Thrombophilia screening: Approach to testing in protein S and antithrombin deficiencies

Welcome everybody, to the next session of the Hemostasis Learning Institute. I'm Doctor John Mitzios, Medical Sciences Partner at Siemens Health and Ears, and I'm your host for the session. Today's speaker is Doctor Marian Rawlins Ravel. Dr. Rawlins Ravel currently serves on the faculty in the Department of Pathology at the University of New Mexico in Albuquerque. Previously, she was a Fellow in the Hemostasis and Hematopathology at the University of North Carolina and the University of Pittsburgh Medical Centers, respectively. Her main research interests include thrombophilia assessments and coagulation disorders. Today's presentation will be on thrombophilia. Thrombophilia is a major reason for thrombotic events in young patients. The current diagnosis for the increased thrombotic risk is essential to treat patients and prevent further thrombotic events. Functional or quantitative deficiencies of antithrombotic protein, such as antithrombin, protein C, or protein S, are often the cause of thrombophilia. This lecture summarizes the diagnosis of these common causes of thrombophilia. Dr. Rollins Ravel, the floor is yours. Hello, I'm Marion Rollins Ravel and today I'll be speaking to you about thrombophilia screening, more specifically an approach to testing and protein S and antithrombin deficiencies. Here are my. Disclosures our objectives today will be. To describe the most commonly recommended approach to screening for thrombophilia. Discuss. The current International Society for Thrombosis and Hemostasis isch guidelines for testing to screen for both protein S and antithromic deficiencies as well as to understand the challenges. With these assays, we'll start with a brief overview looking. At the Mechanisms of Coagulation, discuss the risk of thromboembolism or VTE and as well as a thrombophilia screen, and then we'll talk more. Specifically about. Inherited antithrombin and protein S deficiencies and testing. Looking at both inherited and acquired. Causes the types. And the prevalence? The isth. Guidelines for testing as. Well, as the. Assay inconsistencies, erroneous results, we may see an additional testing that we might need. And then finally, just briefly looking at the external quality assurance for these assays and a brief summary. So to begin, here is the classic simplistic coagulation cascade leading to the ultimate formation of fibrin. This is a helpful model which informs our understanding of the in vitro. Assays. The natural anticoagulants including tissue factor, pathway inhibitor, or. TFPI. Antithrombin and protein C and its cofactor Protein nests. Are highlighted here. Our focus today is on antithrombin, our enzyme regulator, which primarily inhibits factor 10A and thrombin, as well as protein nests, which together with protein C, regulates cofactors 5 and 8. While antithromeda may be the most potent natural anticoagulant, protein S may be the most complex, with other mechanisms of actions not. Illustrated here, however. Deficiencies in either of these anticoagulants may lead to. Decreased. Regulation of our pro coagulants. But what is the risk of thrombosis and what does it mean for our patient? We know that many different factors go into any individual's risk of thrombosis. Shown here is an individual without an inherited thrombophilia who during her lifetime does have several physiologic including age and acquired including pregnancy, oral contraceptive pill, and lupus anticoagulant, all increasing her overall risk for thrombosis, but she never. Actually reaches that threshold to develop AVTE. While this individual, for example, with an inherited deficiency of protein S or antithromic deficiency has an increased baseline risk of thrombosis and given the same life challenges. Reaches the threshold for VTE. During her life. This table with. Data compiled from multiple sources puts the prevalence and relative. Risk for VTE. Of inherited protein S and antithromic deficiency in context of other common inherited and acquired thrombophilias, as you can see. The. Prevalence of protein of inherited protein as an antithrombin deficiency is low. But the relative risk. Of that initial VTE is compared to the general population is really. High. For protein, as compared to other patients with thrombosis, risk of recurrent BTE does not appear to be increased while patients with antithrombin deficiency. Will show a. Slight increase relative risk compared to other patients with thrombosis. So. Let's