Critical Risk Results - An Update on International Initiatives.

Direct communication of significant (often life-threatening) results is a universally acknowledged role of the pathology laboratory, and an important contributor to patient safety. Amongst the findings of a recent survey of 871 laboratories from 30 countries by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM), only 3 tests were noted to be common to 90% of alert lists, and only 48% of laboratories consulted clinicians in developing these alert lists despite ISO15189 recommendations to do so. These findings are similar to previous national surveys demonstrating significant variation worldwide in how critical risk results are managed and also in how these protocols are developed. In order to promote "best practice" and harmonization of critical risk results management, guidelines and recommendations have been published, most recently by Clinical and Laboratory Standards Institute (CLSI) and Australasian Association of Clinical Biochemists (AACB). These statements in particular have placed strong emphasis on patient risk and risk assessment in the management of critical risk results. This focus has resulted in recommendations to adopt new terminology, the consideration of risk assessment when compiling alert tables, consultative involvement of laboratory users in setting up protocols, and the need for outcome-based evidence to support our practices. With time it is expected that emerging evidence and technological improvements will facilitate the advancement of laboratories down this path to harmonization, best practice, and improve patient safety.

Mutational spectrum of smith-lemli-opitz syndrome patients in hungary.

Smith-Lemli-Opitz (SLO) syndrome is an autosomal recessive disorder characterized by multiple congenital abnormalities and mental retardation. The condition is caused by the deficiency of 7-dehydrocholesterol reductase (DHCR7) which catalyzes the final step in cholesterol biosynthesis. Biochemical diagnosis is based on increased concentration of 7-dehydrocholesterol (7-DHC) in the patient serum. Both life expectancy and quality of life are severely affected by the disease. The estimated prevalence of SLO syndrome ranges between 1:20,000 and 1:40,000 among Caucasians. Although the mutational spectrum of the disease is wide, approximately 10 mutations are responsible for more than 80% of the cases. These mutations show a large interethnic variability. There are no mutation distribution data from Hungary to date. Thirteen patients were diagnosed with SLO syndrome in our laboratory. As first-line tests, serum 7-DHC and total cholesterol were measured and, in positive cases, molecular genetic analysis of the DHCR7 gene was performed. Complete genetic background of the disease could be identified in 12 cases. In 1 case only 1 mutation was detected in a heterozygote form. One patient was homozygous for the common splice site mutation c.964-1G>C, while all other patients were compound heterozygotes. One novel missense mutation, c.374A>G (p.Tyr125Cys) was identified.

The most severe forms of Perthes' disease associated with the homozygous Factor V Leiden mutation.

It has recently been postulated that thrombophilia may have a role in the aetiology of Perthes' disease. The published reports, however, remain conflicting. In this study a retrospective analysis of the coagulation parameters was made in 47 patients with Perthes' disease and the results compared with the clinical data. Five patients with Factor V Leiden mutation were found (10.6%) and surprisingly four of them had a homozygous pattern. These four patients showed the most severe form of the disease, Catterall group IV, with flattening of the entire epiphysis, involvement of the metaphysis, shortening and broadening of the femoral neck, trochanteric overgrowth and developed mushroom-shaped aspherical laterally displaced femoral heads in dysplastic acetabula. We would like to suggest that the homozygous form of Factor V Leiden mutation has some role in the clinical course of Perthes' disease and particularly its most severe form.

Anti-factor V auto-antibody in the plasma and platelets of a patient with repeated gastrointestinal bleeding.

Development of autoantibody against coagulation factor V (FV) is a rare clinical condition with hemorrhagic complications of varying severity. The aim of this study was to establish the pathomechanism of an acquired FV deficiency and characterize the FV inhibitor responsible for the clinical symptoms. A 78-year-old female was admitted to hospital with severe gastrointestinal bleeding. General clotting tests and determination of clotting factors were performed by standard methods. FV antigen and FV containing immune complexes were measured by ELISA. The FV molecule was investigated by Western blotting and by sequencing the f5 gene. The binding of patient's IgG to FV and activated FV (FVa) was demonstrated in an ELISA system and its effect on the procoagulant activity of FVa was tested in clotting tests and in a chromogenic prothrombinase assay. Localization of the epitope for the antibody was performed by blocking ELISA. FV activity was severely suppressed both in plasma and platelets. FV antigen levels were normal by ELISA using polyclonal anti-FV antibody or monoclonal antibody against the connecting region of FV, but depressed when HV1 monoclonal antibody against the C2 domain in the FV light-chain was used as capture antibody. The FV molecule was found intact. An IgG reacting with both FV and FVa was present in the patient's plasma and its binding to FV was inhibited by HV1 antibody. FV-containing immune complexes were detected in the patient's plasma and platelet lysate. The patient's IgG inhibited the procoagulant function of FVa. An anti-FV IgG was present in the patient's plasma and platelets. The autoantibody reacted with an epitope in the C2 domain of FV light chain and neutralized the procoagulant function of FVa.

