[Screening for thrombophilia]. / Labormedizinische Aspekte des Thrombophilie-Screenings.

For thrombophilia should be screened, if a first thromboembolic event has occurred at the age of less than 45 years, if family history indicates thrombophilia, or in the case of a special clinical condition. To get valid diagnostic results a few pecularities concerning the physiology of coagulation and laboratory medicine have to be considered. This review gives an overview of pitfalls and options during the preanalytical phase of thrombophilia diagnostics. Furthermore, the laboratory assays to perform a screening are highlighted.

[The Janus face of coagulation factors. High levels, high thromboembolic risk?]. / Das Janusgesicht der Einzelfaktoren. Hohe Spiegel, hohes thromboembolisches Risiko?

Bleeding tendency as a result of clotting factor deficiency is common knowledge. The counterpart, i.e. thrombophilia due to high clotting factor levels, is not surprising, but an association between factor level and thrombosis risk has just recently been described. The role of high factor VIII (FVIII) levels is well documented. The risk of high FVIII levels for the first event is similarly high as that of APC resistance. There is a familiar background of high FVIII levels. Alterations within the FVIII or the von Willebrand factor genes seem to be not causal since polymorphisms of these genes are not associated with venous thromboembolism. An increased FVIII/VWF ratio indicates a reduced FVIII clearance. Probably, the low-density lipoprotein receptor-related protein, i.e. the receptor mediating the hepatic clearance of the FVIII-VWF-complex, is involved. The well known prothrombin G20210A polymorphism is associated with high prothrombin levels perhaps contributing to thrombosis risk via clot resistance against fibrinolysis.

Genetic risk factors in young adults with 'cryptogenic' ischemic cerebrovascular disease.

Mutations such as factor V Leiden G1691A (FVL), prothrombin G20210A (FIIM), methylenetetrahydrofolate reductase (MTHFR) C677T, cystathionine beta-synthase (CBS) 844ins68 and endothelial cell protein C receptor (EPCR) 4031ins23 are risk factors for thromboembolism. To assess the role of these mutations in young adults with cerebral ischemia of otherwise undetermined etiology, 93 patients younger than 50 years old with thromboembolic strokes or transient ischemic attacks were studied. One hundred and eighty-six healthy age-matched and sex-matched blood donors served as controls. The FVL mutation was detected in 15/93 patients and 13/186 controls. After adjustment for smoking, arterial hypertension, and hyperlipidemia, the association of the FVL mutation with cerebral ischemia [odds ratio (OR), 3.19; 95% confidence interval (CI), 1.38-7.39] remained significant. One of 93 patients and 6/186 controls were carriers of FIIM (OR, 0.33; 95% CI, 0.04-2.75). We detected the MTHFR TT677 genotype in 9/93 patients and 26/186 controls (OR, 0.66; 95% CI, 0.30-1.47), a CBS 844ins68 mutation in 12/93 patients and 19/186 controls (OR, 1.30; 95% CI, 0.60-2.81), and an EPCR 4031ins23 mutation in 1/93 patients and in no control individual (P = 0.33). In conclusion, in younger adults the FVL mutation is a risk factor for cerebrovascular disease. FIIM, the MTHFR TT677 genotype and the CBS 844ins68 mutation did not contribute to the risk in this group of patients. The EPCR 4031ins23 mutation is very rare, its possible role needs further investigation.

The impact of dalteparin (Fragmin) on thrombin generation in pregnant women with venous thromboembolism: significance of the factor V Leiden mutation.

Hypercoagulability is observed in patients with inherited thrombophilia, e.g. factor V Leiden (FVL) mutation. Pregnancy represents a hypercoagulable state as well. This study addresses the effects of the FVL mutation on haemostatic activation during pregnancy as indicated by prothrombin fragments (F1+2). 233 pregnant women with no history of venous thromboembolism were studied. Additionally, two patient groups (25 pregnant FVL carriers and 36 pregnant women without thrombophilic diathesis) in whom low molecular weight heparin (dalteparin) was used prophylactically against rethrombosis were investigated. None of the women developed clinical signs of venous thromboembolism during pregnancy or after delivery. Untreated women exhibited substantial hypercoagulability. F1+2 levels were similar in FVL carriers and non-carriers (difference n. s.). After sufficient adjustment for anti-factor Xa activity (> or =0.15; < or =0.4 U/mL), heparinized women without any thrombophilic diathesis had significantly lower levels of F1+2 than untreated pregnant women. This was evident only in the first and second trimenon (p <0.001). F1+2 levels in heparinized FVL carriers were quite similar to the levels observed in untreated pregnant women, however. In conclusion, our data support the thesis that in comparison to asymptomatic patients, thrombin generation is exaggerated in symptomatic FVL carriers. Coagulation activation during pregnancy can be reduced by dalteparin.

