The recommendation for coagulation blood samples is to centrifuge at 4000 revolutions per minute (rpm) for 15 min to produce platelet-poor plasma before analysis. Rapid centrifugation, defined as centrifuging samples at higher speeds for shorter durations, could potentially reduce turn-around times (TAT), provided sample integrity is maintained.
This study assessed the impact of rapid centrifugation on routine coagulation assay results.
Blood samples were collected from volunteers at Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, from September to November 2021. Samples were centrifuged using Method A, the current standard (4000 rpm/15 min), Method B (4000 rpm/10 min), Method C (5000 rpm/10 min) and Method D (5000 rpm/5 min). Platelet count, prothrombin time, activated partial thromboplastin time, thrombin time (TT), fibrinogen and D-dimer levels were analysed and results from Methods B, C and D compared to reference Method A.
Platelet-poor plasma was obtained from all samples (
Rapid centrifugation at 4000 rpm/10 min (Method B) showed results consistent with the reference method. This method could potentially reduce the overall TAT for routine coagulation assays.
Laboratories are expected to provide accurate and reliable results within defined turn-around times (TATs) to facilitate the diagnosis, management and prognostication of patients.
As centrifugation time can be a bottleneck in coagulation testing, there is a need to determine the impact of rapid centrifugation on the laboratory workflow and TAT.
The primary goal of this study was to assess the impact of rapid centrifugation on the accuracy of routine coagulation test results.
The study was approved by the Biomedical Research Ethics Committee of the University of KwaZulu-Natal, South Africa (BREC/00002366/2021). Informed consent was obtained from each study participant and a unique study number was allocated to samples to ensure anonymity. The research data was stored electronically on password-protected devices and was only accessible by the researchers.
This study was conducted at the Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, from September 2021 to November 2021. Patients older than 18 years were included in the study. Samples were excluded if there were insufficient blood volumes, or if clots, fibrin strands, or haemolysis were observed.
Sixty-two participants were included in the study (
Demographics and clinical diagnoses of study participants, Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, September 2021 – November 2021.
Patient demographics and diagnosis | Participants |
|
---|---|---|
% | ||
62 | - | |
60 | - | |
Male | 33 | 55.00 |
Female | 27 | 45.00 |
18–30 | 21 | 35.00 |
31–40 | 15 | 25.00 |
41–50 | 8 | 13.33 |
51–60 | 8 | 13.33 |
61–70 | 8 | 13.33 |
Volunteers | 12 | 20.00 |
Acute Leukaemia | 8 | 13.00 |
Lymphoma | 6 | 10.00 |
Coagulation disorders | 8 | 13.00 |
Plasma cell dyscrasias | 2 | 3.00 |
Myeloproliferative neoplasms | 4 | 7.00 |
Other haematological disorders | 10 | 17.00 |
Non-haematological disorders | 7 | 12.00 |
Unknown | 3 | 5.00 |
, Samples from two participants were excluded due to the specimen being insufficient and tube breakage.
, Of the 60 samples, one was treated as an outlier due to abnormally high D-dimer results (35.2 mg/L) using Method A.
Samples were centrifuged using two table-top Consul 22R (Ortoaltresa®, Madrid, Spain) instruments. The first instrument (Instrument 1) had a maximum rotor speed of 4000 rpm and required a 5 mL tube, while the second instrument (Instrument 2) had a maximum rotor speed of 9000 rpm and required a 50 mL tube (
Speed and time specifications for centrifugation methods, Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, September 2021 – November 2021.
Method | Speed (rpm) | Time (min) | Centrifugation instrument | Tube used |
---|---|---|---|---|
A (reference method) | 4000 | 15 | 1 | 5 mL |
B | 4000 | 10 | 1 | 5 mL |
C | 5000 | 10 | 2 | 5 mL tube inside a 50 mL tube with cotton wool buffer |
D | 5000 | 5 | 2 | 5 mL tube inside a 50 mL tube with cotton wool buffer |
min, minutes; mL, millilitres; rpm, revolutions per minute.
Method A was the Clinical and Laboratory Standards Institute-recommended reference method for centrifugation in the laboratory (4000 rpm for 15 min). Methods B (4000 rpm/10 min), C (5000 rpm/10 min) and D (5000 rpm/5 min) were compared to Method A.
