hematologic profiles, specimens from patients withreactive hematologic abnormalities, and specimens from patientswith known hematologic disorders.

Reference MethodBlood films were prepared by the manual wedge technique

and stained according to a modified Wright technique. Referencedifferential counts were performed in accordance with the Na-tional Committee for Clinical Laboratory Standards (NCCLS)H20-A protocol.7 Briefly, 2 manual, 200-cell WBC-diff countswere performed from each film by 2 independent, qualified med-ical technologists. Films were considered positive for pathologicWBCs if they showed more than 10% band forms and/or $1%metamyelocytes (corresponding to Left Shift), or $1% myelo-cytes and/or promyelocytes (corresponding to Immature Gran),or $1% blast cells, or $5% atypical lymphocytes. Lymphocyteswere classified as atypical if they exhibited either nucleoli or nu-clear shape abnormalities, reactive morphology, or plasmacytoidmorphology. The NRBCs were counted outside the percentagecount of WBCs and were indicated as absolute numbers per 100WBCs.

Automated WBC-Diff CountsThe 5 automated WBC-diff parameters performed by the 2 an-

alyzers were compared with the manual reference counts. Au-tomated WBC-diff counts were considered positive if they con-

tained any of the suspect WBC flags described previously. Toaccount for the different strategies used by the analyzers to createa Left Shift flag, this flag was not analyzed separately, but wascombined with the Immature Gran flag under the heading My-eloid Precursors. After the suspect flags were compared with theresults of the microscopic reference method, they were classifiedas true positive (TP), true negative (TN), false positive (FP), andfalse negative (FN).

StatisticsCorrelations of the 5 WBC-diff parameters were estimated by

regression analyses. The predictive value of instrument flaggingfor the presence of abnormal WBC after clinical review was ex-pressed by the following parameters8:

Sensitivity (%) 5 [TP/TP 1 FN] 3 100

Specificity (%) 5 [TN/TN 1 FP] 3 100

Efficiency (%) 5 Percentage of Subjects Correctly Classified

5 [TP 1 TN/TP 1 FP 1 FN 1 TN ] 3 100

RESULTSCorrelation of Automated 5-Part WBC-Diff ParametersCorrelations of the 5 WBC-diff parameters provided by

the automated analyzers with the manual reference countsare shown in Table 1. Only samples with complete, auto-mated, 5-part WBC-diff counts were included for regres-sion analyses. Correlation coefficients for neutrophils,lymphocytes, and eosinophils were slightly better and cor-relation coefficients for monocytes and basophils wereclearly better for the XE-2100.

Flagging of Abnormal WBCsFour hundred eighty-six differential counts were com-

pared to assess the sensitivity and specificity of the ana-lyzers’ suspect WBC flags. The predictive values of instru-ment flagging for detection of $1% pathologic WBCs (ie,either blasts and/or myeloid precursor cells and/or atyp-ical lymphocytes) are shown in Table 2. One hundred for-ty-nine samples exhibited $1% abnormal WBCs on mi-croscopic examination. The flagging efficiencies were 83%for the XE-2100 and 66% for the NE-8000. This difference


Table 2. Sensitivity, Specificity, and Efficiency ofInstrument Flagging for Detection of $1% Abnormal

WBCs*

AnalyzerAll Samples(n 5 486)

WBC Count

,4 3109/L

(n 5 72)

4–10 3109/L

(n 5 275)

.10 3109/L

(n 5 139)

XE-2100True positive, nTrue negative, nFalse positive, nFalse negative, nSensitivity, %Specificity, %Efficiency, %

1132924536768783

20392

11659582

441991319709488

4954306

896474

NE-8000True positive, nTrue negative, nFalse positive, nFalse negative, nSensitivity, %Specificity, %Efficiency, %

10621712043716466

16271415526660

361733927578276

5417671

982051

* WBC indicates white blood cell.

