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Pre-analytical issues in Hemostasis testing
Date: 31 May , 2017 in India ,

Introduction:

All diagnostic steps which are undertaken before the analytical procedure are termed as the “pre-analytical procedures”. These pre-analytical procedures are a vital part of any laboratory testing which includes formulation of the medical question, patient preparation, sample collection, handling, transportation, processing and storage until the time of analysis.

Even a very small error in pre-analytical procedures may create major interpretative as well as therapeutic issues. The well adopted practice of utilizing internal quality control and external quality assurance has reduced analytical errors many fold in coagulation testing. The consequences arising from pre-analytical errors in diagnostic testing is considered to be less harmful than wrong drug therapy or careless surgery.

Hemostasis can be termed as coagulation process and is a first step of wound healing process. In hemostasis, three steps are involved; primary hemostasis (platelet plug formation), secondary hemostasis (fibrin clot formation) and fibrinolytic pathway. The routine coagulation tests comprise of Prothrombin time (PT)/International Normalized Ratio (INR) and the Activated Partial Thromboplastin Time (APTT), Fibrinogen assays, Thrombin Time (TT) assays and D-Dimer assays. These tests can be performed on specialized “Coagulation Analysers” based on light scatter detection principle and immunogenic turbidimetric assay principle with single or multiple photometric channels.

 

Typical causes for Pre-analytical errors:

  1. Incorrect patient identification and wrong sample collection & labeling: Incorrect identification of patient and sample leads to misdiagnosis and inappropriate therapy.
  2. Inappropriate test selection and ordering: While test results and methodologies comprise analytical issues, the choice of which particular methodologies or test panels to use might best be considered as crucial pre-analytical variables.
  3. Incorrect anticoagulant selection: It may alter the clotting time and all parameter values depending upon it.
  4. Serum sample collected instead of plasma sample: Testing of serum will therefore lead to non-coagulation in tests such as the PT, APTT, and TT, possible diagnosis of coagulation factor deficiencies, and problems with LA identification.
  5. Inadequate separation & mixing of sample: It may lead to activation of platelet and decrease the values coagulation timing.
  6. Hemolysed sample processing: Hemolysis increases the spectrometric absorbance of the plasma sample and leads to high background absorbance readings, which may
    compromise clot detection and affect the accuracy of test times.
  7. Abnormal hematocrit sample processing: Too high a hematocrit will influence the anticoagulant to plasma ratio and test results.
  8. Lipemic sample processing: Lipemia may elevate activity of Factor VIIa, and affect the platelet function & reduce activity of other clotting factors.
  9. Frequently freezing and thawing of sample: This may hamper activity of some labile factor like factor V and factor VIII.
  10. Normal reference range related issues: Use of an inappropriate normal reference ranges may yield abnormal test results.
  11. Demographic variables: Age, gender, ethnicity, and blood group might influence reference values for certain parameters of laboratory hemostasis, and/or generate variable test results for some tests.
  12. Physical activity, illness and stress: Excess physical activity before sample collection leads to certain in vivo events, which may in turn affect hemostasis.
  13. Circadian and diurnal rhythms: Levels of some hemostasis components follow a circadian or diurnal rhythm, with differential levels detectable at different times of
    the day.
  14. Patients on medication: Patients on anticoagulant or any other therapeutic agents may lead to false positive or false negative results.

Pre-analytical guidelines:

  1. Patient & Sample Identification: Patient’s demographical data should be electronically recorded and barcode should be generated accordingly. The samples should be traceable as per the barcode of respective patient. Hence, at least twostep identifier system should be included in patient registration procedure.
  2. Appropriate sample collection: The coagulation testing samples must be drawn into citrate based anticoagulant tubes (3.2% or 3.8%, sodium citrate). If coagulation samples are drawn along with other test samples (i.e., EDTA Whole blood, heparin whole blood, etc.) then Clinical & Laboratory Standards Institute (CLSI) guidelines for “order of draw” sample should be followed. Prior to blood collection expiry of vacutainers should be checked, and it should be filled not less than 90% of total capacity of tube as under filling may cause sample dilution and effectively prolonged clotting time. Once collected, the sample should be mixed “gently” (avoid vigorous mixing) by inverting tube 3 – 4 times. The collected blood should not be transferred from one collection tube to another, in an effort to provide the required complete fill as it may increase the amount of anticoagulant in it.
  3. Sample transport: Sample should be transported as per CLSI guidelines, and testing should be completed within 4 hours of collection. Extreme temperature should be avoided. The plasma should be separated by centrifugation followed by freezing and frozen transport of the plasma should be considered if the testing is done at a different site.
  4. Sample processing and storage: Many of the routine coagulation tests are performed on single centrifuged citrate plasma sample. Samples of Lupus Anticoagulant (LA) testing, should be double centrifuged to remove platelets before freezing. Centrifugation should be at 1500 g for minimum of 10 – 15 minutes. To maintain sample integrity, sample should be processed as soon as possible after collection i.e., within 1-4 hours after collection. For immediate processing sample plasma should be kept in capped tube and can be stored at room temperature. The separated plasma should be frozen in frost-free freezers at -200c – 800c for later processing (i.e., after few weeks to few months). Before processing, samples should be thawed at 370c water bath for 5 -10 minutes and mixed gently, prior to testing.

Comments

Pre-analytical issues in hemostasis testing are an important cause of diagnostic errors and can lead to adverse clinical effects. The increasing global burden of laboratory errors can be reduced by intercepting erroneous results, before they are released to the clinician saving the patient from its harmful effects. The ultimate aim of laboratory practice would be to have no errors or to at least detect and correct all errors before the test result is released. It is recommended that laboratories use appropriate post test guidance to assist the clinician in the interpretation of test results and guide when a repeat, confirmatory and follow-up testing may be required. The new anticoagulants recently introduced in the market will variously affect coagulation and hemostasis and should also be considered in the context of pre-analytical problems associated with hemostasis testing.

References:

  1. Laboratory Variables That May Affect Test Results in Prothrombin Times (PT)/International Normalized Ratios (INR); Laboratory Medicine, February 2003, number 2, volume 34
  2. Pre-analytical Variables in Coagulation Testing Associated With Diagnostic Errors in Hemostasis; Emmanuel J. Favaloro, Dorothy M. (Adcock) Funk, Giuseppe Lippi; Lab Med. 2012;43(2):110

 

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