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ORIGINAL ARTICLE
Year : 2022  |  Volume : 25  |  Issue : 2  |  Page : 41-46

Effect of varying storage time and temperature on unspun blood samples for prothrombin time and activated partial thromboplastin time in a tertiary hospital laboratory in the tropics


1 Department of Haematology and Blood Transfusion, College of Medicine, University of Lagos and Lagos University Teaching Hospital, Idi-Araba, Lagos, Nigeria
2 Department of Haematology and Blood Transfusion, College of Medicine, University of Lagos, Idi-Araba, Lagos, Nigeria
3 Department of Haematology and Blood Transfusion, Lagos University Teaching Hospital, Idi-Araba, Lagos, Nigeria

Date of Submission28-May-2021
Date of Decision26-Aug-2021
Date of Acceptance21-Oct-2021
Date of Web Publication22-Aug-2022

Correspondence Address:
Dr. Oluwafikewa A Oyedele
Department of Haematology and Blood Transfusion, College of Medicine, University of Lagos, Idi-Araba, Lagos State
Nigeria
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/smj.smj_44_21

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  Abstract 


Introduction: Delays in the time of analysis of unspun blood samples stored at varying temperatures received in the laboratory pose a risk for unreliable prothrombin time (PT) and activated partial thromboplastin time (APTT) result; hence, consequent detrimental effect on patient care. This study's aim was thus to determine the optimal storage conditions and the potential effect of various storage times and temperatures on unspun samples for PT and APTT for a reliable test result. Materials and Methods: In a cross-sectional study, 33 eligible apparent healthy volunteers were recruited. Eighteen milliliters (ml) of venous blood were collected into 20 ml plastic bottles containing 2 ml of 0.109 M sodium citrate as an anticoagulant. Each citrated sample was separated into nine 2 ml aliquots. Baseline PT and APTT were determined with a coagulometer immediately and the remaining aliquots were analyzed after 3, 6, 12, and 24 h storage time at refrigerated (4°C) and room temperature (RT), respectively. The Statistical Package for the Social Sciences and Paired student t-test were used for statistical analysis. Results: At 24 h storage time at both RT and 4°C for PT, there was a statistically significant difference (P = 0.000). For APTT, a statistically significant difference was observed at 12 h (P = 0.009) and 24 h (P = 0.000) at RT whereas, at 4°C, all storage time had a statistically significant difference (P < 0.05). Conclusion: Unspun blood samples can be stored maximally for 12 h at RT and 4°C for PT whereas it is 6 h at RT only for APTT.

Keywords: Activated partial thromboplastin time, preanalytical variables, prothrombin time


How to cite this article:
Ogbenna AA, Oyedele OA, Adeyemo TA, Oyewole KM. Effect of varying storage time and temperature on unspun blood samples for prothrombin time and activated partial thromboplastin time in a tertiary hospital laboratory in the tropics. Sahel Med J 2022;25:41-6

How to cite this URL:
Ogbenna AA, Oyedele OA, Adeyemo TA, Oyewole KM. Effect of varying storage time and temperature on unspun blood samples for prothrombin time and activated partial thromboplastin time in a tertiary hospital laboratory in the tropics. Sahel Med J [serial online] 2022 [cited 2022 Nov 30];25:41-6. Available from: https://www.smjonline.org/text.asp?2022/25/2/41/354187




  Introduction Top


Prothrombin time (PT) and activated partial thromboplastin time (APTT) are sensitive coagulation tests frequently ordered together by physicians to evaluate hemostatic disorders. In many clinical practices, samples collected for these tests are transported to the laboratory for undetermined but variable periods after collection. Transport delays may prolong clotting times and in vitro loss of factor activity, especially the labile factors FV and FVIII.[1]

Hence, this study is designed to investigate the potential effect of storing citrated whole blood at varying temperatures and times on coagulation tests. Moreover, to determine the optimal storage conditions for a reliable test result.


  Materials and Methods Top


Study design

A cross-sectional study was carried out in November 2019 in the hematology laboratory of Lagos University Teaching Hospital (LUTH), Lagos State, Nigeria. LUTH is a tertiary hospital established in 1962 affiliated with the University of Lagos.

Participants

Staff and students of LUTH within the period of the research were recruited after obtaining informed consent. Thirty-three apparently healthy adult volunteers ages 18–65 years were recruited. Exclusion criteria included those that did not fall within the age range, were pregnant, people who had breakfast (high in fats), unease due to strenuous exercise, and on any medication (anticoagulant, heparin, and contraceptive).