Table 3. Sensitivity, Specificity, and Efficiency ofInstrument Flagging for Detection of $1% Blasts and

Myeloid Precursor Cells and $5% AtypicalLymphocytes

Analyzer BlastsMyeloid

PrecursorsImmature

GranAtypicalLymph

XE-2100True positive, nTrue negative, nFalse positive, nFalse negative, nSensitivity, %Specificity, %Efficiency, %

18438255

789594

1043035326808584

77327757

928183

84412314369592

NE-8000True positive, nTrue negative, nFalse positive, nFalse negative, nSensitivity, %Specificity, %Efficiency, %

15411528

658988

6124111569476862

273564657328979

94333113419391

was mainly due to the higher flagging specificity of theXE-2100. Both analyzers produced comparable FN results,but the NE-8000 produced almost threefold the numberof FP results produced by the XE-2100. Flagging efficien-cies were best for samples with normal WBC countsthroughout all categories of abnormal WBC flags. On bothanalyzers, the flagging specificity decreased with WBCcounts of .10 3 109/L, an effect that was markedly lesspronounced on the XE-2100. Conversely, the flagging sen-sitivity was lowest among samples with WBC counts ofless than 4 3 109/L, but again, this effect was less markedon the XE-2100 (Table 2).

Blasts. Twenty-three of 486 (5%) blood films showed$1% blasts on microscopic examination (Table 3). Therates of FN results were 1% for the XE-2100 and 2% forthe NE-8000. Among the 5 samples that were missed by

the XE-2100, 2 were flagged by the Immature Gran flag,and 2 were flagged by the Atypical Lymph flag; the fifthsample did not produce a suspect WBC flag. All FN sam-ples exhibited WBC counts below 2.5 3 109/L. The NE-8000 gave twice as many FP results as the XE-2100 (11%vs 5%, respectively). Among the 25 samples with an FPresult determined by the XE-2100, 13 contained $1% cellsof the Immature Gran category, 8 contained singular(,1%) myeloid precursor cells, 1 exhibited singular (,1%)blasts, 1 contained $5% atypical lymphocytes, and 2 didnot show any pathologic WBC on microscopic examina-tion; 16 samples exhibited WBC counts that exceeded 103 109/L, 5 had normal WBC counts, and 4 were leuko-cytopenic. All FP samples were additionally flagged bythe Immature Gran flag.

Immature Gran/Left Shift.—One hundred thirty of 486(27%) samples showed $1% myeloid precursor cells onmicroscopic examination. Eighty-four blood films con-tained $1% cells of the Immature Gran category. The ef-ficiency rates for detection of myeloid precursor cells were84% for the XE-2100 and 62% for the NE-8000 (Table 3).The XE-2100 was highly sensitive in detecting cells of theImmature Gran category: the rate of FN results was only1%, compared with 12% on the NE-8000. Among the 7samples with an FN result determined by the XE-2100, 4were 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-2100were truly negative, without any pathologic WBCs on mi-croscopic examination; 37 samples contained singular(,1%) cells of the Immature Gran category, with or with-out additional cells of the Left Shift category; 17 samplescontained increased numbers of bands and/or metamy-elocytes but no cells of the Immature Gran category; 4samples exhibited blasts; and 5 samples contained atypicallymphocytes.

Atypical Lymph. Twenty-two of 486 (5%) samplescontained $5% atypical lymphocytes. The rates of FP andFN results were identical for both analyzers (Table 3).

Abn Lympho/LpBlasts?. The Abn Lympho/LpBlasts?flag occurred in 31 of 486 (6%) samples. None of thesesamples contained blast cells. Thirteen samples containedatypical lymphocytes; 7 of those were also flagged by theAtypical Lymph flag.

