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The interpretations below are provided by Donna Castellone, MS, MT (ASCP) SH for Aniara Diagnostica.

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Pitfalls in Thrombophilia Testing

Thrombophilia testing has never been easy. Who are the patients who should be tested? When should they be tested? What tests should be performed since testing is expensive? Should one test be performed, or should multiple tests be ordered? Should genetic testing be performed? There are a lot of causes of uncertainty and concern and recommendations for testing are vague. The goal is to minimize these pitfalls by educating clinicians, faculty residents and laboratorians. Implementing prior approval for thrombophilia testing to help ensure the right patient is tested at the right time may help as well as using testing algorithms and a step by step approach.

RECOMMENDATIONS AND GUIDELINES

An initiative called Choosing Wisely was established in 2012. This includes 80 specialty society partners and 700 recommendations of tests and treatments that were deemed to be overused or unnecessary. They have compiled several recommendations for thrombophilia testing. Which includes:

1. Don't test in patients with major transient risk factors for VTE
2. Don't routinely test on patients undergoing routine fertility evaluation.
3. Don't testing on children with venous access associated thrombosis in the absence of a positive family history¹

The ASH Guidelines suggests limiting testing for thrombophilia to specific situations:

1. VTE provoked by non-surgical risk factors, including pregnancy, postpartum, or use of oral contraceptives
2. VTE at unusual sites within the body, if the standard of care is to treat patients for three to six months
3. Family history of VTE and high-risk thrombophilia (AT,C,S) to determine if pharmacological thrombosis prophylaxis during transient thrombosis risk factors is necessary
4. Women with a FH of VTE and high-risk (AT,C,S), to avoid use of hormone treatments, or to determine if postpartum and/or antepartum prophylaxis is necessary
5. Ambulatory cancer patients with a family history of VTE at a low or intermediate risk for VTE, to guide decisions on use of pharmacological thromboprophylaxis.

Only one strong recommendation was stated and that was against testing the general population before starting combined oral contraceptives. In all other instances, the panel suggests not testing for thrombophilia.²

TESTING

While the screening tests of the PT and aPTT can't diagnosis thrombophilia, they are useful in determining possible interfering substances, or elevated levels of fibrinogen or FVIII. If a lupus sensitive reagent is used it can also be helpful in testing for antiphospholipid syndrome. Usually, testing is performed in panels such as Protein C, protein S and AT. Testing should begin with functional testing and if results are abnormal than antigen testing should be performed. Patients with a type I deficiency have a decreased activity and antigen, Type II have a decreased activity and a normal antigen. Genetic testing looks at APCR and Prothrombin mutation.³

Testing provides significant challenges that occur in all phases of testing::Pre-analytical: which includes the sample, Analytical: Understanding your test & Interferences, Post Analytical: What do these results mean.

PRE-ANALYTICAL

Pre analytical issues include collecting the sample in the correct tube, having the correct ratio blood to anticoagulant (9:1), testing and using results obtained from serum or testing a clotted sample which results in low activity. Samples should avoid being thawed during transport which can result in a decreased activity as well as falsely low levels occurring in samples stored in self-defrosting freezers. It is very important for samples to be thawed rapidly in a 37º water bath and mixed well.

If a sample is collected in EDTA versus citrate the PC and PS results are questionable, a false positive can be seen for LA and no clot for APCR. When Na heparin is used versus citrate clot-based PC and PS will be low.

