|Year : 2013 | Volume
| Issue : 1 | Page : 1-4
Effect of plasma storage on prothrombin time and activated partial thromboplastin time at a Nigerian public laboratory
Mohammed A Ndakotsu1, Abdul-Aziz Hassan2, Abubakar U Musa2, Yazid B Gusau1, Ibrahim K Kwaifa1
1 Department of Hematology, UDUTH, Sokoto, Nigeria
2 Department of Hematology, ABUTH, Zaria, Nigeria
|Date of Web Publication||17-May-2013|
Abubakar U Musa
Department of Hematology, ABUTH, Zaria
Source of Support: None, Conflict of Interest: None
Background: Prothrombin time (PT) and activated partial thromboplastin time (APTT) are the tests used in the investigation and monitoring of hemostatic disorders. Plasma is used to perform these tests immediately or stored for later use. The time and storage temperature have been shown to affect the results of these tests. Thus, all coagulation laboratories need guidelines for plasma storage to ensure reliable results. Objective: To determine the effect of varying storage times and temperatures on plasma PT and APTT. Materials and Methods: PT and APTT were run on plasma from 40 healthy adults using a semi-automated coagulometer. PT and APTT were measured at 0, 4, 6, and 24 h on samples stored at room temperature, refrigerated samples, and frozen samples. The values at 0 h were compared with the values at 4, 6, and 24 h. Results: PT and APTT values were within the reference ranges at
0 h. For refrigerated plasma, PT values at 4 h were within normal, but at 6 and 24 h, they were significantly deranged (P < 0.05). PT was significantly different at 4, 6, and 24 h for both room temperature and frozen plasma (P < 0.05). The APTT showed significant differences between 0 h value and values at 4, 6, and 24 h for all the varying temperature conditions. Conclusion: For reliable PT and APTT results, samples should be processed and run immediately after collection. However, plasma for PT can be stored at 2°-4°C for only 4 h.
Keywords: Activated partial thromboplastin time, plasma, Prothrombin time, storage, temperature
|How to cite this article:|
Ndakotsu MA, Hassan AA, Musa AU, Gusau YB, Kwaifa IK. Effect of plasma storage on prothrombin time and activated partial thromboplastin time at a Nigerian public laboratory. Sahel Med J 2013;16:1-4
|How to cite this URL:|
Ndakotsu MA, Hassan AA, Musa AU, Gusau YB, Kwaifa IK. Effect of plasma storage on prothrombin time and activated partial thromboplastin time at a Nigerian public laboratory. Sahel Med J [serial online] 2013 [cited 2022 Aug 10];16:1-4. Available from: https://www.smjonline.org/text.asp?2013/16/1/1/112038
| Introduction|| |
Prothrombin time (PT) and activated partial thromboplastin time (APTT) are tests of hemostasis employed in the evaluation of coagulopathies and monitoring of anticoagulant drug therapy. They measure the extrinsic and intrinsic arms of the coagulation cascade, respectively.  Modern laboratory instrumentation with high levels of test reliability and appropriate quality assurance measures have led to fewer analytical errors in hemostatic testing. However, incorrect test results are still encountered due to pre-analytical events associated with sample collection, processing, and storage, as well as post-analytical events related to reporting and interpretation of test results. 
The Clinical and Laboratory Standards Institute (CLSI) guidelines recommend coagulation tests be performed within 2 h if samples are kept at room temperature, 4 h if stored at 2°-4°C, and 2 weeks if preserved at −20°C.  However, several studies have observed varying stability results of PT and APTT with different plasma storage conditions. ,,,, The recommendations of CLSI may not be generally in keeping with the weather outlook of Sokoto which is located in the Sahel savannah of northern Nigeria with temperatures reaching as high as 45 o C during the dry season. 
The hematology laboratories in most public laboratories in Nigeria are saddled with the responsibility of offering clinical laboratory results and reports to assist the clinician in diagnosing, monitoring, and management of various clinical conditions. The coagulation and hemostasis bench is usually not a busy bench in most general and specialist hospitals; however, in tertiary hospitals like the Usmanu Danfodio University Teaching Hospital (UDUTH) in Sokoto, Northwest Nigeria, the request for hemostatic investigations, particularly PT and APTT, is generally increasing due to the presence of specialists in various aspects of medicine.
