Guidance for storing blood samples in laboratories performing complete blood count with differential

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  • Guidance for storing blood samples in laboratories performing

    complete blood count with differentialE. CORNET*, ,1, C. BEHIER*,1, X. TROUSSARD*,

    *CHU de Caen, Laboratory of

    Hematology, Caen, FranceUniversity of Caen, Medical

    School, Caen, France

    Correspondence:

    Edouard Cornet, Laboratory of

    hematology, CHU de Caen,

    Avenue Cote de Nacre, 14033

    Caen Cedex 9, France.

    Tel.: +33 2 31 06 33 15;

    Fax: +33 2 31 06 50 99;

    E-mail: cornet-e@chu-caen.fr

    doi:10.1111/j.1751-553X.2012.01452.x

    Received 1 February 2012;

    accepted for publication 8 May

    2012

    Keywords

    Blood cell count, laboratories/

    standards, reproducibility of

    results, blood preservation, tem-

    perature

    SUMMARY

    Introduction The complete blood count (CBC) with differential leu-

    kocyte count (DIFF) is an important part of clinical laboratory anal-

    yses and provides crucial data for clinicians. Delivery time after

    blood collection and conditions of storage is known to affect the

    reliability of results of some hematologic parameters. The aim of

    this study was to assess the variations of hematologic parameters

    over time and the influence of storage temperature.

    Methods Blood samples were randomly selected from hospitalized

    patients and stored at room temperature and at 4 C. CBC andDIFF were performed on an automated hematology analyzer and

    the results between the two groups were compared.

    Results Samples stored at room temperature showed an important

    increase in mean corpuscular volume and hematocrit and a

    decrease in mean corpuscular hemoglobin concentration. Neutro-

    phil counts tended to increase, whereas monocyte counts tended to

    decrease.

    Conclusion Storing samples at 4 C improved reproducibility overtime of all quantitative and qualitative parameters. We also

    observed that NEUT-X, a routine parameter useful in detecting

    myelodysplastic syndrome, became unreliable when analyzed 24 h

    after sample collection. Our results led us to recommend that sam-

    ples should be analyzed within 6 h, particularly if samples are

    transported at room temperature. We also recommend storing sam-

    ples at 4 C in case of remote CBC analysis, especially in the con-text of clinical trials.

    INTRODUCTION

    The complete blood count (CBC) with differential leu-

    kocyte count (DIFF) is one of the most routinely per-

    formed laboratory tests in our institution, with about

    190 000 analyses performed in 2011. It is a valuable

    screening tool for a wide variety of medical conditions

    and is an essential routine in the optimal management

    of patients. Compliance with International Organiza-

    tion for Standardization (ISO) 15 189, the international1These authors contributed equally to this work.

    2012 Blackwell Publishing Ltd, Int J Lab Hem. 1

    ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY

    International Journal of Laboratory HematologyThe Official journal of the International Society for Laboratory Hematology

  • standard for the accreditation of medical laboratories

    (1), is becoming progressively accepted as the optimal

    approach to assure quality in medical testing.

    The main objective of any clinical laboratory is to

    produce results that are accurate and precise enough

    for clinical use, and therefore, the correct pre-analyti-

    cal procedures are essential. Many external variables

    can influence laboratory results, including conditions

    of blood collection, storage and delivery time of sam-

    ples to the laboratory. It is particularly critical when

    blood samples are collected at the patients home.

    Delivery time to the laboratory can exceed 8 h.

    The development of clinical trials and centralized

    analyses will impose adoption of strict pre-analytical

    rules. Healthcare laboratories must establish limits

    for delivery time and conditions of storage and the

    impact of pre-analytical conditions on hematologic

    parameters must be known for accurate interpre-

    tation of all parameters provided by an automated

    analyzer.

    This study confirms the impact of pre-analytical

    conditions on variations of CBC-DIFF parameters and

    also highlights new data to interpret NEUT-X, which

    is a routine parameter useful in detecting myelodys-

    plastic syndrome (MDS). Performing CBC/DIFF analy-

    sis within 6 h after sample collection guarantees good

    reliability of the results.

