Pr. P.E. MAKRIS,

Haemostasis & Thrombosis Unit, 1st Prop. Pathological Clinic, AHEPA UniversityUnivercity Hospital,

1, Kiriakidi Str,

Thessaloniki, Greece.

Prof. Dr. JossJos VERMYLEN,

Editor in Chief,

UniversityUnivercity of Leuven,

Campus Gasthuisberg,

O & N, Herestraat 49,

B-3000 Leuven, Belgium.

 

Thessaloniki, 3 February19 Ju1y 1996.

 

 

Dear Sir,

 

    We are in the pleasant situation to submit the present manuscript entitled “Evaluation of two clotting assays for the diagnosis of Activated Protein C Resistance (APCR). APCR is associated with familial thrombiophilia but not with arterial thrombosis.” which deals with the prevalence of Activated Protein C Resistance in the Greek population. This work is an originalorignal first study in Greece and it  has not been published elsewhere.

In this work we evaluated two clotting methods (aPTT and PT based methods) for APCR screening and we used both of them to study the prevalence of APCR in a sample of normal Greek population, in patients suffering familial thrombophilia or arterial thrombosis as well as in patients suffering chronic diabetes or hypertension.

    We hope that you will find this work sufficient enough to be published to Thrombosis and Haemostasis, and if so, it could be assigned to the category “Clinical Studies”.

  Looking forward to your comments, we thank you in advance for your attention.

 

Yours sincerely,

 

 

Pr. P. E. MAKRIS, Dr. G.T. GEROTZIAFAS

 

 

 

 

 

 

 

 

 

 

 

 

Activated Protein C Resistance phenotype, incidence in Greek population and the risk of venous thrombosis and myocardial infarction.

Prevalence diagnosis of Activated Protein C Resistance (APCR) in greek population, in thrombophilic patients  and the risk of arterial thrombosis.

Evaluation of two clotting assays for the diagnosis of Activated Protein C Resistance (APCR)APCR anf factor V-Leiden. APCR is associated with familial thrombophiliathrombiphilia but not with arterial thrombosis.

 

 

 

G. T. Gerotziafas1,2, P. Van Dreden2,3, E. Karavaggeli4, P. Van Dreden Z. Foka11, , A. T. Gerotziafa1, P. E. Makris1.

 

 

1Haemostasis & Thrombosis Unit, 11st Prop. Pathological ClinicClinnic, AHEPA UniversityUnivercity Hhospital, Thessaloniki, Greece.

2Service d’ Hematologied’Hematologie Biologique, Hopital Hotel-Dieu de Paris, France.

3Serbio Laboratories, Gennevillers- France.

4Central Hematological Laboratory, St. Dimitrios Hospital, Thessaloniki, Greece.

 

 

 

 

Correspondence to: Pr P.E. Makris, 1Haemostasis & Thrombosis Unit, 1st Prop. Pathological ClinicClinnic, AHEPA University Hospital, 1, Kiriakidi Str. Thessaloniki, Greece. FAX Number: +3031994777.         

e-mail pemakris@med.auth.gr

 

AbstractSummary

One hundred forty one subjects were screened with two clotting assays for activated Proteinprotein C resistance (APCR). They were separated in normals, patients suffering from familial thrombophilia, patients with personal history of acute myocardial infarction, and patients suffering from diabetes or chronic idiopathic hypertension (high risk factors vascular disease and arterial thrombosis). The Pprevalence of APCR was significant in thrombophilic patients (26,1%) as compared to normals (5%). APCR is not a risk factor for arterial thrombosis since its prevalence among patients suffering from arterial thrombosis (7%) is comparable to normals. Additionally, APCR prevalence is 5% in patients suffering from diabetes or hypertension. APCR was diagnosed using aPTT and PT based clotting assays. In the first part of the study wWe demonstrated that the threshold of abnormality for APCR clotting assays masshouldt be defined according to the way of tests realization (manually or automated instruments) and the reagents used. APCR diagnosis with aPTT based test was not completely cosegregated with the presence of 1691 G--->A mutation detected with molecular biology assay. 50% false negative and 6% false positive cases were found with aPTT APCR-ratio. Normalized aPTT APC-ratio improved the sensitivity and sensibility of the method (16% false negative and no false positive cases). A combination of abnormal APCR-ratio (and normalized ratio) with aPTT and PT based method revealed a perfect cosegregation with molecular biology findings.

 

key  words

activated protein C resistance   (APCR),  factor V Leiden, thrombophilia                      

 

 

 

Introduction

 

The field of familial thrombophilia has been dramatically changed when DahlbäckDahlback discovered the poor anticoagulant response to the action of activated Proteinprotein C as a new cause for familiar thrombophilia and Bertina showed that this phenotype is due to a mutation in the gene of factor V, the factor V Leiden mutation. 1, 2, 3, 4, 5 (1,2,3,4,5)(1,2,3,4,5) .

Venous thrombosis (deep vein thrombosis and pulmonary embolism) tends to be one of the major medical problems in our days. A recent study performed in USA demonstrated that venous thrombosis is associated with approximately 20.000 patients per year, while residivant venous thrombosis is about 33% of the total. Acute myocardial infarction, peripheral angiopathy, malignancies, metabolic diseases like diabetes and autoimmune diseases like lupus anticoagulant and antiphospholipid syndrome, as well as cardiac or respiratory in sufficiency are some of the most important pathological conditions predisposing to venous thrombosis. Obesity, immobilisation or treatment with oestrogens or L-asparaginace are important risk factors to deep vein thrombosis (DVT). The above mentioned risk factors are related to the acquired thrombophilia.

Deficiencies of natural blood anticoagulants are related to the hereditary thrombophilia in patients suffering residivant DVT with a positive familiar history. The field of familial thrombophilia diagnosis has rigidly changed when a new genetic abnormality was discovered by In about 10% of these patients, thrombophilia can be explained by a hereditary deficiency of one of the most important  natural anticoagulants such as antithrombin III (AT III), protein S (PS), protein C (PS) or heparin cofactor II. However, a lot of cases of thrombophilia are still considered as «idiopathic».

Recently, Daahlbaäck et al discovered a new cause of hereditary thrombophilia, associated with an abnormality at the anticoagulant system of PC. When activated PC (APC) is added to normal plasma.. It prolongs aPTT . Dahlback et al, observed that some plasmas obtained from patients suffering residivant DVT with positive familiar history were resistant to the anticoagulant activity of APC. Thus, APC addition did not prolong aPTT as much as in normal plasmas. This  abnormality is characterized by a poor anticoagulant response to called activated protein C (APC) ([1],[2],[3]).  resistance (APCR) Bertina and co-workers demonstrated that APC resistance (APCR) phenotype is associated with heterozygosity or homozygosity for a  the consequence of a single point mutation at the gene of factor V (a G --> A type substitutionsubsitution at nucleotide position 1691). The mutation of factor V gene may be present to one or two alleles.  that predicts the synthesis of factor V molecule (FV Leiden or FV Q506) in which Arg at position 506 (where is located one of the cleavage sites of APC) is substituted by Gln ([4],[5]) . Mutated factor V is also refered as factor V-Leiden. Factor‘s V-Leiden polypeptidic chain is modified at the clivage position by APC (506 Arg ->Gln). Thus, modified FVa FV Leiden (after activation by thrombin (factor IIa) retains its procoagulant activity but it is resistant to the inhibitory effect of it’s not inhibited by APC  and, as a consequence, thrombin production is not regulated by APC. 6 ([6]) accelerated. . Laboratory diagnosis of APCR is performed by the functional clotting assay as described by Dahlbäck .  2(2). . It is based on the study of the prolongation or not of aPTT in the presence and absence of APC in vitro. Diagnosis of FV Leiden abnormality is performed using polymerase chain reaction assay (PCR). The gold standard should be a perfect cosegregation between clotting and molecular  biology assays.  APCR phenotype and FV Leiden mutation is a major risk factor for venous thrombosis whereas its role in arterial thrombosis is still being studied.  7,8 ([7],[8]). .

 

A prevalence of 20-40% among thrombophilic patients has been reported.  ([9],[10],[11],[12],[13]). APCR phenotype as well as FV-Leiden is quite common (2-8 %) in the general population. 14 ([14]). The prevalence of FV Leiden in the general population presents a broad geographical variance.  15([15]). .  

 

Several studies have demonstrated that APCR is of the commonest causes of hereditary thrombophilia. An incidence of about 20-40% among thrombophilic patients has been reported. APC-Resistance is considered to be one of the major risk factor for venous thrombosis. However it is not clear yet if APCR is associated to arterial thrombosis.

