Aging stability of complete blood count and white blood cell differential parameters analyzed by Abbott CELL-DYN Sapphire hematology analyzer

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Aging stability of complete blood count and white bloodcell differential parameters analyzed by Abbott CELL-DYNSapphire hematology analyzerP. HEDBERG, T. LEHTOINTRODUCTIONThe recent trends towards large centralized laborato-ries, and changes in laboratory organizations, havebrought redistribution activity to a new perspective.Laboratories now test specimens that have been dis-patched over a long distance; as a result, testing isoften delayed by 1224 h or more after venipuncture.Although laboratories should still give reliable results,excessive delays in processing might affect the reliabil-ity, accuracy and imprecision analysis. Cellular ele-ments are known to have limited stability inethylenediaminetetraacetic acid (EDTA)-anticoagulat-ed blood (Buttarello, 2004). Also, the different behav-ior of automated counters using impedance andoptical methods may have an effect, and this shouldbe taken into account (Wood et al., 1999).Of the three EDTA salts used for the anticoagula-tion of blood specimens for hematological testing,potassium salts are the most readily soluble (Englandet al. 1993). K3EDTA is dispensed as a liquid and thuscauses a slight dilution of the specimen. This salt alsoaffects the red blood cell size at increased concen-trations and on storage than the dipotassium salt.Department of Clinical Chemistry,Laboratory, Oulu UniversityHospital, University of Oulu, Oulu,FinlandCorrespondence:Pirjo Hedberg, Laboratory, OuluUniversity Hospital, PO Box 500,FI-90029 OYS, Oulu, Finland.Tel.: +358 8 3155453;Fax: +358 8 3154409;E-mail: pirjo.hedberg@ppshp.fidoi:10.1111/j.1751-553X.2007.01009.xReceived 31 May 2007; acceptedfor publication 8 October 2007KeywordsAutomated blood cell counts,hematology, hematology analyzer,stability, WBC differential, WBCflaggingSUMMARYThis study presents the results of an aging stability study of completeblood count (CBC) and leukocyte differential parameters using theAbbott CELL-DYN Sapphire hematology analyzer. Stability studiesshowed no substantial change in CBC parameters up to 2448 h at+23 2 C (room temperature), except for optical platelet count(PLTo). For specimens aged over 24, the value of impedance plateletcount yielded more reliable results than the routine PLTo. White bloodcell (WBC) differential parameters, except eosinophils, were stable forup to 48 h at +23 2 C. CBC parameters were stable for 72 h, exceptmean platelet volume, which slightly increased between 48 and 72 h,at +4 C. WBC differentials were stable 4872 h, with a slight decreaseobserved in absolute neutrophils and lymphocytes at +4 C.ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY 2008 The AuthorsJournal compilation 2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2009, 31, 8796 87Therefore, in 1993, the International Council forStandardization in Haematology recommended thatK2EDTA should be used as the anticoagulant of choicein specimen collection for blood cell counting andsizing (England et al. 1993). However, the cellular ele-ments in hematology specimens have limited stabilityin EDTA-anticoagulated blood during storage at roomtemperature or at +4 C. Of these two temperatures,conservation of specimens at +4 C is known toimprove the stability of some complete blood countand white blood cell differential parameters, but therehave not been any systematic studies that take intoaccount all the modern instruments or that considerednormal specimens, as well as a wide variety of pathol-ogies, where a delay in analysis might give differentresults (Buttarello, 2004).In order to decide whether to accept or reject theaged specimen, laboratory staff needs to be familiarwith the changes known to occur in blood speci-mens during storage. For reticulocyte counts, theNational Committee for Clinical Laboratory Stan-dards (NCCLS) H44-A protocol (National Committeefor Clinical Laboratory Standards, 1997) recom-mended that analyses should be performed within6 h of collection if the specimen is kept at roomtemperature; conserving specimens at +4 C