Study size

The sample size was calculated using power and sample size calculations software version 2.1.31 PlayStation store program for paired test formula with 95% confidence interval. Standard deviation σ = 4.0 according to the literature for paired test formula, desired precision δ = 2 with power 80% was used.[2]

Method

A trained phlebotomist performed venepunctures in the morning following a 12-h fast. From each participant, 18 ml venous whole blood sample was collected into plain bottles containing 2 ml of 0.109 M sodium citrate as an anticoagulant at blood to an anticoagulant ratio of 9:1. Samples were mixed thoroughly (but gently) by 3–6 end over end tube inversions to ensure adequate mixing of the test sample with the anticoagulant. Afterward, the following steps were carried out to each sample:

  1. The blood sample collected was aliquoted evenly into nine plain bottles labeled B0, R3, R6, R12, R24, F3, F6, F12, and F24 in addition to the participant study number
  2. Bottles labeled B0 were tested immediately for baseline result of PT and APTT
  3. Bottles labeled R3, R6, R12, and R24 were stored at room temperature (RT) of 25°C for 3 h, 6 h, 12 h, and 24 h, respectively
  4. Bottles labeled F3, F6, F12, and F24 were stored at 4°C for 3 h, 6 h, 12 h, and 24 h, respectively.


Each sample bottle was centrifuged at 2500 g for 10 min after storage of 3, 6, 12, and 24 h, respectively, including bottle B0 which was not stored. Afterward, the platelet-poor plasma (PPP) obtained was assayed for PT and APTT using a semiautomated blood coagulation analyzer (Sysmex CA-10, Ahrensburg, Germany).

Assay method

PT and APTT reagents; Dade Innovin (LOT 549750C), actin FS (LOT 538552), calcium chloride (LOT 563857) were obtained from Sysmex. PT was determined by preincubating 50 μl of PPP for 30 s at 37°C in the semiautomated coagulometer (Sysmex). The coagulation reaction was then initiated by the addition of the 100 μl Dade Innovin (LOT 549750C). The endpoint result in seconds is displayed when a clot is formed.

For APTT, 50 μl of the PPP is preincubated with 50 μl of actin FS (LOT 538552) for 180 s at 37°C in the semiautomated coagulometer (Sysmex). The coagulation reaction is initiated by the addition of 50 μl of calcium chloride (LOT 563857), and the machine displays the endpoint result in seconds when a clot is formed.

Normal and pathological controls were run for every vial of reagent used. Furthermore, APTT and PT measurements were performed in duplicates and three or four times for that samples, which had a difference of more than 1 s, the final values, were the mean of the results.

Statistical analysis

Data obtained were analyzed using the Statistical Package for the Social Sciences (SPSS) version 22 (IBM Corp., Armonk, NY, USA). Paired student t-test with confidence intervals of 95% was used for comparison between the results at different time intervals with 0 h. Results were expressed as mean values and giving other descriptive statistics. A P ≤ 0.05 was considered significant in all statistical comparisons. To assess stability, the mean percentage changes (result at storage time X– Result at baseline, divided by results at baseline, expressed as a percentage change) compared with the baseline results were calculated and represented graphically. The clinically relevant difference was defined as a percentage change of greater than (>) 10% according to van geest.[3] The effect of a clinically relevant difference >10% observed in <25% of the samples was termed moderate whereas when observed in >25% of the samples was termed large.

Ethical approval

Approval of this study (ADM/DCST/HREC/APP/3260) was obtained from LUTH health research ethics committee before the commencement of the study.


  Results Top


Biodata of study

The mean age of the study population was 34.42 years of which the majority were within the age range 40–44 years (30.3%). There was a high prevalence of males (63.6%) as to the female (36.4%).

Effect of varying storage time and temperature on prothrombin time

[Table 1]a and [Table 1]b show the effect of varying storage time on PT the PT result at RT and 4°C, respectively. The mean PT result (baseline) was 14.22 s which was within the normal range (12.6–17.2 s) of PT in Nigeria.[4]
Table 1:

Click here to view


The mean PT at 24 h at RT had a statistically significant difference (P = 0.000) as shown in [Table 1]a. At 3 h, 6 h, and 12 h, the mean PT was not statistically different from baseline PT (P > 0.05).

A similar trend was observed in the PT results at 4°C storage in [Table 1]b as in the PT RT storage times. A statistically significant difference (P = 0.000) was observed at 24 h storage time.

Effect of varying storage time and temperature on activated partial thromboplastin time

The APTT results at RT and 4°C are shown in [Table 2]a and [Table 2]b. The mean APTT result (baseline) was 30.24 s which was within the normal range (26.92–33.1s) of APTT in Nigeria.[5]
Table 2:

Click here to view


At RT, a statistically significant difference was observed at 12 h (P = 0.009) and 24 h (P = 0.000) storage time as shown in [Table 2]a. However, at 4°C at all the storage times 3, 6, 12, and 24 h a statistically significant difference (P < 0.05) as shown in [Table 2]b.