Laboratory diagnosis of APCR is performed by the functional clotting assay as described by Dahlbäck Dahlback(2). It is based onat the study of the prolongation of aPTT in the presence or absence of APC in vitro. DiagnosisConfirmation of FVfactor V -  Leiden abnormality is performed using polymerase chain reaction assay (PCR).  The gold standard should be a perfect co-segregation between clotting and molecular  biology assays.

Several studies demonstrated that APCR phenotype (defined by clotting test) was not always in perfect cosegregation with the presence of FV Leiden.  16, 17(detected by molecular biology assay)  . The credibility of aPTT based assay  depends on the standardization of the aPTT reagents and the instruments used.  18, 19 ([16],[17]). .  Several studies demonstrated that APCR phenotype (defined by clotting test) was not always in perfect co-segregation with the presence of FV Leiden (detected by molecular biology assay) ([18],[19]).

It has been demonstrated that the clotting assay,  although that is quite sensible and sensitive, is influenced by the anticoagulant treatment (unfractionated heparin or coumarins) and by the CaCl2 or the sodium citrate concentration.  20, 16 , 16. . It has been shown that aPTT based assay (aPTT- APC-SR) is also affected by some acquired clinical conditions (such as inflammatory syndrome, lupus anticoagulant or antiphospholipid syndrome, pregnancy and oral contraceptives). 20,21,22,23 , ). aPTT APC-SR is influenced by pregnancy and oral contraceptives ( I. It is also affected by factor VIII and factor von Willebrand plasma levels, by low levels of PS, factor V,  factor IX or factor X  or by the treatment with heparin or coumarins .  20, 21, 22  . Hematocrit and plasma viscosity has been shown to affect aPTT- APC-SR.  22 . It is also influenced by the CaCl2 or the sodium citrate concentration. Moreover significant variances concerning the lower normal limits of aPTT -APC-SR have been reported (16, 17). These variations have been shown to depend either on differences of aPTT reagents or on variabilities of the different instruments used. Variations between batches obtained from the same source have also been reported.  18, 20 18,  . To minimize such influences alternative methods have been described. 20 ([20]). The presence of antiphopsholipid antibodies or lupus anticoagulant, the plasma levels of factor VIII, vW factor and PS as well as by the anticoagulant treatment (unfractionated heparin or coumarins) and inflammatory syndromesaPTT based assay for APCR screening test is influenced by several conditions (21). A . To minimise such influences on clotting screening test, alternative methods have been described clotting assay like that one which is based on the study of the prolongation of modified prothrombin time (PT) prolongation after addition of APC has been recently describeddiscribed . 21([21]).

 

 However PT based assay may be affected by factor VII levels. .  21 .

The high frequency of APCR raises the possibility that screening for APCR using simple clotting methods may be effectuated even in non specialized laboratories. Thus, we evaluated the credibility of APCR clotting tests (aPTT and PT based tests) using reagents from two different sources. The reproducibility of the tests was studied when they performed manually or on automated instrument. The results of clotting tests, were expressed as APCR ratios (aPTT or PT- APC-SR ratio) and normalized APCR ratios (aPTT or PT APC-NR calculated versus a pool plasma of FV Leiden carriers diagnosed with molecular biology assay). Mean values and threshold normal values were defined for each reagent and each way of tests realization. WeIn the second part of the study, we validated also both assays (aPTT and PT based test) versus diagnosis of FV Leiden mutation performed with molecular biology assay, to improve the sensitivity of APCR clotting test.

Finally, we screened a total population of 1841 individuals to examine the prevalence of APCR in normal Greekgreek population, in thrombophilic patients, and in patients suffering from arterial thrombosis, in. Moreover the prevalence of APCR was studied to patients presenting diabetes and in to a group of patients that suffering fromed idiopathic hypertension - conditions considered as high risk factors for vascular diseases.

 

The incidence of factor V- Leiden in the general population is about 2-8%. Studies using the PCR assay demonstrated that the incidence of factor V-Leiden among general population presents a  broad geographical distribution.

This paper presents the first  Greek study on the incidence of APCR in a sample of Greek population that lives in Greece. Studied population included normal persons, and patients suffering residivant DVT with a positive familial history and no other factor predisposing to thrombosis. APCR was also tested to a group of patients suffering arterial thrombosis. Moreover the incidence of APCR was studied to patients presenting diabetes and to a group of patients that suffered idiopathic hypertension - conditions considered as risk factors for vascular diseases.

Screening for APCR was performed using both the above mentioned functional methods. Both methods were performed manually and using automated instruments and the credibility was controlled. In some APCR positive patients factor V-Leiden was explored using PCR assay.

 

 

 

 

 

materials and methods

 

Materiel

The control group was composed of 20 healthy volunteers (10 males and 10 females) aged 50±14,6  years old. (mean ± SD). All controls did not receive any medication during the last 15 days.

Patients groups

a. Thrombophilic group was consisted of 82 patients (42 males and 40 females), aged 40±10,5 years old. All of them had one or more episode of DVT diagnosed by phlebography (with or without pulmonary embolism) with the first episode at an age less than  40 years old and positive familial history. Patients with  AT-III, PS, PC, Plasminogen and Heparin Cofactor II deficiencies were excluded from the study. In 40 patients among them we determined the factor V Leiden by molecular methods and 12 among them were carrying the mutation.

b. Arterial thrombosis group was consisted of 14 patients (10 male and 4 female) aged 63 ±15 years old. They had one or more episode of acute myocardial infarction during the last 5 years, verified with clinical, electrocardiographic and angiographic evidences.

c. Diabetic patients group consisted of 43 patients (17 male and 26 female) aged 66 ± 9 years old. They presented diabetes during the last 5 years. Nobody had any history of venous or arterial thrombosis.

d. Group of hypertensive patients consisted of  22 patients (9 male and 13 female) aged 65 ±13 years old. They suffered from idiopathic hypertension well regulated by antihypertensive drugs for the last 3 years.

Nobody  had received coumarins or heparin treatment for the last 15 days before venipuncture. Individuals who were found to be APCR positive were negative for lupus anticoagulant and antiphospholipid antibodies.

Plasma from controls was tested manually and with STA. Clotting times of plasmas from thrombophilic patients were tested manually whereas clotting times from all the other groups were assayed with the STA.

 

Blood sampling

 

After atraumatical veinipuncture, whole blood (4,5 ml) was collected in siliconized glass tubes containing 0,13 M sodium citrate ( 0,5 ml). Platelet poor plasma (PPP) was prepared after double centrifugation at 2.000 g for 20 min. Plasma samples were aliquoted and stored at -30oC until they were analyzed, for 6 months the maximum.. Studied clotting times were measured on plasma samples thawed at 37oC.  All tests were performed in the same day using the same lot of reagents. Pool PPP from our laboratory served as reference normal plasma. Pool PPP from APCR patients (FV Leiden was characterized by molecular biology assay) prepared at Serbio Laboratory was used as reference APCR plasma.

 

Laboratory assays

 

  Screening for APCR was performed using the following clotting assays :

a. Assay based on aPTT, as described by Dahlbäck, was performed using the Coatest APC Resistance kit (Chromogenix Sweden).

b. Assay based on aPTT using the Automated (PTT-A) reagentPTT-A reagent. This test was performed as follows: 100 ìl test plasma were mixed with 100 ìl of PTT-A reagentPTT- Automated (PTT-A) reagent ( ( Diagnostica Stago, France). After 3 min incubation at 37oC, clotting was triggered by addition of 100 ìl of a solution containing 25 mM CaCl2 with or without APC, in a final concentration of 16 nM (Serbio laboratories France).

c. Assay based on PT clotting method, as described by Vasse et al.  21(21). Briefly, 50 ìl of test plasma were incubated for 3 min at 37oC and then they were mixed with 100 ìl of a mixture of Neoplastin (Diagnostica Stago, France) diluted 1/60 in CaCl2 (12 mM final concentration) in presence or absence of 16 nM APC (Serbio France)

 

  FV-Leiden mutation was identified on genomic DNA isolated from 5 ml citrated blood. FV gene exon 10 was amplified with polymerase chain reaction by standard procedures and the final product was digested by the restriction enzyme Mnl1 4.  4(4)(4).

 

Determination of APCR - ratio

 

   Reproducibility control was checked by performed using normal pool PPP. Clotting tests were repeated 20 times on the same sample. Tests were performed manually by an experienced technician in water bath at 37oC and on automated instrument (STA automate, Diagnostica Stago). Normal limits were defined for each test and each way of performance. Results were expressed as the ratio of clotting time in the presence of APC divided by the respective clotting time in the absence of APC (aPTT and PT APC-SR). To sensibilise the method clotting times in the presence of APC were compared to the clotting times of a pool plasma from APCR patients (carriers of factor V Leiden) and normalized ratio was calculated (aPTT and PT APC-NR).