Familial clustering of high factor VIII levels in patients with venous thromboembolism.

High levels of factor VIII (FVIII) but not von Willebrand factor (vWF) are known to increase the risk for venous thromboembolism. Whether high FVIII levels originate from hereditary defects or from acquired conditions remains unanswered. The objective of our study was to investigate whether there is evidence for familial clustering of elevated FVIII levels in families in which >/=1 member has been affected by a thromboembolic event and had reproducibly high FVIII levels. We investigated FVIII levels in 361 patients with previous venous thromboembolism. FVIII levels were measured by a chromogenic assay; the cutoff value was defined as the 98th percentile of FVIII plasma levels of 266 blood donors. vWF levels were determined by an enzyme immunoassay. After exclusion of known causes of FVIII elevation, such as the acute thrombotic event itself; inflammation; malignancy; liver, renal, or vascular disease; surgery; or pregnancy, we included 17 patients with unexplained, reproducibly high FVIII levels. The investigation was also extended to these patients' relatives. Multiple regressive analysis of blood donors and asymptomatic family members showed that the affiliation with a family in which 1 member suffered from venous thromboembolism and had reproducibly high FVIII levels is the second most important predictor for FVIII levels. Familial clustering was analyzed by the Houwing-Duistermaat familial aggregation test. After adjustment for the influence of age, sex, blood group, and vWF, FVIII levels were significantly (P:=0.038) clustered within families. In conclusion, FVIII levels seem to be familially determined in families in which a member showed high FVIII levels after previous venous thromboembolism.

Transient alkaline hyperphosphatasaemia in an adult: biochemical peculiarities.

We report on a 27-year-old healthy female with transient hyperphosphatasaemia of adulthood (it is the eighth case ever recorded). A maximum alkaline phosphatase activity of 1950 U/l, 11-fold the upper reference limit, was measured. The activity normalized within 11 weeks. Electrophoresis revealed the typical pattern for alkaline phosphatase isoenzymes observed in transient hyperphosphatasaemia of infancy: a fast-migrating liver isoenzyme and a bone isoenzyme. Contrary to the findings in transient hyperphosphatasaemia of infancy the liver isoenzyme did not precipitate with wheat-germ lectin whereas the bone isoenzyme partially bound to lectin. Biochemical features of transient hyperphosphatasaemia in an adult may be different from those in infancy. Recognition of an atypical pattern could help avoid unnecessary extensive investigations.

Selective screening for the Factor V Leiden mutation: is it advisable prior to the prescription of oral contraceptives?

The cumulative thrombotic risk of Factor V (FV) Leiden and oral contraceptives (OC) recommends screening for the mutation. Assuming that a family history of thrombosis increases the patient's likelihood of bearing FV Leiden, a selective rather than universal screening would be performed. We studied the utility of a family history of thrombosis for screening of FV Leiden before prescription of OC and, furthermore, the utility of screening even if oral contraception is favoured. 101 patients who had their first and single thromboembolic event while using OC were interviewed. 609 women without any history of thromboembolism recruited by gynecologists completed a standard questionnaire. 101 of these women, age-matched and currently using OC, were selected for a case-control study. Regarding patients with previous thromboembolism, a family history in a first-degree relative had a positive predictive value (PPV) of only 14% for FV Leiden. A PPV of 12% was calculated by investigating the 609 thrombosis-free women. Inherited FV Leiden (odds ratio = 4.9) and acquired risk factors (odds ratio = 10.1) were both found to be the most prominent, but independent additional risks. Nevertheless, FV Leiden carriers, both heterozygotes and homozygotes, did not suffer earlier from thromboembolism than patients without the mutation. In conclusion, family history is an unreliable criterion to detect FV Leiden carriers. Screening for factor V Leiden can be worthwhile even if the advantages of oral contraception are higher assessed than the thrombotic risk. Affected women knowing about their additional risk could contribute to the prevention of thrombosis in risk situations.

