497.Full Evaluacion Del Celldyn 4000

Hematopathology / EVALUATION OF THE ABBOTT CELL-DYN 4000 HEMATOLOGY ANALYZER

Evaluation of the Abbott CELL-DYN 4000 HematologyAnalyzer

Ernesto Grimaldi, MD, and Francesco Scopacasa, PhD

Key Words: Abbott CD 4000; Blood cell counter; Automated hematology analyzer; Accuracy; Precision; Interference

A b s t r a c t

A new generation hematology analyzer, AbbottCELL-DYN 4000 (CD 4000), capable of providing 26parameters, including fully automated reticulocyte,nucleated RBC, blast, band, and immature granulocyte,and variant lymphocyte counts, was evaluated by usingthe National Committee for Clinical LaboratoryStandards H20-A protocol and compared with theBayer-Technicon H-2 analyzer, which is used routinelyin our laboratory. A lipid interference experiment and asample aging study also were performed. Linearity,carryover, and precision were within the limitsestablished by the manufacturer, and satisfactoryagreement was found with the H-2 analyzer. Theevaluation of leukocyte differential counts indicated anexcellent correlation with the manual reference methodfor neutrophils and lymphocytes, a good correlation formonocytes and eosinophils, and a poor correlation forbasophils in samples with low counts; for basophilcounts of 2% or higher, we found an improvement of thecorrelation coefficient. In the lipid interferenceexperiment, only hemoglobin determination wasinfluenced significantly on the CD 4000, but by using anew Abbott hemoglobin reagent, the interference waseliminated. The CBC and differential counts were stableand reportable up to at least 24 hours. Intrasampleviability information on leukocytes provided a qualitycheck on each individual specimen.

The Abbott CELL-DYN 4000 (CD 4000) (Abbott Diag-nostics, Abbott Park, IL) is a new generation fully automatedhematology analyzer that uses 4-angle argon laser light scatterand 2-color fluorescence flow cytometry.1 The analyzer providesthe laboratory with up to 26 blood count parameters, includingreticulocytes, immature reticulocyte fraction, nucleated RBCs,blasts, bands, immature granulocytes, and variant lymphocytes;in addition, fluorescence technology2 also provides a newintrasample quality control feature, the WBC viable fraction.3 Aconfidence fraction for blasts, bands, immature granulocytes,and variant lymphocytes also was provided.1

During a 3-month period, we evaluated, on the basis of theH20-A protocol of the National Committee for Clinical Labora-tory Standards,4 the CBC and differential WBC count perfor-mance of the CD 4000 in comparison with a reference fullyautomated hematology analyzer in use in our laboratory, theBayer-Technicon H-2 (Bayer-Technicon, Tarrytown, NY) andwith the reference manual method (differential WBC countonly). We also evaluated the lipid interference on both instru-ments and the sample aging effects on CD 4000 measurements.

Materials and Methods

System DescriptionAll analyses were performed on the Abbott CELL-DYN

4000 using software version R4-12F without individualizedlaboratory settings for the laser scatter channels (Abbott setup).The system incorporates 4 different measurementtechnologies,1 including fluorescence flow cytometry, and usesan argon laser to undertake analyses of reticulocytes andnucleated RBCs in particular. The analyzer fluorometer section

Am J Clin Pathol 2000;113:497-505 497 American Society of Clinical Pathologists

Grimaldi and Scopacasa / EVALUATION OF THE ABBOTT CELL-DYN 4000 HEMATOLOGY ANALYZER

has 2 fluorescence emission laser optics (4 angles of lightscatter). Blasts, immature granulocytes, bands, and variantlymphocytes are detected by a multiparameter, multiweighted,discriminant function; this function generates a flag andreports a confidence fraction from 0.50 to 0.99 (ie, the proba-bility that these cells were classified correctly). After detectionby the discriminant function, cell counts are estimated byusing light scatter. Determination of the hemoglobin concen-tration is based on spectrophotometry. The default systemmeasurements of the RBC and platelet counts are by imped-ance and optical methods, respectively; in addition, a simulta-neous determination of optical RBC and impedance plateletcounts are performed. Discrepancies between the 2 measure-ments provide an alert suggesting the presence of sampleinterferences.