The APCR ratios below the mean value - 2 SDsd were arbitrary defined as pathological (95% confidence interval).

 

 

Studied populations

Validation of APC Resistance clotting assays versus FV Leiden diagnosis with molecular biology assay.

 

APCR clotting tests were performed on plasmas from 28 normal individuals who were preselected as negative for FV Leiden and 12 thrombophilic patients preselected to be positive for FV Leiden (1 homozygous and 11 heterozygous). FV Leiden had been previously was tested bywith PCR assay.

 

MaterielStudied population

 

 

The Ccontrol group was composed ofincluded 20 healthy volunteerssubjects (10 males and 10 females) with a mean aged of  50±14,6  years old. (mean ± SDmean±SD). All controls did not receive any medication during the last 15 days.

  Patients groups

a. Thrombophilic patients group was consisted ofincluded 842 patients (1428 males and 4024 females), with a mean aged of 40±10,5 years old mean age. All of them had one or more episode ofpresented residivant DVT  diagnosed by phlebography (with or without pulmonary embolism) with the first episode at an age less than  before the age of 40 years old age and positive familial history. Thrombophilic Ppatients with  AT -III, PS, PC, Plasminogen and Heparin Cofactor II  deficiencies were excluded from the study. In 40 patients among them we determined the factor V Leiden by molecular methods and 12 among them were carrying the mutation.

b. Arterial thrombosis group was consisted ofincluded 14 patients (10 male and 4 female) with a mean aged of with 63 ±15 years old mean age. They hadpresented one or more episodes of acute myocardial infarction during the last 5 years, verified with clinical, electrocardiographic   and angiographic evidences.

 c. Diabetic patients group consisted of included 43 patients (17 male and 26 female) with a  mean aged of 66 ± 9 years old. They presented diabetes during the last 5 years. Nobody had any history of venous or arterial thrombosis.

d. Group of hypertensive patientspatientsc consisted of. It included  22 patientspts (9 male and 13 female) with mean aged of 65  ±13 years old old. They suffered from idiopathic hypertension well regulated byequilibrated with antihypertensive drugs forduring the last 3 years.

Nobody  had received any coumarins or heparin treatment forduring the last 15 days before veinipuncture. IndividualsSubjects who were found to be APCR positive were negative for lupus anticoagulant and antiphospholipidantiphospholipide antibodies.

 

 

Plasma from controls was tested manually and with STA. Clotting times of plasmas from thrombophilic patients were tested manually whereas clotting times from all the other groups were assayed with the STA.

 

 

 

 

Blood samples and reagents

After atraumatical veinipuncture, whole blood (4,5 ml) was collected in siliconized glass tubes containing 0,13 M citrate ( 0,5 ml). Platelet poor plasma (PPP) was prepared after double centrifugation at 2.000 g for 20 min. Plasma samples were aliquoted and congealed at -30oC until they were analysed. Studied clotting times were measured on plasma samples decongeled once. All tests were performed in the same day using the same lot of reagents.

Pool PPP from our laboratory served as reference normal plasma. Pool PPP from APCR patients (with factor V Leiden detected with molecular biology assay) prepared at Serbio Laboratory was used as reference APCR plasma.

Screening for APCR was performed using two clotting assays :

a. Classic assay bested on aPTT, as described by Dahlback. 100 ìl test plasma were mixed with 100 ìl of aPTT Automated reagend (Diagnostica Stago, France). After 3 min incubation at 37oC, clotting was triggered by addition of 100 ìl of a solution containing 25 mM CaCl2 with or without APC, in a final concentration of 16 nM (offered by Serbio laboratories France). In a preliminary step we compared aPTT based method for the determination of APCR, using the commercially available diagnostic kit Coatest APC-Resistance (offered by Chromogenix-Sweden) with the regents obtained by Serbio Laboratories.

b. Clotting assay based on PT, as described by Vasse et al. Briefly, 50 ìl of test plasma were incubated for 3 min at 37oC and then they were mixed with 100 ìl of a solution containing CaCl2 (12 mM final concentration) and diluted thromboplastin (1/60) (Neoplastine, Diagnostica Stago, France). This solution contained or not APC (16 nM final concentration).

 

 

 

Determination of APCR - ratio

 

Reproducibility control was performed using normal pooled PPP. Both tests were repeated 10 times on the same sample. Tests were performed manually, on semiautomatic instrument (Behring Fibrintimer A, BFA; AG Behringwerke) and on automated instrument (ST 888, Diagnostica Stago). Normal limits were defined for each apparatus. Plasmas from control subjects were tested with the three different ways. Clotting times of plasmas from thrombophilic patients were tested manually and with Behring Fibrintimer whereas clotting times from all the other groups were assayed with the ST 888.

Results were expressed as the ratio of clotting time (aPTT or PT) in the presence of APC divided by the respective clotting time in the absence of APC. To sensibilise the method clotting times (aPTT and PT) in the presence of APC were compared to the clotting times of a pool plasma from APCR patients and normalised ratios were calculated (aPTT and PT normalised ratio).

To calculate the inferior normal limits from the control group we excluded the subjects having both ratios (aPTT-APC ratio and PT-APC ratio) inferior than the mean - 2 standard deviations (sd). The APCR ratios below the mean - 2 sd were considered pathological (95% confidence interval).

All test plasmas with pathological or limited normal ratios were tested using the diagnostic kit Coatest APCR (Chromogenix) and the results were correlated.

 

 

Statistical analysis

 

 

Regression analysis and coefficient correlationof correlation coefficient were applicatedapplicated. Comparison of mean values was donerealised using the Students’ t-test. Chi-square test was used to compare the prevalenceincidence of APCR among studied populations. Statistical analysis was realizedrealiszedrealised using the SPSS software.

 

 

 

 

 

 

 

Results

 

A preliminary study using normal and patients plasmas, demonstrated that APCR aPTT based assay performed with the commercially available kit Coatest APCR (Chromogenix) was significantly correlated with results obtained using reagents obtained by Serbio (data not shown).

 

Reproducibility of aPTT based test and PT based test was acceptable when assays were performed manually and automatically. Measurements were repeated 10 times using normal pool plasma and  Tthe coefficients of variation (C.V.) for each test isare shown in Ttable 1I.

  aPTT based assays (Coatest and PTT-A assays) were well-correlated (r=0,7 and p<0,05) when they performed on plasma from 28 normal individuals without FV Leiden mutation (fig 1). The correlation was also significant between Coatest and PT based assays (r=0,66 and p<0,0001) as well as between PTT-A and PT based assays (r=0,8 and p<0,001).

 

Table I. Reproducibility control of clotting assays for APCR diagnosis. Tests were performed manually and on semiautomatic instrument (Fibrintimer) and automated (ST 888). Normal pool plasma was tested 10 times and coefficients of variation (CV) were calculated.

 

In figure 1 is demonstrated that APCR ratios obtained with PT based assay were significantly correlated with ratios of aPTT based assay (r=0,8 and p<0,001). Similarly, aPTT  APC-NR - normalised ratios were significantly correlated with PT APC -NR normalised ratios (r=0,8 and p<0,001). Moreover aPTT and PT-ratios were highly correlated with aPTT and PT -  normalised ratios respectively (r=0,8 and p<0,001).

 

Mean values of APCR ratios (aPTT and , PT based tests and the respective normalizednormalised ratios) were different for each assay and for each way of assay realization (manually or STA automate). Mean values of studied APCR ratios were significantly lower when tests were were performed manually than with automated instruments (p<0,05). (Ttable 2II). As a consequence, normal limits were lower for the manual assay than for the automated one. When the normal range was defined by mean values -2SD the lower normal limits for each assay are presented in Table 2.As a consequence, normal limits were lower for the manual assays than for the automated All ratios inferior than the limits are presented in the Table 2.  were arbitrary defined as pathological for APCR. (table III).

 

Sensitivity of APCR clotting assays versus molecular biology assay for diagnosis of FV Leiden.

 

Among the 28 normal individuals that had been preselected as negative for FV Leiden mutation, one was found to have pathological aPTT- APC-SR with Coatest assay (2,08). Coatest aPTT APC-NR as well as PTT-A and PT- APC-SR (and the respective normalized ratios) were in the normal zone for all the studied individuals.