stabilizesparameters for 72 h. Also, for the differential leuko-cyte count, it was recommended that smears shouldbe prepared for microscopic analysis within 4 h(National Committee for Clinical Laboratory Stan-dards, 1996).Several studies have been published during recentyears that include data concerning the stability ofcomplete blood count and automated differentialcount using different analyzers (Warner & Reardon,1991; Briggs, Harrison & Grant, 1999; Wood et al.,1999; Walters & Garrity, 2000; Gulati et al., 2002).Our study investigated the stability of completeblood count (CBC), reticulocyte and white blood cell(WBC) differential parameters in EDTA-anticoagulatedblood at room temperature (+23 2 C) and at +4 C.The WBC viability fraction (WVF) and leukocyte flag-ging changes were also followed during these condi-tions. The measurements were undertaken using anAbbott CELL-DYN Sapphire hematology analyzer (Ab-bott Laboratories, Diagnostic Division, Abbott Park, IL,USA) at Oulu University Hospital Laboratory, Oulu,Finland.MATERIALS AND METHODSAnalytical methodsThe Abbott CELL-DYN Sapphire Hematology Systemuses multi-angle polarized scatter separation (MAPSS)and focused-flow impedance technologies, combinedwith three-color fluorescent flow cytometry. This sys-tem has a fully automated reticulocyte analysis withimmature reticulocyte fraction (IRF), a five-part WBCdifferential, fluorescent DNA staining of nucleated redblood cell absolute count (NRBC), optical and imped-ance platelet measurement and fully automatedmonoclonal antibody testing for CD 3/4/8 and CD61.A cyanide-free method is used to measure hemoglobin(HGB) colorimetrically.SamplesFor these studies, residual fresh (Sarstedt, D-51588) at +4 C. One aliquot from eachsample was analyzed after 6, 24, 48 and 72 h. However,it is not known how aliquoting into nonblood collec-tion tubes could affect any analysis. It is difficult tomaintain specimens at +4 C when using automatedsample loading. Samples stored at +4 C were initiallycold, taken from the refrigerator and then reanalyzedafter a brief warming period (15 min) at 23 2 C. Allof the time measurement groups (6, 24, 48 and 72 h)included pathologic specimens according to the dataattained from the first measurements. At baselinemeasurements, six of the samples in the group storedfor 6 h at +4 C were flagged as abnormal, with onevariant lymphocyte (VARLYM), three IG, one uniden-tified fluorescent population (FP?) or NRBC and oneBLAST flags. Of the samples stored for 24 h, ninewere abnormal (five IG and four BAND flags) andeight of the samples in the group stored for 48 h wereabnormal (five VARLYM, one IG, one FP? or NRBCand one BAND flags). Of the samples stored for 72 h,eight were abnormal (four IG, one FP? or NRBC andthree BLAST flags). In the third study, 20 differentsamples (six were abnormal from the beginning; fourIG, one BLAST and one FP? or NRBC flags) werestored at +4 C in between the measurements (6 h,24 h and 48 h) and warmed to room temperaturebefore analysis (altogether four cycles). Additionally,the stability of the leukocyte flagging was studied. Thesuspect population flags BAND, IG, BLAST, VARLYMand FP? were followed up, as were any positive NRBCresults. Stability profiles were derived from longitudi-nal comparisons of parameter changes.Statistical analysisStatistical processing was performed using personalcomputer EXCEL software. The statistical significance ofthe differences between the means was assessed byStudents t-test as appropriate. P < 0.05 was consid-ered statistically significant.RESULTS AND DISCUSSIONAging stability at +23 2 C (room temperature)Changes observed in the CBC results of blood speci-mens stored at +23 2 C are summarized in Table 1.At this temperature, the WBC, RBCi (impedance mea-surement of red blood cells), RBCo (optical measure-ment of RBC), HGB, MCH (mean corpuscularhemoglobin), RDW (red cell distribution width) andPLTi (impedance measurement of PLT) were found tobe stable over the time, with mean percentagechanges of less than 10%. However, a statisticallysignificant change (P < 0.05) was found for RDW at48- and 72-h storage. The MCV (mean corpuscularvolume), HCT (hematocrit) and MPV (mean plateletvolume) increased