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 Table of Contents  
Year : 2022  |  Volume : 9  |  Issue : 1  |  Page : 57-61

Effect of room temperature and refrigerated storage on automated complete blood count: A longitudinal study

1 Department of Pathology, Dr. Somervell Memorial C.S.I. Medical College and Hospital, Karakonam, Kerala, India
2 Department of Pathology, MM Medical College and Hospital, Kumarhatti, Solan, Himachal Pradesh, India
3 Department of Clinical Haematology, Haemato-Oncology and Bone Marrow (Stem Cell) Transplantation, Christian Medical College and Hospital, Ludhiana, Punjab, India

Date of Submission04-Oct-2020
Date of Decision29-Jul-2021
Date of Acceptance29-Jul-2021
Date of Web Publication18-Oct-2022

Correspondence Address:
Naveen Kakkar
Department of Pathology, Maharishi Makandeshwar Medical College and Hospital, Kumarhatti - 173 229, Solan, Himachal Pradesh
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/cjhr.cjhr_140_20

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Objective: Delay in complete blood count (CBC) analysis is likely in laboratories with very high workload or when the samples are collected off site for testing in centralized laboratories. This research was done to study the stability of blood cell parameters in automated CBC at the room temperature (20°C–24°C) storage after 24 h and refrigerated (2°C–8°C) storage after 24 and 48 h. Materials and Methods: Automated CBC was done initially on 500 randomly selected blood samples in a 3-part hematology analyzer. Of these, 250 samples were stored at the room temperature (20°C–24°C), and the rest were refrigerated at 2°C–8°C. Re-analysis after 24 h was done for both groups. Data were analyzed using the Pearson correlation coefficient, ANOVA, and repeated-measure ANOVA. Results: Refrigerated samples stored at 2°C–8°C showed good stability for all hematological parameters (P > 0.5), except for mean platelet volume (MPV) in comparison with those at the room temperature storage. MPV showed a statistically significant (P < 0.001) rise on refrigerated storage. In the room temperature storage group, hematocrit (Hct), mean corpuscular volume, mean corpuscular hemoglobin concentration, red cell distribution width, MPV, and granulocyte percentage showed significant difference compared to the initial values. In the extended 48 h refrigerated storage group, all CBC parameters except Hct showed acceptable stability. Conclusion: The storage of blood samples at the room temperature and on refrigeration (2°C–8°C) for 24 h has shown acceptable stability of most of the automated CBC parameters. Refrigeration retained the true values over the room temperature storage and should be recommended if a delay in testing is expected.

Keywords: Complete blood count, refrigeration, room temperature, stability, storage

How to cite this article:
Jaya A, Kakkar N, John M. Effect of room temperature and refrigerated storage on automated complete blood count: A longitudinal study. CHRISMED J Health Res 2022;9:57-61

How to cite this URL:
Jaya A, Kakkar N, John M. Effect of room temperature and refrigerated storage on automated complete blood count: A longitudinal study. CHRISMED J Health Res [serial online] 2022 [cited 2022 Nov 27];9:57-61. Available from: https://www.cjhr.org/text.asp?2022/9/1/57/358812

  Introduction Top

Complete blood count (CBC) is one of the most commonly ordered investigations in clinical practice and is an important guide to the diagnosis in many disorders.[1] Maintaining optimal specimen quality for testing is vital for accurate test results.[2] The use of automated analyzers for CBC testing in hematology laboratories has been increasing steadily. Ideally, the blood samples for CBC should be processed as soon as possible or within 4 h.[3] However, on many occasions, blood samples need to be transported to centralized laboratories for testing. Furthermore, in busy laboratories with high throughput, there may be delay in sample processing. As cellular elements of blood have limited stability in anticoagulated blood, a delay in testing leads to suboptimal specimen quality that affects test results and may prompt re-analysis.[4] This in turn leads to delay or difficulty in clinical decision making and also adds to the health-care expense.[5]