Among 12 thrombophilic patients preselected as carriers of FV Leiden mutation, 6 (50%) had normal Coatest aPTT- APC-SR (2,86, 2,57, 2,38, 2,32, 2,31, and 2,29) and one had a borderline value (2,2). Only 2 (16%) of these patients had the respective normalized ratio in the normal zone (0,82 and 0,91) whereas the rest 4 patients had abnormal aPTT APC-NR (<0,7). When the 12 plasmas were tested with PTT-A reagent and PT based method, there was not found any case with ratio in the normal zone according to the normal limits defined in Table 2. Similarly, all normalized ratios were abnormal. Results are summarized in Table 3. The combination of aPTT APC-SR  ratio with the aPTT APC-NR ratio improved the sensitivity of clotting assay, 50% false negatives in the first case and 16% in the last one. A perfect co-segregation between abnormal results with clotting assays and molecular biology assay was observed when abnormal aPTT- APC-SR (and NR) was combined with abnormal PT -APC-SR  (and NR) to diagnose APCR.

 

APC resistance prevalence in normals and patients groups

Analytical results are presented in Table 5.

Mean values of the studied ratios at the different patients groups  were not  different as compared to the appropriate controls (Table 4). A total of 141 individuals  were tested with both aPTT (using PTT-A reagent) and PT based assays (results are summarized in Table 5). Moreover , Mmean values of the studied ratios at the different patients groups  were not  different as compared to the appropriate controls (Ttable 4II).  No significant differences of APCRR ratios was found between the males and females (p>0,5). APCRR ratios were not correlated with the age of the examined individuals (for each studied ratio p>0,05).

  

Table II. Mean values and sd of studied APCR ratios for the studied groups. The referred controls were the same population. Tests for arterial thrombosis, diabetics and hypertension groups were performed with automated instrument (ST - 888).

 

 

Table III. Normal limits for the studied ratios. Normal limits were calculated as mean value - 2 sd of the control group after elimination of the subjects whose ratios were pathological. When tests were performed manually, normal limits were significantly lower than those obtained by automated assays (p<0,05).

 

 

 

Figure 1. Correlation between aPTT and PT-ratios (n=99, many points are overlapped).

 

 

APCR aPTT - ratio as well as PT ratio (and the normalised ratios) were not correlated with the age of the studied subjects. Mean values of the studied ratios were similar for both sexes.

When APCR clotting assays were performed manually, ratios below 1,4 and 1,52 for aPTT and PT based test respectively were considered as pathological. For normalised ratios all values inferior than 0,47 and 0,5 (for aPTT and PT respectively) were considered pathological. When tests were performed automatically, as pathological were defined all values inferior than 2,4 for aPTT based test and 2,12f or PT based test (for normalised ratio, normal limits were 0,84 and 0,82 respectively) (table III).

 

According to the normalpathological limits defined above (Table 2), it was found that 22 out of 20 controls (10% had abnormal aPTT -APC-SR ratio(2,32 and 2,07) and one had borderline ratio (2,73). All test were performed with STA automate. aPTT APC-NR was abnormal for two individuals (0,76 and 0,68 respectively). The individual with the borderline aPTT APC-SR had normal aPTT APC-NR. When samples were tested with PT- APCRPT based assay it was found that only one normal subject had pathological values for PT APC-SR as well as for PT APC-SR (1,53 and 0,69 respectively). The second subject with abnormal aPTT ratios had PT ratios above the inferior normal limits. As it was defined previously, the combination of abnormal results in both aPTT-APCR and PT-APCR based tests has a high likelihood to predict FV Leiden. Thus, the prevalence of APCR and probably of FV Leiden in a random sample of Greekgreek population was 5% (1 out of 20 normals). of normal population) were APCR positive with pathological values of all the studied ratios.

 

   Among thrombophiliacs patients, 15 patients out of 42 had abnormal aPTT- APC- SR and NR. PT- APC-SR and NR were abnormal in 11 patients. There was not found any patient with normal aPTT and abnormal PT ratio. APCR prevalence in the group of thrombophilic patients was 26,1% (11 out of 42 patients).

APCR were found to be 11 out of 42 (26,1% of the thrombophilic group). These patients had pathological values for aPTT and PT ratios as well as for normalised ratios.  Other 4 patients, (9% of the thrombophilic group) had pathological only the aPTT - ratio (and the normalised aPTT - ratio) and normal PT - ratio.

In patients group presented arterial thrombosis APCR incidence was 14 % (2 out of 14 patients). In the group of patients suffering from acute myocardial infarction, it was found that one out of 14 had abnormal aPTT- APC-SR -ratio(2,16) and a  second one had a borderline aPTT APC-SR (2,34).. aPTT APC-NR was abnormal for the first patient (0,72) and normal for the second one (0,79). With PT-APCR PT based assay it was found that one patient had a borderline PT APC-SR ratio(1,77) and clearly abnormal the PT APC-NR (0,63). It was the same patient who had abnormal aPTT- ratios. APCR prevalence in the group of patients with acute myocardial infarction was 7,1% (1 out of 14 patients).

In the group of diabetics group, 24 out of 43 patients had abnormal aPTT -APC-SR (2,25 both of them) whereas one had a borderline aPTT APC-SR (2,34). The aPTT APC-NR was abnormal for the two patients (0,75 both of them) whereas the third one was found to have a normal value (0,78). Withhen the PT-APCR ratioall plasmas were tested with the PT based assay it  it was found that one them had abnormal ratioPT APC-SR (1,63) and the second one (who had abnormal aPTT APC-SR) was found to have a borderline value (0,77). Examination with the PT APC-NR revealed that both patients had clearly abnormal ratios (0,62 and 0,63). APCR prevalence in the diabetic patients group was 4,6% (2 out of 43 patients). were

.

    found to be APCR with pathological all the studied ratios (9,3 % of the diabetic patients). There was not found any cases with discordant results for the studied ratios. The incidence of APCR was the same among insulin and non insulin dependant diabetic patients.

Among the 22 patients with idiopathic hypertension, 12 (9 %) werewas found to have abnormal aPTT- APC-SR ratio(2,28 and 2,16). aPTT APC-NR was borderline for the first patient (0,77) and abnormal for the second one (0,72). When plasmas were tested with the PT based method it was found that only the patient having abnormal aPTT APC-NR had abnormal PT APC-SR and NR (1,76 and 0,69) whereas all the other patients had normal ratios. APCR prevalence among patients suffering from chronic idiopathic hypertension, was 4,5% (1 out of 22 patients).

APCR was significantly more frequent in thrombophilic patients group as compared to controls (26,1% versus 5% respectively, p<0,05). APCR prevalence was slightly increased in the group of patients suffering from arterial thrombosis (7,1%) but the difference versus the controls was not significant (p>0,05).

APCR prevalence was not significantly different among the studied groups of patients having high risk factors for arterial disease, as compared to control group  (Table 5).

pathological values for both aPTT and PT ratio as well as for normalised ratios. Another one patient (4,5%) had pathological PT - ratio (and PT - normalised ratio) whereas aPTT based test was normal.

Moreover,  APCR incidence in the total population (except thrombophilic patients) was 5,510 % (510 out of 99 subjects).

 

 

The incidence of APCR was not significantly different among the studied patient groups as compared to control group  (table IV). Comparison between APCR incidence in thrombophilic group (11 out of 42 patients) and the rest population (10 out of 99 subjects) reveals a slightly significant difference.

 

 

 

Table IV. APCR incidence in normal population and patients groups.

 

 

 

 

Discussion.

 

Activated Protein C Resistance (an abnormality of PC anticoagulant pathway) is a new hereditary cause of thrombophilia that has been recently described by DahlbäckDahlback et al. It has been demonstrated that a point mutation on factor V gene results to a change on factor V polypeptidic chain (substitution of Arg 506 by Gln) which is the cleavage site of APC. Thus fModified factor Va (FV Leiden or FV Q506) cannot be inhibited by APC and thrombin production is insufficiently regulated ([22]) continued. In vitro, when APC is added to patients plasma the presence of this defectabnormality, causes diminished prolongation of aPTT, as compared to control. Laboratory screening for the presence of APCR is performed with thea clotting assay based on aPTT. APCR diagnosis with clotting assay is not always well co-segregated with the presence of FV Leiden mutation detected with molecular biology assay. Moreover significant variances concerning the lower normal limits of aPTT APC-SR have been reported (16, 17). These variations have been shown to depend either on differences of aPTT reagents or on variabilities of the different instruments used. Variations between batches obtained from the same source have also been reported([23]). It has been shown that aPTT based assay (aPTT APC-SR) is affected by some acquired clinical conditions (such as inflammatory syndrome, lupus anticoagulant or antiphospholipid syndrome)(21,[24]). aPTT APC-SR is influenced by pregnancy and oral contraceptives ([25],[26],[27]). It is also affected by factor VIII and factor vW plasma levels, by low levels of PS, factor V,  factor IX or factor X or by the treatment with heparin or coumarins (21,[28],[29],[30]). Hematocrit and plasma viscosity has been shown to affect aPTT APC-SR(22). It is also influenced by the CaCl2 or the sodium citrate concentration. A clotting assay based on PT prolongation by APC has been described in order to avoid influences on endogenous coagulation pathway. However PT based assay may be affected by factor VII levels (21). Moreover normalization of aPTT APC-SR has been suggested that it could improve the specificity and sensitivity of the clotting assay.However, it has been demonstrated that aPTT bases assay is affected by some acquired clinical factors which result to factor VIII and vW plasma levels (such as inflammatory syndrome), lupus anticoagulant or antiphospholipid syndrome. Thus an assay based on PT prolongation by APC has been described.