Capturing of results, statistical tests and construction of Bland Altman plots were done using Microsoft® Excel 2016 (Microsoft®, Redmond, Washington, United States). The EP Evaluator software version 8 (Informer Technologies Inc, Los Angeles, California, United States) and Stata version 17 (StataCorp®, College Station, Texas, United States) were used for statistical analysis. For descriptive statistics, numerical data were summarised as means, medians, standard deviations or percentages.
Platelet-poor plasma was produced in all samples (
Platelet counts, prothrombin time, activated partial thromboplastin time, thrombin time, fibrinogen and D-dimer levels using four centrifugation methods, Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, September 2021 – November 2021.
Test | Method A 4000 rpm/15 min |
Method B 4000 rpm/10 min |
Method C 5000 rpm/10 min |
Method D 5000 rpm/5 min |
||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
No. | Mean | s.d. | Range | Mean | s.d. | Range | Mean | s.d. | Range | Mean | s.d. | Range | ||||
Platelet count (×109/L) | 60 | 1.18 | 1.23 | 0–5 | 1.77 | 1.63 | 0–8 | 0.02 | 9.35 | 7.66 | 0–38 | < 0.001 | 10.53 | 12.65 | 0–90 | < 0.001 |
Normal (sec) | 49 | 10.71 | 0.60 | 9.5–12.4 | 10.69 | 0.63 | 9.8–12.7 | 0.45 | 10.62 | 0.60 | 9.4–12.6 | 0.002 | 10.62 | 0.59 | 9.6–12.3 | 0.005 |
Abnormal (sec) | 11 | 19.26 | 10.39 | 9.2–42.4 | 19.22 | 10.51 | 9.3–43.0 | 0.70 | 19.25 | 10.34 | 9.4–41.8 | 0.86 | 19.30 | 10.26 | 9.4–41.9 | 0.80 |
Normal (sec) | 38 | 28.26 | 2.48 | 25–36.5 | 27.97 | 2.68 | 22.5–36.3 | 0.13 | 28.02 | 2.35 | 24.2–35.8 | 0.08 | 28.16 | 2.66 | 24.2–37 | 0.51 |
Abnormal (sec) | 22 | 37.48 | 25.36 | 21.1–94.5 | 37.27 | 24.34 | 21–91.8 | 0.56 | 37.44 | 24.47 | 20.6–93.8 | 0.91 | 37.19 | 24.11 | 21.4–89.1 | 0.47 |
Normal (sec) | 46 | 17.47 | 0.85 | 16–19 | 17.37 | 0.81 | 15.7–19 | 0.26 | 17.31 | 0.88 | 15.6–19.1 | 0.08 | 17.34 | 0.87 | 15.7–18.9 | 0.17 |
Abnormal (sec) | 14 | 16.72 | 2.44 | 13.1–20.2 | 16.69 | 2.42 | 13.1–20.1 | 0.70 | 16.57 | 2.53 | 13.1–20.4 | 0.21 | 16.46 | 2.46 | 13.0–20.2 | 0.03 |
Normal (g/L) | 47 | 3.41 | 0.75 | 1.86–4.5 | 3.46 | 0.77 | 1.86–4.66 | 0.08 | 3.52 | 0.90 | 1.86–6.63 | 0.11 | 3.45 | 0.76 | 1.84–4.59 | 0.14 |
Abnormal (g/L) | 13 | 5.28 | 2.15 | 1.21–8.24 | 5.18 | 2.38 | 1.20–8.9 | 0.63 | 5.44 | 2.32 | 1.22–8.9 | 0.06 | 5.37 | 2.31 | 1.21–8.9 | 0.26 |
Normal (mg/L) | 19 | 0.20 | 0.02 | 0.19–0.25 | 0.27 | 0.26 | 0.19–1.34 | 0.29 | 0.21 | 0.03 | 0.19–0.27 | 0.04 | 0.21 | 0.03 | 0.19–0.28 | 0.02 |
Abnormal (mg/L) | 40 | 1.5 | 2.05 | 0.26–9.06 | 1.49 | 2.05 | 0.27–8.9 | 0.89 | 1.44 | 2.06 | 0.23–8.93 | 0.52 | 1.47 | 2.10 | 0.22–9.04 | 0.74 |
min, minute; No., number; rpm, revolutions per minute; s.d., standard deviation; sec, seconds.