PC levels increase with age. They are low at birth and adult values are seen at the age of 17yr. PC chromogenic levels are higher in women on OC. AT is low at birth and increases at 1 year. Working with platelet poor plasma (PPP) is important in particular in LA testing. PPP (<10 x 109/L) is critical, residual platelets contain phospholipids and they may neutralize LA. Frozen/thawed sample, platelets will burst, shortening clotting times. For APC testing PPP should be used to prevent loss of sensitivity caused by the release of FV from platelet α-granules.⁴

Hemolysis alters coagulation due to release of , tissue factor, phospholipids, & ADP which activates coagulation & platelets which shortens clotting time. If there is consumption of factors, results will be prolonged results. Analyzers may use (mechanical clot detection, wave-form analysis) to compensate, but the quality of these specimens should be questioned. Icteric samples are less significant using dedicated wavelengths (> 650 nm), samples with a bilirubin concentration up to 20 mg/dL can still be analytically reliable. Lipemia: interference of lipid particles can be prevented using wavelengths >650 nm,/or using high speed microcentrifugation. Study showed no interference of AT, PC, PS after a light meal.5

ANALYTICAL

When is the best time to test a patient? For sure not during an event, results will reflect the consumptive process and not accurately determine the specific cause. Testing should be performed after the initial treatment, however the patient should be off OAC, warfarin should be stopped 2 weeks prior, DOAC 48 hours prior (earlier in renal impairment), LMWH tested 12 hours after last dose and UFH in the therapeutic range (<0.7) In patients on DOACS, protein C and S clot based, APCR and AT assays will be falsely increased and may be misclassified as normal. Lupus testing will result in a false positive result, misclassifying a patient as LA positive. OAC has no impact on genetic testing.⁶

When testing for Activated Protein C resistance (APCR) the principle of the test is an aPTT assay to which APC is added. The result will be prolonged in healthy patients but shortened in APCR patients. The test is sensitive for acquired forms of APCR including pregnancy, OC, LA, but is affected by anticoagulation. It is less specific and sensitive to the Factor V Leiden mutation (FVL). The modified-APCR adds V deficient plasma APCR is less influenced by factor deficiencies, oral anticoagulation, and lupus inhibitors. However, 10%–20% of cases of APCR are not carriers of the FVL mutation, presence of other mutations that cause the same phenotype (FV Cambridge and FV Hong Kong). The test is performed as part of a molecular panel which includes FVL, Prothrombin Mutation and MTHFR. Molecular testing is not impacted by anticoagulation.⁷

Chromogenic protein C (PC) assay is recommended as screen for PC deficiency because it is highly specific, but it will miss type 2b deficiency (rare). Can be falsely increased due to activation (XIIa, plasmin, thrombin) caused by substrate. To determine this, the substrate can be replaced with water and retested, if still high need new sample.⁸

If normal and thrombophilia is strongly suspected may need PC clot assay. Clot based PC detect Type 1, 2a & 2b, APTT based PC may have falsely low levels due to increased FVIII levels. APC may also decrease PC levels, can be prevented by pre-diluting in PC def plasma. RVV based assays are okay because they activate the cascade at FX. LA can overestimate PC due to anticoagulant effect of prolonged clotting time and may underestimate due to interference with APC anticoagulant activity- depe PC Antigen ELISA is most sensitive vs radial immunodiffusion (RID)as this can be overestimated due to rheumatoid factor.⁸

Patients who present with an Antithrombin (AT) deficiency are at the highest risk for thrombosis. It is important to be aware that the type of assay and incubation time can influence sensitivity. IIa based assay uses bovine thrombin and all Xa based assays, and are not affected by Heparin Cofactor II. While Xa based assays: may not detect some type II deficiencies compared to IIa assays. Antigen testing includes ELISA, RID, immunoturbidometric- antigen levels measured by latex agglutination can be falsely reduced in pregnancy. Genetic testing can also be performed. There are 399 different mutations, AT Dublin & AT Wibble can have normal antigen and activity testing & decrease only during fever or pregnancy.⁹

Protein S (PS) clot based assay detects Type I, II, & III defects (I,III quantitative) and has a 40-70% specificity. There are up to 10-15% falsely decreased values in normal subjects due to the variability in bound and unbound PS making accurate assessment difficult.¹⁰ Immunologic Total & Free PS can be performed by ELISA or latex agglutination assays. FPS is the initial test to assess plasma phenotype. Decreased PS activity correlated with amount of (unbound) FPS-but will not detect most Type II deficiencies. TPS measures free and bound PS may aid in differentiating some types of PS deficiency but it is not performed in all labs due to cost and minimal clinical info. Genetic testing can be performed, the PROS1 mutational analysis in homozygous Type 1 rare cases.¹⁰

POST ANALYTICAL

What do these results mean? Do they need to be repeated? Do I need more testing? If a patient has an abnormal test result, should that be the only test performed. Most testing should screen with functional testing and if abnormal then antigen testing should be performed. It is important to distinguish between an acquired disorder or thrombophilia. This will impact treatment as well as how long the patient is placed on anticoagulants.