talk a little bit more. Specifically about. Antithromic deficiency and testing inherited the antithromic deficiency. Is rare. The age of onset may vary. There is a. Higher risk of thrombosis than all other commonly evaluated inherited thrombophilias. It is an autosomal dominant inheritance pattern. Most patients with homozygous mutations will lead leading to a quantitative. As well. As likely, most qualitative deficiencies are considered incompatible. With life, so we usually see. Patients who are. Heterozygotes. With antithrombin levels between 40 and 60%, Serpent C1 mutation is identifiable in the majority of these patients, but many different ones have been described. Acquired antithrombin deficiency is also unfortunately very common, making it very challenging sometimes. To evaluate. For an inherited deficiency, some of the most common causes of acquired antithrombin deficiency are listed. Here liver. Dysfunction may be identifiable by looking at a combination of protein, C&S as well as antithrombin. Based on their half lives, you may be you may see different levels of of decrease. Poor nutrition is one of the. Causes of liver. Dysfunction that may not. Always be thought about but that. Lack of proteins for synthesis, so for instance any disease. That could include diseases. With that nutritional absorption, including things. Like ulcerative colitis, you could also. Have consumption of antithrombin and this would. Also obviously. Consume protein, CNS and so you. Could. Think about surgery or thrombosis, DIC or anything that would have happened free analytically in vitro to the to the sample proteinuria, for instance, nephrotic syndrome and medication. So some of the examples of medication that may lead to acquired antithrombin deficiency are LS, barogenase, heparin, which may cause mild decreases, and then frutizuran which is. Made actually targeted. To decrease the synthesis of antithrombin, you may also have dilutional effects, and then Kylo thorax has also been described after. Pediatric surgery to lead to. Decreased antithrombin levels. So there are two types of. Assays we have. Our functional assays and also our immunologic or quantitative. Assays. For the functional assays, the most commonly. Used are. The Heparin cofactor. Assays. And in. Here we have. Heparin with an excess of thrombin which are combined with the patient's endogenous antithrombin. Antithrombin will inactivate thrombin with the remaining thrombin cleaving a chromogenic substrate. Antithrombin activity is inversely proportional to the spectrophotometric signal detected. There are lots of differences in reagents, including 10A and thrombin based assays. But also. Boven bovine versus human components or differences in incubation time can lead to varying ability to detect different deficiencies. There's. Also the progressive antithrombin assay and this. Is has a decreased actually specificity? For antithrombin deficiency. Because due to. This long incubation it can be affected by other inhibitors such as trypsin inhibitor or A2 macroglobulin. On the other hand, antigen testing is less commonly available in labs overall, and is mostly available in. Specialized Laboratories. Here you can. See. The inherited antithrombin deficiencies maybe either type 1 quantitative or. Type 2. Qualitative. Based on mostly the results. Of our laboratory assays in type. One deficiencies both antigen and. Activity assays are low. In type 2 deficiencies, the heparin cofactor. Activity assays are low. But the antigen levels are more often normal or higher than the activity levels with an EAT. With a decreased activity to. Antigen ratio, the three generally described. Type 2 defects are reactive site. Heparin binding site or a combination thereof. Which is pleiotropic. Not listed here in this. Table are so. Called transient variants, which have also been described with mutations that may lead to a mild effect on the folding or function of antithrombin but show no deficiency under normal conditions. However, these variants are more sensitive than the wild type antithrombin to acquired factors or conditions imposing A conformational stress leading to the development of thrombosis and carriers of these mutations under specific conditions such as the raised body temperature of fever. If the sample of these patients is not collected under these particular conditions, all functional assays will fail to detect an antithromic deficiency. Although in the general population the prevalence of type. 2. Antithromic deficiency outnumbers type one