, One sample was treated as an outlier due to abnormally high D-dimer results (35.2 mg/L) using Method A.
Forty-nine (49/60; 82%) samples had a normal PT level (Method A: mean [seconds] = 10.71 s, median [seconds] = 10.6 s; Method B: mean = 10.69 s, median = 10.5 s; Method C: mean = 10.62 s, median = 10.5 s; Method D: mean = 10.62 s, median = 10.5 s). Eleven (11/60; 18%) samples had an abnormal prothrombin result (Method A: mean = 19.26 s, median = 15.6 s; Method B: mean = 19.22 s, median = 15.1 s; Method C: mean = 19.25 s, median = 15.5 s; Method D: mean = 19.30 s, median = 15.4 s). There were statistically significant differences for Method C (
Comparison of prothrombin time results between the reference centrifugation method (Method A) and Methods B, C and D, Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, September 2021 – November 2021. Linear regressions for: (a) Method A vs Method B, (b) Method A vs Method C, (c) Method A vs Method D.
Thirty-eight (38/60; 63%) of the samples had normal APTT values (Method A: mean [seconds] = 28.26 s, median [seconds] = 27.9 s; Method B: mean = 27.97 s, median = 27.6 s; Method C: mean = 28.02 s, median = 27.7 s; Method D: mean = 28.16 s, median = 27.7 s). Twenty-two (22/60; 37%) samples had abnormal APTT results (Method A: mean = 37.48 s, median = 24.1 s; Method B: mean = 37.27 s, median = 24.4 s; Method C: mean 37.44 s, median = 24.3 s; Method D: mean = 37.19 s, median = 24.2 s). There were no statistically significant differences in the APTT results obtained using Method A versus Methods B, C and D; APTT results from the three processing methods strongly correlated with the results of the reference method (
Forty-six (46/60; 77%) samples had normal TT levels (Method A: mean [seconds] = 17.47 s, median [seconds] = 17.5 s; Method B: mean = 16.69 s, median = 17.3 s; Method C: mean = 17.37 s, median = 17.3 s; Method D: mean = 17.34 s, median = 17.5 s). Fourteen (14/60; 23%) had abnormal TT levels (Method A: mean = 16.72 s, median = 15.6 s; Method B: mean = 16.69 s, median = 15.6 s; Method C: mean = 16.57 s, median = 15.5 s; Method D: mean = 16.46 s, median = 15.6 s). Abnormal TT levels were significantly lower among samples analysed using Method D compared to Methods A, B and C. All three methods correlated poorly (
Comparison of thrombin time results between the reference centrifugation method (Method A) and Methods B, C and D, Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, September 2021 – November 2021. Linear regressions for: (a) Method A vs Method B, (b) Method A vs Method C, (c) Method A vs Method D.
Forty-seven (47/60; 78%) samples had normal fibrinogen levels (Method A: mean [g/L] = 3.41, median [g/L] = 3.63; Method B: mean = 3.46, median = 3.61; Method C: mean = 3.52, median = 3.64; Method D: mean = 3.45, median = 3.57). Thirteen (13/60; 22%) samples had abnormal fibrinogen levels (Method A: mean = 5.28, median = 5.31; Method B: mean = 5.18, median = 5.43; Method C: mean = 5.44, median = 5.60; Method D: mean = 5.37, median = 5.40). The results of all three test methods correlated strongly with the reference method.
Nineteen (19/59; 32%) samples had normal D-dimer levels (Method A: mean [mg/L] = 0.20, median [mg/L] = 0.19; Method B: mean = 0.27, median = 0.19; Method C: mean = 0.21, median = 0.19; Method D: mean = 0.21, median = 0.20). Forty (40/59; 62%) samples had abnormal D-dimer levels (Method A: mean = 1.5, median = 0.58; Method B: mean = 1.49, median = 0.57; Method C: mean = 1.44, median = 0.56; Method D: mean = 1.47, median = 0.60). D-dimer levels were significantly lower for Methods C (
Comparisons of D-dimer results between the reference centrifugation (Method A) and Methods B, C and D, Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital, Durban, KwaZulu-Natal, South Africa, September 2021 – November 2021. Linear regressions for: (a) Method A vs Method B, (b) Method A vs Method C, (c) Method A vs Method D.