    MATERIALS AND METHODS

    Samples and analyzer

    We studied 64 venous peripheral blood samples,

    randomly selected among hospitalized patients in

    Caen University Hospital. Samples were collected in

    vacutainers containing dipotassium ethylene diamine-

    tetraacetate (K2EDTA BD Vacutainer; Becton and

    Dickinson, Le Pont-De-Claix, France), which is the

    anticoagulant of choice in specimen collection for

    blood cell counting and sizing, as recommended by

    the International Council for Standardization in

    Hematology (2). This salt also has been shown to

    affect red blood cell size less than tripotassium salt

    (3). Thirty-two samples were stored at room tempera-

    ture (RT) (Group 1) and 32 at +4 C (Group 2). CBC/

    DIFF was processed at different time points during a

    period of 3 days, that is, at delivery (t0) then 6, 12,

    24, 48 and 72 h (t6, t12, t24, t48 and t72). The maxi-

    mum time interval between blood collection and sam-

    ple analysis at t0 (reference values) was 4 h. All

    samples were normal blood samples, without any

    qualitative abnormalities.

    Laboratory methods

    Analyses were performed in automatic mode on a

    Sysmex XE-2100 automated multiparameter hematol-

    ogy analyzer (Sysmex Corporation, Kobe, Japan) in

    accordance with manufacturers recommendations.

    The analyzer was calibrated by the manufacturer.

    Two levels of controls (internal quality control) were

    run on a daily basis (e-check XE control) according

    to manufacturers recommendations. Only the data

    from the CBC, DIFF and Immature Myeloid Informa-

    tion channels were taken into account, we did not

    consider reticulocyte count or nucleated red blood

    cell count (NRBC) in this analysis. Parameters of

    CBC measured in this study were the following:

    white blood cell count (WBC), red blood cell count

    (RBC), hemoglobin concentration (HB), hematocrit

    (HCT), mean corpuscular volume (MCV), mean corpus-

    cular hemoglobin (MCH), mean corpuscular hemo-

    globin concentration (MCHC) and platelet count (PLT).

    The study assessed relative percentages of clusters: neu-

    trophil (NEU%), eosinophil (EOS%), basophil (BASO

    %), lymphocyte (LYMPH%), monocyte (MONO%)

    and immature granulocyte (IG%). All presented per-

    centages were automatically generated parameters.

    Statistical analysis

    As previously described in the literature, stability of

    an analyte was defined in relation to the imprecision

    of the respective analytical method. A parameter was

    considered stable when its average change was smaller

    than one coefficient of variation (CV, %) of the

    assessed method, allowing a 5% risk of error (4, 5).

    CV was obtained with daily controls. Except for EOS

    %, BASO% and IG%, we evaluated the variability of

    each parameter at each time point by calculating the

    mean percent change from the initial value (t0). We

    evaluated the variability of EOS%, BASO% and IG%

    by calculating the mean absolute difference from the

    initial value rather than percent change because some

    initial values were at zero. Statistical analyses were

    performed for the two groups. Mean, standard

    2012 Blackwell Publishing Ltd, Int J Lab Hem.

    2 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES

  • deviation (SD) and 95% confidence interval (95% CI)

    were calculated with MicrosoftTM Excel software.

    RESULTS

    Samples characteristics

    All samples were normal blood samples. Characteris-

    tics of the two groups were comparable for all mea-

    sured parameters (P > 0.05) (Table 1). We considered

    values at t0 as reference values.

    CBC parameters

    Many parameters of CBC were stable throughout the

    study period. No significant difference was observed

    for WBC, RBC, HB, MCH and PLT in Group 1 and

    Group 2 until 72 h. Conversely, there was a signifi-

    cant MCV difference from t0 to t6 in Group 1. At 6 h,

    the percent change was 4.2% (95% CI, 1.91; 6.42)

    and regularly increased, reaching 17.5% (16.6418.42)

    at 72 h (CV for MCV was 0.96%) (Figure 1). In

    Group 2, percent changes at t6 and t72 were, respec-

    tively, 1.2% (1.08; 1.31) and 1.5% (1.14; 1.85).

