Five-Minute Preparation of Platelet-Poor Plasma for Routine Coagulation Testing A
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املجلة الصحية لرشق املتوسط املجلد السادس عرش العدد الثاين Five-minute preparation of platelet-poor plasma for routine coagulation testing A. Sultan1 إعداد يستغرق 5دقائق للبﻻزما الفقرية بالصفيحات من أجل اﻻختبار الروتيني للتخثر أمحد حسن سلطان تتطلب اخلﻻصـة:الدﻻئل اﻹرشادية ملؤسسة املعايري اﻹكلينيكية ومعايري املختربات أن جترى اﻻختبارات الروتينية للتخثر ببﻻزما فقرية بالصفيحات )أقل من 000 10 صفيحة/مكرولرت(، وحترض من كامل الدم املضاف إليه السرتات وجرى تنبيذه برسعة منخفضة ملدة 10 – 30 دقيقة. وملقارنة النتائج التي تم احلصول عليها من البﻻزما التي خضعت للتنبيذ ملدة 5دقائق برسعة 3000جاذبية أو ملدة 10 دقائق برسعة 2000 جاذبية، أجريت مقايسة شملت 46عينة مأخوذة من بالغني أصحاء أسوياء بقصد التعرف عىل زمن الربوثرومني، وزمن الثرومبوبﻻستني اجلزئي املفعّل لواملعدَّ الدويل املسوَّ ى. ﻻ يوجد اختﻻفات يعتد هبا يف نتائج اﻻختبار، ُواستنتج ِأن النبذ ملدة مخس دقائق بمقدار g 3000يعترب ًاختيارا ًموثوقا فيه ًومفيدا للحد من الوقت املستغرق يف هذه اﻻختبارات. ABSTRACT Clinical and Laboratory Standard Institute guidelines require that routine coagulation tests are performed with platelet-poor plasma (< 10 000 platelets/µL) and prepared from whole citrated blood centrifuged at low speed for 10–30 minutes. To compare results obtained from plasma centrifuged for 5 minutes at 3000 g or for 10 minutes at 2000 g, 46 blood samples from normal healthy adults were assayed for prothrombin time, international normalized ratio and activated partial thromboplastin time. No significant differences were found in test results and it was concluded that 5 minutes centrifugation at 3000 g is a reliable and useful option to reduce the turnaround time for these tests. Préparation en cinq minutes d’un plasma pauvre en plaquettes pour les tests d’hémostase de routine RÉSUMÉ Les recommandations du CLSI (Clinical and Laboratory Standard Institute) imposent que les tests d’hémostase soient réalisés avec du plasma pauvre en plaquettes (< 10 000 plaquettes/µL) et préparés à partir de sang total citraté, centrifugé à vitesse lente pendant 10 à 30 minutes. Afin de comparer les résultats obtenus à partir de plasma centrifugé pendant 5 minutes à 3000 g ou pendant 10 minutes à 2000 g, 46 échantillons de sang prélevés sur des adultes en bonne santé ont été analysés pour évaluer le temps de prothrombine, le rapport international normalisé et le temps de thromboplastine partielle activée. Nous n’avons constaté aucune différence significative dans les résultats des tests et avons conclu que la centrifugation à 3000 g pendant cinq minutes était une possibilité fiable et utile pour réduire le temps nécessaire à la réalisation de ces tests. 1Department of Medical Laboratory Technology, College of Health Sciences, University of Sharjah, Sharjah, United Arab Emirates (Correspondence to A. Sultan: [email protected]). Received: 24/07/07; accepted: 11/11/07 233 EMHJ • Vol. 16 No. 2 • 2010 Eastern Mediterranean Health Journal La Revue de Santé de la Médiderranée orientale Introduction prepared PPP obtained at each speed lipaemia and haemolysis (plasma with was used to measure platelet count, PT, pink to red colour) in the second and/ Prothrombin time (PT) and activated international normalized ratio (INR) or third tubes. partial thromboplastin time (APTT) are and APTT. A fixedangle, tabletop centrifuge the 2 most commonly requested rou- [CenturionK40 model, Centurion tine coagulation tests for the screening Scientific, Sussex, United Kingdom of acquired and inherited coagulation Method (UK)] was used for the preparation of disorders and the monitoring of anti- PPP at room temperature within 1 h of Between 25 February and 28 May 2006, coagulation therapy [1]. Quality care collection by centrifuging the second blood samples were taken from 46 requires reliable test results and prompt draw tubes at 3000 g for 5 min and the turnaround times. Reliable results can healthy volunteers at the University of Sharjah, Sharjah, UAE. The sample con- third draw tubes at 2000 g for 10 min. be achieved by carefully controlling pre sisted of 38 female and 8 male students The instrument was checked twice a analytical variables, which are responsi- and staff members who gave informed year (once per academic semester) with ble for 64% of all errors in testing [2,3]. consent, age range 18–33, with a mean a digital photo tachometer (Tenma PT and APTT tests represent about age of 22.3 years. Precollection verbal 726633, Springboro, Ohio, USA) and 90% of coagulation tests ordered, and it questions to the volunteers revealed a sports stopwatch to ensure that the takes 40–180 minutes or more for re- that they did not have any coagulation obtained speed and time did not exceed porting results with standard laboratory disorders, had not ingested aspirin 5% of the expected speed [15]. Before methods [4,5]. Such long turnaround within the previous 3–7 days and were the study