Stability studies of prothrombin time and activated partial thromboplastin time

The stability of the PT and APTT assay was investigated using the mean percentage change from baseline at the different storage times and temperatures as shown in [Figure 1].
Figure 1: Mean percentage change in prothrombin time/activated partial thromboplastin time from baseline over time

Click here to view


The mean percentage change for PT at both RT and 4°C were all positive following a 3, 12, and 24 h storage. A negative change was observed at 6 h storage at both RT and 4°C. On the other hand, the mean percentage for APTT at both RT and 4°C were all positive except at 3 h storage at RT.

Clinical relevance

[Table 3]a and [Table 3]b show the range of positive and negative differences from the baseline result observed in the PT and APTT results, respectively.
Table 3:

Click here to view


For PT at RT, the percentage of samples with positive changes that exceeded the 10% limit was moderate (<25% of sample size) after 3 h, 6 h, and 12 h and large (>25% of sample size) after 24 h as shown in [Table 3]a. At 4°C, the percentage of samples with positive changes that exceeded the 10% limit was moderate (<25% of samples) at 6 h storage time and large (>25% of samples) at 3, 12, and 24 h.

The result from [Table 3]b at RT APTT shows that the percentage of samples with positive changes that exceeded the 10% limit was moderate (<25% of samples size) at 3 h and 6 h storage. At 12 h and 24 h storage the effect was large (>25% of sample size).

Whereas at 4°C, the percentage of samples with positive changes that exceeded the 10% limit was moderate (<25% of samples) at 6 h storage time and large (>25% of samples) at 3, 12, and 24 h.


  Discussion Top


The result of this study demonstrates that the storage time and temperature affect routine screening coagulation test PT and APTT. For PT, the changes observed at 3 h, 6 h, and 12 h were not statistically significant but were clinically relevant with moderate to large effect. However, at 24 h of storage at both RT and 4°C, the results were both statistically significant and clinically relevant with large effect.

Contrarily, the results of APTT showed that the maximum storage time as unspun blood was 6 h at RT. After 6 h of storage, a statistically significant difference was observed. Moreover, storage at the refrigerator is unacceptable as all the results were statistically significant with clinical relevance of large effect.

The findings of this study when whole blood was stored unspun was in partial agreement with that of a previous study done by Salvagno et al.,[6] which reported that APTT can be stored uncentrifuged for 6 h at RT but in variance with 6 h storage for refrigerated sample. In addition, Oddoze et al.,[7] evaluated the potential stability of APTT at 6 h and 24 h and states that the acceptable delay time for whole blood samples for APTT 6 h at RT which is in line with the findings of this study. However, their finding of APTT stability at 6 h storage at 4°C contradicts the result of this study.

Furthermore, similar studies on unspun samples by van Geest-Daalderop et al.[3] is in variance with the result of this study. They reported that the optimal storage of whole blood samples for PT/INR is 6 h. This is shorter than our findings of 12 h this could be attributed to the fact that the study evaluated 3 h., 6 h, and 24 h. Moreover, their study sample was patients on anticoagulant therapy whereas this study involved healthy individuals, not on an anticoagulant.

A study by Toulon et al.[8] in France investigated four storages that are, below 2, 4, 6, and 8 h on both healthy and patients on vitamin K-antagonists, at a RT in the range between +18 and +25°C. Their result report on the maximal storage time at +25°C of unspun blood samples for the determination of PT/INR and is APTT is 8 h. This storage time is shorter than this study result for PT, which is 12 h and higher APTT maximum storage of 6 h at RT. The short storage time investigated for PT could be attributed to their conclusion of maximum storage time for PT. On the contrary, Rao et al. report[9] samples stored as whole blood can give a reliable result with a maximum storage of 24 h for PT and 12 h. For APTT at 4°C and RT. However, statistical analysis of the 24 h storage result at RT and 4°C for PT was statistically significant (P = 0.000) in this study. In addition, the 12 h storage for APTT at RT and refrigerator were statistically significant.

The variation to this study highlighted earlier could be due to variation in the population sample used in the study and the times investigated. Similar studies carried out on PT and APTT using plasma storage at different storage conditions report a longer storage time for PT as 24 h [9],[10],[11],[12] while some reported shorter storage time of 2 h,[13] 4 h,[2] and 12 h.[14] Nevertheless, this study was on whole blood storage at different storage conditions; hence, no statement can be made about the optimal storage conditions on plasma storage. An additional limitation of this study was that this study investigated only the effects of RT and 4°C on PT and APTT for up to 24 h and healthy participants not on heparin or warfarin therapy. Thus, no statement can be made about the effects of these storage conditions, time, and temperature on such samples.


