INTRODUCTION
One of the important objectives of quality assurance for a laboratory is to deliver accurate, timely, and clinically relevant diagnostic reports to the customers i.e. the clinicians and patients. The quality of this service is thus related, among other factors, to the accurateness and timeli-ness of the reports. For a pathology re-port to be beneficial to the clinicians, re-sults should be available on time espe-cially for urgent requests from critical units.
The turn-around time (TAT) can be de-fined as the time taken from when the specimen arrives at the laboratory to the time results are released. It is one of the indicators of quality and efficacy, princi-pally in laboratories performing urgent analyses (Hilborne et al. 1989). This is of importance especially when processing samples for coagulation tests where standard processing methods may not be fast enough for urgent requests.
A survey in the United States showed that the prothrombin time (PT) and acti-vated partial thromboplastin time (APTT) tests represent more than 90% of co-agulation tests ordered (Shahangian et al. 2005) which mirrors the requested tests in our laboratory. In coagulation testing, platelet poor plasma (PPP) is required to prevent interference by the phospholipid surface on the platelets. With standard laboratory methods (Ad-cock et al. 2008), the duration for reporting coagulation tests is about 40–180 minutes. A long TAT may cause adver-sity in medical situations associated with critical, surgical and emergency cases, and good medical care could be denied (Steindel & Howanitz 2001).
Strategies to reduce the time essential for specimen processing without affecting quality should be recognized, especially for laboratories performing urgent ana-lyses. In our laboratory, urgent cases are mainly requested by the Emergency Department. The standard methods for processing coagulation tests do not meet the current TAT for urgent requests (30 minutes) in our centre. This study was performed to determine whether a higher centrifugation speed and a shorter centrifugation time would affect coagulation results as compared to centrifugation using the standard time (10-15 minutes) for PPP preparation (Ad-cock et al. 2008).
MATERIALS AND METHODS
Blood samples for routine coagulation test were randomly selected from patients from November through December 2010. The age range for the study population was from 28 to 83-years-old.
Platelet-poor plasma (PPP) was pre-pared at room temperature within one hour of sample collection using two table- top centrifuge machines (Rotina 38, Hettich Zentrifugen, Germany). Each sample was thoroughly mixed and separated into equal volumes in two separate plastic vacuum tubes (with 3.2% sodium citrate). The first tube was centrifuged at 4000 rpm for five minutes (protocol A), while the second tube was centrifuged at 3580 rpm for 15 minutes (protocol B) simulta-neously. A plastic pipette was used to carefully transfer approximately 1ml of supernatant plasma from the middle of the sample away from the buffy coat to avoid contamination with excess platelets.
All paired samples were prepared and tested for PT, APTT and fibrinogen at room temperature immediately after cen-trifugation using a coagulation analyzer with the required controls and reagents (STA-Compact, All-Eights, France). The PPP samples were tested for platelet count within 30 minutes of collection us-ing a haematology analyser (LH 750, Beckmann Coulter, USA). Data were entered using Microsoft Ex-cel and descriptive statistical analyses (mean, range, standard deviation (SD) and paired Student t-test) were computed using SPSS, version 14.
RESULTS
A total of 79 samples were randomly selected for this study. Of these 79 samples, 49 samples showed abnormal PT, 30 samples showed normal PT, 34 samples showed abnormal APTT and 44 samples showed normal APTT readings. One APTT reading (21.5s) was much lower than the normal reference range and was thus excluded from the data analysis. The normal reference range for PT and APTT are 11.4-14.2 seconds and 31.3-46.1 seconds respectively. Platelet counts less than 10x109/L were produced from all 79 PPP samples centrifuged for 15 minutes at 3580 rpm ex-cept for two samples (2.5%) which showed a platelet count of more than 10x109/L (12x109/L and 14x109/L). However, when samples were centrifuged for five minutes at 4000 rpm, only 15 sam-ples (19%) showed platelet counts less than 10x109/L. The difference between these two groups were statistically significant (p<0.001).
Descriptive statistics for platelet count, PT, APTT and fibrinogen levels prepared for five minutes at 4000 rpm (protocol A) and 15 minutes at 3850 rpm (protocol B) are shown in Table 1. We found a significant difference (p=0.01) between the normal PT levels when comparing centrifugation between 15 minutes (standard time) and five minutes. However, there was no significant difference in the results of normal APTT, abnormal PT and APTT between the two protocols. There was also good correlation between the results of PT, APTT and fibrinogen levels of protocol A and B respectively (Figure 1, 2 and 3).
DISCUSSION
Preparation of blood specimens can be a major time limiting factor in the laboratory throughput. In order to obtain a faster TAT, while maintaining sample quality and accuracy of results, the time con-sumed on coagulation sample collection, handling, processing and testing should be looked into (Lawrence 2003). This is especially so in critical areas where results are needed urgently.
In our laboratory, it takes about 60 minutes to release routine coagulation results to the wards or clinics. The time may be further prolonged if repeat testing is necessary. This will not fulfill the TAT requirement for urgent tests for critical units such as the Emergency Department and Intensive Care Unit. The shortest recommended centrifugation time to produce PPP with platelet counts less than 10x109/L is 10-15 minutes at 3850 rpm using regular centrifuges (Adcock et al. 2008). In this study, we looked at whether centrifugation at five minutes would affect coagulation results (PT/APTT), as compared to centrifugation at 15 minutes recommended time.
We found that 15-minutes centrifugation, achieved a better PPP than a five minutes centrifugation. A shorter centrifugation time (five minutes) causes an increase in platelet count in the sample tubes in 81% of cases. Our results dif-fered from a previous report by Sultan (2010) on 46 healthy volunteers which showed nearly all samples centrifuged at five minutes had platelet count less than 10x109. Barnes and colleagues (2002) showed that 10 minutes was the minimum centrifugation time required to consistently meet the recommended standards.
However, although platelet counts were significantly different in both protocols, the coagulation results between 15 minutes recommended centrifugation time and five minutes centrifugation time did not differ significantly for abnormal PT, normal APTT, abnormal APTT and fibrino-gen results (Table 1). This is in agreement with a previous study (Barnes et al. 2002). For normal PT results, the difference between 15 minutes centrifugation and five minutes centrifugation had a p value of 0.01. Nevertheless, the mean (13.1s vs 13.3s) and SD (0.58 vs 0.57) did not differ much. In our opinion, this unexpected result may be due to statistical reason as a result of a small number of samples for this subgroup, rather than the platelet content in the PPP itself. APTT is more sensitive to platelet con-tamination than PT. Therefore platelet contamination will affect the APTT result more than the PT result, a finding of which was not evident in this study.
When we combined both normal and abnormal PT and APTT results, we observe a good correlation between the two different protocols used in this study (Figure 1 and 2). This is also true for fibrinogen levels (Figure 3). In view of this, we feel that although the platelet count can be high when a sample is centrifuged for five minutes, it does not affect coagulation results in general.
CONCLUSION
This study showed that, although there was presence of residual platelet counts in PPP prepared with five minutes cen-trifugation at 4000 rpm, no significant difference was found for the results of PT, APTT and fibrinogen values. We conclude that this centrifugation time can be used for urgent test requests without compromising the quality and reliability of the results. This contributes to reducing the overall turnaround time for coagula-tion STAT results and thus provides bet-ter medical care for patients with coagulation test requests.