Fluorescence technology also provides a new intrasamplequality control feature, the WBC viable fraction, determinedfrom the ratio of unstained to total WBCs. By combining theWBC viable fraction results with the fluorescence informa-tion, the CD 4000 can flag specimens (nonviable WBCs) forpotential sample deterioration.3

The CD 4000 and H-2 analyzers were calibratedfollowing the manufacturers guidelines and using their owncalibration samples; the instruments were controlled byroutine quality control methods. The low, normal, and highcontrol samples were supplied by the manufacturers.

Blood SamplesThe evaluation of the instrument was performed by

analyzing blood specimens from routine samples afterobtaining patients informed consent.

Specimens were selected to span the full range of concen-trations expected in clinical practice, and at least half thesamples were from patients with blood disorders giving resultsin abnormal (low and high) ranges. In particular, for differen-tial WBC count abnormalities, 10% of these samples hadblasts, 20% immature granulocytes, 10% variant lymphocytes,5% nucleated RBCs, and 5% progranulocytes; 50% had noabnormalities; bands ranged from 0% to 20%. All sampleswere collected in evacuated 3.5-mL tubes containing EDTAK3 and were analyzed within 2 hours after obtaining the bloodsample. For the aging study, the samples were stored at roomtemperature or refrigerated (4 C) and analyzed within 24, 48,and 72 hours.

Carryover AssessmentCarryover assessment was performed according to

method of Broughton5: a sample in the high range was tested 3consecutive times (H1, H2, H3), and a sample in the low rangewas tested 3 consecutive times (L1, L2, L3). The percentageof carryover for each parameter was calculated as follows:[(L1 L3)/(H3 L3)] 100.

LinearityLinearity was evaluated by analyzing serial dilutions of 5

specimens in platelet-free autologous plasma.6 The resultswere evaluated in accordance with the International Councilfor Standardization in Haematology recommendation.5

ImprecisionFor the between- and within-batch imprecision study, 20

samples in triplicate and in 2 batches on the same day wereanalyzed. The instrument was switched off and recalibratedbetween the 2 batches.

The results were expressed as mean, SD, and coefficientof variation percentage.

ComparabilityWe selected 120 blood samples on the basis of the recom-

mended International Council for Standardization in Haema-tology range of values7 and analyzed them side-by-side withthe CD 4000 and H-2 instruments. Calibration was donefollowing the manufacturers guidelines. Measured WBC,RBC, hemoglobin, mean corpuscular volume, platelet, anddifferential WBC count parameters were compared by linearregression analysis, and correlation coefficients (r) were calcu-lated. Since bands and immature granulocytes are countedtogether with neutrophils by the H-2 analyzer, these cells, welldefined by the CD 4000, were added to neutrophil counts inthe statistical evaluation.

We also compared 112 normal blood samples with themanual differential WBC count; all films were examined inde-pendently by 4 experienced technologists4; results werecompared by linear regression, and correlation coefficientswere calculated.

Interference StudyLipid interference was evaluated in 10 blood samples

added with increasing amounts of Intralipid (soya lipids)(Pharmacia AB, Uppsala, Sweden); they were analyzed side-by-side by using the 2 instruments, and each test wasperformed in triplicate. The results were expressed as themean value of 3 determinations. The lipid concentration wasmeasured as triglycerides.

At the end of the evaluation period, Abbott supplied anew hemoglobin reagent, Tri-Methyl-Tetra-Decyl-ammoniumchloride free (TTAB free), and a new software version (R8-1)that are able to eliminate any interference on CD 4000 hemo-globin determination. The lipid interference experiment alsowas performed with these new components.

Time (Aging) StudyWe analyzed 25 normal samples in duplicate within 2,

24, 48, and 72 hours after venipuncture and storage at roomtemperature (25 C) or under refrigeration (4 C).

498 Am J Clin Pathol 2000;113:497-505 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE

Results

Carryover

Carryover data for the WBC, RBC, hemoglobin, andplatelet values are given in Table 1. The results for high tolow carryover testing were less than 0.5%.

