Laboratory issues in coagulation testing

Laboratory issues in coagulation testing Pre analytical Analytical Post analytical Biological variation (inter and intra individual variability)

Pre analytical phase: Lack of standardized procedures for sample collection patient preparation specimen acquisition handling and storage often outside the control of the laboratory performing the test.

Patient preparation Testing for haemostasis should be avoided drugs interfering with the function of platelets and coagulation factors closer to any transfusion of blood, blood products and factor concentrates. For factors like FXIII it needs to be avoided even upto 1 month. Biological variation in tests of haemostasis: Plasma clotting times - constant within and between subjects because proteins such as fibrinogen, clotting factors and antithrombin show a low biological variability. Fibrinolytic parameters such as PAI 1 and fibrinopeptide A show very high variability.

Issues related to sample collection Under filling - dilution of plasma resulting in underestimation of clotting factor levels overfilled standard vacuum tube will not give erroneous results until it is overfilled to more than 120%. Under mixing may affect tests downstream specialized hemostasis assays performed after some time. Vigorous mixing (shaking of tubes) might lead to hemolysis or spurious test activation and false shortening of test clotting times and even false elevation of clotting factor activity(factor VII). Poor venesection :- Not so uncommon error is the plasma may be a sample collected in EDTA. EDTA plasma may result in falsely prolonged plasma clotting times and will show an inhibitor effect and if the requested test is a Lupus anticoagulant (LA) this will cause a false positive result. Unseparated samples for VWF could lead to loss of high molecular weight multimers during transportation. Filtered plasma might produce spurious hemostasis tests results, false diagnosis of vWD could occur due to loss of factor VIII and vWF

Issues related to Storage Inadeqautely thawed samples would lead to inhomogeneous sampling Cryoprecipitate portion would be selectively sampled very high levels of FVIII:C, VWF, Fibrinogen or FXIII. Cryo poor part very low levels of FVIII:C, VWF, Fibrinogen or FXIII. Frozen samples should be thawed and mixed at 370C waterbath before testing.

Proper Collection Use a blood collection system that collects the specimen directly into a tube containing the anticoagulant Order or draw for evacuated venous blood collection tube system the blood coagulation tube is always first when multiple containers are to be collected except when a blood culture too is needed. Prothrombin Time/Internal normalized ratio (PT/INR) and activated partial thromboplastin time (APTT) results are not affected if tested on the first tube drawn When using a winged blood collection set for venipuncture and a coagulation tube is the first tube to be drawn a discard tube should be drawn first. Syringe draws using a hypodermic needle/syringe may have increased risk of hemolysis and apparent safety issues. If a double-syringe technique is used, blood from the second syringe should be used for the coagulation specimen.

Proper Collection Blood should be added to the appropriate volume of anticoagulant within one minute of completion of draw. All tubes should be inverted at least four times to mix. Excessive mixing can cause hemolysis and/or platelet activation, leading to erroneous results. If the blood in the syringe is to be transferred to an evacuated tube system the rubber stopper of the evacuated tube is pierced with the needle. Use the same “order of draw” as for evacuated blood collection tube system. 105 to 109 mmol/L, 3.13% to 3.2% (commonly described as 3.2%) of the dihydrate form of trisodium citrate (Na3C6H5O7 • 2H2O), buffered or nonbuffered. The proportion of blood to the sodium citrate dihydrate anticoagulant volume is 9:1. Correction for hematocrit values above 0.55 L/L (55%). X mL = 60/(100-PCV) x Z mL

Proper acquisition and Processing The whole blood specimen should be checked for clot formation by gentle inversion and observation. To obtain a plasma sample, the capped specimen tube should be centrifuged at a speed and time required to consistently produce platelet poor plasma (platelet count <10 x 109/L) (10,000/μL). This may be accomplished by centrifuging at 1,500 g for no less than 15 minutes at room temperature. Swing-out bucket rotor should be used to minimize remixing of the plasma and platelets, particularly with plasma removal. While it is crucial that an essentially platelet-free sample be obtained if the specimen will be frozen for subsequent testing, APTT, PT/INR, and TT performed on fresh plasma samples are not affected by platelet counts of at least up to 200 x 109/L (200,000/μL). Platelet counts >10 x 109/L are not acceptable for lupus anticoagulants, other phospholipid antibodies, and heparin monitoring. The reliability of the centrifugation procedure should be validated every six months or after modification of the centrifuge to ensure plasma platelet counts are within acceptable limits. Samples that have visible hemolysis should not be used because of possible clotting factor activation and end point measurement interference.