    Hence, MCV increased at both RT and +4 C but this

    change was more limited in case of refrigerated stor-

    age. As a consequence, we also observed a significant

    variation of MCV-related parameters at RT with a pro-

    gressive decrease in MCHC and a progressive increase

    in HCT.

    DIFF parameters

    No significant variations of the WBC count were

    detected between t0 and t72. Only samples from

    Group 1 showed an increase in NEU% from t48

    (Figure 1). Percent change at t72 was +8.2% (5.84;

    10.54) (CV for NEU% was 1.62%). In contrast,

    MONO% decreased from t48 (28.4%, 36.87;19.88) (CV for MONO% was 7.08%). No significantvariation was detected in Group 2. Both groups

    showed no LYMPH% variation, and no significant

    variation was observed for EOS%, BASO% and IG%.

    DISCUSSION

    A few studies have analyzed variations of CBCDIFF

    with regard to the storage of blood specimens at room

    temperature (4, 69). All of these studies described an

    increase in MCV and a variation of its related

    parameters (HCT and MCHC). These observations

    were independent of the type of automated analyzer.

    Similarly, our study demonstrated a significant varia-

    tion of many hematologic parameters over 3 days

    related to storage conditions of blood. The largest vari-

    ation concerned erythrocyte parameters. MCV

    increased dramatically from 6 h if samples were kept

    at RT, but was limited in case of refrigerated storage

    (+4 C). This observation could be related to a swell-

    ing of red blood cell because of a decrease in mem-

    brane resistance and an increase in cell permeability.

    This phenomenon seemed to depend on storage dura-

    tion and temperature. As expected, these modifica-

    tions also affected MCV-related parameters, such as

    HCT and MCHC, which are calculated from the MCV,

    with an increase in HCT and a decrease in MCHC. As

    previously described (6), RBC, HB, MCH and PLT

    measurements were unmodified until 72 h at RT.

    Table 1. Initial characteristics (t0) of the 32 samplesstored at room temperature (Group 1) and the 32

    samples stored at +4 C (Group 2)

    Group 1 (RT)

    Group 2 (+4C)

    PMean SD Mean SD

    White blood cell

    count (109/L)

    9.75 3.83 8.50 4.83 0.25575 (NS)

    Red blood cell

    count (1012/L)

    4.04 0.83 3.86 0.76 0.38470 (NS)

    Hemoglobin (g/dL) 12.30 2.58 11.66 2.15 0.28535 (NS)Hematocrit (%) 36.3 7.2 34.7 6.0 0.34182 (NS)

    Mean corpuscularvolume (fl)

    90.3 5.9 90.4 5.5 0.94268 (NS)

    Mean corpuscularhemoglobin (pg)

    30.6 2.3 30.4 2.4 0.72081 (NS)

    Mean corpuscularhemoglobin

    concentration (g/dL)

    33.8 1.3 33.5 1.4 0.39222 (NS)

    Platelet count(109/L)

    224 87 225 87 0.97345 (NS)

    Neutrophils (%) 67.73 13.56 62.65 16.46 0.18334 (NS)Eosinophils (%) 2.22 1.79 3.33 2.85 0.06595 (NS)Basophils (%) 0.34 0.26 0.50 0.47 0.11342 (NS)

    Lymphocytes (%) 18.22 10.84 22.36 14.12 0.19332 (NS)Monocytes (%) 10.75 4.28 10.15 5.33 0.61772 (NS)

    Immaturegranulocytes (%)

    0.74 0.92 1.01 1.49 0.38757 (NS)

    Results are expressed as mean and standard deviation

    (SD). NS: Not significant.