Our study found that centrifuging samples at 4000 rpm for 10 min yielded PPP in 100% of samples compared to centrifugation at 5000 rpm for 10 min and 5 min, which only yielded PPP in 55% of samples. The higher centrifugation speed caused an increase in platelet count in 45% of cases. This study has shown that Method B (4000 rpm/10 min) is superior to Methods C and D as it did not have a significant impact on the coagulation assay results. Hence, Method B could be an alternate method of processing samples for coagulation tests.
A study by Barnes et al showed that 10 min was the minimum centrifugation time required to consistently meet the recommendations for PPP.
Statistically significant differences were observed in the normal PT and normal D-dimer assay results when centrifuged at 5000 rpm compared to the reference method. Abnormal TT levels were significantly lower when measured with Method D compared to Method A; however, this was not the case for Method C which was also a 5000-rpm method. Although the results for normal PT were found to be statistically significantly different between the reference method and Methods C and D, the differences in the actual mean, median and standard deviation values between the groups were minimal. When combined, the normal and abnormal PT results showed a good correlation between the different methods (
There were statistically significant differences in the abnormal TT (Method D) and normal D-dimer values (Methods C and D) when compared to the reference method; however, there was minimal variation in the mean, standard deviation and median values. Furthermore, the TT results showed poor correlation (
The APTT assay is usually more sensitive to platelet contamination than the PT assay.
A single D-dimer result from Method A had an outlying value when compared with the results from Methods B, C and D. It was confirmed that the sample was collected following standard practice guidelines, labelled correctly and processed using the standard operating procedure of the laboratory.
Our study results encourage further research on rapid centrifugation of coagulation samples to verify the reliability of the results and explore the potential benefits it could have in a clinical laboratory setting. These findings could have practical applications and serve as a basis for additional research to establish local centrifugation protocols in laboratories.
The results of this study (sample size = 60) need to be validated with a larger case-control study. A larger number of healthy individuals (controls) should be included. The PT, TT and fibrinogen assays had low abnormal sample numbers ranging from 20% to 30% and the TT results did not reflect extreme abnormal ranges. Participants were recruited on a voluntary basis; therefore, some assays showed a bias in the normal-to-abnormal ratios.
This study demonstrates that Method B is superior to Methods C and D as it produced results that were most consistent with those obtained using the reference method. Methods C and D produced statistically significant differences in results for the PT, TT and D-dimer assays. We show that the centrifugation of whole blood samples in 5 mL citrate tubes at 4000 rpm for 10 min is suitable for routine coagulation testing. This rapid centrifugation method provides consistent and reliable results and could potentially reduce the overall TAT. These findings may assist experts in revising the current recommendations for the centrifugation of coagulation samples.
The authors wish to thank: Rookaya Mahomed – Senior technologist, National Health Laboratory Services Inkosi Albert Luthuli Central Hospital, for the processing of the study samples; Catherine Connolly – Statistician University of KwaZulu-Natal, for assisting with the statistical analysis of the results; National Health Laboratory Services Haematology Laboratory, Inkosi Albert Luthuli Central Hospital, staff for the technical assistance; Inkosi Albert Luthuli Central Hospital and King Edward VIII Hospital: Department of Clinical Haematology staff for their assistance during the sample collection process.
The authors wish to declare that they have no personal or financial interests that may have had an influence on the writing of this article.
R.H. conceived and presented the idea of the study to D.P. R.H. and D.P. developed the theory, methodology and protocol for this study. N.R. reviewed the work and advised on the study design and implementation of the research. D.P. supervised and N.R. co-supervised the project. All authors contributed to the analysis of the results and to the writing of the final manuscript.
The authors acknowledge the receipt of financial support for the research from the National Health Laboratory Services Department of Haematology, Inkosi Albert Luthuli Central Hospital.
The data generated during the study is available on request from the corresponding author, R.H., due to privacy/ethical restrictions.
The views expressed in this article are those of the authors and not of the institution (University of KwaZulu-Natal) or the funder (National Health Laboratory Services).