The credibility of APCR clotting methods In the first part of the present study wase evaluated the credibility of APCR clotting methods (aPTT and PT based assays) when they performed manually versus onresults obtained using automated instrument. The reproducibility of the tests when they performed manually was comparable to that when the tests were performed on STA automate (the CV were 5-7% and 3-8% respectively Table 1).

Two different reagents for aPTT based test were controlled. Our results demonstrated that aPTT-APCR based m method either performed with Coatest reagents or PTT-A reagents as well as PT-APCR based test performed with Neoplastin reagent, have an important credibility when performed manually. The reproducibility of the tests when they performed manually was comparable to that when the tests were performed on STA automate (the CV were 5-7% and 3-8% respectively Table 1). We did not fiound any discordant results for all the controls tested manually and on automated instrument. However, the mean values and the inferior normal zone for all the tests Coatest aPTT APC-SR, PTT-A aPTT APC-SR and PT APC-SR (and the respective NR) were significantly lower when the tests were performed manually as compared to the values obtained with the automated instrument. Moreover, inferior normal limits were lower when aPTT-APCR based test was performed with PTT-A reagents as compared to Coatest reagents (Table 2). PT- APC-SR inferior normal limits were lower for PT based test as compared to aPTT-APCR based tests  (with Coatest and PTT-A). Inferior normal limits in test performed with STA automate presented here, were different to those reported by Vasse et al 21, 21 (21), who used the same reagents and apparatus. This could be explained as a consequence of differences between batches or as a result of variations among the different control plasma used to define the normal ranges.

   AaPTT- APCR based test has been already reported that is not perfectly co-segregated with molecular biology assay for the detection of FV Leiden mutation 18,19. 18,19  (18,19). In our series we found 3,5% false negative aPTT- APC-SR with Coatest reagents. The use of aPTT APC-NR improved the sensibility of the method (no false negatives were found). Using PTT-A reagents as well as PT-APCR based  method we did not found any false negative cases. However we cannot conclude if this is due to the reagents used or to the hazard. Screening patients carriers of  FV Leiden mutation, revealed a 50% false negative cases with Coatest aPTT- APC-SR and 8,3% of cases with borderline values. Similar results have been published by Lerroy-Matheron et al, 18 (18) who found that 43% of heterozygous patients had normal aPTT- APC-SR while 6% of the normals had abnormal aPTT- APC-SR. aPTT APC-NR improved the sensitivity of the test since the false negative cases were 16% and the borderline values were eliminated. WAll samples tested with PTT-A reagent as well as with PT-APCR based method we did not find had abnormal values (any0% false negative results). This confirms that aPTT based clotting assay is insufficient for a firm diagnosis. On the other hand our results allow to conclude that a combination of abnormal results with clotting tests that examine intrinsic and extrinsic coagulation pathway (aPTT- APC-SR and PT- APC-SR) improves the likelihood that functional tests predict FV Leiden mutation. Moreover it in Tables 3 and  5 is shown that normalized ratio contributes to discriminate borderline values. Screening for APCR phenotype can be performed using reagents from different verse sources, but it is obligatory to define the normal ranges according to the reagents and the instrument used. It is essential to define the cut threshold of abnormality by testing a normal population for each laboratory since important interinteri-individual variances have been reported. Inter-batch variability shouldmast also be taken in to account 22. 22  (22). Although safe diagnosis of FV Leiden mutation can be performed mainly with molecular biology methods, it is important to standardize clotting assays for APCR diagnosis. Usually screening for APCR phenotype precedes that of FV Leiden genotype. Since APCR is a quite common genetic cause of familial thrombophilia, andit is probable that screening for APCR phenotype shouldwould be realized even in non specialized laboratories, our results demonstrate that APCR clotting tests performed manually are as credible as those performed on automated instruments.

Very soon it was demonstrated that APCR and mutation on factor V gene, is one of the most frequent underlying cause of familial thrombophilia and it is quite frequent in normal population. Some geographic differences have been observed.

In the presentthis study we demonstrated that the prevalenceincidence of APCR phenotype among normal Greeksgreeks subjects is 5% (1 out of 20 normals or 5 out of 99 individuals - total population except thrombophilic patients). Rees et al (15) reported that the factor V Leiden prevalence among Greeksgreeks that live in Britain is 13% (the higher as compared to other ethnic groups). 15.  However our results show a prevalence of APCR phenotype comparable to that reported for Netherlands (4,7% )10Koster et al (10), Sweden (6,9%) Svenson and Dahlbäck (3 3), and USA (5%) 7Griffin et al (7). and in Greeks living in their country (4,8% )Lampropoulos et al (). Considering that in our study we applied a combination of clotting tests for APCR screening which improved the  likelihood to predict FV Leiden mutation, this discrepancy could be explained by the fact that the Greekgreek population studied by Rees becomes from a closed national community that lives in Britain and the possibility of family relative relationships is important.raises to 10 %, which is one of the higher incidence reported in the literature.

  

Our finding is in accordance with a recent study which demonstrates that the incidence of factor V Leiden among greeks that live in Britain is the higher as compared to other ethnic groups.

The prevalence of APCR phenotype among thrombophilic patients iswas found to be 26,1 % and is in agreement with previous reports. APCR isThis incidence found to be slightly significantly more frequent among thrombophilic patients  as compared either to controlss or to the rest population (all studied groups except thrombophiliacsthrombophilics). It is noteworthy that  4 unrelated thrombophilic patients, (9% of the thrombophilic group),  had pathological only the aPTT-  APC-SR, (and the aPTT APC-NR) whereas PT- APC-SR test and NR wasere normal. This allows to hypothesize either the existence of an abnormality on factor VIII - which probably has a non negligible prevalence - or an augmentation of factor VIII plasma levels. A more detailed study is performed toin our laboratory.

   Thus, APCR phenotype, as detected by global clotting tests, seems to be a moderate risk factor for venous thrombosis and familial thrombophilia when the prevalence of the abnormality in the general population is quite high (10 %). Moreover, we have some data (which are going to be published) that demonstrate an important prevalence of factor V Leiden (about 32 %) among thrombophilics  which is not correlated with APCR clotting test. Our data raise some questions concerning the relationship between aPTT based APCR clotting test and factor V-Leiden. It is noteworthy that  4 unrelated thrombophilic patients, (9% of the thrombophilic group) had pathological only the aPTT - ratio (and the normalised aPTT - ratio) whereas PT - ratio was normal. This allows to hypothesise the existence of an abnormality on factor VIII which probably has a non negligible prevalence.

APCR prevalence in patients with myocardial infarction was 714 % and it was not significantly different as compared to controls (59 %). In agreement with previous studies we dido not fiound any relationship between APCR and arterial thrombosis  . 31, 32, 33, 34.([31],[32],[33],[34]).  Furthermore, even among patients who did not present arterial thrombosisany thrombotic episode but suffered from diabetes or hypertension (risk factors for vascular disease and arterial thrombosis) APCR prevalence was 59 %. Thus APCR and probably FV Leiden mutation is not associated with arterial thrombosis even in patients that accumulate important risk factors for vascular disease such as chronic diabetes or hypertension.

   Our data suggest that APCR phenotype is athe most common clotting defectcoagulation abnormality in Greekgreek in the general population which is strongly related to familial thrombophilia but it is not a risk factor for arterial thrombosis even in patients having important risk factors for vascular disease and arterial thrombosis.  and its prevalence is the highest reported among european peoples. However APCR screening disorder has to be performedverified using well standardized reagents and well defined normal ranges considering that many factors (laboratory artifacts or clinical conditions or inter-i-ndividual variabilities) may influence APCR clotting tests. The application of  two clotting tests (aPTT and PT based test) that examine intrinsic and extrinsic coagulation pathway improves the sensitivity and the specificiensitivity of the method in order to predict FV Leiden mutation. and to accept as APCR positive those subjects with both tests pathological. In greek population APCR phenotype seems to be a moderate risk factor to venous thrombosis while it is not related to myocardial infarction. Its high prevalence among normal population as well as among patients presenting important risk factors for vascular disease (as diabetes and hypertension)

 

 

 

 

 

References

 

1.    Dahlbäck B, Hildebrand B. Inherited resistance to activated protein C is corrected by anticoagulant cofactor activity found to be a property of factor V. Proc Natl Acad Sci USA 1994; 91: 1396-400.