Enumeration of NRBCs

NRBC counts were determined for 544 samples, includ-ing the 486 samples that were analyzed for flagging ofabnormal WBC. One hundred six samples containedNRBCs on microscopic examination (range, 0.5–428 /100WBCs). The analyzer gave a positive NRBC result in 91samples (77 TP and 14 FP). In 453 samples, the XE-2100counted zero NRBC (424 TN and 29 FN). Among the 29FN samples, 23 showed #1 NRBCs /100 WBCs, and 6showed 2 NRBCs /100 WBCs on microscopic examina-tion. The correlation between microscopic NRBC countsand NRBC counts provided by the XE-2100 was excellent(r 5 .97) for 540 samples containing fewer than 100NRBCs/100 WBCs (Figure 4). There was a trend towardlower NRBC counts on the XE-2100 for samples contain-ing more than 15 NRBCs /100 WBCs. Four samples ex-hibited 428, 340, 214, and 213 NRBCs/100 WBCs; the re-spective counts on the XE-2100 were 312, 258, 96, and 60.The analyzer flagged all NRBC counts above 100/100


Figure 4. Correlation data of NRBC counts (XE-2100 vs manual). Thecorrelation coefficient (r) is .97, the slope of the linear regression is0.62, and the y-intercept is 10.09.

WBCs, as well as the corresponding WBC and WBC-diffcounts.

NRBC Counts and Abnormal WBC FlagsAmong the 91 samples with positive automated NRBC

counts, 83 exhibited at least 1 additional abnormal WBCflag. Among the remaining 8 samples without additionalabnormal WBC flags, 5 were TP and 3 were FP. Four ofthe TP samples were from severely anemic patients; thefifth sample was obtained from a patient with knownhairy cell leukemia.

COMMENT

The Sysmex XE-2100 is a new hematology analyzer thatprovides 32 parameters, including reticulocyte and NRBCcounts.5 Data on precision, reproducibility, linearity, carryover, and stability have been provided.9–11 The presentstudy focused on the performance of the novel WBC-difftechnology that combines an optical laser/fluorescencemethod with the impedance/IMI method, and on the ac-curacy of NRBC enumeration. The results were comparedwith those of standardized, microscopic 400-cell countsand with results obtained from the established hematol-ogy analyzer NE-8000.

Correlation coefficients for neutrophils, lymphocytes,monocytes, eosinophils, and basophils were consistentlybetter for the XE-2100 than for the NE-8000. The differencewas marked for monocyte counts, which have been re-ported to be less accurate on the NE-8000,12,13 and for ba-sophil counts, which have generally been reported topoorly correlate with manual reference methods.12,14,15

The sensitivity of flagging $1% pathologic WBC was76% on the XE-2100, which is similar to the flagging sen-sitivities that we have previously observed for Sysmex an-alyzers of the NE, SF, and SE series.13,16 The flagging spec-ificity was markedly better for the XE-2100 when com-pared with the NE-8000 in the present study and also best

when compared with previously established specificityrates for the SE-9000 and the SF-3000.12,16 Thus, among allhitherto evaluated Sysmex analyzers, we obtained the bestflagging efficiency with the XE-2100.

We have previously shown that the flagging efficienciesof hematology analyzers are associated with total WBCcounts.12,16 The flagging sensitivities were rather poor inleukocytopenic samples and highest in samples with morethan 10 3 109 WBC/L. Conversely, the flagging specifici-ties markedly decreased with rising WBC counts. Thistrend was also recognizable on the XE-2100. However, forsamples containing fewer than 4 3 109 WBC/L, the sen-sitivity rate of the XE-2100 was markedly better than therates observed with the other analyzers.12,16 Thus, the XE-2100 seems more suitable for screening for pathologicWBC in leukocytopenic samples. Furthermore, when com-pared with all other tested analyzers, the XE-2100 exhib-ited a better flagging specificity in samples with leuko-cytosis.12,16

The flagging sensitivity of the XE-2100 for blasts wasexcellent for samples with normal and elevated WBCcounts. However, the presence of $1% blasts was missedin 5 samples with WBC counts of less than 2.5 3 109/L.This observation further stresses the importance of micro-scopic clarification of samples with unexplained leukocy-topenia.16 Most of the FP samples exhibited elevated WBCcounts, with myeloid precursor cells on microscopic ex-amination, most probably representing reactive condi-tions. Combined evaluation of the Blast flag with the AbnLympho/LpBlasts flag on the XE-2100 did not increase thesensitivity of blast cell flagging but resulted in an increaseof FP results.

