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Furthermore, water baths must be properly maintained to make certain they are not inadvertently maintained at a higher temperature because this may lead to deterioration of coagulation factor activities and spurious coagulation test results. Once samples are thawed, it is imperative they are thoroughly and adequately mixed prior to testing. Patient misidentification errors are potentially associated with the worst clinical outcome due to the potential for misdiagnosis and inappropriate therapy.

Whenever misidentification is suspected, the laboratory might be able to identify this as being the case by investigation, but the safest approach is generally recollection and retesting. Although serum, heparin, or EDTA samples provided as a primary collection tube can be quickly identified as unsuitable by the laboratory, collection into the wrong anticoagulant may be missed when samples are provided in secondary tubes, or if samples have been mixed or transferred or added to a primary citrate tube. For example, testing for routine coagulation tests such as the PT and APTT will result in no clot or prolonged clotting times, but effects on other test results may be more subtle.

Thus, testing of normal serum for VWF tests will not provide extreme changes but might instead give rise to patterns consistent with Type 2 VWD. Samples in which the blood is slow to fill the collection container, where there is prolonged use of a tourniquet, or considerable manipulation of the vein by the needle may be prone to develop a clot in vitro.

Clots may also develop when samples are incompletely mixed immediately following collection or in under-filled tubes. The presence of a clot is a cause for rejection of the specimen. Alternatively, test results using serum might be normal with some other tests. Table has been adapted and updated from reference 4.

To determine if the sample is serum, a TT can be performed. Non-clotting will suggest either serum or heparin contamination, which can then be differentiated by mixing studies ie, TT performed on sample mixed with normal plasma; if the mixed plasma clots, then serum is confirmed, whereas if the mixed plasma does not clot, this would suggest heparin contamination. The presence of heparin can also be determined by use of a heparin anti-FXa assay or by measuring a TT or APTT before and after the addition of a heparin-neutralizer.

In some cases it may also lead to false identification of weak LA. As before, some test results might be normal eg, VWF:Ag, D-dimer , and so, the ability of a laboratory to recognize an EDTA plasma sample will depend on the tests performed. Assessment of potassium extremely increased or calcium very low to absent will usually identify the presence of an inappropriate EDTA collection. Within a hospital setting heparin contamination is much more common than hemostasis assays incorrectly collected in either EDTA or submitted as serum.

Effects on hemostasis tests depend on the heparin concentration or level of contamination and the test performed. There is also a potential for false identification of LA and factor inhibitors and reduced antithrombin. Although useful, this can lead to complex patterns of test results and laboratories are sometimes falsely reassured that these tests are not influenced by heparin.

Results might be normal with some other tests eg, D-dimer, VWF:Ag , and so whether the laboratory recognizes a heparin-contaminated sample as such will again depend on the tests performed. Heparin contamination can be provisionally identified by testing of select clot-based assays especially APTT and TT , and then by mixing studies see end of serum section above , and confirmed by using an anti-FXa assay or by repeat APTT or TT testing after addition of a heparin neutralizer.

Badly processing samples can lead to hemolysis or platelet activation, falsely prolonging or shortened clotting times, depending on the extent of hemolysis vs platelet activation. Alternatively, inadequate mixing may lead to clotting or partial clotting and prolongation or shortening of clotting times and elevated or diminished clotting levels. Freezing of plasma contaminated with cellular material may also lead to hemolysis or activation events, as well as the potential for false-negative LA. This results from cellular destruction within whole blood and the release of cellular lysis products including hemoglobin into the plasma.

Although in vitro hemolysis might be a byproduct of a problematic collection or the result of poor handling of blood post collection, hemolysis can also derive from in vivo blood cell lysis eg, from hereditary, acquired, and iatrogenic conditions such as autoimmune hemolytic anemia, severe infections, intravascular disseminated coagulation, or transfusion reactions. Instruments utilizing mechanical means of clot detection are not affected by this interference, but the test result may still be compromised since cell lysis products include tissue factors that may activate coagulation.