2.    Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to previously unrecognised mechanism characterised by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-8.

3.    Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: 517-22.

4.    Bertina RM, Koeleman BPC, Koster T. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.

5.    Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg 506 Gln mutation in factor Va. Lancet 1994; 343: 1361-62.

6.    Dahlbäck B, Shen Lei. Factor V and protein S as synergistic Cofactors to activated protein C in degradation of factor VIIIa. Journal Biol Chem. 1994; 269: 18735-38.

7.    Griffin JH, Evatt B, Wideman C, Fernandez JA, Anticoagulant protein C pathway defective in majority of thrombophilia patients. Blood 1993; 82: 1989-93.

8.    Lindblad B, Svensson PJ, Dahlbäck B. Arterial and venous thromboembolism with fatal outcome and resistance to activated protein C. Lancet 1994; 343: 917.

9.    Dahlbäck B. Inherited thrombophilia : Resistance to activated protein C as a pathogenic factor for venous thromboembolism. Blood 1995; 85: 607-14.

10. Koster T,  Rosendaal FR, de Ronde H, Briet E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden thrombophilia study. Lancet 1994; 342: 1503-6.

11. Sun X, Evatt B, Griffin JH. Blood coagulation factor Va abnormality associate with resistance to activated protein C in venous thrombophilia. Blood 1994; 83: 3120-5.

12. Koelman BPC, Reitsma PH, Allaart CF, Bertina RM. Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficiency families. Blood 1994; 84: 1031-5.

13. Zeller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venous thrombosis. Lancet 1994; 343: 1536-8

14. Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. High risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 1995; 85: 1504-8.

15. Rees DC, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet 1995; 346: 1133-4.

16. Rosén S, Johansson K, Lindberg K, Dahlbäck B. Multicenter evaluation of a kit for activated protein C resistance on various coagulation instruments using plasma from healthy individuals. Thromb Haemost 1994; 72: 255-60.

17. Kraus M, Wagner C. Evaluation of APC-sensitivity in normal blood donors using different reagents and instruments. Thromb Res 1994; 76: 231-6.

18. Leroy-Matheron C, Levent M, Pignon JM, Mendonça, Gouault-Heilmann M. The 1691 G- A Mutation in the Factor V Gene : Relationship to Activated Protein C (APC) Resistance and Thrombosis in 65 Patients. Thromb Haem 96; 75:4-10.

19. Zöller B, Svensson PJ, Xuhua H, Dahlbäck B. Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J Clin Invest 1994; 94: 2521-2524.

20. Trossaërt M, Conard J, Horellou MH, Samama MM, Ireland H, Bayston TA, Lane DA. Modified APC resistance assay for patients on oral anticoagulants. The Lancet 1994; 344: 1709.

21. Vasse M, Leduc O, Borg JY, Chrétien MH, Monconduit M. Resistance to activated protein C: evaluation of three functional assays. Thromb Res 1994; 76: 47-59.

22. Zöller B, Holm J, Svensson PJ, Dahlbäck B. Elevated levels of prothrombin activation fragment 1+2 in plasma from patients with heterozygous Arg 506 to Gln mutation in the factor V gene (APC-Resistance) and/or inherited protein S deficiency. Thhromb Haem 1996; 75: 270-4.

23. Freyburger G, Bilhou-Nabera C, Dief S, Javorschi S, Labrouche S, Lerebeller MJ, Boisseau MR. Technical and Biological Conditions Influencing the Functional APC Resistance Test. Thromb Haem 96; 75:460-5.

24. Ehrenforth S, Radtke KP, Scharrer I. Activated protein C-resistance  in patients with lupus anticoagulant. Thromb Haemost 1995; 74:797-8.

25. Vandenbroucke JP, Koster T, Briët E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet 1994; 344: 1453-7.

26. Østerud B, Robertsen R, Åsvang GB, Thijssen F. Resistance to activated protein C is reduced in women using oral contraceptives. Blood Coag Fibrinol 1994; 5:853-4.

27. Hellgren M, Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Am J Obstet Gynecol 1995;173:210-3.

28. Cooper PC, Hampton KK, Makris M, Abuzennadah A, Paul B, Preston FE. Further evidence that activated protein C resistance can be misdiagnosed as inherited functional protein S deficiency. Brit J Haematol 1994; 88: 201-2.

29. Rao AK, Elliot D, Stadnicki A, De La Cadena R, Sherry S. High levels of plasma factor VIIIc impair anticoagulant response to activated protein C. Thromb Haemost 1995; 7:1126.

30. Colluci M, Ciavarella N, Giliberti MG, Semeraro N. Resistance to activated protein C (APC) influence of factor V levels. Thromb Haemost 1994; 72: 880-6.

31. Ridker PM, Hennekes CH, Lindpaintner K, Stampfer MJ, Eisenberg PR, Miletich JP. Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke and venous thromboembolism in apparently healthy men. N Engl J Med 1995; 332:912-7.

32. Emmerich J, Poirier O, Evans A, Marques-Vidal P, Arveiler D, Luc G, Aiach M, Cambien F. Myocardial infarction, Arg 506 to Gln factor V mutation, and activated protein C resistance. The Lancet 1995; 345: 321.

33. Marz W, Seydewitz H, Winkelmann B, Chen M, Nauck M, Witt I. Mutation in coagulation factor V associated with resistance to activated protein C in patients with coronary artery disease. The Lancet 1995; 345:526-7.

34. Samani NJ, Lodwick D, Martin D, Kimber P. Resistance to activated protein C and risk of premature myocardial infarction. The Lancet 1994; 344: 1709-10.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 1. Reproducibility control of clotting assays for APCR diagnosis.

 

Coatest APCR

(aPTT - based assay)

PTT-A APCR

(aPTT - based assay)

PT -APCR

 based assay

manually

7%

5%

5%

STA

8%

6%

3%

 

Table 1. Reproducibility control of clotting assays for APCR diagnosis. Tests were performed manually and on automated instrument (STA automate). The same normal pool plasma was tested 20 times and coefficients of variation (CV) were calculated.

 

Table 2. Mean values ± SD of APCR ratios for each method.

 

Coatest APCR ratio

PTT-A APCR ratio

PT based APCR ratio

manually

2,3±0,4

<1,5*

2,4±0,5

<1,4*

2,18±0,33

<1,52*

STA

2,88±0,38

<2,12*

3 ±0,33

<2,34*

2,4±0,32

<1,76*

* limits for the pathological values manually or automated for each method.

Values Table 2. Mean values±SD of APCR ratios for each method. Values below the mean - 2SD were arbitrary defined as pathological for APCR (95% confidence interval). APCR ratios were always significantly lower for the tests performed manually than on STA      (n= 48 control individuals, p<0,05).

 

 

Table 3. Comparison of APCR tests sensitivity to PCR results.

 

FV-Leiden -/-neg (n=28)

false positive APCR ratio

FV Leiden +/+ or +/- (n=12)

false negative APCR-ratio

Coatest aPTT-APCSR

1 (3,5%)

6    (50%)

1  with borderline ratio              (8,3%)

PTT-A-APCSR

0

0

PT-APCSR

0

0

All tests combined

0

0

+/+ = homozygote for the mutation, +/- heterozygote, -/- normal for the mutation

Table 3. Control of specificity and sensitivity APCR aPTT and PT based methods and the combination of them versus the diagnosis of FV Leiden with molecular biology assay. Plasmas with discordant results of aPTT and PT APC-SR were considered as normals.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 4. Mean values and SD of APC ratios for the studied groups.

 

n

aPTT-APC R-rratio

PT-APCR-r ratio

thrombophilia  group (manually)

42

2,3 ± 0,4

2,17 ± 0,30

arterial thrombosis group

14

3,1 ± 0,5

2,45 ± 0,33

diabetics group

43

3,2 ± 0,4

2,55 ± 0,32

hypertension group

 22

3,1± 0,4

2,56 ± 0,26

 

Table 4. Mean values and sd of APCR ratios for the studied groups. Tests for arterial thrombosis, diabetics and hypertension groups were performed with STA automate whereas plasmas of thrombophilic patients were assayed manually. Mean values of the control group are presented in Table 2. Mean values of APCR ratios were not significantly different between controls and each patients’ group (p>0,05).

 

 

Table 5. Abnormal and borderline APC-ratios and APCR prevalence in the studied groups.