Severe coagulation factor V deficiency caused by 2 novel frameshift mutations: 2952delT in exon 13 and 5493insG in exon 16 of factor 5 gene.

A male infant with severe bleeding tendency had undetectable factor V activity. Sequence analysis of the proband's DNA revealed one base deletion in exon 13 (2952delT) and one base insertion in exon 16 (5493insG) in heterozygous form. Both mutations introduced a frameshift and a premature stop at codons 930 and 1776, respectively. The proband's father and mother were heterozygous for 2952delT and for 5493insG, respectively. Both mutations would result in the synthesis of truncated proteins lacking complete light chain or its C-terminal part. In the patient's plasma, no factor V light chain was detected by enzyme-linked immunosorbent assay. The N-terminal portion of factor V containing the heavy chain, and the connecting B domain was severely reduced but detectable (1.7%). A small amount of truncated factor V-specific protein with a molecular weight ratio of 236 kd could be immunoprecipitated from the plasma and detected by Western blotting. This protein, factor V(Debrecen), corresponds to the translated product of exon 16 mutant allele.

Natural coagulation inhibitor proteins in young patients with cerebral ischemia.

Disturbances of coagulation and fibrinolytic pathways were studied in 53 young patients with cerebral ischemia. Upon admission 26 of 53 patients had abnormality in at least one of the antithrombin-III, protein C, protein S activities or in activated protein C (APC) ratios. Three months after the first examination the majority of the previously detected abnormalities returned to normal values and the most frequent alterations were decrease in protein S activity (3 patients) and APC resistance (3 patients). Conditions resulting in impaired fibrinolysis were frequently detected upon admission. Elevation of plasminogen activator inhibitor-1, lipoprotein (a), and alpha-2-antiplasmin was present in 23, 10, and 4 cases, respectively. It is concluded that abnormalities of coagulation as well as of the fibrinolytic systems are prevalent in the acute phase of cerebral ischemia, however, the results may be significantly influenced by the disease process or the acute phase effect.

Enzyme-linked immunosorbent assay for the determination of blood coagulation factor XIII A-subunit in plasma and in cell lysates.

A new one-step ELISA was developed for the determination of the concentration of blood coagulation factor XIII subunit A (FXIII-A) in plasma and in cell lysates. Monoclonal antibodies directed against different epitopes on FXIII-A were used for the assay. The capture antibody was biotinylated on its carbohydrate moiety and the detection antibody was labelled with horseradish peroxidase. The antigen-antibody reaction was carried out in the well of a streptavidin-coated microplate. Complex formation with FXIII subunit B (FXIII-B) and association to fibrinogen did not influence the accessibility of the antibodies to FXIII-A. The method could be performed within 2 h and demonstrated good reproducibility, recovery and sensitivity. Plasma samples could be assayed after storage at -20 degrees C for at least 6 months. However, in the case of platelet lysates freezing and rethawing resulted in a significant loss of FXIII-A. FXIII-A concentrations measured in the plasma samples of healthy individuals and patients correlated well with the concentrations of complexed plasma FXIII (A2B2) and with the results of FXIII activity measurements. A reference range of 46-82 fg/platelet was established for platelet FXIII-A.

Factor V Leiden as a risk factor for miscarriage and reduced fertility.

The Leiden mutation is a recent discovery. It is the main cause of inherited thrombophilia and has been found in 20-60% of deep vein thrombosis cases. More recently it has been found in a significant number of cases of obstetric complications attributable to placental thrombosis. Current patient management practice for dealing with the Leiden mutation is based mainly on information about deep vein thrombosis because there is little information on pregnancy complications. There are no prospective studies examining the risk of developing pregnancy complications for Leiden mutation carriers. The aim of this study is to do that by comparing the frequency of unfavourable pregnancy outcomes among carriers with those among controls. The number of women developing miscarriages, intrauterine deaths, or infertility problems among 128 Leiden mutation carriers was compared with the number among 461 controls. The risk of having at least one miscarriage or infertility problems was 1.5 times greater for Leiden mutation carriers than controls. This result was statistically significant (95% CI 1.2, 2.7). The risk of having at least two miscarriages or infertility problems was 2.5 times greater for Leiden mutation carriers than controls. This was also statistically significant (95% CI 1.2, 5.13).

