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

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<ul><li><p>Guidance for storing blood samples in laboratories performing</p><p>complete blood count with differentialE. CORNET*, ,1, C. BEHIER*,1, X. TROUSSARD*,</p><p>*CHU de Caen, Laboratory of</p><p>Hematology, Caen, FranceUniversity of Caen, Medical</p><p>School, Caen, France</p><p>Correspondence:</p><p>Edouard Cornet, Laboratory of</p><p>hematology, CHU de Caen,</p><p>Avenue Cote de Nacre, 14033</p><p>Caen Cedex 9, France.</p><p>Tel.: +33 2 31 06 33 15;</p><p>Fax: +33 2 31 06 50 99;</p><p>E-mail:</p><p>doi:10.1111/j.1751-553X.2012.01452.x</p><p>Received 1 February 2012;</p><p>accepted for publication 8 May</p><p>2012</p><p>Keywords</p><p>Blood cell count, laboratories/</p><p>standards, reproducibility of</p><p>results, blood preservation, tem-</p><p>perature</p><p>SUMMARY</p><p>Introduction The complete blood count (CBC) with differential leu-</p><p>kocyte count (DIFF) is an important part of clinical laboratory anal-</p><p>yses and provides crucial data for clinicians. Delivery time after</p><p>blood collection and conditions of storage is known to affect the</p><p>reliability of results of some hematologic parameters. The aim of</p><p>this study was to assess the variations of hematologic parameters</p><p>over time and the influence of storage temperature.</p><p>Methods Blood samples were randomly selected from hospitalized</p><p>patients and stored at room temperature and at 4 C. CBC andDIFF were performed on an automated hematology analyzer and</p><p>the results between the two groups were compared.</p><p>Results Samples stored at room temperature showed an important</p><p>increase in mean corpuscular volume and hematocrit and a</p><p>decrease in mean corpuscular hemoglobin concentration. Neutro-</p><p>phil counts tended to increase, whereas monocyte counts tended to</p><p>decrease.</p><p>Conclusion Storing samples at 4 C improved reproducibility overtime of all quantitative and qualitative parameters. We also</p><p>observed that NEUT-X, a routine parameter useful in detecting</p><p>myelodysplastic syndrome, became unreliable when analyzed 24 h</p><p>after sample collection. Our results led us to recommend that sam-</p><p>ples should be analyzed within 6 h, particularly if samples are</p><p>transported at room temperature. We also recommend storing sam-</p><p>ples at 4 C in case of remote CBC analysis, especially in the con-text of clinical trials.</p><p>INTRODUCTION</p><p>The complete blood count (CBC) with differential leu-</p><p>kocyte count (DIFF) is one of the most routinely per-</p><p>formed laboratory tests in our institution, with about</p><p>190 000 analyses performed in 2011. It is a valuable</p><p>screening tool for a wide variety of medical conditions</p><p>and is an essential routine in the optimal management</p><p>of patients. Compliance with International Organiza-</p><p>tion for Standardization (ISO) 15 189, the international1These authors contributed equally to this work.</p><p> 2012 Blackwell Publishing Ltd, Int J Lab Hem. 1</p><p>ORIGINAL ARTICLE INTERNATIONAL JOURNAL OF LABORATORY HEMATOLOGY</p><p>International Journal of Laboratory HematologyThe Official journal of the International Society for Laboratory Hematology</p></li><li><p>standard for the accreditation of medical laboratories</p><p>(1), is becoming progressively accepted as the optimal</p><p>approach to assure quality in medical testing.</p><p>The main objective of any clinical laboratory is to</p><p>produce results that are accurate and precise enough</p><p>for clinical use, and therefore, the correct pre-analyti-</p><p>cal procedures are essential. Many external variables</p><p>can influence laboratory results, including conditions</p><p>of blood collection, storage and delivery time of sam-</p><p>ples to the laboratory. It is particularly critical when</p><p>blood samples are collected at the patients home.</p><p>Delivery time to the laboratory can exceed 8 h.