among the symptomatic patients. Type 1 is much more prevalent. Well, about. Half of patients with type 1 antithromic deficiency will present with a. VTE. Type 2 patients have a much more heterogeneous phenotype. Type 2B. Or the heparin binding site antithrombin deficiency appear to be much less thrombogenic than the quantitative type 1 deficiencies. This is the one of the reasons why additional testing including the progressive antithrombin assay as seen in this table where it will be normal, as well as potentially molecular testing maybe it'd be maybe especially helpful to distinguish. With these types of qualitative. Variants. Here is the. 2020 ish recommendation for testing for antithrombin deficiency and they recommend starting with the antithrombin activity. If this is normal, then you need to exclude that the patient is on some sort of direct thrombin inhibitor or a 10A inhibitor. If they are. Then it's recommended to repeat this testing after this has been discontinued or to use an alternate assay and we'll talk about that a little more on the next. Slide if. These are not present then Essentially it looks like there's no evidence of an anti trauma deficiency, but if this is clinical suspicion is high, you may want to go ahead and test the anti thrombin antigen because there are some type 2 deficiencies that will present with a low normal activity. Level if you test the. Activity level and it's low then you need to. Exclude acquired causes. This can include heparin and if your patients on heparin therapies and you should discontinue that. For at least a week. Prior to testing again other acquired deficiencies. Could be evaluated. And and you could potentially consider additional testing to help. Exclude those. If you have one of those present, then really. You should be. Waiting until that acquired deficiency. Is resolved if. That's not possible. Then you may want to consider a testing family members or molecular testing. If there's no acquired deficiency present, then you could go on to measure that antithrombin antigen if if indicated. If that's low, then basically you're. Looking at it a type one deficiency where your antigen in your activity are are similar. If it's not low, then you're thinking about some sort of type 2 deficiency, and in that case is where you might consider additional testing for the progressive antithrombin assay. All of these results should be. Confirmed at A at a subsequent time usually. About four weeks after. And you could consider testing relatives also to to confirm this diagnosis. There are several. Erroneous results for antithromic assays, some of which. I've already alluded. To so you can have direct thrombin inhibitors which will be present and that'll increase. Your 2A. Base but not your 10A base results and the opposite is true for the 10A inhibitors. You could also have impaired specimen integrity or some other pre analytic variable present which may interfere. With your assay. Per your manufacturer's guidelines such as hemolysis, Icarus or lipaemia. You could consider. Additional testing and as we've said, you know you could screen for these direct thrombin inhibitors or 10A inhibitors. You could. You should probably look at the. Activity to antigen ratio and anti thrombin and usually. These would be established. Or a reference range, but. Typically if you have. Less than 0.7 that. Would suggest a. Type 2 deficiency and even if you have a normal. Activity typically. A low normal, you could consider running an antigen as we mentioned there are some variants. That will have. A normal activity level, but the discrepancy between the antigen activity levels will be high enough to make you consider some sort of antithromant deficiency. You could also consider protein CNS testing, which is. Helpful to evaluate for acquired causes. PT and PTT. Will help to verify the. Integrity of your sample and also. Evaluate for acquired causes. You could also consider that progressive antithrombin activity test, keeping in mind that it's better as a confirmatory test and a screening test given its. Lack of specificity, but that it will. Help to distinguish the heparin binding site mutation and that may be helpful given the lower prothrombotic risk in that population. Molecular testing is not routinely available, but may also be helpful in cases with high clinical suspicion or underlying acquired deficiencies which will not resolve. OK. So now we'll move on to proteinous deficiency and testing in this case if we're looking at inherited protein S deficiency. This is also. Rare. The age of onset is similar to antithrombin deficiency. It's also an autosomal dominant inheritance. But we do see homozygotes and they often present with neonatal purpura fulminants. These are mostly present as heterozygotes, so in the population, and they have a range of disease manifestation with their plasma protein S levels ranging from about 30. 