 

Abnormal limits

Controls

n=20

Vein thromb.T

n=42

ATarter. thromb

n=14

Diabetics

n=43

Hyprtensivese

n=22

aPTT

APC-SR

<2,34

borderline

2 (10%)

1(5%)

15 (36%)

0

1 (7%)

1 (7%)

2 (4,6%)

1 (2,3%)

2 (9%)

0

PT-

APCSR

<1,76 borderline

1 (5%)

0

11 (26%)

0

0

1 (7%)

1 (2,3%)

1 (2,3%)

1 (4,5%)

0

APCR prevalence)

all tests abnormal

1 (5%)

11 (26%)

1 (7%)

2 (4,6%)

1 (4,5%)

 

Table 5. Abnormal and borderline APCR-ratios and APCR prevalence in the in the studied groups. As APCR were defined the patients having values for all the studied APC ratios.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Legends to tables

 

 

Table 1. Reproducibility control of clotting assays for APCR diagnosis. Tests were performed manually and on automated instrument (STA automate). The same normal pool plasma was tested 20 times and coefficients of variation (CV) were calculated.

Table 2. Mean values ± SD of APCR ratios for each method. Values below the mean -2SD were arbitrary defined as pathological for APCR (95% confidence interval). APCR ratios were always significantly lower for the tests performed manually than on STA (n= 48 control individuals, p<0,05).

Table 3. Comparison of APCR tests sensitivity to PCR results. Control of specificity and sensitivity APCR aPTT and PT based methods and the combination of them versus the diagnosis of FV Leiden with molecular biology assay. Plasmas with discordant results of aPTT and PT APCR were considered as normals.

Table 4. Mean values and SD of APC ratios for the studied groups. Tests for arterial thrombosis, diabetics and hypertension groups were performed with STA automate whereas plasmas of thrombophilic patients were assayed manually. Mean values of the control group are presented in Table 2. Mean values of APCR ratios were not significantly different between controls and each patients’ group (p>0,05).

Table 5. Abnormal and borderline APC-ratios and APCR prevalence in the studied groups. As APCR were defined the patients having values for all the studied APC ratios.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.

 

0.Fig. 1

 

0. 

0.Fig. 1 Linear regression between aPTT based APCR ratio measured with PTT-A reagent and APCR ratio measured with Coatest. Plasmas from 28 normal individuals (without the mutation of FV Leiden)  were tested with both reagents on STA 888. Only one plasma gave discordant result.

 

0. 

0.

0.Figure 2. The correlation between aPTT and PT-ratios was highly significant (r=0,8, p<0,05) (n=99, many points are overlapped). References

0.Holm J, Zöller B, Svensson PJ, Berntorp E, Erhardt L, Dahlbäck B. Myocardial infarction associated with homozygous resistance to activated protein C. The Lancet1994; 344:952-953.

 

0.Nathwani AC, Tuddenham EGD. Epidemiology of coagulation disorders. Bailliere’s Clin Haematol 1992; 5: 383-439.

 

0.Rintelen C, Pabinger I, Knöbl P, Lechner K, Mannhalter C. Probability of recurence of thrombosis in patients with and without factor V Leiden. Thhromb Haem 1996; 75: 229-232

 

0.Deep vein thrombosis and pulmonary embolism are among the most common and serius medical problems in our days. A recent study performed in USA demonstrated that venous thrombosis is associated with approximately 200.000 patients per year, while residivant venous thrombosis is about 33% of the total1.

0.Several pathological circumstances such as acute myocardial infarction, peripheral angiopathy, malignancies, metabolic diseases like diabetes and autoimmune diseases like lupus anticoagulant and antiphospholipid syndrome, as well as cardiac or respiratory in sufficiency, obesity, prolonged immobilisation or treatment with oestrogens or L-asparaginace increse the risk for venous thrombosis2.

0.Hereditary deficiencies of natural blood anticoagulants such as antithrombin III (AT III), protein S (PS), protein C (PS) or heparin cofactor II are related to familial thrombophilia in patients suffering residivant vein thrombosis  with a positive familiar history.  However the prevalence of these genetic disorders of coagulation inhibitors among thrombophilic patients is about 8 - 15 %3. Therefore for the vaste majority of these patients, familial thrombophilia remains undetermined.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

0.References

0.[1] Dahlbäck B, Hildebrand B. Inherited resistance to activated protein C is corrected by anticoagulant      cofactor     activity found to be a property of factor V. Proc Natl Acad Sci USA 1994; 91: 1396-400.

0.[1] Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to previously unrecognised mechanism characterised by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-8.

0.[1] Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: 517-22.

0.[1] Bertina RM, Koeleman BPC, Koster T. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-7.

0.[1] Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg 506 Gln mutation in factor Va. Lancet 1994; 343: 1361-62.

0.[1] Dahlbäck B, Shen Lei. Factor V and protein S as synergistic Cofactors to activated protein C in degradation of factor VIIIa. Journal Biol Chem. 1994; 269: 18735-38.

0.[1] Griffin JH, Evatt B, Wideman C, Fernandez JAFernandezJA, Anticoagulant protein C pathway defective in majority of thrombophilia patients. Blood 1993; 82: 1989-93.

0.[1] Lindblad B, Svensson PJ, Dahlbäck B. Arterial and venous thromboembolism with fatal outcome and resistance to activated protein C. Lancet 1994; 343: 917.

0.[1] Dahlbäck B. Inherited thrombophilia : Resistance to activated protein C as a pathogenic factor for venous thromboembolism. Blood 1995; 85: 607-14.

0.[1] Koster T,  Rosendaal FR, de Ronde H, Brieët E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden thrombophilia study. Lancet 1994; 342: 1503-6.

0.[1] Sun X, Evatt B, Griffin JH. Blood coagulation factor Va abnormality associate with resistance to activated protein C in venous thrombophilia. Blood 1994; 83: 3120-5.

0.[1] Koelman BPC, Reitsma PH, Allaart CF, Bertina RM. Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficiency families. Blood 1994; 84: 1031-5.

0.[1] Zeöller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in venousvevous thrombosis. Lancet 1994; 343: 1536-8

0.[1] Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. HighHIgh risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 1995; 85: 1504-8.

0.[1] Rees DC, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet 1995; 346: 1133-4.

0.[1] Rosén S, Johansson K, Lindberg K, Dahlbäck B. Multicenter evaluation of a kit for activated protein C resistance on various coagulation instruments using plasma from healthy individuals. Thromb Haemost 1994; 72: 255-60.

0.[1] Kraus M, Wagner C. Evaluation of APC-sensitivity in normal blood donors using different reagents and instrumentsinstrumens. Thromb Res 1994; 76: 231-6.

0.[1] Leroy-Matheron C, Levent M, Pignon JM, Mendonça, Gouault-Heilmann M. The 1691 G- A Mutation in the Factor V Gene : Relationship to Activated Protein C (APC) Resistance and Thrombosis in 65 Patients. Thromb Haem 96; 75:4-10.

0.[1] Zöller B, Svensson PJ, Xuhua H, Dahlbäck B. Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J Clin Invest 1994; 94: 2521-2524.

0.[1] Trossaërt M, Conard J, Horellou MH, Samama MM, Ireland H, Bayston TA, Lane DA. Modified APC resistance assay for patients on oral anticoagulants. The Lancet 1994; 344: 1709.

0.[1] Vasse M, Leduc O, Borg JY, Chrétien MH, Monconduit M. Resistance to activated protein C: evaluation of three functional assays. Thromb Res 1994; 76: 47-59.

0.[1] Zöller B, Holm J, Svensson PJ, Dahlbäck B. Elevated levels of prothrombin activation fragment 1+2 in plasma from patients with heterozygous Arg 506 to Gln mutation in the factor V gene (APC-Resistance) and/or inherited protein S deficiency. Thhromb Haem 1996; 75: 270-4.

0.[1] Freyburger G, Bilhou-Nabera C, Dief S, Javorschi S, Labrouche S, Lerebeller MJ, Boisseau MR. Technical and Biological Conditions Influencing the Functional APC Resistance Test. Thromb Haem 96; 75:460-5.

0.[1] Ehrenforth S, Radtke KP, Scharrer I. Activated protein C-resistance  in patients with lupus anticoagulant. Thromb Haemost 1995; 74:797-8.

0.[1] Vandenbroucke JP, Koster T, Briët E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet 1994; 344: 1453-7.

0.[1] Østerud B, Robertsen R, Åsvang GB, Thijssen F. Resistance to activated protein C is reduced in women using oral contraceptives. Blood Coag Fibrinol 1994; 5:853-4.

0.[1] Hellgren M, Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Am J Obstet Gynecol 1995;173:210-3.

0.[1] Cooper PC, Hampton KK, Makris M, Abuzennadah A, Paul B, Preston FE. Further evidence that activated protein C resistance can be misdiagnosed as inherited functional protein S deficiency. Brit J Haematol 1994; 88: 201-2.