</p><p>The development of clinical trials and centralized</p><p>analyses will impose adoption of strict pre-analytical</p><p>rules. Healthcare laboratories must establish limits</p><p>for delivery time and conditions of storage and the</p><p>impact of pre-analytical conditions on hematologic</p><p>parameters must be known for accurate interpre-</p><p>tation of all parameters provided by an automated</p><p>analyzer.</p><p>This study confirms the impact of pre-analytical</p><p>conditions on variations of CBC-DIFF parameters and</p><p>also highlights new data to interpret NEUT-X, which</p><p>is a routine parameter useful in detecting myelodys-</p><p>plastic syndrome (MDS). Performing CBC/DIFF analy-</p><p>sis within 6 h after sample collection guarantees good</p><p>reliability of the results.</p><p>MATERIALS AND METHODS</p><p>Samples and analyzer</p><p>We studied 64 venous peripheral blood samples,</p><p>randomly selected among hospitalized patients in</p><p>Caen University Hospital. Samples were collected in</p><p>vacutainers containing dipotassium ethylene diamine-</p><p>tetraacetate (K2EDTA BD Vacutainer; Becton and</p><p>Dickinson, Le Pont-De-Claix, France), which is the</p><p>anticoagulant of choice in specimen collection for</p><p>blood cell counting and sizing, as recommended by</p><p>the International Council for Standardization in</p><p>Hematology (2). This salt also has been shown to</p><p>affect red blood cell size less than tripotassium salt</p><p>(3). Thirty-two samples were stored at room tempera-</p><p>ture (RT) (Group 1) and 32 at +4 C (Group 2). CBC/</p><p>DIFF was processed at different time points during a</p><p>period of 3 days, that is, at delivery (t0) then 6, 12,</p><p>24, 48 and 72 h (t6, t12, t24, t48 and t72). The maxi-</p><p>mum time interval between blood collection and sam-</p><p>ple analysis at t0 (reference values) was 4 h. All</p><p>samples were normal blood samples, without any</p><p>qualitative abnormalities.</p><p>Laboratory methods</p><p>Analyses were performed in automatic mode on a</p><p>Sysmex XE-2100 automated multiparameter hematol-</p><p>ogy analyzer (Sysmex Corporation, Kobe, Japan) in</p><p>accordance with manufacturers recommendations.</p><p>The analyzer was calibrated by the manufacturer.</p><p>Two levels of controls (internal quality control) were</p><p>run on a daily basis (e-check XE control) according</p><p>to manufacturers recommendations. Only the data</p><p>from the CBC, DIFF and Immature Myeloid Informa-</p><p>tion channels were taken into account, we did not</p><p>consider reticulocyte count or nucleated red blood</p><p>cell count (NRBC) in this analysis. Parameters of</p><p>CBC measured in this study were the following:</p><p>white blood cell count (WBC), red blood cell count</p><p>(RBC), hemoglobin concentration (HB), hematocrit</p><p>(HCT), mean corpuscular volume (MCV), mean corpus-</p><p>cular hemoglobin (MCH), mean corpuscular hemo-</p><p>globin concentration (MCHC) and platelet count (PLT).</p><p>The study assessed relative percentages of clusters: neu-</p><p>trophil (NEU%), eosinophil (EOS%), basophil (BASO</p><p>%), lymphocyte (LYMPH%), monocyte (MONO%)</p><p>and immature granulocyte (IG%). All presented per-</p><p>centages were automatically generated parameters.</p><p>Statistical analysis</p><p>As previously described in the literature, stability of</p><p>an analyte was defined in relation to the imprecision</p><p>of the respective analytical method. A parameter was</p><p>considered stable when its average change was smaller</p><p>than one coefficient of variation (CV, %) of the</p><p>assessed method, allowing a 5% risk of error (4, 5).</p><p>CV was obtained with daily controls. Except for EOS</p><p>%, BASO% and IG%, we evaluated the variability of</p><p>each parameter at each time point by calculating the</p><p>mean percent change from the initial value (t0). We</p><p>evaluated the variability of EOS%, BASO% and IG%</p><p>by calculating the mean absolute difference from the</p><p>initial value rather than percent change because some</p><p>initial values were at zero. Statistical analyses were</p><p>performed for the two groups. Mean, standard</p><p> 2012 Blackwell Publishing Ltd, Int J Lab Hem.