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  • Aging stability of complete blood count and white blood

    cell differential parameters analyzed by Abbott CELL-DYN

    Sapphire hematology analyzerP. HEDBERG, T. LEHTO

    INTRODUCTION

    The recent trends towards large centralized laborato-

    ries, and changes in laboratory organizations, have

    brought redistribution activity to a new perspective.

    Laboratories now test specimens that have been dis-

    patched over a long distance; as a result, testing is

    often 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 in

    ethylenediaminetetraacetic acid (EDTA)-anticoagulat-

    ed blood (Buttarello, 2004). Also, the different behav-

    ior of automated counters using impedance and

    optical methods may have an effect, and this should

    be 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 (England

    et al. 1993). K3EDTA is dispensed as a liquid and thus

    causes a slight dilution of the specimen. This salt also

    affects the red blood cell size at increased concen-

    trations and on storage than the dipotassium salt.

    Department of Clinical Chemistry,

    Laboratory, Oulu University

    Hospital, University of Oulu, Oulu,

    Finland

    Correspondence:

    Pirjo Hedberg, Laboratory, Oulu

    University Hospital, PO Box 500,

    FI-90029 OYS, Oulu, Finland.

    Tel.: +358 8 3155453;

    Fax: +358 8 3154409;

    E-mail: pirjo.hedberg@ppshp.fi

    doi:10.1111/j.1751-553X.2007.01009.x

    Received 31 May 2007; accepted

    for publication 8 October 2007

    Keywords

    Automated blood cell counts,

    hematology, hematology analyzer,

    stability, WBC differential, WBC

    flagging

    SUMMARY

    This study presents the results of an aging stability study of complete

    blood count (CBC) and leukocyte differential parameters using the

    Abbott CELL-DYN Sapphire hematology analyzer. Stability studies

    showed 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 platelet

    count yielded more reliable results than the routine PLTo. White blood

    cell (WBC) differential parameters, except eosinophils, were stable for

    up 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 87

  • Therefore, in 1993, the International Council for

    Standardization in Haematology recommended that

    K2EDTA should be used as the anticoagulant of choice

    in specimen collection for blood cell counting and

    sizing (England et al. 1993). However, the cellular ele-

    ments in hematology specimens have limited stability

    in EDTA-anticoagulated blood during storage at room

    temperature or at +4 C. Of these two temperatures,conservation of specimens at +4 C is known toimprove the stability of some complete blood count

    and white blood cell differential parameters, but there

    have not been any systematic studies that take into

    account all the modern instruments or that considered

    normal specimens, as well as a wide variety of pathol-

    ogies, where a delay in analysis might give different

    results (Buttarello, 2004).

    In order to decide whether to accept or reject the

    aged specimen, laboratory staff needs to be familiar

    with the changes known to occur in blood speci-

    mens during storage. For reticulocyte counts, the

    National Committee for Clinical Laboratory Stan-

    dards (NCCLS) H44-A protocol (National Committee

    for Clinical Laboratory Standards, 1997) recom-

    mended that analyses should be performed within

    6 h of collection if the specimen is kept at room

    temperature; conserving specimens at +4 C stabilizesparameters for 72 h. Also, for the differential leuko-

    cyte count, it was recommended that smears should

    be prepared for microscopic analysis within 4 h

    (National Committee for Clinical Laboratory Stan-

    dards, 1996).

    Several studies have been published during recent

    years that include data concerning the stability of

    complete blood count and automated differential

    count 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 complete

    blood count (CBC), reticulocyte and white blood cell

    (WBC) differential parameters in EDTA-anticoagulated

    blood 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 an

    Abbott CELL-DYN Sapphire hematology analyzer (Ab-

    bott Laboratories, Diagnostic Division, Abbott Park, IL,

    USA) at Oulu University Hospital Laboratory, Oulu,

    Finland.

    MATERIALS AND METHODS

    Analytical methods

    The Abbott CELL-DYN Sapphire Hematology System

    uses multi-angle polarized scatter separation (MAPSS)

    and focused-flow impedance technologies, combined

    with three-color fluorescent flow cytometry. This sys-

    tem has a fully automated reticulocyte analysis with

    immature reticulocyte fraction (IRF), a five-part WBC

    differential, fluorescent DNA staining of nucleated red

    blood cell absolute count (NRBC), optical and imped-

    ance platelet measurement and fully automated

    monoclonal antibody testing for CD 3/4/8 and CD61.

    A cyanide-free method is used to measure hemoglobin

    (HGB) colorimetrically.

    Samples

    For 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 to

    maintain specimens at +4 C when using automatedsample loading. Samples stored at +4 C were initiallycold, taken from the refrigerator and then reanalyzed

    after 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 data

    attained from the first measurements. At baseline

    measurements, six of the samples in the group stored

    for 6 h at +4 C were flagged as abnormal, with onevariant lymphocyte (VARLYM), three IG, one uniden-

    tified fluorescent population (FP?) or NRBC and one

    BLAST flags. Of the samples stored for 24 h, nine

    were abnormal (five IG and four BAND flags) and

    eight of the samples in the group stored for 48 h were

    abnormal (five VARLYM, one IG, one FP? or NRBC

    and one BAND flags). Of the samples stored for 72 h,

    eight were abnormal (four IG, one FP? or NRBC and

    three BLAST flags). In the third study, 20 different

    samples (six were abnormal from the beginning; four

    IG, one BLAST and one FP? or NRBC flags) were

    stored at +4 C in between the measurements (6 h,24 h and 48 h) and warmed to room temperature

    before analysis (altogether four cycles). Additionally,

    the stability of the leukocyte flagging was studied. The

    suspect population flags BAND, IG, BLAST, VARLYM

    and FP? were followed up, as were any positive NRBC

    results. Stability profiles were derived from longitudi-

    nal comparisons of parameter changes.

    Statistical analysis

    Statistical processing was performed using personal

    computer EXCEL software. The statistical significance of

    the differences between the means was assessed by

    Students t-test as appropriate. P < 0.05 was consid-

    ered statistically significant.

    RESULTS AND DISCUSSION

    Aging 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 corpuscular

    hemoglobin), RDW (red cell distribution width) and

    PLTi (impedance measurement of PLT) were found to

    be stable over the time, with mean percentage

    changes of less than 10%. However, a statistically

    significant change (P < 0.05) was found for RDW at

    48- and 72-h storage. The MCV (mean corpuscular

    volume), HCT (hematocrit) and MPV (mean platelet

    volume) 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 at

    72-h storage for MPV. The MCHC (mean corpuscular

    hemoglobin concentration) trended downward over

    time, with change of 13.1% within 48 h. Statistically

    significant changes were seen at all storage time

    points. There was a proportional 12.1% decrease at

    24-