5 to 60. 5% of normal. In contrast to anti thrombin deficiencies, there are multiple gene mutations present in these patients, many of whom do not have an identifiable PROSE mutation. Acquire protein nest deficiency. Is also relatively. Common and here you can see some of these clinical conditions that will. Lead to the. These conditions are. Are are acquired? Anti thrombin deficiency, again complicating the evaluation for an inherited deficiency and these affect both the activity and the antigen. Assays as. You can see protein as has numerous described functions, both anticoagulant and non anticoagulant, focusing on its critical anticoagulant functions in addition to previously discussed. Roles of cofactor. In the activated protein C complex. It also acts as a cofactor for TFPI as well. As having TFPI and APC independent roles, together these functions allow protein nests to regulate coagulation both during the initiation phase of the extrinsic pathway. As the TFPI. Cofactor function as well as during the propagation phase and APC dependent and independent activities. There's still more functions that have. Yet to be elucidated. And even many of the functions described here on this. Slide have. Not been completely defined in terms of their contribution to coagulation, or we don't? Have widely available. Commercial testing to evaluate them so. Here are our types of assays that we have. For protein ass testing. We have activity. Assays and immunologic assays. I'm focusing on the commercially available protein. ASS activity assays. These are clot. Based mostly using a, the role of protein S as a. Cofactor for APC. To inactivate factor 5 and factor. 8. We add patient plasma with protein S deficient plasma and activated. Protein C with. Reagents for the clot based assays and that will. Lead to clot. And the clotting time is? Theoretically. Directly. Proportional to the amount of protein S in the sample. Then we have our commercially available immunologic assays. There's free protein S antigen, which is typically a monoclonal antibodies. That is. To the unbound or functional anticoagulant form of protein S and these are latex immunoassays more frequently than alysis we. Also have the total. Protein S antigen and these are polyclonal or monoclonal antibodies. That are. Meant to detect both bound and unbound protein S. So here. Just to kind of describe. To you the. In circulation, typically the majority or approximately 60% of protein S is bound to the. C4B. Binding protein and is inactive. The total. Protein S antigen measures all protein S in the sample, including that inactive form bound to C4 binding protein free Protein S antigen only measures the unbound or active form of the protein S, which in most. Cases closely. Approximates that plasma protein S. Activity here are the. 3. Plasma based assays. Commonly used to evaluate protein S deficiencies. There are three types of disease. As measured. By these assays. Type 1 is a quantitative defect leading to decreased production of the protein and as you can. See all three assays will be decreased type 3. Protein as deficiency demonstrates a protein as with increased binding affinity for protein as to C4B binding protein. So while total. Protein as antigen will be normal or even high. Both. Free protein as and protein as activity will be. Decreased. Type 2 is again a qualitative defect leading to normal levels of dysfunctional protein. Antigen levels by both free and total protein as assays are normal, but protein as. Activity will be decreased. Of note and in contrast to anti trauma deficiency. This is a rare type of. Protein S deficiency accounting for no more than 5% of protein S deficiencies, similar to the anti trauma deficiency we need. To exclude acquired causes if possible for a. Protein S testing according to the ISTH recommendations. Given the numerous challenges with protein S activity, which I will cover in the next two slides, Free protein S antigen assay is recommended. As the initial test. If this is low. Then it would be suspicious for a type 1. Or a type. 3 deficiency, which should necessitate a repeat confirmatory test in four weeks or more. Additional testing for the