Introduction

Knowledge of the effects of temperature on blood sample stability is important in a haematology laboratory and blood bank setting. Exposure to high environmental temperatures (e.g. above 30 °C), which are quite common in under-resourced settings in tropical countries, may lead to variation and errors in the reporting of different haematology parameters. Guidelines have stated that processing of samples should be done within 6 to 8 hours (1, 2). However, there is scant evidence that samples maintain their analytical stability when stored for such time at high environmental temperatures. Most studies have shown that haemoglobin (Hb), red blood cell count (RBC), white blood cell count (WBC), platelet count (Plt) and mean corpuscular haemoglobin (MCH) are stable for at least 24 hours at 4 °C and between 20 °C and 25 °C (3). Mean corpuscular volume (MCV), mean corpuscular haemoglobin concentration (MCHC), red cell distribution width (RDW) and haematocrit (Hct) were stable for at least 6 hours at 4 °C and between 20 °C and 25 °C. However, we found only one study in English on analytical stability providing numerical data for most haematological parameters on samples stored above 30 °C (4).

Therefore, the present study estimated the analytical stability of different haematological parameters over a period of 72 hours in samples stored at 33 °C.

Materials and methods

Results

Platelets, Hct, MCV, MCHC, RDW and MPV showed unacceptable bias (which was both clinically as well as statistically significant) within 4 hours of storage at 33 °C (Tables 1 and 2{ label needed for table-wrap[@id='t2'] }). The mean and median of the percent differences and their 95% confidence intervals are given in Supplementary tables 1 and 2{ label needed for table-wrap[@id='supplementary_table2'] }.

When tested for equivalence, the stable parameters were found to be RBC till 72 hours (P_holm: less than 0.001 at all time-points) and WBC, Hb and MCH till 24 hours (P_holm for WBC count and Hb: less than 0.001 up to 24 hours, P_holm for MCH: 0.009, 0.002, less than 0.001 and less than 0.001 at 4, 6, 12 and 24 hours respectively). WBC and Hb at 48 hours failed the equivalence tests.

Discussion

The present study demonstrates the appearance of systematic bias for Hct, MCV, MCHC, RDW, Plt and MPV when stored for 4 hours at 33 ˚C, as well as the stability of WBC, RBC, Hb and MCH results when stored for 24 hours at the same temperature. The rapid appearance of bias at 33 ˚C shows that the present recommendation of sample storage up to 6 or 8 hours is inadequate at high temperatures (1, 2). This study also shows the importance of temperature-controlled storage and transport boxes in blood camps and extra-institutional sample collection centres in areas exposed to high temperatures. In case such precautions cannot be taken (e.g. in a resource poor setting), interpretation should be done cautiously. RDW and Hct become unreliable markers. MCH may be preferable to MCV or MCHC. For Plt counts, an initial decrease of about 10% within the first few hours may be taken into account.

Other studies have also found rapid appearance of significant bias in Plt counts on storage above 30 °C (4, 8). Significant shift in RDW and MCHC within 6 hours has also been reported (4). In contrast to the present study, clinically significant bias in MCV was not reported after storage above 30 °C for 6 to 8 hours, possibly due to a lower precision in these studies as a result of not taking replicates. Also, a comparison of the present study with some of the previously published studies is not possible due to lack of numerical data for most results, and due to taking a lenient equivalence threshold of 10% (8, 9). Also, equivalence for analytes on storage for ostensibly stable parameters was not analysed in these studies.

In the present study, the samples stored at 33 °C were brought to room temperature at the time of analysis during the duration of the study, and then again restored to 33 °C. Such repeated “heat-cold” cycles may affect the analytical results. However, the primary conclusion of the study (i.e. the appearance of unacceptable bias even before 4 hours for most of the parameters after storage at 33 °C), remains unaffected by this limitation since the bias in these cases appeared before any “heat- cold “cycle. The acceptable bias for RBC, Hb, MCH and WBC for at least 24 hours at 33 °C should also be a robust finding; these parameters remain stable despite the potentially destabilizing influence of the “heat-cold” cycles.

The samples were taken from a healthy population; however, differences from patient samples are likely to be mild and should not affect the conclusions of the study. Also, storage stability may depend on the analyser used (10). Sysmex XP-100 uses impedance counts; counters using other techniques (e.g. optical counts) may show different results.

The present study has tried to fill a gap in published knowledge about the extent of systematic error arising due to sample storage arising in warm ambient temperatures using tests of difference as well as equivalence. In doing so, it demonstrates the stability of Hb, RBC, WBC and MCH for at least 24 hours. More importantly, it demonstrates rapid appearance of bias in RBC volume related parameters (Hct, MCV, RDW, MCHC) as well as Plt and MPV after sample storage at 33 °C, even within the currently published guideline of 6 hours. Thus the present study points out the necessity of special haematological sample storage guidelines for laboratories where such high ambient temperatures are common.