LinearityLinearity data are given in Table 2. Excellent correla-

tion was found between expected and obtained values forall parameters sensitive to dilution, also in a very lowrange. Statistical analysis revealed a correlation coefficienthigher than 0.997 for WBC, RBC, hemoglobin, and hemat-ocrit values and a lower correlation for platelet values(0.992). We also found that the unflagged percentage forthe WBC differential count results were reproducible andlinear down to a level of 0.20 109/L (data not shown).

ImprecisionAs shown in Table 3, there is good within- and

between-batch reproducibility for all parameters, includingthe CBC and WBC differential counts. Only for basophilswas the coefficient of variation higher than 20%.

ComparabilityAs shown in Table 4, the CD 4000 gave CBC and

differential WBC count results that compared very wellwith data obtained by the H-2. The results for basophilsshowed poor agreement (r = 0.297) between the 2 instru-ments in the low range of counts and good agreement (r =0.920) for high basophils counts.

The results of correlation and linear regressionbetween the CD 4000 and manual differential are given inTable 5. Correlation was excellent for neutrophils andlymphocytes, and monocytes and eosinophils showed agood correlation, while basophils results yielded a lowercorrelation in the low range of values (



Table 2Linearity of the CELL-DYN 4000 Analyzer*

Range r Intercept Slope

WBC (109/L) 0.15-100 0.998 0.030 0.998RBC (1012/L) 0.17-7.0 0.997 0.091 1.035Hemoglobin (g/dL) 0.50-20.0 0.999 0.125 1.012Hematocrit (%) 1.5-60.0 0.999 0.452 1.000Platelets (109/L) 6.20-1,000 0.992 3.460 0.955

* Abbott Diagnostics, Abbott Park, IL.

Table 3Within- and Between-Batch Precision Observed With the Abbott CELL-DYN 4000* in 20 Samples

Within Batch Between Batch

Mean SD CV (%) Mean SD CV (%)

WBC (109/L) 8.02 0.19 2.44 8.10 0.20 2.47 RBC (1012/L) 4.10 0.10 1.98 5.00 0.10 2.02 Hemoglobin (g/dL) 14.70 0.08 0.54 14.70 0.81 0.55 Hematocrit (%) 45.20 0.82 1.82 45.20 0.85 1.88 Mean corpuscular volume (fL) 90.50 0.28 0.31 90.30 0.27 0.30 Platelets (109/L) 269.10 6.40 2.38 270.00 5.95 2.20 Neutrophils (%) 49.35 0.65 1.32 50.10 0.67 1.34 Lymphocytes (%) 40.79 0.63 1.55 41.00 0.63 1.53 Monocytes (%) 6.57 0.25 3.80 6.60 0.26 3.93 Eosinophils (%) 3.00 0.10 3.32 3.06 0.15 4.90 Basophils (%) 0.27 0.07 24.80 0.30 0.06 21.33

* Abbott Diagnostics, Abbott Park, IL.

Table 4Comparability Test Between Abbott CELL-DYN 4000* and Bayer-Technicon H-2* for 120 Samples

Intercept Slope r

WBC (109/L) 0.053 0.971 0.998RBC (1012/L) 0.073 1.034 0.997Hemoglobin (g/dL) 0.035 1.018 0.998Mean corpuscular volume (fL) 6.606 1.019 0.990Platelets (109/L) 2.147 0.911 0.986Neutrophils (%) 0.061 0.970 0.981Lymphocytes (%) 0.042 1.031 0.976Monocytes (%) 0.028 1.206 0.996Eosinophils (%) 0.285 1.053 0.975Basophils (%) Range of values, 0% to


For the CBC and differential WBC counts, we found agood correlation with H-2 analyzer. Blasts and variantlymphocytes were both counted as large unstained cells bythe H-2, while they were well defined by the CD 4000;

therefore only neutrophil, lymphocyte, monocyte,eosinophil, and basophil data were comparable.

High nucleated RBCs would give invalid WBC compar-ison data (interference with the WBC count on the H-2


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Figure 1 Percentage of basophils with the CELL-DYN 4000(Abbott Diagnostics, Abbott Park, IL) compared with 400-cell manual differential count for basophil counts in the lowrange. The identity line is displayed with the 95% binomialconfidence limits. Few outliers are identified.