</p><p>2 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES</p></li><li><p>deviation (SD) and 95% confidence interval (95% CI)</p><p>were calculated with MicrosoftTM Excel software.</p><p>RESULTS</p><p>Samples characteristics</p><p>All samples were normal blood samples. Characteris-</p><p>tics of the two groups were comparable for all mea-</p><p>sured parameters (P &gt; 0.05) (Table 1). We considered</p><p>values at t0 as reference values.</p><p>CBC parameters</p><p>Many parameters of CBC were stable throughout the</p><p>study period. No significant difference was observed</p><p>for WBC, RBC, HB, MCH and PLT in Group 1 and</p><p>Group 2 until 72 h. Conversely, there was a signifi-</p><p>cant MCV difference from t0 to t6 in Group 1. At 6 h,</p><p>the percent change was 4.2% (95% CI, 1.91; 6.42)</p><p>and regularly increased, reaching 17.5% (16.6418.42)</p><p>at 72 h (CV for MCV was 0.96%) (Figure 1). In</p><p>Group 2, percent changes at t6 and t72 were, respec-</p><p>tively, 1.2% (1.08; 1.31) and 1.5% (1.14; 1.85).</p><p>Hence, MCV increased at both RT and +4 C but this</p><p>change was more limited in case of refrigerated stor-</p><p>age. As a consequence, we also observed a significant</p><p>variation of MCV-related parameters at RT with a pro-</p><p>gressive decrease in MCHC and a progressive increase</p><p>in HCT.</p><p>DIFF parameters</p><p>No significant variations of the WBC count were</p><p>detected between t0 and t72. Only samples from</p><p>Group 1 showed an increase in NEU% from t48</p><p>(Figure 1). Percent change at t72 was +8.2% (5.84;</p><p>10.54) (CV for NEU% was 1.62%). In contrast,</p><p>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</p><p>showed no LYMPH% variation, and no significant</p><p>variation was observed for EOS%, BASO% and IG%.</p><p>DISCUSSION</p><p>A few studies have analyzed variations of CBCDIFF</p><p>with regard to the storage of blood specimens at room</p><p>temperature (4, 69). All of these studies described an</p><p>increase in MCV and a variation of its related</p><p>parameters (HCT and MCHC). These observations</p><p>were independent of the type of automated analyzer.</p><p>Similarly, our study demonstrated a significant varia-</p><p>tion of many hematologic parameters over 3 days</p><p>related to storage conditions of blood. The largest vari-</p><p>ation concerned erythrocyte parameters. MCV</p><p>increased dramatically from 6 h if samples were kept</p><p>at RT, but was limited in case of refrigerated storage</p><p>(+4 C). This observation could be related to a swell-</p><p>ing of red blood cell because of a decrease in mem-</p><p>brane resistance and an increase in cell permeability.</p><p>This phenomenon seemed to depend on storage dura-</p><p>tion and temperature. As expected, these modifica-</p><p>tions also affected MCV-related parameters, such as</p><p>HCT and MCHC, which are calculated from the MCV,</p><p>with an increase in HCT and a decrease in MCHC. As</p><p>previously described (6), RBC, HB, MCH and PLT</p><p>measurements were unmodified until 72 h at RT.</p><p>Table 1. Initial characteristics (t0) of the 32 samplesstored at room temperature (Group 1) and the 32</p><p>samples stored at +4 C (Group 2)</p><p>Group 1 (RT)</p><p>Group 2 (+4C)</p><p>PMean SD Mean SD</p><p>White blood cell</p><p>count (109/L)</p><p>9.75 3.83 8.50 4.83 0.25575 (NS)</p><p>Red blood cell</p><p>count (1012/L)</p><p>4.04 0.83 3.86 0.76 0.38470 (NS)</p><p>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)</p><p>Mean corpuscularvolume (fl)</p><p>90.3 5.9 90.4 5.5 0.94268 (NS)</p><p>Mean corpuscularhemoglobin (pg)</p><p>30.6 2.3 30.4 2.4 0.72081 (NS)</p><p>Mean corpuscularhemoglobin</p><p>concentration (g/dL)</p><p>33.8 1.3 33.5 1.4 0.39222 (NS)</p><p>Platelet count(109/L)</p><p>224 87 225 87 0.97345 (NS)</p><p>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)</p><p>Lymphocytes (%) 18.22 10.84 22.36 14.12 0.19332 (NS)Monocytes (%) 10.75 4.28 10.15 5.33 0.61772 (NS)</p><p>Immaturegranulocytes (%)</p><p>0.74 0.92 1.01 1.49 0.38757 (NS)</p><p>Results are expressed as mean and standard deviation</p><p>(SD). NS: Not significant.