Of all the preanalytical variables which affect the state of the sample, age of the specimen and ambient temperature are the vital factors that affect the test results.[3] Various studies have shown differences in the stability of hematology parameters following prolonged storage at the room temperature and on refrigeration. Previous studies have shown that automated hematological parameters such as hematocrit (Hct) and mean corpuscular volume (MCV) increase when blood is allowed to stand at the room temperature while white cell count and platelet count fall. The parameters show better stability on refrigerated storage.[2],[3],[4],[6],[7],[8],[9],[10],[11],[12],[13] This study was conducted to evaluate the effect of room temperature and refrigerated storage on automated CBC.

  Materials and Methods Top

Study setting

This prospective study was done in the hematology section of a tertiary care hospital in North India over a period of 1 year and has been approved by the institutional research and ethics committee. Details of the patients, including age, sex, and clinical diagnosis, were recorded from the test request forms.

Sample size and study groups

Five hundred blood samples from patients sent for CBC for various hematological and nonhematological disorders were included. These were divided into two study groups: (a) 250 samples: 24 h room temperature (20°C–24°C) storage and (b) 250 samples: 24 h refrigerated (2°C–8°C) storage. Of the refrigerated group, 50 samples were taken up for extended 48 h refrigerated storage [Figure 1]. The sample size was calculated for a confidence interval of 95% and a 90% power of the study. The samples with blood volume less than that recommended for the sample container were excluded.
Figure 1: Study design for the samples (n = 500) included in the study

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The selection of samples was done by using random tables. Further allocation to the room temperature or refrigerated storage group was done by block randomization. Venous blood was collected in evacuated containers containing dipotassium ethylenediaminetetraacetic acid. Automated CBC was done on Mindray, BC3000 plus, a three-part automated hematology analyzer. The instrument generates 18 numerical parameters and three histograms for red cells, white cells, and platelets. The WBC histogram generated by the analyzer divides the white cell population into three subpopulations based on cell volumes-lymphocytes (30–80 fl), mid zone (monocytes and eosinophils) (60–120 fl), and granulocytes (120–250 fl).

The initial automated CBC was done within 30 min of sample collection. For the 24 h room temperature storage (20°C–24°C) group, the samples were then kept at the room temperature for 24 h and re-analyzed. For the 24 h refrigerated storage (24 h at 2°C–8°C) group, samples were stored at 2°C–8°C in the refrigerator for 24 h. After 24 h, the samples were equilibrated at the room temperature for 15 min and re-analyzed. Of these samples, 50 samples were refrigerated for extended storage and re-analyzed after 48 h [Figure 1].

Since in three part differential hematology analyzers, the mid population does not accurately reflect the monocyte and eosinophil population, only the major granulocyte and lymphocyte zones were considered for the comparative analysis.

Quality control measures

Daily temperature monitoring for room as well as refrigerator temperature was done. A precision check (coefficient of variation) for the automated analyzer was done at the beginning of the study and at 6 months for all parameters to be assessed during the study. This was found to be <2.5% for all parameters.

Statistical analysis

Data were analyzed using ANOVA, repeated-measure ANOVA, and Pearson correlation coefficient.

  Results Top

Of the 500 patients included in the study, 86% were adults. The male: female ratio was 1.2:1. The automated parameters encompassed patients with cytopenias, normal and raised values across various disease spectra. The automated CBC parameters of the patients included in the study are shown in [Table 1]. In the room temperature storage group, Hct, MCV, mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), mean platelet volume (MPV), and granulocyte percentage showed significant difference compared to the initial values. Refrigerated samples stored at 2°C–8°C showed good stability among all the hematological parameters (P > 0.5), except for MPV which showed a significant rise (P < 0.001) in comparison with room temperature storage value [Table 2].
Table 1: Baseline automated complete blood count parameters of patients included in the study (n=500)

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Table 2: Comparison of complete blood count parameters between initial, 24 h room temperature (n=250) and 24 h refrigerated group (n=250)