    2012 Blackwell Publishing Ltd, Int J Lab Hem.

    E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES 3

  • RBC

    t6 t12 t24 t48 t72 t6 t12 t24 t48 t72 t6 t12 t24 t48 t72

    t6 t12 t24 t48 t72t6 t12 t24 t48 t72t6 t12 t24 t48 t72

    t6 t12 t24 t48 t72 t6 t12 t24 t48 t72 t6 t12 t24 t48 t72

    t6 t12 t24 t48 t72t6 t12 t24 t48 t72

    2

    1

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    1

    RT+4 C

    1 CV%(0.62%)

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    lue

    (t 0)

    % -

    Mea

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    HCT

    0

    5

    10

    15 RT+4 C

    1 CV%(1.05%)P

    erce

    nt c

    hang

    e

    % -

    Mea

    n (C

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    MCV

    0

    5

    10

    15

    20RT+4 C

    1 CV%(0.96%)P

    erce

    nt c

    hang

    e

    % -

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    MCH

    1

    0

    1

    RT+4 C

    1 CV%(0.81%)

    Perc

    ent c

    hang

    e

    % -

    Mea

    n (C

    I95%

    )

    MCHC

    20

    15

    10

    5

    0

    5

    RT+4 C

    1 CV%(1.25%)

    Perc

    ent c

    hang

    e

    % -

    Mea

    n (C

    I95%

    )

    PLT

    5

    0

    5

    10RT+4 C

    1 CV%(2.81%)

    Perc

    ent c

    hang

    e

    % -

    Mea

    n (C

    I95%

    )

    WBC

    2

    0

    2

    4

    6RT+4 C

    1 CV%(2.15%)

    Perc

    ent c

    hang

    e

    % -

    Mea

    n (C

    I95%

    )

    NEU%

    10

    5

    0

    5

    10 RT+4 C

    1 CV%(1.62%)

    Perc

    ent c

    hang

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    % -

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    )

    MONO%

    60

    40

    20

    0

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    1 CV%(7.08%)

    Perc

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    )

    LYMPH%

    15105

    05

    1015

    RT+4 C

    1 CV%(2.91%)

    Perc

    ent c

    hang

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    % -

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    )

    * **

    **

    * **

    * *

    * **

    **

    **

    **

    Figure 1. Average percent changes from reference value (t0) of complete blood count and differential leukocytecount (DIFF) parameters for Group 1 (RT) and Group 2 (+4 C). Percent changes were calculated from referencevalues at t0. Each data point represents the mean percent change and each bar represents the 95% confidence

    interval (95% CI). A parameter was considered as unstable if its average change (with 95% CI) was higher than

    the coefficient of variation (CV) of the method. Results are presented for samples stored at room temperature (RT)

    and under refrigeration (+4 C). CV% was obtained with daily controls. WBC indicates white blood cell count;RBC, red blood cell count; HB, hemoglobin concentration; HCT, hematocrit; MCV, mean corpuscular volume;

    MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet count;

    NEU%, neutrophils (%); LYMPH%, lymphocytes (%); MONO%, monocytes (%). *Significant variation ascompared to CV of the method. All parameters were stable until 72 h at +4 C. Significant variations wereobserved at RT from t6, for erythrocyte volume-related parameters, such as HCT, MCV and MCHC. Significant

    variations were observed at RT for NEUT% (increase) and MONO% (decrease) from t48.

    2012 Blackwell Publishing Ltd, Int J Lab Hem.

    4 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES

  • In our study, leukocyte count (WBC) was

    unchanged until 72 h at both RT and +4 C. Modifica-

    tions of DIFF were exclusively observed at RT, with

    an increase in NEU% and a decrease in MONO%.

    Indeed, modification of cellular structure over time

    could alter detection and identification of cells, partic-

    ularly if blood specimens are stored at RT. To evaluate

    this progressive cellular deterioration, we tested sec-

    ondarily eight normal blood samples at RT (data not

    shown). In addition to the usual DIFF parameters, we

    also considered the neutrophil structural and matura-

    tion parameters provided by our analyzer: NEUT-X

    (10). NEUT-X is the direct measurement of side scatter

    diffraction and is representative of neutrophil struc-

    ture. We compared NEUT-X with manual differential

    and cell morphology analyzed by microscopic meth-

    ods. Microscopic DIFF count became unreliable from

    48 h, with a decrease in monocyte count and an

    increase in neutrophil count. We observed a progres-

    sive decrease in NEUT-X from 48 h, which was corre-

    lated with an increase in neutrophil cellular

    deterioration on blood smears (karyorrhexis)