started the centrifuge was times can be very costly in some medi- not on anticoagulant therapy. checked for the required speed (3000 cal situations associated with critical, Duplicate 2.7 mL plastic vacuum g) and time (1, 5 and 10 min) and surgical and emergency cases, when tubes (with 3.2% sodium citrate) and the results were acceptable. The selec- good care could be denied [6]. Faster 21gauge needles [BectonDickinson, tion of 3000 g speed was determined turnaround time, while maintaining Rutherford, New Jersey, United States of empirically after a number of trials of sample quality and accuracy of results, america (USA)] were used to collect the centrifuging duplicate 2.7 mL sodium depends mainly on the time consumed samples. Capillary haematocrit values citrated samples at speeds that ranged on coagulation sample collection, han- ranged from 32 to 42 mg/dL. To control from 2500–3000 g and achieving re- dling, processing and testing [7,8]. The preanalytical variables and protect the producible platelet counts < 10 000/µL shortest recommended centrifugation quality of the tested samples the venous (data not shown). time to produce the recommended blood collection techniques and sample About 1 mL supernatant plasma was plateletpoor plasma (PPP) with plate- handling and processing conformed to carefully removed with a plastic transfer let counts < 10 000 /μL is 10 minutes the CLSI guidelines [14]. These were: pipette from the middle of the sample at 2000 g using regular centrifuges [9]. the tourniquet was placed for under 1 Highspeed centrifuges have been used and away from the buffy coat to avoid min to locate the vein and release it as contamination with excess platelets. for the rapid preparation of PPP in 2–3 soon as the blood entered the first tube minutes but these are unavailable in All paired PPP samples were prepared without any needle probing to ensure and tested for platelet count within 0.5 most laboratories. In addition, most of nontraumatic venepuncture; second them are microcentrifuges with limited h of collection using a semiautomated and third tube draws were used follow- haematology analyser (Medonic CA capacity (up to 12 samples) and require ing a 3 mL redtop plain tube to avoid 620, Stockholm, Sweden). The PT, INR the transfer of aliquots from the collect- contamination with tissue thromboplas- and APTT were tested at room tem- ed blood into conical microcentrifuge tin; collected samples were immediately perature within 2 h of collection using a tubes [10–13]. mixed gently 3–5 times by inversion; all coagulation analyser with the required This study in the United Arab Emir- collected samples were visually checked controls and reagents (Dade BFT II, ates (UAE) used readily available labo- for complete filling against a sample tube ratory centrifuges to compare 5minute to ensure 9:1 ratio of blood to antico- Dade Behring, Marburg, Germany). centrifugation at 3000 g with 10minute agulant; tubes were gently inverted 2–5 Data were entered using Microsoft centrifugation at 2000 g to prepare PPP times before centrifugation to facilitate Excel and descriptive statistical analy- in accordance with the approved guide- residual platelet suspension and to check ses—mean, range, standard deviation lines of the Clinical and Laboratory for the presence of clots; all centrifuged (SD) and paired Student ttest—were Standards Institute (CLSI) [14]. The samples were visually checked for computed using SPSS, version 14. 234 املجلة الصحية لرشق املتوسط املجلد السادس عرش العدد الثاين Results citrate) vacuum tubes with 5 min centrif- III, heparin, Ddimer and Russell dilute ugation at 3000 g in a tabletop centrifuge venom time values [11]. Platelet counts < 10 000 /μL were with fixed angle. These residual platelet produced from all 46 PPP samples cen- counts met the approved CLSI guidelines trifuged for 5 minutes and only 1 sample for PPP used for coagulation tests [14] Conclusion out of the 46 (2%) had a platelet count and showed no significant differences in > 10 000 /μL (13 000 /μL) and was platelet count, PT, INR and APTT values This study showed that residual excluded as an outlier (Z-score > 3.9) compared with PPP prepared by 10 min platelet counts in PPP prepared with [16]. centrifugation at 2000 g. 5 min centrifugation at 3000 g were < 10 000 /μL, as recommended by CLSI Descriptive statistics for platelet Our platelet counts prepared in 5 guidelines for PPP used for coagulation count, PT, INR and APTT for PPP pre- min were lower than the range reported tests [14]. No significant difference was pared for 5 min at 3000 g and 10 min by previous studies (5–79 × 109/L) that found for healthy individuals between at 2000 g centrifugations are shown in used patients’ blood collected in 4.5 mL platelet count, PT, INR and APTT val- Table 1. The mean and SD values of the tubes and centrifuged for 5 min and 10 ues produced by PPP prepared in 5 and parameters were very close and were min at regular speeds.