The net effect is that detected fibrinogen levels may fall with increasing hemolysis, whereas D-dimer levels may increase. Prothrombin time values may fall in line with decreasing fibrinogen, whereas APTTs may increase or decrease depending on the net effect of activation vs the loss of fibrinogen. Hemolysis may also influence other test results eg, decrease antithrombin levels. If possible, grossly hemolyzed specimens should be rejected.


If testing must be pursued eg, if in vivo hemolysis is present , testing using a mechanical end point detection system is recommended, although the potential effect of activation should also be noted. Samples appearing hemolyzed due to the presence of a hemoglobin substitute are not a cause of specimen rejection, and these samples should be evaluated using a mechanical or electromechanical method for clot detection. The presence of significant anemia has not been shown to influence test results. It is not easy to dichotomize the biological and analytical effect of lipemia on coagulation tests.

Analytical interferences in some laboratory assays especially those based on optical clot detection also occur but are minimized using mechanical or electromechanical-based procedures or using analyzers comparing the absorption of samples at 2 wavelengths or performing coagulation assays at alternative wavelengths.

Since it is not always clear how many times a sample has been thawed and refrozen prior to testing, retesting using a fresh sample is always indicated should an unexpected low factor result be obtained. Laboratories use normal reference ranges NRRs to identify whether a test result is within the normal range or outside this range and to thus identify an abnormal result. Use of an inappropriate NRR may mean some normal individuals will yield apparently abnormal test results.

Another way to consider this is to recognize that a standard laboratory NRR will correctly identify only 95 out of every normal test results. Put into clinical context, 5 in every or 1 in every 20 tests a clinician orders using such NRR estimates will likely reflect a false abnormal test result, again simply based on the statistical model used to generate the NRR.

The relative false positive to true positive rate increases substantially for rare disorders and is a particular problem with congenital disorders such as protein C, protein S, and antithrombin, especially when patient cohorts are inappropriately selected for testing. Thus, interpretation of test results should consider these issues to prevent misdiagnosis.

The INR is the most common test performed by coagulation laboratories. This is because the process leads to loss of other plasma components, including FVIII and VWF, and may cause potential problems in LA detection because it artificially elevates the baseline clotting times observed using some LA clot-based assays, and may also lead to a false conclusion of an elevated APTT.

Hemostasis practice: state-of-the-art

In extreme cases, microfiltration may even generate false weak positive LA findings. Moreover, in many routine hemostasis laboratories, LA testing is requested not only for specific investigation of APS but also for investigation of unexplained prolongation of APTT test times, a common incidental finding in a laboratory practice. Accordingly, the double centrifugation approach for preparation of hemostasis samples prior to freezing is now strongly favored. Excess physical activity in patients immediately prior to collection leads to certain in vivo events eg, plasma volume expansion and increased basal metabolism , which may in turn lead to significant effects on hemostasis.

However, perhaps the best-known acute effects are related to acute phase reactants, which may rise due to physical activity, illness or stress, and include fibrinogen, VWF, and FVIII.


In the worse case scenario, these elevations may result in a misdiagnosis of mild hemophilia A or VWD Type 1 patients as a non-hemophilia or non-VWD false negatives. Blood collection may sometimes be stressful for some patients particularly children leading to acute phase changes in proteins secondary to the phlebotomy itself. Levels of some hemostasis components follow a circadian or diurnal rhythm, with differential levels detectable at different times of the day.

PFA closure times and possibly VWF may also provide different values throughout a hour period. Although most changes tend to be fairly subtle, in the worse case scenario this might also lead to some clinically significant differences.

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Testing for thrombophilia is often performed in patients who have recently suffered a thrombotic event. Patients are placed on anticoagulant therapy after a thrombosis. Testing while on anticoagulant therapy will affect both biologically and analytically many of the tests undertaken in this context, including LA, activated protein C resistance APCR , anti-thrombin, protein C, and protein S.