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The automated WBC differential for granulocytes and lymphocytes showed good stability when initial counts were compared with the room temperature and refrigerated storage. Refrigerated storage showed better stability compared to the room temperature storage for both granulocytes (r - 0.86 vs. 0.96) and lymphocytes (r - 0.91 vs. 0.96) [Figure 2]. Granulocytes showed a significant decrease (P < 0.5) at the room temperature.
Figure 2: Comparison of automated differential count for granulocytes (a and b) and lymphocytes (c and d) after 24 h storage. Granulocytes showed good stability at room temperature (a) (r = 0.86) which improved on refrigeration (b) (r = 0.96). Lymphocytes also showed good stability on 24 h room temperature storage (c) (r = 0.91) which improved on refrigerated storage (d) (r = 0.96)

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Automated CBC parameters for the 50 patients in whom extended 48 h refrigerated storage at 2°C–8°C was done showed good stability (P > 0.05) for most hematological parameters. Only Hct showed a statistically significant difference (P = 0.03).

  Discussion Top

Our study has shown statistically insignificant change for all automated CBC parameters with 24 h storage on refrigeration except for MPV. MCV, RDW, Hct, and MPV increased on room temperature storage while MCHC and granulocytes showed a fall. In the extended 48 h storage group, all CBC parameters except Hct showed acceptable stability.

A study of 102 CBC samples storage for 48 h at the room temperature showed findings similar to our study. The authors reported an increase in MCV, RDW, and MPV and a decrease in MCHC.[6] Another study by Cornet et al. in 64 CBC samples stored at the room temperature showed an increase in MCV and Hct and a decrease in MCHC. Similar to our study, the stability of the parameters improved with refrigerated storage.[7]

In a similar study done by Gulati et al. on 40 blood samples after 24 h room temperature storage, Hb, RBC count, MCH, WBC, and platelet counts showed no significant change. MCV, Hct, and RDW showed a statistically significant increase (P < 0.05).[5]

Studies by Cohle et al. and de Baca et al. also showed statistically significant increase in MCV at the room temperature after storage for 24 h.[8],[9] In a study on 30 samples, Mahmoodi et al. concluded that Hb, Hct, MCH, WBC, and platelet counts were stable till 24 h room temperature with a decrease in MCV.[14] Wood et al. in their study of 113 patients and 21 healthy volunteers also had similar findings with Hb, RBC count, and platelet count stable for 24 h with an increase in MCV. However, unlike our study, they observed a significant decrease in WBC count on refrigerated storage.[2]

Another study in 117 samples showed Hb, RBC count, WBC, and platelet count to be stable for 24 h at the room temperature with rise in MCV, RDW, and MPV while MCHC decreased compared to the initial values. On refrigerated storage, lower mean WBC and platelet count with an increase in MCV, MPV, and MCH were seen at 24 h.[15] This was in contrast to our study which showed a significant increase of MPV alone. Oliveira et al. studied 651 samples at 2, 24, 48, and 72 h. They reported RBC count, Hb, and MCH to be stable up to 72 h at room temperature and at 4°C whereas the rest of the parameters showed imprecision irrespective of the temperature.[16]

In a meta-analysis of 89 studies, Wu et al. reported that except MPV, most parameters were stable for 24 h at the room temperature with WBC, Platelets, HCT, Hb, and MCH stable up to 3 days.[17]

Other studies have showed variation in stability different parameters after 24 h room temperature storage.[10],[18],[19]

In our study, following room temperature storage, the automated differential showed good correlation when initial counts were compared with the 24 h storage values. However, the granulocyte percentage showed a significant decrease. Refrigeration improved the granulocyte stability. Lymphocyte percentage showed good correlation (r. 0.90) and no statistically significant variation between the room temperature and refrigerated groups when compared with the initial values.