Figure 2 Percentage of basophils with the CELL-DYN 4000(Abbott Diagnostics, Abbott Park, IL) compared with 400-cell differential count for the basophil counts in the highrange. The identity line is displayed with the 95% binomialconfidence limits. No outliers are identified.

Figure 3 Interference of hyperlipidemic samples on thehemoglobin determination. Both the Abbott CELL-DYN 4000(Abbott Diagnostics, Abbott Park, IL) (squares) and Bayer-Technicon H-2 (Bayer-Technicon, Tarrytown, NY) (diamonds)show significant overestimation (1 g/dL or more) startingfrom a triglyceride value of 450 mg/dL or more.

Figure 4 Interference of hyperlipidemic samples on theRBC count. The RBC count is stable on both instruments(Abbott CELL-DYN 4000, Abbott Diagnostics, Abbott Park, IL[squares]; and Bayer-Technicon H-2 Bayer-Technicon, Tarry-town, NY [diamonds]) up to a triglyceride value of 200mg/dL. With higher lipid concentrations, only the H-2analyzer shows an RBC count overestimation.


analyzer). Because only a few samples with nucleated RBCspassed through the laboratory during this evaluation, a statisti-cally insignificant number of these samples was included in ourstudy (5%), and no interference with comparison was observed.

We also found a good correlation with the referencemanual differential for all WBC subpopulations except forbasophils in the low range (0% to


between the 2 instruments are not related primarily to poorperformance of the analyzers for the WBC differentialcount but to the low number of basophils counted. The lowcorrelation with the reference method could be due to thelow number and irregular distribution of the cells in the

peripheral blood films or, more probably, to the interfer-ence of nonviable WBCs, which were present in all theidentified outliers. Finally, when analyzing samples withhigh basophil counts, a dramatic increase of the correlationcoefficient was found.


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Figure 9 Differential WBC count in stored samples. A, Only minor reduction in the proportion of eosinophils was observedin samples stored at room temperature. B, No significant changes were observed in samples stored at 4 C. Ne, neutrophils;Ly, lymphocytes; Mo, monocytes; Eo, eosinophils; Ba, basophils.

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Figure 10 Time course of neutrophil subpopulations insamples stored at room temperature. Significant changes(P < .001) were observed in the segmented neutrophil andband fractions: the time course shows a progressive reduc-tion in the proportion of segmented neutrophils and a side-by-side, progressive increase in the proportion of bands. Nochange in the immature granulocyte fraction was observed.Diamonds, segmented neutrophils; squares, bandneutrophils; triangles, immature granulocytes.

Figure 11 Time course of neutrophil subpopulations insamples stored at 4 C. In samples stored at 4 C, the timecourse shows a greater reduction in the fraction ofsegmented neutrophils and a greater progressive increasein the band fraction than in samples stored at room temper-ature. No change in the immature granulocyte fraction wasobserved. Diamonds, segmented neutrophils; squares,band neutrophils; triangles, immature granulocytes.


An excellent correlation for monocytes was found withthe H-2 analyzer (r = 0.990) and a moderate correlation withthe reference method (r = 0.830). This finding is in agree-ment with those of other reports8,9 and seems to be due toartifact distribution for these cells during preparation of theblood films and to difficulties in differentiating small mono-cytes from large lymphocytes.

In the lipid interference study, we observed, as expected,a significant (P < .001) inaccuracy of hemoglobin determina-tion on both instruments starting from a lipid value of 450mg/dL, while very high lipid amounts (4,800 mg/dL)showed no interference with the WBC, RBC, and plateletcounts performed by the CD 4000. These data suggest theadequacy of the Abbott CELL-DYN 4000 algorithm fordiscriminating high amounts of hyperlipidemic particles andexcluding them from counts as well. Finally, the AbbottTTAB free hemoglobin reagent improves the accuracy of thehemoglobin determination by the CD 4000 analyzer,including in samples with very high lipid amounts.

In stored samples, the increase in the percentage ofnonviable cells was much slower in refrigerated samples(4 C), while storage at higher temperatures induced an accel-eration of the process.

The apparent change in the relationship of segmentedneutrophils to bands (Figures 10 and 11) with storage time isopposite what might be expected with the passage of time.This apparent change should be due to the changes in

neutrophil nuclear morphologic features (loss of lobularity)in aged samples, as evident when examining the blood films.