Thus, false-positive and false-negative diagnoses can both occur, depending on the extent of the anticoagulant effect, and the test performed. A variety of therapeutic agents may cause spurious coagulation results due to variable mechanisms. This effect is not always intuitive based on the pharmaceutical product. The concept of clinical ordering patterns as a pre-analytical issue is also worth mentioning, in particular for the case of inappropriate clinical orders.

Following a thrombotic event, some loss consumption of the natural anticoagulants might arise; hence, testing too soon after a thrombosis might lead to false conclusion of a deficiency. Alternatively, anticoagulant therapy will affect the detected levels of the natural anticoagulants, as mentioned previously viz, heparin therapy may influence antithrombin detection, warfarin therapy may influence protein C and protein S levels, and heparin and warfarin therapy may influence APCR testing.

Heparin and warfarin therapy may also influence the appropriate identification of LA. While test results and methodologies comprise analytical issues, the choice of which particular methodologies or test panels to use might best be considered as pre-analytical variables. For example, VWD may be misdiagnosed or missed if the test panel does not include tests for VWF activity, such as a collagen-binding assay. In another example, different laboratories and even experts use different tests or methodologies and test panels for the identification or exclusion of APS.

Pre-analytical issues in hemostasis testing are an important cause of diagnostic error summarized in Tables 2 and 4 and can lead to significant adverse clinical events. However, the burden of laboratory errors is estimated to remain globally modest ie, 1 in every — patients or every — laboratory results.

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. Accordingly, several tools might assist in their identification, including a comprehensive education of all personnel regarding types and sources of errors, the accurate evaluation of sample quality ie, volume, blood to anticoagulant ratio, presence of potential interferents, or contaminants , and the systematic recording of suspect results along with pertinent clinical information.

The most reliable approach to deal with laboratory errors is to establish a total quality management system. We also strongly recommend that laboratories use appropriate post-test guidance to assist clinicians in the interpretation of test results, as well as to guide when repeat, confirmatory, and follow-up testing may be required.

There have been several new oral anticoagulants recently released onto the market most notably Dabigatran and Rivaroxaban for a variety of clinical indications including prevention of venous thromboembolism after major orthopaedic surgery or secondary prevention in atrial fibrillation. These agents will variously affect coagulation and hemostasis tests as described elsewhere, 53 , 54 but should now also be considered within the context of preanalytical problems associated with hemostasis testing.

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Oxford University Press is a department of the University of Oxford. Here are a few:. Intrinsic vs. How come the extrinsic and intrinsic pathways are named that way? Why do you need the intrinsic pathway when you have the extrinsic pathway? Coagulation Quiz. For more on the coagulation cascade, see Robbins 9th edition, page or 8e, page When I got up, my head hit the corner of an open locker. As it was merely painful at first, I continued what I was doing.

And one second later I felt some drops of a warm liquid falling on my right shoulder. Useless to say that the right part of my torso turned red. The guy in the showers looked at me as if I were a weirdo… What? I had to clean it up at some point, before entering the pool! I realized I was dozing in and out of sleep while reading that bit on the nitty-gritty of the coagulation cascade.

External image. The hemostasis laboratory has a vital role in the diagnosis and management of patients with familial and acquired haemorrhagic and thrombotic disorders. Its role in the monitoring of traditional anticoagulant therapy, as well as therapy using new anticoagulants, presents new challenges to the laboratory.

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    The product may be sprayed on a bleeding surface, or applied internally using a patch. Skip to content. Search for books, journals or webpages All Pages Books Journals. View on ScienceDirect. Editors: Beth Shaz Christopher Hillyer. Paperback ISBN: Imprint: Elsevier Science. Published Date: 11th June Page Count: Flexible - Read on multiple operating systems and devices.

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    Institutional Subscription. Free Shipping Free global shipping No minimum order. Updated content covers new coagulation tests, cellular therapies, and quality control issues Easy to use, with focused, well-defined chapters in a standardized format throughout Offers quick "cross-reference" lists at the end of each chapter Includes lists of common abbreviations and indexes that cross reference diagnostic, clinical and therapeutic commonalities.

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