Other studies have also shown a decrease in automated granulocyte and an increase in lymphocyte percentages after sample storage at the room temperature for 24 h.[2],[5],[9],[14],[20]

Wood et al. attributed the fall in automated granulocyte percentage due to expansion and loss of definition of the neutrophil cluster, blurring the boundary with the lymphocyte cluster and resulting in inclusion of neutrophils with the lymphocyte population.[2]

Hill et al. in their study of 166 samples observed excellent correlation with no significant change for automated granulocytes and lymphocytes at 24 h room temperature storage.[19] Tangvarasittichai and Tangvarasittichai also reported acceptable stability of the automated leukocyte differential for a period of 72 h at 4°C.[11] Few studies have reported a slight increase in the neutrophil population with a proportional decrease in the lymphocyte population on 24 h refrigerated storage.[2],[12] A slight decrease in neutrophils and lymphocytes at 24 h refrigerated storage was observed by Hedberg et al unlike our study.[13] Another study done by Grimaldi and Scopacasa observed a statistically significant progressive decrease in the number of neutrophils especially at 4°C, unlike the present study.[21]

The variation in stable automated CBC parameters after storage in different hematology analyzers and even across different laboratories may differ. This could be a reflection of local environmental conditions and different instruments used in these studies. The comparison of parameter stability on similar analyzers may provide more robust data. Although many studies have evaluated the effect of prolonged sample storage, it is encouraged that laboratories define their own criteria for parameter stability in their local conditions.

Even though the variation in some of the hematological parameters in the study was statistically significant, it may not always translate to clinical significance. However, the findings of our study support those of similar studies that refrigeration be preferred over room temperature storage if a delay in analysis of the sample is expected.

In tropical countries, where temperature variations within laboratories are extreme, the findings from the room temperature storage group might not be reproducible in real-life scenarios, and refrigeration of the sample is always a safer option. This is relevant when there are offsite laboratories which perform these tests between 4 and 24 h after the collection of sample without appropriate storage conditions. While most of changes in RBC parameters have not much routine clinical relevance, increase in MCV and RDW could possibly mask an underlying iron deficiency state or overlook a possible suspicion of thalassemia carrier state in populations with a high prevalence of these conditions.

A limitation of our study is that we did not consider the mid zone population for changes after prolonged storage. This was done as three part differential hematology analyzers do not reflect the monocyte and eosinophil population accurately unlike five part analyzers. Similar studies on five part analyzers may be able to enumerate the stability of monocytes, eosinophils, and basophils.

  Conclusion Top

Storage of blood samples at the room temperature and refrigeration 2°C–8°C for 24 h has shown acceptable stability of most of the automated CBC parameters across normal and pathological ranges. Refrigeration improved the stability over room temperature storage and should be recommended, especially if evaluating for iron-deficiency anemia or thalassemia carrier state. Blood samples that are likely to have a delayed analysis should be refrigerated.

  Acknowledgment Top

The study was conducted as a thesis in the Department of Pathology at Christian Medical College and Hospital, Ludhiana (Punjab).