Hypercoagulability in various autoimmune diseases: no association with factor V Leiden mutation.

The coagulation factor V Leiden mutation, leading to resistance to activated protein C (APC), is the most common inherited risk factor for venous thrombosis. In various systemic autoimmune diseases the hypercoagulable state was shown to be associated with the presence of antiphospholipid antibodies (aPL). Our aim was to determine the prevalence of both, Leiden mutation and aPL in autoimmune diseases and their impact on the occurrence of venous thrombosis. The dataset consists of results from 137 patients having Sjögren's syndrome (n = 50), progressive systemic sclerosis (n = 43) (PSS), undifferentiated connective tissue disease (n = 24) (UCTD) and mixed connective tissue disease (n = 20) (MCTD) with or without venous thromboembolic complications. The Leiden mutation was detected with polymerase chain reaction (PCR), aPL, such as lupus anticoagulant (LA) with screening and confirmatory procedures and others with enzyme linked immunosorbent assay (ELISA). The prevalence of mutation ranged between 8.3% and 18.0% (13.1%). The thromboembolic risk was found to be increased in the presence of aPL. Eight patients (5.84%) (4 heterozygous) experienced thromboembolic events and 3 out of 4 heterozygous showed aPL positivity, too. There were no difference between the frequencies of Leiden mutation in examined systemic autoimmune diseases and unselected populations.

The Leiden mutation of coagulation factor V in Hungarian SLE patients.

We studied the prevalence and the effect of coagulation factor V Leiden mutation on the occurrence of thrombotic episodes in 120 Hungarian patients having systemic lupus erythematosus (SLE) with or without antiphospholipid antibody. The frequency of the factor V Leiden mutation in Hungarian SLE patients was 13%, which is comparable with those found previously in a healthy Caucasian population. The incidence of venous thrombosis among factor V Leiden carriers has been found to be higher (odds ratio [OR] 1.7) than it is in patients without Leiden mutation (38% vs 29%). In addition, the frequency of venous thrombosis in the heterozygous SLE patients (OR 8.4 [confidence interval (CI) 0.8-83.9] P = 0.06) is dependent on the coexistence of other risk factors, such as antiphospholipid antibody. Moreover, among heterozygous factor V SLE patients, the Leiden mutation could explain the tendency to have significantly higher prevalence of fetal losses (OR 3.9 [CI 1.2-12.0] P = 0.02) and higher prevalence of cerebrovascular lesions, cardiac valvular abnormalities, and Raynaud's syndrome than that found in individuals without factor V Leiden mutation of those having antiphospholipid antibody. Systemic lupus erythematosus patients with combined defects suffer more severely from thrombosis than those with a single risk factor do, suggesting that thrombophilia is a multifactorial disorder in SLE, also. Although, the factor V Leiden mutation does not seem to be a significant risk factor for venous thrombosis in SLE, these data demonstrate that Leiden mutation can be regarded as an additive thrombogenic factor providing higher predisposition to several vasoocclusive disorders in SLE.

[Hemostatic abnormalities in ischemic stroke]. / Haemostasis eltérések ischaemiás stroke-ban.

The authors studied whether haemostatic abnormalities connected with the development of cerebral circulatory disturbances can be demonstrated in young stroke patients in whom Doppler and angiographic examination failed to reveal deviations indicative of stroke. They determined the in vivo activation of the coagulation system (TAT, F 1 + 2), the degree of secondary fibrinolysis (D-dimer), the plasma levels of the markers of fibrinolysis, with special regard to inhibitors: plasminogen activator inhibitor (PAI-1), alpha 2 antiplasmin (alpha 2 AP), alpha 2 macroglobulin (alpha 2 M), the frequency of pathologic serum lipoprotein (a)-Lp(a)-values and the association of PAI-1 and Lp(a) with the fibrinolytic system. They conclude that in the acute phase of the disease, the TAT and F 1 + 2 values were significantly elevated compared to the control, without change in the D-dimer value. The results suggest that in the tested period increased thrombin generation dominated and it significantly surpassed plasmin activity since the D-dimer values of that period did not indicate substantial increase in secondary fibrinolysis. The results of the study were separately analyzed in acute, chronic TIA and stroke groups. In the TIA and acute group the F 1 + 2 values, while in stroke the TAT values were more elevated. The in vitro fibrinolytic capacity of the patients significantly decreased compared to controls, showing significant correlation with the Lp(a) level, but not with the PAI value. Examination of the marker molecules renders possible to assess the degree of hypercoaguability and of endogenous lysis. Their knowledge is held important for judging the progression of the disease and the therapeutic consequences.