Finally the WBC viable fraction, the nonviable WBCflag, and the confidence fraction are sensitive intrasamplequality checks, and they could contribute to increased accu-racy of the counts, particularly when the discriminationbetween mature granulocytes and bands is of clinicalinterest. In particular, the confidence fraction, which indi-cates the probability that a cell population was classifiedcorrectly, in our study was always 0.76 or more in samplesthat were not stored, whereas the confidence fraction wasalways less than 0.76 in stored samples for all WBC subsets.In fact, in the presence of high band counts, a WBC viablefraction of 95% or less and/or a nonviable WBC flag and/ora confidence fraction less than 0.76 could indicate sampledeterioration and, therefore, the need for a fresh sample.

Conclusions

Our evaluation data show that the Abbott CELL-DYN4000 is a precise device, and it is at least as accurate as theBayer-Technicon H-2. The results are linear to extremecounts, and no significant carryover was observed.

Derived from a DNA fluorescence analysis,2 the avail-ability of quantitative and qualitative viability informationfor WBCs is a new feature on a hematology analyzer and is avery useful tool for analyzing aged blood specimens. In addi-tion, we found that ease of use contributes to the CELL-DYN 4000 performance, which may prove very useful in theclinical laboratory. The Abbott CD 4000 analyzer representsa substantial advance in hematology instrumentation and hasconsiderable potential for extending the clinical role ofhematologic studies.

From the Department of Laboratory Medicine, UniversitFederico II, Naples, Italy.

The CELL-DYN 4000 analyzer, reagents, and calibration andcontrol samples were provided free of charge for this evaluationby Abbott Diagnostics, Abbott Park, IL.

Address reprint requests to Dr Grimaldi: DipartimentoAssistenziale di Medicina di Laboratorio, PoliclinicoUniversitario Federico II, Via S Pansini 5, 80131 Napoli, Italy.

References

1. CELL-DYN 4000 [product manual]. Santa Clara, CA:Abbott Diagnostics; 1997.

2. Ormerod MG. Estimating cell viability, application in cellbiology. In: Ormerod MG, ed. Flow Cytometry: A PracticalApproach. New York, NY: IRL Press at Oxford UniversityPress; 1990:265-282.


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Figure 12 Time course of the confidence fraction in storedsamples. In samples stored at room temperature andsamples stored at 4 C, the confidence fraction (ie, the prob-ability that the cells were classified correctly) for segment-ed neutrophils, bands, and immature granulocytes wasalways less than 0.76, whereas the fraction was always0.76 or more in samples that were not stored.


3. DOnofrio G, Zini G, Tommasi M, et al. Integration offluorescence and hemocytometry in the CELL-DYN 4000:reticulocyte, nucleated blood cell and white blood cellviability study. Lab Hematol. 1996;2:131-138.

4. National Committee for Clinical Laboratory Standards.Reference Leukocyte Differential Count (Proportional) andEvaluation of Instrumental Methods; Approved Standard.Villanova, PA: National Committee for Clinical LaboratoryStandards; 1992. NCCLS document H20-A. Vol 12.

5. Broughton PMG, Gowenlock AH, McCormach JJ, et al. Arecommended scheme for the evaluation of instruments forautomated analysis in the clinical biochemistry laboratory. JClin Pathol. 1969;22:278-285.

6. Shinton NK, England JM, Kennedy DA. Guidelines for theevaluation of instruments used in haematology laboratories.J Clin Pathol. 1982;35:1095-1100.

7. International Council for Standardization in HaematologyExpert Panel on Cytometry. Guidelines for the evaluation ofblood cell analyzer including those used for differentialleukocyte and reticulocyte counting and cell markerapplications. Clin Lab Haematol. 1994;16:157-174.

8. Lebeck LK, Amst BJ, Houwen B. Flow cytometric whiteblood cell differentials: a proposed alternate referencemethod. Sysmex J Int. 1993;3:61-65.

9. Goossens W, Van Hove L, Verwilghen RL. Monocytecounting: discrepancies in results obtained with differentautomated instruments. J Clin Pathol. 1991;3:44-49.


497.Full Evaluacion Del Celldyn 4000

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