</p><p> 2012 Blackwell Publishing Ltd, Int J Lab Hem.</p><p>E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES 3</p></li><li><p>RBC</p><p>t6 t12 t24 t48 t72 t6 t12 t24 t48 t72 t6 t12 t24 t48 t72</p><p>t6 t12 t24 t48 t72t6 t12 t24 t48 t72t6 t12 t24 t48 t72</p><p>t6 t12 t24 t48 t72 t6 t12 t24 t48 t72 t6 t12 t24 t48 t72</p><p>t6 t12 t24 t48 t72t6 t12 t24 t48 t72</p><p>2</p><p>1</p><p>0</p><p>1</p><p>RT+4 C</p><p>1 CV%(0.62%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>HB</p><p>3</p><p>2</p><p>1</p><p>0</p><p>1</p><p>RT+4 C</p><p>1 CV%(0.68%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>efr</p><p>om re</p><p>fere</p><p>nce </p><p>valu</p><p>e (t 0</p><p>)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>from</p><p> refe</p><p>renc</p><p>e va</p><p>lue </p><p>(t 0)</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>HCT</p><p>0</p><p>5</p><p>10</p><p>15 RT+4 C</p><p>1 CV%(1.05%)P</p><p>erce</p><p>nt c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>MCV</p><p>0</p><p>5</p><p>10</p><p>15</p><p>20RT+4 C</p><p>1 CV%(0.96%)P</p><p>erce</p><p>nt c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>MCH</p><p>1</p><p>0</p><p>1</p><p>RT+4 C</p><p>1 CV%(0.81%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>MCHC</p><p>20</p><p>15</p><p>10</p><p>5</p><p>0</p><p>5</p><p>RT+4 C</p><p>1 CV%(1.25%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>PLT</p><p>5</p><p>0</p><p>5</p><p>10RT+4 C</p><p>1 CV%(2.81%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>WBC</p><p>2</p><p>0</p><p>2</p><p>4</p><p>6RT+4 C</p><p>1 CV%(2.15%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>NEU%</p><p>10</p><p>5</p><p>0</p><p>5</p><p>10 RT+4 C</p><p>1 CV%(1.62%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>MONO%</p><p>60</p><p>40</p><p>20</p><p>0</p><p>20</p><p>RT+4 C</p><p>1 CV%(7.08%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>LYMPH%</p><p>15105</p><p>05</p><p>1015</p><p>RT+4 C</p><p>1 CV%(2.91%)</p><p>Perc</p><p>ent c</p><p>hang</p><p>e</p><p>% - </p><p>Mea</p><p>n (C</p><p>I95%</p><p>)</p><p>* **</p><p>**</p><p>* **</p><p>* *</p><p>* **</p><p>**</p><p>**</p><p>**</p><p>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</p><p>interval (95% CI). A parameter was considered as unstable if its average change (with 95% CI) was higher than</p><p>the coefficient of variation (CV) of the method. Results are presented for samples stored at room temperature (RT)</p><p>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;</p><p>MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PLT, platelet count;</p><p>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</p><p>variations were observed at RT for NEUT% (increase) and MONO% (decrease) from t48.</p><p> 2012 Blackwell Publishing Ltd, Int J Lab Hem.</p><p>4 E. CORNET, C. BEHIER AND X. TROUSSARD | GUIDANCE FOR STORING BLOOD SAMPLES</p></li><li><p>In our study, leukocyte count (WBC) was</p><p>unchanged until 72 h at both RT and +4 C. Modifica-</p><p>tions of DIFF were exclusively observed at RT, with</p><p>an increase in NEU% and a decrease in MONO%.</p><p>Indeed, modification of cellular structure over time</p><p>could alter detection and identification of cells, partic-</p><p>ularly if blood specimens are stored at RT. To evaluate</p><p>this progressive cellular deterioration, we tested sec-</p><p>ondarily eight normal blood samples at RT (data not</p><p>shown). In addition to the usual DIFF parameters, we</p><p>also considered the neutrophil structural and matura-</p><p>tion parameters provided by our analyzer: NEUT-X</p><p>(10). NEUT-X is the direct measurement of side scatter</p><p>diffraction and is representative of neutrophil struc-</p><p>ture. We compared NEUT-X with manual differential</p><p>and cell morphology analyzed by microscopic meth-</p><p>ods. Microscopic DIFF count became unreliable from</p><p>48 h, with a decrease in monocyte count and an</p><p>increase in neutrophil count....</p></li></ul>


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