total protein antigen could be considered, but it's unlikely to change management and so and this testing will. Allow for. Diagnosis of greater than 95% of all these patients with protein S deficiency. On the other hand, if the free protein as antigen is normal, even the low prevalence in most populations of type 2 protein S deficiencies, Additional testing could be considered for patients with a strong family history. Or. Specific populations that. Have a high. Prevalence for type 2 mutations. If this activity assay is. Still normal. You may have interfering factors such as a lupus anticoagulant or a 10A inhibitor that could. Cause. False elevations which. Should be excluded. Here are some of these assay inconsistencies that I've been alluding to. So for instance protein S activity even when you compare in normal individuals. The the. Protein S. Activity to the. Free protein S 10 to 20% of normal individuals. Will show a low activity. And it's unclear. If this is due to elevated factor 8. Or other causes. But on repeat testing, if you test these patients again or, then you'll find that the protein S. Activity actually. Is more comparable to the initial free protein S level. And if on the third round of testing you repeat again, most of the rest of the normal individuals will show that normal free protein S, there are some protein S activity. Levels. That will be significantly elevated and that could be due to interferences like lupus anticoagulants. While this activity assay has a high. Sensitivity has a. Really low specificity for these. Reasons. So if we look at our. Free protein as antigen. It looks it's the recommended initial. Step but it. Doesn't detect some of our dysfunctional protein as molecules. Although that is. Estimated to be 2% or fewer protein as deficiencies. And it is. A really good. Surrogate for protein as activity, our total protein as antigen. This. Includes an evaluation of that protein ass that's bound to that. C4B. Binding protein which isn't active and it really doesn't add very much for information for clinical management and you can see there are a lot of erroneous results for protein. Assays, particularly the activity assays. And the antigen assays are not influenced by a lot of these possible interferences and so. That's why the. Again, the free protein as assay. Was picked as. A initial testing we may see some. Effect due to. Pre analytic variables such as improper processing or the incorrect specimen and that will influence both. Of our assays. So how do these assays? Perform compared to each other and within labs or external quality assurance data. Well, this information is a little older, but it nicely compares Antithrombin. Activity directly to. Protein S, protein S assays and the antithrombin antigen was not reported in the study, reflecting its. Overall lower usage. As we can see, Antithrombin. Activity shows better. Enter and intralab precision as compared to all protein S assays. Free protein S antigen consistently shows better precision than than protein S activity and total antigen, as is often seen in both antithrombin and protein assays as well. As other factor. Assays they do show more imprecision at these at the lower concentrations and it would be really nice to have a a newer. Data review so hopefully that our EQ. Agencies will be working on that, but this I just thought was a really nice representation. Overall, the published. CAP, Nescola and ecat. Data shows that. Diagnostic accuracy is much better for free protein S than protein S. Activity. And of the laboratories that only reported a single protein S value, 70% reported the free protein S antigen. So this supports. The. Algorithmic approach to protein S testing recommended by the. Isth. And it also, it's interesting to see that. The accuracy. Was much better for antithrombin assays than it was for either protein C or protein assays. So in summary, the new St. IS. TH guidelines are helpful to guide antithrombin and protein as testing in a cost and clinically efficient approach. Type 2 qualitative antithrombin and protein S deficiencies may still present challenges in diagnosis. Additional testing, including testing of affected family members and molecular testing may be indicated. Anti trauma testing shows much higher precision and accuracy than protein S testing overall and just because. You can see all of these issues with. Testing as well as the acquired causes and the potential interferences. For these assays. Thrombophilia testing really should only be. Approached with the. Appropriate patient population and timing in mind, and that's all I have. For you today.