over time, producing mean per-centage changes over 10% within 72 h (10.6%,11.1% and 11.1%, respectively). The statistically sig-nificant changes were found at 24-, 48- and 72-h stor-age for MCV, at 48- and 72-h storage for HCT and at72-h storage for MPV. The MCHC (mean corpuscularhemoglobin concentration) trended downward overtime, with change of 13.1% within 48 h. Statisticallysignificant changes were seen at all storage timepoints. There was a proportional 12.1% decrease at24-h storage, 19.8% decrease at 48-h storage and24.4% decrease at 72-h storage for PLTo (optical mea-surement of PLT). The R% (reticulocyte percent) andRETC (reticulocyte absolute count) and IRF (imma-ture reticulocyte fraction) fell slightly at 72 h, but notat earlier time points, at +23 2 C. A statisticallysignificant change (P < 0.05) was found for R% at24- and 72-h storage.The results of the automated differential countfor specimens stored at +23 2 C are summarizedin Table 1. Storage of specimens at +23 2 Cresulted in a fast decrease in WVF (leukocyte viabil-ity fraction) (P < 0.05 at 6-, 24-, 48- and 72-h stor-age). After 24 h, the change was already )29.4%and tended to decrease further with time. Storage ofspecimens for 6 h resulted in decreased absolutebasophil counts ()31.1%). In contrast, prolongedstorage up to 72 h showed an increase in basophils.Basophils represented the smallest fraction of all theWBC. The standard deviations of the percentagechanges of the absolute basophil counts were veryhigh. Also, there was a poor analytical day-to-dayprecision of the basophil counting (data not shown).Additionally, one of the samples in this study withvery low absolute basophil counts measured afterthe 6-h storage differed from the first measurementmore than the other samples thus giving a highoverall bias%. If this sample was not included inthe analysis, the decrease in the absolute counts of 2008 The AuthorsJournal compilation 2008 Blackwell Publishing Ltd, Int. Jnl. Lab. Hem. 2009, 31, 8796P. HEDBERG AND T. LEHTO AGING STABILITY OF AUTOMATED HEMATOLOGY PARAMETERS 89basophils would be 2%. Eosinophil counts haddecreased by 78.8% during 24-h storage andshowed a progressive decline over the time. Statisti-cally significant (P < 0.05) changes were found foreosinophils at 24-, 48- and 72-h storage. Also thechanges over 10% were seen for basophils andmonocytes at 72 h.The stability of NRBC values were assessed andany NRBC value from the analyzer was consideredas a positive result with discrimination level of>0%. None of the samples analyzed at baseline, at6-h and 24-h time points had a positive NRBC value.Four of the samples analyzed after 48- and 72-hstorage had positive NRBC values (48 h: 0.35, 0.25,0.27, 0.25 109/l and 72 h: 0.54, 0.44, 0.23,0.39 109/l).Aging stability at +4 CThe effects of +4 C storage on the CBC and automatedWBC differential parameters are shown in Table 2 andFigures 1 and 2. Prolonged storage of specimens for upto 72 h at +4 C revealed stability of the CBC parame-ters, reticulocyte parameters and monocyte countswithin 10% of their original values. However, a statisti-cally significant change (P < 0.05) was seen in theMCHC at 48 and 72 h. A decrease in PLTo at +23 2 Cwas corrected by storage at +4 C. A small increase inMPV was seen during storage (up to 11.8%). A statisti-cally significant change (P < 0.05) was observed forMPV at 24-, 48- and 72-h storage. A substantialdecrease in the absolute counts of eosinophils and inWVF with storage at +23 2 C was not observedTable 1. Changes of CBC parameter and WBC differential values induced by storage of blood at +23 2 C. Data arepresented as means of parameter values, changes (% from the original value, which is 100%) at different time pointsand standard deviation (SD) of changes in parenthesis. Statistically significant changes (P < 0.05; Students t-test)between the means are marked with asterisks (*)Table2.ChangesofCBCparameterandWBCdifferentialvaluesinducedbystorageofbloodat+4C.Dataarepresentedasmeansofparametervalues,changes(%fromtheoriginalvalue,whichis100%)atdifferenttimepointsandSDsofchangesinparenthesis.Thestatisticallysignificantchanges(P

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