This study is thus aimed to find out the ideal ways of handling samples for hemostatic tests in order to set up a standard procedure and temperature for plasma specimen handling at the hematology laboratory of UDUTH and probably other coagulation laboratories in Northwest Nigeria.
| Materials and Methods|| |
This was a prospective study carried out on apparently healthy adults among the staff and students of UDUTH Sokoto. Subjects with chronic liver disease, pregnancy, hypertension, sickle cell disease, or on drugs that could affect coagulation were excluded. It was conducted at the hematology laboratory of UDUTH Sokoto from May 2012 to June 2012. Approval was obtained from the Ethical and Research Committee of UDUTH Sokoto, and informed consent was obtained from all the subjects.
Venous blood was collected after aseptic procedure from each subject and 4.5 ml of this was dispensed into a specimen bottle containing 0.5 ml of 3.2% trisodium citrate to conform to a specimen mixture of 9 parts of blood to 1 part of the anticoagulant. This was gently mixed and then centrifuged at 2000 g for 15 min to obtain platelet-poor plasma. PT and APTT were run on the plasma at 0 h (i.e. within an hour of sample collection). The plasma was divided into seven aliquots of 0.4 ml each. One aliquot was kept at room temperature (20°-29°C); one batch of three aliquots was refrigerated (2°-4°C) and another batch of three aliquots was kept frozen (−10° to −14°C). Using a mercury thermometer, the storage temperatures were taken before running each batch of tests to ensure that there were no fluctuations in the predetermined storage temperatures.
PT was measured semi-automatically by pipetting 50 μl of plasma into a cuvette and prewarming at 37°C for 3 min. Then 100 μl of prewarmed hemostat thromboplastin reagent was added and the time taken for clot formation was detected automatically by HumaClot Juniour Coagulometer. , Similarly, APTT was measured by initially pipetting 50 μl plasma in a cuvette and prewarming for 2 min. Fifty microliters of Hemostat aPTT-EL was then added to the plasma and incubated for an additional 5 min. Finally, 50 μl of prewarmed 0.02 mol/l calcium chloride was added and the time taken to clot formation was detected using the coagulometer.
Measurement of both PT and APTT for each sample was carried out in duplicates and the mean values recorded. Equally, both normal and abnormal control samples were run alongside each batch of samples to be run at a time.
Results of the measured PT and APTT were entered and analyzed using the Statistical Package for the Social Sciences (SPSS) software version 17. Using paired sample t-test, the values for PT and APTT at h were compared with each of those obtained at 4, 6, and 24 h at the various storage temperatures. The level of significant statistical relationship was set at 0.05.
| Results|| |
A total of 40 results were analyzed. The mean values and ranges of PT and APTT at 0 h were found to be within the reference range of our facility (10.0-16.5 sec for PT and 30.0-48.0 sec for APTT), with mean values of 13.52 ± 1.79 sec and 37.50 ± 4.65 sec, respectively. The PT and APTT results of the aliquot samples stored at various temperatures showed that only the PT of refrigerated plasma at 4 h with a mean value of 14.05 ± 1.66 was found to be within the reference range and there was no significant difference found between PT at 0 h and PT at 4 h in the refrigerated sample (P > 0.05). However, there was a significant difference found between PT at 0 hand that at 6 h (P < 0.001) and 24 h (P < 0.001) in the refrigerated samples.
There were also significant differences between PT at 0 h and the values at 4, 6, and 24 h for both room temperature and frozen plasma (P < 0.05) [Table 1].
On the other hand, APTT showed statistically significant differences between 0 h and at 4, 6, and 24 h for all the storage temperatures [Table 2]. There was also an observed progressive prolongation of the PT and APTT with time irrespective of the storage temperature. This was most prominent with storage at room temperature [Figure 1] and [Figure 2].
| Discussion|| |
The quality of any laboratory result is dependent on the quality of sample collection, transportation, and storage. This is due to the fact that erroneous results may arise from simple avoidable mistakes such as inappropriate storage temperatures. These pre-analytical sources of error can be eliminated or reduced to a bearable minimum by taking precaution through conforming to set-down guidelines during blood sample collection and storage.  These guidelines are usually put up as Standard Operating Procedures (SOP) in most laboratories.
