Activated Protein C Resistance (APCR), Factor V Leiden (FVL), and a Case Report of a Family with the presence of FVL over 5 generations.

Prior to 1992 the special coagulation laboratory performed testing on three inherited thrombotic risk factors. Antithrombin (formerly known as ATIII), Protein C, and Protein S deficiencies were implicated in venous thromboembolism (VTE). Altogether these only detected approximately 7% of inherited recurrent VTE.1 . However none of these factors were implicated in arterial thrombosis.

In 1993 Dahlback and associates reported on activated protein C resistance (APCR). In 1994, Bertina characterized the factor V Leiden (FVL) mutation as well as its cause and effect. Factor V Leiden was found to be the most common genetic variation among the blood coagulation pathway leading to a prothrombotic state, and is considered an important gene for understating the stroke mechanism.2,3

The presence of the factor V Leiden (FVL) mutation results in the resistance of FVa to degradation by APC., one of the most common risk factors for thrombosis.4,5
The APC resistance phenotype is, in more than 90% of cases of thromboses, due to a mutation in the Factor V gene, resulting in a replacement of Arg506® with Gln(Q) in the Factor V protein. The FVL heterozygous mutation is most common in Caucasians (2-15%, depending on the geographic population) particularly in Northern European populations.

The selectivity for the Factor V:Q506 or other mutations in the Factor V gene will render the protein resistant to inactivation by APC. Then increased by normalizing the concentrations of other plasma proteins involved in formation and regulation of thrombin. To accomplish this, sample plasma is prediluted with Factor V Reagent plasma and incubated with the APTT reagent for a standardized period of time. Coagulation is then triggered by the addition of CaCl2 in the absence and presence of APC and the time of clot formation is recorded.9,10,11

The clot-based assay is based on the principle that the addition of APC to a plasma sample induces a prolongation of the APTT mediated by the inactivation of FVa and FVIIIa in the plasma sample. The sensitivity and specificity of the screening test has been improved by predilution of the patient plasma with FV-deficient plasma; this permits the evaluation of patients receiving either heparin or warfarin, or who have an abnormal APTT from factor deficiencies other than FV. The testing proceeds with the performance of an APTT with and without the addition of APC. The APC ratio is calculated using the clotting time (CT) of the sample with APC (results should be prolonged because of the destruction of FVa and FVIIIa) divided by the CT for the sample without APC. A cut-off is established, and APCR is indicated by if the ratio is less than that cut-off, for the laboratory using a particular instrument and reagent combination. Acquired conditions, such as pregnancy, oral contraceptives use, elevated FVIII and a stroke aftermath can also produce APCR, in which case an assay without the FV-deficient plasma can be used to detect APCR phenotype.

The APCR functional assay (as described) is a screening test, not a diagnostic test, for FVL. Other rare congenital conditions including FV Cambridge and homozygosity for the HR2 haplotype can result in APCR. Because 10% of individuals with APCR do not have the FVL mutation, clinical diagnosis of genetic FVL requires both the clotting and molecular (PCR) tests for the FVL genetic mutation. If FVL mutation is not identified, PCR-based confirmatory tests for APCR resulting from other genetic mutations are available in research laboratories.4,12

To find out more about the APCR and FVL Diapharma has on their web site a tremendous section of complete information about the FVL disorder and testing. This will take you to a lot of information on the theory and performance and history of the APCR testing and FVL information. Check it out. It is extremely well written and informative on this topic.

The rest of this communication will deal with a very interesting Case Report.

This is a family that is very close to me. I have always wanted have them evaluated for a thrombotic issue. They have a long history of cerebral ischemia, transient ischemia attack (TIA), atrial fibrillation, aphasia, and myocardial infarction. They all have great lipid profiles such as total cholesterol levels of 140 mg/ml and HDL’s of 100 mg/dl. However, they still have severe health issues starting at around 50 years old. Also, these events occur primarily in the male gender.

In this family, the father, two sons, one brother, uncle, and 3 male cousins, and several other male cousins from related lines, have all experienced strokes and heart attacks, but with no evidence of deep vein thrombosis or pulmonary embolus. However, several have had atrial fibrillation.

I am going to focus on one of subjects because we have found the risk factor in his lab work that probably explains the family history of these life-threatening events. It is interesting that the ethnic background of the family is German-American which is prominent in South Texas.