Proper Storage and Transportation PT assays - uncentrifuged or centrifuged with plasma remaining on top of the cells in an unopened tube kept at 18 to 24°C should be tested within 24 hours from time of specimen collection. Storage at 2 to 4°C may result in cold activation of Factor VII and therefore alter PT results. APTT assays - uncentrifuged or centrifuged with plasma remaining on top of the cells in an unopened tube kept at 2 to 4 °C or 18 to 24°C should be tested within four hours from time of specimen collection. Same for other assays - thrombin time, protein C, Factor V, and Factor VIII APTT assays - for heparin kept at 2 to 4°C or 18 to 24°C should be centrifuged within one hour of collection and the plasma tested within four hours from time of specimen collection. Frozen at -200C for up to two weeks or -700C for up to six months. A frost-free freezer should not be used. If testing cannot be performed immediately, the specimen may be held for a maximum of two hours at 40C until tested. Cold activation at 2-40C is not known to occur in 4 hrs. Cold activation will result in activation of FVII and also FIX resulting in falsely decreasing the times in PT and APTT tests respectively.

In the case of any unexplained abnormal coagulation test result, a new specimen should be obtained and the test repeated

Preparation of reagents Improper reconstitution of reagents. Reagent stability. Contamination of reagents. Lot variation. Wrong labeling of reagents. Deterioration of buffer. Stability of factor deficient plasma. Inadequate preparation of adsorbed plasma and aged serum for correction studies. They should be Quality controlled where in adsorbed plasma should have a PT of more than 60 seconds and less than 90 seconds to ensure adequate adsorption and prevent over adsorption that leads to loss of FVIII and FV. Aged serum contains activated factors so should include three separate serum samples for mixing as there is a risk of activated factors clotting and correcting FVIII / FV deficiency.

Issues Related to Bleeding time Time taken by a Standard skin wound to stop bleeding

Bleeding Time (Modified Ivy’s Method) BP cuff to 40 mm Hg Select an area avoiding any vein or angiomas Clean the Volar aspect Stab confidently three times and start Stop watch at the end of the third wound. At least one wound is STANDARD

(Skin) bleeding times: Disadvantages: Time consuming. Operator variability / subjectivity. Invasive. Difficult to standardise. Establishment of a normal range ???? Relatively insensitive to mild defects (mild platelet dysfunction, mild VWD). No specificity (an abnormal result won’t diagnose a particular defect … further testing).

PFA-100 – Screening test Whole blood - 5 min platelet function test. High shear stress flow system (‘pseudo-physiological’). Blood added to reservoir – vacuum – drawn into a capillary. Capillary has a membrane with aperture. Membrane coated with Collagen Active platelets adhere to membrane ... platelet activation & release ... platelet aggregation … blocks capillary device … instrument detects this as a ‘closure time’ (CT). Assesses cessation of blood flow (closure time = CT). Two cartridge types (C/ADP & C/Epi) with differing sensitivities.

PFA-100: Advantages: Only requires small amount of blood. Very simple … with proper training - anyone can do it (but typically lab person). Quick test (5-10 min) No real operator variability / subjectivity. Able to standardise. Able to establishment a normal range. Very sensitive and specific

The PFA-100 closure time should be considered optional in the evaluation of platelet disorders and function, and its use in therapeutic monitoring of platelet function is currently best restricted to research studies and prospective clinical trials. Von Willebrand disease

Utility of BT in severe bleeding disorder Among 852 patients evaluated for primary haemostatic defect at our centre from 2004 The sensitivity of Ivy’s method was 100% - Glanzmanns thrombasthenia, 85% - Bernard Soulier syndrome, 68% - platelet secretion defect 63% - von Willebrand disease 100% - VWD-3 52% - VWD-1

Utility of BT in severe bleeding disorder (developing countries) Majority of the patients - more severe defects BT by Ivy method if done as per reference specifications can be a good screening test for primary haemostatic disorder. BT by modified Ivy’s method compares well with any Template bleeding time method provided is done by the described reference method.

Standardization of Ivy’s BT Site - Select a site lateral one-third of the forearm, 2 to 3 cm distal to the antecubital crease, in an area devoid of hair, scars, tattoos, bruises, surface veins, infected skin, moles, or other lesions. Direction of incision – perpendicular (vertical) or parallel (horizontal) to the antecubital crease. One direction, horizontal to be used consistently. A horizontal incision gives a longer bleeding time when compared to a vertical incision. The vertical incision may produce less scarring. Both procedures have a similar degree of reproducibility. The horizontal incision is more sensitive to the effects of aspirin. Blot from the side only a fewer technologists and more so experienced ones to perform and 2-3 incision.

Plasma Clotting tests Automation have permitted an exceptional degree of analytical quality over past 50 years and there is significant decrease in error rates. In Semiautomated instruments tests should be resulted only after duplicate tests. The difference between duplicate tests should agree within 10% of their mean value. Except for automated detection of the clot formation rest of the process involved in semiautomated method is the same as manual therefore the need for duplicate testing on samples.