PIP: The cumulative thrombotic risk of Factor V Leiden (FVL) and oral contraceptive (OC) use raises the possibility of either selective or universal screening for this mutation before OCs are prescribed. Family history of venous thromboembolism as a criterion to detect FVL carriers was evaluated in a case-control study of 101 women from Bavaria, Germany, who had their first and single thromboembolic event while using OCs and 101 healthy age-matched OC users. A questionnaire was administered to a broader group of 609 OC users without a history of thromboembolism. Analysis of the 609 women revealed a 7.4% prevalence of FVL, but no association between this mutation and a family history of thromboembolism. Among women with a previous thromboembolism, a family history in a first-degree relative had a positive predictive value of only 14% for FVL. The sensitivity of family history was under 50%. 35% of cases compared with 8% of controls carried the FVL mutation. The most significant independent risk factors of thromboembolism were inherited FVL (odds ratio, 4.9) and acquired risk factors--i.e., surgery, leg fractures, distortions, confinement to bed for more than 1 week, or a restricted sitting position more than 6 hours in the 4 weeks before the index date (odds ratio, 10.1). Both heterozygote and homozygote FVL carriers did not suffer earlier from thromboembolism than patients without the mutation. These findings indicate that family history is not an effective predictor of FVL. However, even if the advantages of OC use are greater than the thrombotic risk, screening for FVL may be indicated to permit high-risk women to take preventive action.

Characterization of myeloma cells by means of labeling index, bone marrow histology, and serum beta 2-microglobulin.

The relationship between two cellular prognostic parameters of multiple myeloma, the plasma cell labeling index (LI%) and bone marrow histology was studied. The LI% as the percentage of monoclonal plasma cells in the S-phase was measured by bromodeoxyuridine incorporation using the anti-bromodeoxyuridine antibody BU-1. The histologic classification was based on six plasma cell types that allow prognostic grading as the Marschalko, small cell, cleaved, polymorphous, asynchronous, and blastic types. The biopsies also were used for estimating the degree of infiltration. Beta 2-microglobulin (IMx assay, Abbott, North Chicago, IL), the most significant serum parameter for myeloma also was measured for comparison. Bone marrow specimens and sera were obtained from 50 myeloma patients. Fourteen patients with smoldering myeloma were characterized by well-differentiated Marschalko or small cell type cells, a low LI% and a low beta 2-microglobulin concentration. Considering all myeloma patients, plasma cell type and degree of infiltration showed a significant correlation with LI% and beta 2-microglobulin concentration. Patients with plasma cells that were not mature cells of the Marschalko or small cell type revealed a high LI% or high beta 2-microglobulin level in 16 of 19 cases. However, a high LI% or high beta 2-microglobulin level could be detected in only 8 of 31 patients with plasma cells of the Marschalko or small cell type. Three of 21 stage I patients did not show the typical finding of a low LI%, low beta 2-microglobulin level, and a favorable grade. Stage II patients were not uniformly characterized by LI%, beta 2-microglobulin and plasma cell morphology. The percentage of nucleolated plasma cells was not associated with the LI%. Bone marrow histology, LI%, and beta 2-microglobulin concentration appear to be supplementary prognostic factors. If LI% and beta 2-microglobulin levels are not measured, a higher risk could be overlooked in cases with mature plasma cells.

Methodological and clinical comparison of the ACS prostate-specific antigen assay and the Tandem-E prostate-specific antigen assay in prostate cancer.