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Mitsios PhD, Medical Sciences Partner Siemens Healthineers Thrombophilia Screening: Approach to Testing in Protein S and Antithrombin Deficiencies Marian Rollins-Raval MD MPH Associate Professor of Pathology Director, Coagulation Laboratories University of New Mexico/TriCore Reference Laboratory SIEMENS Healthineers Financial Disclosures Siemens Healthineers, Educational Speaker Precision Biologic, Site Primary Investigator Sanofi, Advisor/Consultant Objectives Describe the most commonly recommended approach to screening for thrombophilia Discuss the current International Society for Thrombosis and Hemostasis (ISTH) guidelines for testing to screen for Protein S and antithrombin deficiencies Understand the challenges with these assays Outline Mechanisms of Coagulation . Risk of venous thromboembolism (VTE) and Thrombophilia Screen · Inherited Antithrombin (AT) and Protein S (PS) Deficiencies and Testing Inherited vs. Acquired Types and Prevalence ISTH Guidelines for Testing Assay inconsistencies, erroneous results, and additional testing External Quality Assurance (EQA) Summary XIIa TFPI XIa Classic VIII IXa VIIa TF Coagulation Cascade with Natural Anticoagulants Xa AT Thrombin Fgn Fibrin Courtesy of E. Lockhart VIIa TF Prothrombotic Threshold Paradigm No Genetic Defect, No Clinical Manifestation Threshold for VTE LA Pregnancy OCP V Xa Risk of Thrombosis Normal Baseline Time/Age Adapted from Lancet. 1999 3;353(9159):1167 PS or AT Def Prevalence and Risk of VTE in Thrombophilia Thrombophilia Prevalence Relative risk of Initial VTE recurrent VTE Factor V Leiden (FVL) Heterozygous 2-7% FVL Homozygous Prothrombin Gene Mutation (PGM) Heterozygous PGM Heterozygous Rare Uncertain Compound FVL&PGM Heterozygote Protein C deficiency PS deficiency AT deficiency Antiphospholipid Syndrome Adapted from J Thromb Thrombolysis. 2016;41(1):154 Antithrombin Deficiency and Testing Inherited Antithrombin (AT)Deficiency Rare (<1% of general population) Age of Onset:10 to 50 years Higher risk of thrombosis than all other commonly evaluated inherited thrombophilias Autosomal dominant inheritance Patients with homozygous mutations leading to quantitative, as well as likely most qualitative, deficiencies, are considered incompatible with life · Most patients are heterozygotes with plasma AT levels between 40-60% SERPINC1 mutation identifiable in majority of patients (>80%), but many different ones described J Thromb Haemost. 2020;18:17 J Thromb Haemost. 2018;16(4):680 Thromb Haemost 1997; 77: 197 Acquired Antithrombin Deficiency Liver dysfunction (T1/2 PC1 resolved* and/or week NORMAL DTI/Xa-Inhibitor NORMAL DTI/Xa-Inhibitor Impaired specimen integrity Hemolysis, icterus, and lipemia per J Thromb Haemos Erroneous Results for Antithrombin Assays Possible Interferences · Will increase lla-based, but not Xa-based results Xa Inhibitors (Rivaroxaban/Apixaban/Edoxaban) · Serum or clotted samples (decreased activity, normal antigen) Samples stored in self-defrosting freezers Progressive /Hemolysis, icterus, and lipemia per manufacturer Additional Testing? TT and Anti-Xa assay . Need to establish reference range, but typically 0.7 or less would suggest type 2 deficiency Protein C and S Testing PT/PTT Evaluate for acquired causes Distinguish HBS mutation, since lower prothrombotic risk Molecular testing . Not routinely available, but may also be helpful in cases with high clinical suspicion and/or underlying acquired causes which may not resolve Antithrombin Testing AT activity:antigen ratio . Even if normal activity (typically low normal), could consider running antigen (e.g ., AT Cambridge II) · Helpful to evaluate for acquired causes Verify integrity of sample Progressive AT Activity Test Protein S Deficiency Inherited Protein S Deficiency Age of Onset: 15 to 50 years Homozygotes may present with Neonatal Purpura Fulminans Mostly present as heterozygotes and Range of disease manifestation Plasma RS levels range from 35-65% of normal Multiple gene mutations, many of whom do NOT have an identifiable PROS mutation Clinical Conditions Activity Assays Antigen Assays Possible decrease Decrease Acute Phase reaction (some) Decreased free PS Vitamin K antagonists Decreased PS Decreased PS, but less affected than activity Liver disease Consumption (i.e. DIC, recent surgery, recent thrombosis) Oral Contraception containing estrogen Systemic Lupus Erythematosus J Thromb Haemost. 