  Conclusion Top


An inference from this study using healthy individuals shows that the PT result was reliable only within 12 h of storage of unspun blood samples at both 4°C and RT. After which further storage time elicit a statistically significant difference with clinical relevance. On the other hand, the APTT result was reliable within 6 h storage as unspun blood at RT. At 4°C, all storage times after phlebotomy showed a statistically significant difference. Hence, this study recommends that unspun blood samples for PT and APTT be stored for a maximum of 6 h as physicians often request these tests together. In addition, more studies need to be carried out on unhealthy populations as this study was limited to only healthy Individuals.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
Emmanuel F, Dorothy A, Giuseppe L. Pre-analytical Variables in Coagulation Testing Associated With Diagnostic Errors in Hemostasis. Laboratory Medicine 2012;43:1-10. doi: 10.1309/LM749BQETKYPYPVM.  Back to cited text no. 1
    
2.
Mohammed Saghir SA, Al-Hassan FM, Alsalahi OS, Abdul Manaf FS, Baqir HS. Optimization of the storage conditions for coagulation screening tests. J Coll Physicians Surg Pak 2012;22:294-7.  Back to cited text no. 2
    
3.
van Geest-Daalderop JH, Mulder AB, Boonman-de Winter LJ, Hoekstra MM, van den Besselaar AM. Preanalytical variables and off-site blood collection: Influences on the results of the prothrombin time/international normalized ratio test and implications for monitoring of oral anticoagulant therapy. Clin Chem 2005;51:561-8.  Back to cited text no. 3
    
4.
Alao O, Damulak D, Joseph D, Puepet F. Haemostatic Profile of Patients with Type 2 Diabetes Mellitus in Northern Nigeria. The Internet Journal of Endocrinology. 2009;6.  Back to cited text no. 4
    
5.
Ifeanyichukwu MO, Ibekilo Sylvester N, John Aja O' Brien C, et al. Activated Partial Thromplastin Time, Thrombin Time and Platelet Count Study in HIV Seropositive Subjects at Nnamdi Azikiwe Teaching Hospital Nnewi. Transl Biomed 2016,7:2.  Back to cited text no. 5
    
6.
Salvagno GL, Lippi G, Montagnana M, Franchini M, Poli G, Guidi GC. Influence of temperature and time before centrifugation of specimens for routine coagulation testing. Int J Lab Hematol 2009;31:462-7.  Back to cited text no. 6
    
7.
Oddoze C, Lombard E, Portugal H. Stability study of 81 analytes in human whole blood, in serum and in plasma. Clin Biochem 2012;45:464-9.  Back to cited text no. 7
    
8.
Toulon P, Metge S, Hangard M, Zwahlen S, Piaulenne S, Besson V. Impact of different storage times at room temperature of unspun citrated blood samples on routine coagulation tests results. Results of a bicenter study and review of the literature. Int J Lab Hematol 2017;39:458-68.  Back to cited text no. 8
    
9.
Rao LV, Okorodudu AO, Petersen JR, Elghetany MT. Stability of prothrombin time and activated partial thromboplastin time tests under different storage conditions. Clin Chim Acta 2000;300:13-21.  Back to cited text no. 9
    
10.
Collection, Transport, and Processing of Blood Specimens for Testing Plasma Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline Fifth edition. CLSI document H21 A5 (ISBN 1-56238-657-3). Clinical and Laboratory Standards Institute, 950 West Valley Road, Suite 2500, Wayne, Pennsylvania 19087 USA, 2008.  Back to cited text no. 10
    
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Zhao Y, Lv G. Influence of temperature and storage duration on measurement of activated partial thromboplastin time, D-dimers, fibrinogen, prothrombin time and thrombin time, in citrate-anticoagulated whole blood specimens. Int J Lab Hematol 2013;35:566-70.  Back to cited text no. 11
    
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Feng L, Zhao Y, Zhao H, Shao Z. Effects of storage time and temperature on coagulation tests and factors in fresh plasma. Sci Rep 2014;4:3868.  Back to cited text no. 12
    
13.
Ikhuenbor D, Aghedo F, Isah I, Iwueke I, Oladigbolu R, Egenti N, et al. The effect of time and temperature variables on some routine coagulation tests among subjects of African descent in Sokoto, North Western Nigeria. Open J Blood Dis 2016;6:79-88. [doi: 10.4236/ojbd. 2016.64011].  Back to cited text no. 13
    
14.
Thabet NM, Galal SH, Sayed Refae AA. Effect of some preanalytical variables on some screening tests of coagulation: a single center experience. J Curr Med Res Pract 2019;4:209-15.  Back to cited text no. 14
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    Figures

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    Tables

  [Table 1], [Table 2], [Table 3]



 

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