The subject is a 69 year old white male with a long history of atrial fibrillation. It first started out as paroxysmal, then intermittent and finally continuous. This is in spite of 3 atrial cardiac ablations. None of the ablation surgeries corrected the arrhythmias longer than 3 weeks.

In November of 2019, the subject informed his Primary Care Physician (PCP) that he was experiencing numbness and tingling in arms and fingers and other neurological symptoms such as speech issues, balance problems, and memory problems. He was referred to a Neurologist who ordered an MRI of the neck and brain. The MRIs showed cervical stenosis and the possibility of two mini-strokes and/or metastatic process. The PCP then consulted with his Cardiologist and referred him to a Hematology Oncologist. Before that appointment occurred the subject experienced a transient ischemic attack (TIA, a mild stroke). This concerned all of his physicians because he was taking apixaban for his chronic atrial fibrillation. They then added 81 mg aspirin to his medication regimen.

It was reported to his PCP that he was taking his aspirin at the same time as his NSAID. He was informed him that the NSAID could negate the effects of his aspirin. He was informed that he should take the aspirin either 30 minutes before or 8 hours after administering his NSAID which was naproxen. Just after this his team stopped his NSAID dosing. He does have a long medication regimen, including aspirin and apixaban.

Blood work was ordered and sent to a reference laboratory that specializes in special coagulation testing.

His results from the blood work-up are as follows:

Flow Cytometry for PNH profile No evidence of PNH presence Antibodies used were CD15, CD45, CD64, and CD235a
Antithrombin activity 132% 80-120%
Antithrombin antigen 27 mg/dl 19-30 mg/dl
Β2 glycoprotein IgG, IgM, IgA <9 for all OR <20 for all
Protein S antigen Total 124% 70-140%
Protein S antigen Free 146% 57-171%
Protein S activity 146% 70-150%
Protein C activity 150% 70-180%
Protein C antigen 102% 70-140%
Activated Protein C Resistance 1.5L OR=2.1 ratio
Homocysteine 8.5 umol/L <11.4umol/L
PTT-LA Screen for LA 35 sec, < OR=40 sec.
DRVVT Screen for LA 49 sec. <OR=45 sec.
DRVVT confirm for LA 56 sec. <OR=45 sec.
DRVVT ratio 0.88 <1.21
Prothrombin 20210A Variant not detected normal
Factor V Leiden (R506Q) Positive for one copy of variant-heterozygous Positive result evident of inherited thrombophilia
Lipoprotein (a) <10nmol/L <75nmol/L
LDH 280U/L 135.0-225.0 U/L
CBC/CMP No significant abnormal results.

The abnormal APCR and the molecular test finding of the FVL heterozygous mutation suggest the cause of his thrombotic issues.

The subject is writing a book on the history of his family’s immigration and life going back 5 generations in South Texas, where there is a large ethnic German population. He has investigated over 5 generations by seeking death certificates, autopsy reports and any other information he could get his hands on. T It’s too bad that genetic counseling did not exist until recently. The finding of the FVL mutation in this family explains a lot about the issues he and his family have experienced. After finding this genetic mutation, it was suggested all the family look into getting tested for the presence of this abnormality.

The below information comes from the American Board of Internal Medicine recent post on the Choosing Wisely website on Factor V Leiden.

Don’t order a factor V Leiden (FVL) mutation assay as the initial test to identify a congenital cause for a thrombotic event. First, order a phenotypic activated protein C resistance (APCR ) ratio assay.

Support: there exist several acquired APCR conditions such as elevated factor VIII and antibody-mediated APCR that can lead to thrombotic events such as deep venous thrombosis or pulmonary embolism. Further, several factor V Leiden-independent mutations may be associated with thrombosis. Best practice guidelines recommend testing for APCR using one of several phenotypic clot-based APCR ratio assays as an initial assay and following up positive APCR ratio results with the molecular factor V Leiden assay. Most currently available phenotypic tests are economical, have a greater than 95% concordance with molecular testing and up to 99% clinical sensitivity. Based on Medicare reimbursement rates, switching to initial-phase phenotypic testing and relying on its negative predictive value with follow-up genotypic testing on APCR-positive samples could result in a 75% reduction in costs. Although the FVL mutation assay is often ordered to determine the cause of venous thromboembolic disease, the APCR ratio assay provides greater clinical sensitivity at a lower cost. In instances when clot-based thrombosis risk testing is indicated during acute thrombosis, line-associated thrombosis, or anticoagulant therapy, the APCR is compromised and the FVL mutation assay is used as a primary assay.


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