2021; 19: 68 Protein S: Functions and Mechanisms Non-Anticoagulant Functions of Protein S Cofactor for APC to regulate FVIlla Binding C4 BP- ability to interact with negative Direct APC independent inhibition of PL to localize complement regulation Prothrombinase Binding apoptotic cells and micro-participles Direct APC independent inhibition of Factor Activation of TAM receptors Xase Stimulating phagocytes of apoptotic cells Cofactor for TFPlo inhibition of FXa · (Commercially available) · Free PS antigen · Clot-based using role of PS as a . Monoclonal antibody to the unbound cofactor for APC to inactivate (functional/anticoagulant) form of PS Latex immunoassays more frequently · Patient plasma + PS-def plasma + used than ELISAs APC + reagents for clot-based assays · Total PS antigen (e.g. PT, PTT, RVVT)->clot antibodies to detect both bound and · The clotting time (seconds) is unbound PS directly proportional to the amount of PS in the sample Protein S Assays Free (Active): 40% Bound (Inactive): 60% directly proportional to the amounttein S Antigen Adapted by E. Lockhart from R. Marlar Free Protein S Antigen Protein S Activity C4b BP Protein S Total Protein S Antigen Protein S Deficiencies Type PS Activity: Free PS antigen Total PS antigen Decreased Up to 80% Up to 20% Am J Hematol 2011; 86:418 2021 ISTH Recommendations for Testing for Protein S Deficiency 2021 Assess for causes of No Free PS Low acquired deficiencies or interfering factors antigen assay Yes Delay Diagnose testing excluded* Strong clinical suspicion Total PS for PS deficiency# assay Evaluate for acquired causes and repeat ≥ 4 weeks to confirm deficiency No further testing PS deficiency confirmed On repeat, >95% low PS Act compared to Free PS Some PS Act levels are significantly elevated free PS Antigen: tional PS molecules (estimated 2% or fewer PS Surrogate for PS activity Total PS Antigen: Adds cost, but little information for clinical management PS deficiency confirmed thol 2012;1371 Assay Inconsistencies False low PS activity compared to Free PS Ag is 10-20% even in "normal" individuals Due to elevated FVII, or other causes .. High sensitivity, but low specificity (40-70%) Does not detect some dysfunctional PS molecules (estimated 2% or fewer PS deficiencies) Includes evaluation of PS bound to C4b BP, which is not active Am J Clin Pathol 2012;137:173 J Clin Ligand 2005; 28:130 Possible Erroneous Interferences Lupus May falsely elevate Not influenced results for PS anticoagulants results Elevated Factor May falsely decrease assays results in APTT-based Factor V Leiden Direct thrombin inhibitors Direct factor Xa May interfere; most assay kits contain heparin inhibitors (check effective level in specific kit) Improper May falsely processing decrease results Incorrect May falsely diagnose specimen deficiency Protein C results (only in assays where endogenous PC is activated) How do these assays perform compared to each other within and between labs? EQA Data Published Above lower limit reference interval Below lower limit reference interval ECAT Data Test samples Interlab CV (%) Concentration Analyte Number range (UdL-) Median 95% CI Antithrombin (activity) Protein S (activity) Protein S (total antigen) Protein S (free antigen) Intralab CV (%) Number of laboratories J Thromb Haemost. 2003;1:748 · Diagnostic accuracy is better for free PS than PS activity . Of laboraNumber antigen Thromb Haemost 2016;116(1):50 Am J Clin Pathol 2005;123(5):778 · Published CAP, NASCOLA, ECAT Data · Of laboratories that only reported a single PS value, 70% reported the free PS · Accuracy was better for antithrombin assays than for protein C or protein S Arch Pathol Lab Med. 2011;135:227 ntithrombin and Protein S test . Type 2 Qualitative Antithrombin and Protein S deficiencies may still present family members and molecular testing, may be indicated . Antithrombin testing shows higher precision and accuracy than Protein S Testing overall . Thrombophilia testing should only be approached with appropriate patient population and timing in mind, especially due to interfering substances and acquired conditions which may lead to false positive or negative results . New ISTH guidelines are helpful to guide Antithrombin and Protein S testing in a cost and clinically efficient approach challenges in diagnoses and additional testing, including testing of affected Siemens Healthcare Diagnostics Inc ., 2025 The products and features mentioned here are not commercially available in all countries. Their future availability cannot be guaranteed. SLS-24-3800-76 QR700016259