The sensitivity of flagging of Immature Gran was sig-nificantly higher for the XE-2100 than for the NE-8000 inthe present study and also significantly higher than sen-sitivities we obtained on other hematology analyzers.12,16

The detection of Immature Gran was least sensitive insamples with low WBC counts. The better sensitivity ofthe XE-2100 was slightly outweighed by a decreased spec-ificity at a positivity threshold of .1% pathologic cells.However, among the 75 FP samples, only 12 were trulynegative, without any pathologic WBC on microscopic ex-amination, whereas the majority of samples contained ei-ther few numbers of Immature Gran or pathologic WBCsof other categories. Thus, the Immature Gran flag on theXE-2100 seems to be highly sensitive for the presence oflow numbers of myeloid precursor cells.

The sensitivity of flagging for the presence of $5%atypical lymphocytes was poor on both analyzers. Thisobservation corresponds to our previous experiences withother hematology analyzers and might best be explainedby the rather broad definition of an atypical lympho-cyte.12,16 To screen for abnormalities of the peripheralblood lymphoid system, it might thus be more effectiveto check the absolute lymphocyte numbers. The new AbnLympho/LpBlasts flag detected few additional sampleswith atypical lymphocytes that had not been flagged bythe Atypical Lymph flag.

The XE-2100 is the second hematology analyzer, afterthe Abbott Cell Dyn 4000 (Abbott Diagnostics Division,Mountain View, Calif), that provides numerical NRBC re-sults.3 In the present evaluation, the correlation of micro-scopic and automated NRBC counts was excellent for sam-ples with fewer than 100 NRBCs/100 WBCs. The ob-served trend toward lower NRBC counts in samples with


more than 15 NRBCs/100 WBCs turned into a markeddifference for the 4 samples with NRBC counts of morethan 200/100 WBCs. A similar trend has been reportedfor NRBC counts on the Cell Dyn 4000.3 Thus, the valueof automated NRBC enumeration in terms of concomitantcorrection of the WBC count is limited, since the necessityfor correction of WBC counts increases with increasingNRBC counts. The clinical importance of automatedNRBC enumeration has to be established. Beyond the neo-natal period, the presence of NRBCs in the peripheralblood is highly indicative of a pathologic condition. Theautomated NRBC method offers a tool to screen for suchpathologic samples. However, the additional request for anautomated NRBC count on the XE-2100 would double thecost of each complete blood count. Thus, the automatedenumeration of NRBCs cannot reasonably be consideredfor screening as long as the costs overshadow the possiblebenefits of this method. Moreover, in the present study, 83of the 91 samples with positive automated NRBC countsexhibited at least 1 additional abnormal WBC flag andwould therefore have been further investigated anyway.The remaining 5 TP samples without an additional WBCflag exhibited numerical complete blood count abnormal-ities that would also have prompted further diagnosticworkup. Automated NRBC enumeration might be benefi-cial in neonatology, to facilitate the estimation of WBCcounts on the one hand and to provide prognostic infor-mation on the other hand.17–20 Monitoring the course ofhemolytic disorders and the treatment of renal anemiawith erythropoietin and iron in patients undergoing he-modialysis might be additional fields of application forautomated NRBC counts.21–23

In summary, our data suggest that when compared withother analyzers of the Sysmex series that we have tested,the new combined WBC-diff technology of the XE-2100 issuperior in terms of predicting the presence of abnormalWBCs. We have already noted that, relative to the NE se-ries, the SE and SF series have greater sensitivity for thedetection of abnormal WBCs12,16; the XE-2100 representsfurther improvement in terms of specificity. The perfor-mance of automated NRBC counts on the XE-2100 com-pares very well with that reported for the Cell Dyn 4000.3

The authors wish to thank our laboratory staff for excellenttechnical assistance.

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017 the New Hematology Analyzer Sysmex XE-2100