OBJECTIVES: The new ACS prostate-specific antigen (PSA) assay was methodologically and clinically compared with the established Tandem-E PSA assay. We intended to find a possible advantage in primary diagnosis and monitoring the recurrence of prostate cancer due to the additional better recognition of the free PSA form by the ACS PSA assay. METHODS: sera of 51 healthy men, 127 patients with hyperplasia, and 82 untreated patients with prostate cancer were analyzed by means of the Tandem-E PSA assay (Hybritech) and the ACS PSA assay (Ciba Corning). Follow-up was done on 12 cancer patients with recurrences. RESULTS: Both assays correlated very well (r = .98 for all studied men or hyperplasia patients or cancer patients). However, both assays did not yield comparable values: The ACS assay was characterized by nearly doubled values compared with the Tandem-E assay. At 95% specificity versus patients with benign hyperplasia, cutoff values were obtained as follows: 28.8 ng/mL for the ACS PSA assay and 15.2 ng/mL for the Tandem-E assay. At 95% specificity versus hyperplasia patients, we calculated sensitivities of 60% (ACS PSA) and 63% (Tandem-E PSA). Our longitudinal study revealed more prominent slopes for the ACS assay in patients with recurrent cancer disease. However, using either the ACS assay or the Tandem-E assay, the PSA increase started at the same time. In 1 patient, the increasing ACS PSA accelerated 3 months earlier than the increasing Tandem-E PSA did. CONCLUSIONS: The ACS assay has obviously higher PSA levels. The clinician is not familiar with such high PSA levels. The specificity-sensitivity profile nonetheless remains unchanged. If the PSA concentration is measured by the ACS assay, patients who relapse will reveal a more rapid PSA increase. Then, recurrent cancer disease may be detected earlier in some cases.

Evaluation of the COBAS CORE Immunoassay for measuring prostate-specific antigen (PSA)--multi-centre study results. The PSA Study Group.

Hoffmann-La Roche has introduced a fully automated COBAS CORE EIA for the measurement of prostate specific antigen (PSA). A regular and an ultrasensitive version of the assay are available. Both versions of the COBAS CORE PSA EIA were compared with the established IMx PSA assay from Abbott. Sera from 98 apparently healthy males, 224 patients with benign prostate hyperplasia, 17 patients with prostatitis and 111 patients with prostate cancer were determined using the COBAS CORE PSA EIA in comparison with the IMx PSA assay. The sera were drawn before treatment. Sera from 26 patients were also monitored through follow-up testing. The COBAS CORE analyser allows rapid analysis of large series of samples. Intra-assay imprecision (CV) was between 1.7% and 4.9% (IMx PSA: between 2.4% and 2.7%). The coefficient of variation for inter-assay imprecision was between 3.4% and 6.5% (IMx PSA: between 3.2% and 3.3%). The analytical detection limit was determined as 0.2 microgram/l for the regular COBAS CORE PSA EIA and 0.05 microgram/l for the ultrasensitive version. A biological detection limit of 0.1 microgram/l was determined for the ultrasensitive version. Results obtained using the COBAS CORE PSA EIA and IMx PSA assays were in excellent correlation: coefficient of correlation r = 0.99 and slope = 0.92, using prostate-specific antigen values from the complete study. Only in the measuring range below 10 micrograms/l did the coefficients of correlation vary between 0.82 and 0.93.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma cell proliferation in monoclonal gammopathies: measurement using BU-1 antibody in flow cytometry and microscopy: comparison with serum thymidine kinase.

AIMS: The labelling index as defined by the percentage of bone marrow plasma cells doubling their DNA in the S phase is a useful prognostic factor in multiple myeloma. The aim of this study was to examine the specificity and sensitivity of a new flow cytometric method for measuring the labelling index. METHODS: Bone marrow specimens from five patients with monoclonal gammopathy of undetermined significance and 61 patients with multiple myeloma were investigated. The labelling index (LI%) was determined by means of a microscopic and flow cytometric method using the antibromodeoxyuridine antibody BU-1. Serum thymidine kinase, another index of proliferation, was measured by radioimmunoassay. RESULTS: Good comparability (r = 0.83) and nearly equal imprecision (CV < 20%) were found with microscopic and flow cytometric methods of LI% measurement. However, 1000 or more cells had to be counted by microscopy around the cutoff value to avoid an unacceptable imprecision. Plasma cells with increased S phase (LI% > 1%) were characterised by their reduced light chain fluorescence intensity ratio between plasma cells and nonspecifically stained cells (7.9 v 14.8, p < 0.002), that is, by their generally lowered cytoplasmic immunoglobulin content. There was a moderate correlation between thymidine kinase and labelling index (r = 0.56, p < 0.001). At 100% specificity, myelomas with proliferating plasma cells were more sensitively detected by the labelling index than by serum thymidine kinase (55% v 32% sensitivity). CONCLUSIONS: The labelling index represents a more specific and sensitive proliferation marker than serum thymidine kinase. Flow cytometry does not result in greater precision.