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Tefferi A, Hanson CA, Inwards DJ. How to interpret and pursue an abnormal complete blood cell count in adults. Mayo Clin Proc 2005;80:923-36.  Back to cited text no. 1
Wood BL, Andrews J, Miller S, Sabath DE. Refrigerated storage improves the stability of the complete blood cell count and automated differential. Am J Clin Pathol 1999;112:687-95.  Back to cited text no. 2
Buttarello M. Quality specification in haematology: The automated blood cell count. Clin Chim Acta 2004;346:45-54.  Back to cited text no. 3
Lewis SM, Tatsumi N. Collection and handling of blood. In: Lewis SM, Bain BJ, Bates I, editors. Dacie and Lewis Practical Haematology. 12th ed. New Delhi: Elsevier; 2017. p. 5-9.  Back to cited text no. 4
Gulati GL, Hyland LJ, Kocher W, Schwarting R. Changes in automated complete blood cell count and differential leucocyte count results induced by storage of blood at room temperature. Arch Pathol Lab Med 2002;126:336-42.  Back to cited text no. 5
Gunawardena D, Jayaweera S, Madhubhashini G, Lokumarakkala DD, Senanayake SJ. Reliability of parameters of complete blood count with different storage conditions. J Clin Lab Anal 2017;31:e22042.  Back to cited text no. 6
Cornet E, Behier C, Troussard X. Guidance for storing blood samples in laboratories performing complete blood count with differential. Int J Lab Hematol 2012;34:655-60.  Back to cited text no. 7
Cohle SD, Saleem A, Makkaoui DE. Effects of storage of blood on stability of hematologic parameters. Am J Clin Pathol 1981;76:67-9.  Back to cited text no. 8
de Baca ME, Gulati G, Kocher W, Schwarting R. Effects of storage of blood at room temperature on hematologic parameters measured on Sysmex XE-2100. Lab Med 2006;37:28-36.  Back to cited text no. 9
Tendulkar A, Jain P, Gujral S, Tambe M, Kenjale R, Ganesh B. Stability of selected hematological parameters in stored blood samples. J Cell Sci Ther 2015;6:5.  Back to cited text no. 10
Tangvarasittichai O, Tangvarasittichai S. Predictive WBC count and differential WBC on time variation. Siriraj Med J 2008;60:248-50.  Back to cited text no. 11
Imeri F, Herklotz R, Risch L, Arbetsleitner C, Zerlauth M, Risch GM, et al. Stability of haematological analytes depends on the hematology analyzer used: A stability study with Bayer Advia 120, Beckman Coulter LH 750 and Sysmex XE 2100. Clin Chim Acta 2008;397:68-71.  Back to cited text no. 12
Hedberg P, Lehto T. Aging stability of complete blood count and white blood cell differential parameters analyzed by Abbott CELL-DYN Sapphire hematology analyzer. Int J Lab Hematol 2009;31:87-96.  Back to cited text no. 13
Mahmoodi M, Hajizadeh M, Rashidinejad H, Asadikaram G, Khaksari M, Mirzaee M, et al. Survey of changes in complete blood count and red cell indices of whole blood incubated in vitro at different temperatures up to 48 hours. J Ayub Med Coll Abbottabad 2006;18:14-6.  Back to cited text no. 14
Turhan T, Sezer S, Yücel C, Koca Y. Effects of storage conditions on complete blood cell count parameters. Turk J Biochem 2011;36:165-74.  Back to cited text no. 15
Oliveira LR, Simionatto M, Cruz BR, Bittencourt JI, Krum EA, Moss MF, et al. Stability of complete blood count in different storage conditions using the ABX PENTRA 60 analyzer. Int J Lab Hematol 2018;40:359-65.  Back to cited text no. 16
Wu DW, Li YM, Wang F. How long can we store blood samples: A systematic review and meta-analysis. EBioMedicine 2017;24:277-85.  Back to cited text no. 17
Obeidi N, Safavi E, Emami H. Evaluation of the effect of temperature and time of incubation on complete blood count (CBC) tests. Afr J Biotechnol 2012;11:1761-3.  Back to cited text no. 18
Hill VL, Simpson VZ, Higgins JM, Hu Z, Stevens RA, Metcalf JA, et al. Evaluation of the performance of the Sysmex XT-2000i Hematology Analyzer with whole blood specimens stored at room temperature. Lab Med 2009;40:709-18.  Back to cited text no. 19
Vogelaar SA, Posthuma D, Boomsma D, Kluft C. Blood sample stability at room temperature for counting red and white blood cells and platelets. Vascul Pharmacol 2002;39:123-5.  Back to cited text no. 20
Grimaldi E, Scopacasa F. Evaluation of the Abbott CELL-DYN 4000 hematology analyzer. Am J Clin Pathol 2000;113:497-505.  Back to cited text no. 21


  [Figure 1], [Figure 2]

  [Table 1], [Table 2]


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