Choice of Reagent Though WHO recommend the use of PT reagent that have an ISI between 0.9-1.7. PT reagents with ISI of less than 1.4 is good and quite easily available. For APTT there are no such standards since it has to be a screening test for either Factor deficiency or Heparin or for LA. It will be a good practice to be able to validate the sensitiveness and responsiveness (degree to which the time gets prolonged when an abnormality is present) of the APTT reagent by doing some ex-vivo invitro spiking studies. Either have clinical samples to reflect various levels of factors/heparin/LA or spike commercially available or in-house PNP/Deficient plasma e.g, for various FVIII levels mix PNP with FVIII deficient plasma to get different levels of FVIII. The least Risk involved is going in for a Lupus sensitive reagent

Lupus Anticoagulant Use of APTT has increased and usage of DRVVT the most commonly performed test for LA is stable. Reporting a prolonged or a positive screening tests (either APTT or dRVVT) as lupus anticoagulant without mixing studies which is the only way to demonstrate the inhibition or the anticoagulant effect of Lupus anticoagulant is unethical and against the standard as it reports a lot of false positives consistently. 47 yrs old PE on OAT. INR – 2.8. dRVVT Screen – 72 secs; dRVVT Confirm – 43 secs. Screen/Conf = 1.67. – Moderate Positive ISI 1.0 – 32.2 Secs (MNPT=11.5 secs) INR = 2.8; ISI 1.8 – 20.7 Secs (MNPT=11.5 secs) INR = 2.88: ISI 1.0/ISI 1.8 = 32.2/20.7 = 1.56 - Moderate Positive. Mix with Normal Plasma – Complete correction.

APTT:-A major pitfall of APTT is the involvement of FXII in the determination of the time FXII is not part of a procoagulant pathway FXII deficiency is not that uncommon as has been considered. It is a good practice to have an additional APTT reagent in the laboratory with another contact activator (Ellagic acid if the laboratory is using silica) since this could overcome it to some extent. Reporting :- The laboratory should report the results of PT and APTT to the nearest half of a second with the normal reference interval and not with a time of a normal or control sample. INR is reported as such and not with a reference interval. TT is a good test in the laboratory in a hospital that deals with conditions that could cause acquired dysfibrinogenaemia, e.g, Liver or renal disease and possibly indicate use of Cryoprecipiatae instead of FFP in a bleeding patient when the TT is prolonged and Fibrinogen levels are normal. Controls:- 2 levels are adequate and a must. It is recommended to run after every 40 samples in laboratories that have a heavy workload, otherwise beginning of the day, at least once in each shift or with each group of tests.

Factor Assays Factor deficient plasma are the most important part of the factor assays as they help in most of the end-point clot formation. Standard evaluation will require assaying the concentration of individual factors, residual FVIII or low titre inhibitors. An easy way to realize if this is defective is by looking at the 1:10 dilution timing of the standard graph. 48-55 secs > 60-65 seconds - deteriorated. The same applies to FVIII inhibitor assay residual FVIII levels of PNP (most common source of FVIII ) have decreased by more than 20% - false positive results upto 4 BU. Introduction of the Nijmegen modification makes this assay more specific. When ever a moderate to mild deficiency of factors are detected it is a good idea to do other appropriate factors to rule out a familial multiple factor deficiency combination though the screening tests may indicate such. One-stage FVIII assay for determination of recovery levels of FVIII concentrate infusion could result in 20-30% reduced levels compared to assays done by chromgenic method. This is overcome by using a standard graph made of known levels of Factor VIII concentrate spiked to close to 1.0 iu/mL in FVIII deficient plasma for “Like vs Like’ principle.

Thrombophilia testing These are tests to identify risk factors for VTE introduce tests recognizing their sensitivity and specificity Based on this the following information are useful:- Protein C – Use of clot based method has decreased and chromogenic assay has increased Protein S – free antigen assay is more used especially by LIA. ELISA has a risk of antigen fall out during incubation leading to falsely low levels or exaggerating a low level. Clot based assay is wrought with irreproducibility. Antithrombin – use of chromogenic tests have increased and antigenic have considerably decreased. Activated Protein C Resistance – APTT based is decreased APTT with predilution of sample in FV deficient plasma APCR based tests that have shorter cascade steps are used e.g, dRVVT, direct activation of FX. This is useful if the laboratory is not doing molecular testing for Factor V Leiden Mutation - make them specific for the FV Leiden mutation. If the laboratory also does molecular testing for Factor V Leiden Mutation then the traditional APTT based APCR is highly recommended as this APCR is a significant risk factor for VTE independent of Factor V Leiden Mutation.

D-Dimer Use of reagents that has been established through good literature review to be sensitive and almost 100% negative predictive value should be employed. Even if the laboratory is not able to establish a cut-off for VTE/DVT use of 500 ng/mL or 500µg/L or 0.5 µg /mL or 0.5 mg/L reported widely in literature. Certain physiological conditions like pregnancy the cut-off could be raised to 600 ng/mL or 600µg/L or 0.6 µg /mL or 0.6 µg/mL. A good knowledge of assay limitations and the effects of interfering substances are most important aspect of interpretation of complex haemostasis results.