    (Figure 2). Analysis of the DIFF scattergram confirmed

    that the position of neutrophils on the x-axis had

    shifted to the left (NEUT-X) and showed that position

    of monocytes on the y-axis tended to shift down into

    the neutrophil cluster (Figure 2). Lymphocyte mor-

    phology seemed to be less affected by this cellular

    deterioration. As recently demonstrated (11) that

    NEUT-X value is correlated with hypogranularity of

    neutrophils and therefore is useful in detecting MDS.

    Our observations indicated the unreliability of NEUT-

    X when samples were analyzed 24 h after collection.

    Checking the time between sample collection and

    analysis should be given high priority before reporting

    results, because significant variations were observed for

    RBC volumetric indices from 6 h and from 48 h for dif-

    ferential WBC. These variations may have conse-

    quences on diagnostic classification and clinical

    strategies (12). For instance, a false increase in MCV,

    especially if associated with a false decrease in NEUT-X,

    could be interpreted as a sign of MDS. Such a mistake

    could result in unnecessary complementary tests, espe-

    cially invasive procedures such as medullar biopsy. In

    Figure 2. Influence of time on differential leukocyte count (DIFF) parameters and neutrophils cellular structure fora sample stored at RT. DIFF parameters and neutrophils cellular structure were analyzed by automated method

    (NEUT-X) and microscopic method. Analyzer graphs, automated and manual DIFF are represented. We observed a

    progressive increase in neutrophils (NEU%), a progressive decrease in monocytes (MONO%). Microscopic analysis

    revealed a progressive neutrophil cellular deterioration on blood smears from t48 (karyorrhexis at t96), correlated

    with a degradation of neutrophils structure (side scatter on x-axis) identifiable by the progressive decrease in

    NEUT-X. LYMPH%, lymphocytes (%).

    2012 Blackwell Publishing Ltd, Int J Lab Hem.

    E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES 5

  • contrast, this false increase in MCV could hide a real

    microcytosis in case of iron deficiency anemia.

    Limits for delivery time and storage condition could

    be defined with goals for desirable bias for each

    parameter. Quality specifications have been published:

    (13) proposed recommendations for maximum allow-

    able storage time on EDTA-anticoagulated blood: 6 h

    for CBC and DIFF at RT and 24 h at +4 C. Various

    systems were used to propose tolerance limits for

    analytic bias (13). The method based on variations in

    population test distribution seemed to be the best,

    even though it is based on relatively complicated

    calculations. It is the method that best meets clinical

    needs (14). For example, they proposed a desirable

    bias of 0.7% for MCV (15). Our MCV results did not

    comply with these recommendations from 6 h at RT

    and +4 C. For MONO%, the decrease did not comply

    with specifications from 48 h only at RT (desirable

    specification for bias is 3.5%). Regarding the other

    parameters for which percent changes were calculated,

    the bias was not higher than that recommended.

    To conclude, we recommend that CBC and DIFF

    analysis should be performed within 6 h after blood

    sample collection. In samples analyzed 24 h after col-

    lection, a decreased NEUT-X, which is associated with

    morphological degradation of neutrophils, has to be

    interpreted cautiously and, in particular, should not

    be used for detecting MDS. This recommendation has

    to be taken into account when blood samples are col-

    lected at home or in clinical trials, where analysis is

    sometimes performed in remote analytical platforms.

    In cases where the time between collection and analy-

    sis is longer than 6 h, we recommend that samples

    should be stored at +4 C to maintain the stability of

    CBC/DIFF parameters and a good reliability of results.

    Complying with ISO requirements, especially for con-

    trolling the pre-analytical phase, appears to be imper-

    ative to avoid erroneous conclusions.

    DISCLOSURES

    The authors disclosed no potential conflicts of interest.

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    6 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES

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