When should genetic testing be recommended? To identify causative variants responsible for phenotypically identified deficiencies of AT, PC, PS when the results will influence management. For variants in genes (e.g., MTHFR, SERPINE1 variants (PAI- 1) without a clinically significant link to thrombosis testing is not recommended.¹¹

Another important post-analytical measure is the information from proficiency testing. What are the most utilized tests and how does your laboratory compare to your peers. Enrolling in these exercises helps to ensure quality results for thrombophilia testing.

CONCLUSION:

It is important to communicate with hematologists and to understand the patient population you are testing. This should be the focus when deciding on methodology of testing. It is beneficial to implement education, algorithms, and approval processes to guide testing, Thrombophilia testing should only be performed on the correct set of patients. It is beneficial to familiarize everyone with the impact of pre-analytical variables and the limitations of laboratory testing should be understood. All of these efforts will help to stop unnecessary thrombophilia testing and ensure that the right patient is tested with the right test at the right time.

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REFERENCES:

1. https://www.hematology.org/education/clinicians/guidelines-and-quality-care/choosing-wisely
2. Middeldorp S, Nieuwlaat R, Baumann Kreuziger L, Coppens M, Houghton D, James AH, Lang E, Moll S, Myers T, Bhatt M, Chai-Adisaksopha C, Colunga-Lozano LE, Karam SG, Zhang Y, Wiercioch W, Schünemann HJ, Iorio A. American Society of Hematology 2023 guidelines for management of venous thromboembolism: thrombophilia testing. Blood Adv. 2023 Nov 28;7(22):7101-7138.
3. Nakashima MO, Rogers HJ. Hypercoagulable states: an algorithmic approach to laboratory testing and update on monitoring of direct oral anticoagulants. Blood Res. 2014 Jun;49(2):85-94.
4. https://insights.omnia-health.com/technology/regulating-healthcare-ai-fast-evolving-world
5. Lippi G, Plebani M, Favaloro EJ. Semin Thromb Hemost. 2013;39(3):258-66.
6. Albert, T., Who, What and Where of Thrombophilia Testing, CAP today,Jan 2024
7. Transfusion Medicine and Hemostasis (Second Edition), 2013
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8. Cooper PC, Pavlova A, Moore GW, Hickey KP, Marlar RA. Recommendations for clinical laboratory testing for protein C deficiency, for the subcommittee on plasma coagulation inhibitors of the ISTH. J Thromb Haemost. 2020 Feb;18(2):271-277
9. Van Cott EM, Orlando C, Moore GW, Cooper PC, Meijer P, Marlar R; Subcommittee on Plasma Coagulation Inhibitors. Recommendations for clinical laboratory testing for antithrombin deficiency; Communication from the SSC of the ISTH. J Thromb Haemost. 2020 Jan;18(1):17-22.
10. Richard A. Marlar, Jana N. Gausman, Hiroko Tsuda, Marian A. Rollins‐Raval, Herm Jan M. Brinkman, Recommendations for clinical laboratory testing for protein S deficiency: Communication from the SSC committee plasma coagulation inhibitors of the ISTH, Journal of Thrombosis and Haemostasis,Volume 19, Issue 1,2021, Pages 68-74.
11. Arachchillage DJ, Mackillop L, Chandratheva A, Motawani J, MacCallum P, Laffan M. Thrombophilia testing: A British Society for Haematology guideline. Br J Haematol. 2022; 198: 443–458.

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