The findings in this study showed that reliable results for both PT and APTT are best obtained at 0 h, and on storage for 4 h at 2°-4 o C for PT only. This suggests that samples for PT and APTT should be analyzed immediately or within 4 h of storage at 2°-4°C for PT in order to ensure reliable results.
Saghir et al. in Malaysia reported that PT test was stable only with plasma stored at room temperature for 4 h, but thereafter, the samples deteriorated, and for APTT, the test results were only reliable at 2 h only and deteriorated thereafter. These findings are similar to our study findings and may be as a result of the similarity in the temperatures between Malaysia and Nigeria which are both tropical countries.
This result is at variance with the findings of Neofotistos et al. who reported no significant difference in the PT results for up to 8 h when stored at room temperature, while the APTT was prolonged, albeit not clinically significant. The difference may be due to the difference in what is called room temperature; while it is 20°-22°C in Chicago, it is 29°C in Sokoto. Heil,  working in Germany, observed that when stored at room temperature, APTT test was stable for 24 h, whereas PT was only stable for 8 h. This is significantly at variance with our finding in Sokoto and is likely due to the higher room temperature in Sokoto that can accelerate the decay of coagulation factors.
Rao et al. found that the results of PT tests were stable up to 12 h if refrigerated, while APTT samples remained stable up to 24 h if refrigerated. This finding is significantly at variance with our findings and may still be due to difference in room temperature between Sokoto and Huntsville.
| Conclusion|| |
The duration and temperature of plasma storage affect PT and APTT test results. Therefore, it is advisable to run plasma samples immediately after separation. However, plasma for PT can be stored at 2°-4°C for only 4 h. Provision of appropriate and standard storage facilities and SOP is crucial for reliable PT and APTT results. Point-of-care testing can obviate the necessity for plasma storage.
| References|| |
|1.||Laffan M, Manning R. Investigation of haemostasis. In: Dacie JV, Lewis SM, editors. Practical Haematology 10 th ed. Philadelphia: Churchill Livingstone Publishers; 2006. p. 379-440. |
|2.||Favaloro EJ, Funk DM, Lippi G. Preanalytical Variables in Coagulation Testing associated with diagnostic errors in haemostasis. Lab Med 2012;43:1-10. |
|3.||Clinical and Laboratory Standards Institute. Collection, Transport, and Processing of Blood Specimens for Testing Plasma-Based Coagulation Assays and Molecular Hemostasis Assays; Approved Guideline 4 th ed. CLSI document 2003; H21-A3. |
|4.||Rao LV, Okorodudu AO, Petterson JR, Elghetany MT. Stability of PT and APTT tests under different storage conditions. Clin Chem Acta 2000;300:13-21. |
|5.||Adcock D, Kressin D, Marlar RA. The effect of time and temperature variables on routine coagulation tests. Blood Coagul Fibrinolysis 1998;9:463-70. |
|6.||Koepke JA, Rodgers JL, Ollivier MJ. Pre-instrumental variables in coagulation testing. Am J Clin Pathol 1975;64:591-6. |
|7.||Heil W, Grunewald R, Amend M, Heins M. Influence of time and temperature on coagulation analytes in stored plasma. Clin Chem Lab Med 1998;36:459-62. |
|8.||Saghir SAM, Al-Hassan FM, Alsalahi OS, Abdul-Manaf FS, Baqir HS. Optimization of the storage conditions for coagulation screening tests. J College Physicians Surg Pak 2012;22:294-7. |
|9.||Sokoto. Wikipedia encyclopedia. Available from: http//www.wikipedia.org/wiki/sokoto. [accessed on 2012 Nov 23]. |
|10.||HumaClot Junior Coagulometer. Human GmbH. Max- Planck- Ring 21. D-65205 Weisbaden Germany. 2009. |
|11.||Product Inserts. Manual and Automated Determination of PT, APTT, TT, Fibrinogen and D-dimer. Human GmbH Diagnostics. Max-Planck-Ring 21. D-65205 Weisbaden Germany 2009. |
|12.||Neofotistos D, Oropeza M, Tsao CH. Stability of plasma for add-on PT and APTT tests. Am J Clin Pathol 1998;109:758-63. |
[Figure 1], [Figure 2]
[Table 1], [Table 2]