PRE-ANALYTIC VARIABLES IN COAGULATION TESTS Dr Susheela Innah Professor and HoD Dept of Transfusion Medicine Jubilee Mission Medical College, Thrissur

Analytic The Quality Assurance Cycle Pre-Analytic Post-Analytic Patient/Client Prep Sample Collection Personnel Competency Test Evaluations Reporting Data and Lab Management Safety Customer Service Post-Analytic Sample Receipt and Accessioning Record Keeping Sample Transport Quality Control Testing Analytic

Principles of Pre-Analytical Variables A. Sample Collection -Patient identification - Sample labeling - Phlebotomy technique - Sample volume - Sample collection tube

B. Sample Handling -Storage - Agitation, Centrifugation Transport conditions Delay in transport to the laboratory for processing

C. Patient Related Factors Pathological states Physiological variables

Sample Collection Variable Sources of Error Patient Identification Incorrect patient identification Venepuncture -Drawing blood downstream from an IV infusion -Applying the tourniquet for more than a minute -Slow venepuncture

Venepuncture ( contd) - An inappropriately narrow gauge needle - Failing to discard the initial sample from an indwelling catheter - Incorrect tubes Samples, if drawn through a needle into a syringe, should be drawn into plastic or siliconised glass Tubes should be filled without foam.

Variable Sources of Error Inadequately filling the sample collection tube Inadequate filling results in an incorrect anticoagulant : plasma ratio and inaccurate results. - The ratio for blood taken into sodium citrate should be 1 part anticoagulant:9 parts blood. Most tubes are calibrated to collect 2.7ml of whole blood into 0.3ml of 3.2% (0.109M) sodium citrate to give a final volume of 3.0ml and a ratio of whole blood : anticoagulant of 9:1.

Inadequate filling of tube - When the haematocrit exceeds 0.55 (55%) the reduced plasma volume requires a decrease in the volume of anticoagulant used to maintain the ratio of 9:1. Amount of calcium available to chelate the citrate is less. The formula C (ml) = 1.85 x 10-3 x (100-Hct (%)) x V (ml) Or for HCT of more than 60%, volume of citrate adjusted according to formula 60X4.5/100-pt’s HCT=Amount of blood collected in 0.5ml anticoagulant

The graph below shows an alternative method for calculating the concentration of 3.2% trisodium citrate to be used as an anticoagulant depending upon the haematocrit and which will ensure a ratio of 1 part anticoagulant:9 parts blood.

Sample Handling Variable Sources of Error Temperature and agitation - Exposure of samples to excess heat can cause denaturation of plasma proteins resulting in the formation of variably sized protein microparticles the smaller of which may be misread by automated analysers as platelets giving spurious elevation of the platelet count. - Delays of >6 hours between collection and processing of INR or prothrombin time samples can cause significant (i.e. >10%) changes in measured values. Mechanical agitation e.g. a long car or van journey can also contribute to this.

Variable Sources of Error Temperature and agitation The temperature at which archived samples are stored affects their shelf-life. For most coagulation tests storage at -35°C or less gives a shelf life of several years but storage at -20°C is inadequate. Repeated freeze-thaw cycles may affect factor level, for example, a reduction in vWF:CB activity and FXII levels. Freeze-thawing may also produce phospholipid rich membrane microvesicles from platelet damage which may then mask the presence of a lupus anti-coagulant

Patient related factors Variable Sources of Error Age At birth the levels of the vitamin K dependent clotting factors are reduced and only reach adult values at approximately six months of age. Physiological variation Pregnancy affect components of the haemostatic pathway, in this case a rise in the levels of factor VIII, Von Willebrand Factor, fibrinogen, a fall in protein S and often a mild fall in platelet count

Variable Sources of Error Platelet Clumping due to EDTA (Pseudo- thrombocyto-penia) Platelet clumping is a phenomenon occurring due to EDTA dependent antibodies against platelet surface glycoproteins and may result in a falsely low platelet count. Collection of samples for platelet counting into sodium citrate can sometimes eliminate this Elevated bilirubin or lipid concentration High levels of bilirubin or lipids may result in turbid plasma which can interfere the with optical density measurements

Preparation of Samples Most tests use PPP (Platelet Poor Plasma) Samples must be processed within 2 hours For platelet function tests PRP (Platelet Rich Plasma) must be used (Samples centrifuged at 1000-1200rpm for 10min at room temp) If low and Bernard Soulier syndrome suspected then do not centrifuge. Keep at room temp

Transfer PRP for platelet function tests in transfer tubes Transfer PRP for platelet function tests in transfer tubes. Platelet count should be adjusted to 200 to 250 thousand Centrifuge tube and take the PPP for other tests. Platelet count should be less than 10000/cumm Check platelet count at least once in a batch Always run control sample with test sample

Do not use grossly hemolyzed specimens Samples for thrombotic workup must be frozen at -800C within 2 hours

Possible causes of Increased INR and aPTT Short draw – excess of citrate in plasma Elevated HCT (>55%) – commonly in newborn – excess citrate in plasma Clotted sample Sample more than 4 hours old : aPTT Sample more than 24 hours old : INR Hemodilution – drawn above IV line site, drawn via arterial line without proper clearance

Possible causes of decreased INR and aPTT Low hematocrit (<25%) Elevated Calcium Poorly collected sample (decrease due to activation of sample) Sample placed on ice – decreases INR Sample > 2hours old for patients on UFH – Decrease aPTT due to platelet degranulation and release of PF4 release which neutralizes heparin

Summary Most coagulation screening tests are crude in nature All coagulation results should be interpreted with caution until pre-analytical and analytical variables have been excluded to prevent false positive/negative results

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