0.[1] Rao AK, Elliot D, Stadnicki A, De-La-Cadena R, Sherry S. High levels of plasma factor VIIIc impair anticoagulant response to activated protein C. Thromb Haemost 1995; 7:1126.

0.[1] Colluci M, Ciavarella N, Giliberti MG, Semeraro N. Resistance to activated protein C (APC) influence of factor V levels. Thromb Haemost 1994; 72: 880-6.

0.[1] Ridker PM, Hennekes CH, Lindpaintner K, Stampfer MJ, Eisenberg PR, Miletich JP.     Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, strokestorke and venous thromboembolism in apparently healthy men. N Engl J Med 1995; 332:912-7.

0.[1] Emmerich J, Poirier O, Evans A, Marques-Vidal P, Arveiler D, Luc G, Aiach M, Cambien F. Myocardial infarction, Arg 506 to Gln factor V mutation, and activated protein C resistance. The Lancet 1995; 345: 321.

0.[1] Marz W, Seydewitz H, Winkelmann B, Chen M, Nauck M, Witt I. Mutation in coagulation factor V associated with resistanceresitance to activated protein C in patients with coronarycoronaty artery disease. The Lancet 1995; 345:526-7.

0.[1] Samani NJ, Lodwick D, Martin D, Kimber P. Resistance to activatedactivaated protein C and risk of premature myocardial infarction. The Lancet 1994; 344: 1709-10.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 


[1] Dahlbäck B, Hildebrand B. Inherited resistance to activated protein C is corrected by anticoagulant      cofactor     activity found to be a property of factor V. Proc Natl Acad Sci USA 1994; 91: 1396-400.

[2]  Dahlbäck B, Carlsson M, Svensson PJ. Familial thrombophilia due to previously unrecognised mechanism characterised by poor anticoagulant response to activated protein C: Prediction of a cofactor to activated protein C. Proc Natl Acad Sci USA 1993; 90: 1004-1008.

[3] Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thrombosis. N Engl J Med 1994; 330: 517-522.

[4] Bertina RM, Koeleman BPC, Koster T. Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994; 369: 64-67.

[5] Greengard JS, Sun X, Xu X, Fernandez JA, Griffin JH, Evatt B. Activated protein C resistance caused by Arg 506 Gln mutation in factor Va. Lancet 1994; 343: 1361-1362.

[6] Dahlbäck B, Shen Lei. Factor V and protein S as synergistic Cofactors to activated protein C in degradation of factor VIIIa. Journal Biol Chem. 1994; 269: 18735-18738.

[7] Griffin JH, Evatt B, Wideman C, FernandezJA, Anticoagulant protein C pathway defective in majority of thrombophilia patients. Blood 1993; 82: 1989-1993.

[8] Lindblad B, Svensson PJ, Dahlbäck B. Arterial and venous thromboembolism with fatal outcome and resistance to activated protein C. Lancet 1994; 343: 917.

[9] Dahlbäck B. Inherited thrombophilia : Resistance to activated protein C as a pathogenic factor for venous thromboembolism. Blood 1995; 85: 607-614.

[10] Koster T,  Rosendaal FR, de Ronde H, Briët E, Vandenbroucke JP, Bertina RM. Venous thrombosis due to poor anticoagulant response to activated protein C: Leiden thrombophilia study. Lancet 1994; 342: 1503-1506.

 

[11] Sun X, Evatt B, Griffin JH. Blood coagulation factor Va abnormality associate with resistance to activated protein C in venous thrombophilia. Blood 1994; 83: 3120-3125.

[12] Koelman BPC, Reitsma PH, Allaart CF, Bertina RM. Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficiency families. Blood 1994; 84: 1031-1035.

[13] Zöller B, Dahlbäck B. Linkage between inherited resistance to activated protein C and factor V gene mutation in vevous thrombosis. Lancet 1994; 343: 1536-1538

[14] Rosendaal FR, Koster T, Vandenbroucke JP, Reitsma PH. HIgh risk of thrombosis in patients homozygous for factor V Leiden (activated protein C resistance). Blood 1995; 85: 1504-1508.

[15] Rees DC, Cox M, Clegg JB. World distribution of factor V Leiden. Lancet 1995; 346: 1133-1134.

[16] Rosén S, Johansson K, Lindberg K, Dahlbäck B. Multicenter evaluation of a kit for activated protein C resistance on various coagulation instruments using plasma from healthy individuals. Thromb Haemost 1994; 72: 255-260.

[17] Kraus M, Wagner C. Evaluation of APC-sensitivity in normal blood donors using different reagents and instrumens. Thromb Res 1994; 76: 231-236.

[18] Leroy-Matheron C, Levent M, Pignon JM, Mendonça, Gouault-Heilmann M. The 1691 G- A Mutation in the Factor V Gene : Relationship to Activated Protein C (APC) Resistance and Thrombosis in 65 Patients. Thromb Haem 96; 75:4-10.

[19] Zöller B, Svensson PJ, Xuhua H, Dahlbäck B. Identification of the same factor V gene mutation in 47 out of 50 thrombosis-prone families with inherited resistance to activated protein C. J Clin Invest 1994; 94: 2521-2524.

[20] Trossaërt M, Conard J, Horellou MH, Samama MM, Ireland H, Bayston TA, Lane DA. Modified APC resistance assay for patients on oral anticoagulants. The Lancet 1994; 344: 1709.

[21] Vasse M, Leduc O, Borg JY, Chrétien MH, Monconduit M. Resistance to activated protein C: evaluation of three functional assays. Thromb Res 1994; 76: 47-59.

[22] Zöller B, Holm J, Svensson PJ, Dahlbäck B. Elevated levels of prothrombin activation fragment 1+2 in plasma from patients with heterozygous Arg 506 to Gln mutation in the factor V gene (APC-Resistance) and/or inherited protein S deficiency. Thhromb Haem 1996; 75: 270-274.

[23] Freyburger G, Bilhou-Nabera C, Dief S, Javorschi S, Labrouche S, Lerebeller MJ, Boisseau MR. Technical and Biological Conditions Influencing the Functional APC Resistance Test. Thromb Haem 96; 75:460-5.

[24] Ehrenforth S, Radtke KP, Scharrer I. Activated protein C-resistance  in patients with lupus anticoagulant. Thromb Haemost 1995; 74:797-8.

[25] Vandenbroucke JP, Koster T, Briët E, Reitsma PH, Bertina RM, Rosendaal FR. Increased risk of venous thrombosis in oral-contraceptive users who are carriers of factor V Leiden mutation. Lancet 1994; 344: 1453-1457.

[26] Østerud B, Robertsen R, Åsvang GB, Thijssen F. Resistance to activated protein C is reduced in women using oral contraceptives. Blood Coag Fibrinol 1994; 5:853-854.

[27] Hellgren M, Svensson PJ, Dahlbäck B. Resistance to activated protein C as a basis for venous thromboembolism associated with pregnancy and oral contraceptives. Am J Obstet Gynecol 1995;173:210-213.

[28] Cooper PC, Hampton KK, Makris M, Abuzennadah A, Paul B, Preston FE. Further evidence that activated protein C resistance can be misdiagnosed as inherited functional protein S deficiency. Brit J Haematol 1994; 88: 201-2.

[29] Rao AK, Elliot D, Stadnicki A, De-La-Cadena R, Sherry S. High levels of plasma factor VIIIc impair anticoagulant response to activated protein C. Thromb Haemost 1995; 7:1126.

[30] Colluci M, Ciavarella N, Giliberti MG, Semeraro N. Resistance to activated protein C (APC) influence of factor V levels. Thromb Haemost 1994; 72: 880-6.

[31] Ridker PM, Hennekes CH, Lindpaintner K, Stampfer MJ, Eisenberg PR, Miletich JP.     Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, storke and venous thromboembolism in apparently healthy men. N Engl J Med 1995; 332:912-917.

[32] Emmerich J, Poirier O, Evans A, Marques-Vidal P, Arveiler D, Luc G, Aiach M, Cambien F. Myocardial infarction, Arg 506 to Gln factor V mutation, and activated protein C resistance. The Lancet 1995; 345: 321.

[33] Marz W, Seydewitz H, Winkelmann B, Chen M, Nauck M, Witt I. Mutation in coagulation factor V associated with resitance to activated protein C in patients with coronaty artery disease. The Lancet 1995; 345:526-527.

[34] Samani NJ, Lodwick D, Martin D, Kimber P. Resistance to activaated protein C and risk of premature myocardial infarction. The Lancet 1994; 344: 1709-1710.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

The field of familial thrombophilia has been dramatically changed when Dahlback discovered the poor anticoagulant response to the action of activated protein C as a new cause for familiar thrombophilia and Bertina showed that this phenotype is due to a mutation in the gene of factor V, the factor V Leiden mutation.(1,2,3,4,5)