[Prevention of thromboembolic side effects of oral contraceptives: methodologic recommendations]. / Orális fogamzásgátlás thromboemboliás szövödményeinek megelözése: módszertani javaslat.

The authors analyse the incidence of thromboembolic complications during combined oral contraception. Among genetic factors predisposing to venous thrombosis and thromboembolic disease, they present, with the help of three case histories, the molecular biology of activated protein-C resistance, the newly discovered and most frequently implicated pathogenetic factor of inherited thrombophilia. Among 350 asymptomatic women requesting prescription for an oral contraceptive they found a 9.1% frequency of heterozygote Leiden-mutation carriers. For the prevention of a rare, however, almost always life-threatening complication the authors recommend activated protein C-resistance analysis to be carried out whenever an oral contraceptive is prescribed for the first time. Positive results will provide life-time aid in avoiding factors which increase the risk of thromboembolic disease. Negative results may help reducing the number of those who, risking an unwanted pregnancy, are hesitant to start on oral contraceptives for their implication in thromboembolic disease.

Induction of apoptosis by retinoids and retinoic acid receptor gamma-selective compounds in mouse thymocytes through a novel apoptosis pathway.

Retinoic acids are morphogenic signaling molecules that are derived from vitamin A and involved in a variety of tissue functions. Two groups of their nuclear receptors have been identified: retinoic acid receptors (RARs) and retinoic acid X receptors (RXRs). All-trans retinoic acid is the high affinity ligand for RARs, and 9-cis retinoic acid also binds to RXRs with high affinity. In cells at high concentrations, all-trans retinoic acid can be converted to 9-cis retinoic acid via unknown mechanisms. It was previously shown that retinoic acids prevents activation-induced death of thymocytes. Here, we report that both all-trans and 9-cis retinoic acid induce apoptosis of mouse thymocytes and purified CD4+CD8+ cells in ex vivo cultures, with 9-cis retinoic acid being 50 times more effective. The induction of apoptosis by retinoic acids is mediated by RARgamma because (a) the phenomenon can be reproduced only by RARgamma-selective retinoic acid analogs, (b) the cell death induced by either retinoic acids or RARgamma analogs can be inhibited by RARgamma-specific antagonists, and (c) CD4+CD8+ thymocytes express RARgamma. In vivo administration of an RARgamma analog resulted in thymus involution with the concomitant activation of the apoptosis-related endonuclease and induction of tissue transglutaminase. The RARgamma pathway of apoptosis is RNA and protein synthesis dependent, affects the CD4+CD8+ double positive thymocytes, and can be inhibited by the addition of either Ca2+ chelators or protease inhibitors. Using various RAR- and RXR-specific analogs and antagonists, it was demonstrated that stimulation of RAR alpha inhibits the RARgamma-specific death pathway (which explains the lack of apoptosis stimulatory effects of all-trans retinoic acid at physiological concentrations) and that costimulation of the RXR receptors (in the case of 9-cis retinoic acid) can neutralize this inhibitory effect. It is suggested that formation of 9-cis retinoic acid may be a critical element in regulating both the positive selection and the "default cell death pathway" of thymocytes.

[Thrombocyte dysfunction in children with albinism]. / Thrombocytafunkció-zavar albínó gyermekekben.

Seven children with albinism were examined for haemorrhagic diathesis. In 6 patients coagulation screening tests (prothrombin time, activated partial thromboplastin time, thrombin time) were normal, in 1 patient prolongation of activated partial thromboplastin time due to mild factor XI deficiency was found. Platelet counts were in the reference range, however, the template bleeding time of 6 out of 7 patients was prolonged. Evaluation of platelet aggregation and secretion in a lumi-aggregometer demonstrated that only the child with normal bleeding time had normal platelet function, and 6 children with prolonged bleeding time had impaired aggregation and release reaction. Five out of these 6 patients had severe decrease of ATP secretion even when high dose of thrombin was used as agonist. The number of dens granules in the platelets of these patients, as measured by the uptake of mepacrine, was significantly less than in normal controls. These findings clearly suggested storage pool deficiency. In one of these patients storage pool deficiency was associated with mild factor XI deficiency. The high frequency of haemorrhagic diathesis in albino children emphasizes, the importance of the screening of patients with albinism for bleeding diathesis.

Efficacy of prevention of thromboembolic complications with LMW-heparin in experiment.

The authors used an antithrombotic agent (Nadroparin Calcium) with anti-Xa effect in their experiments to prevent thromboembolic complications in the model of endoprosthetic replacement of the hip joint in mongrel dogs. 10 experimental animals (Group I.) were given doses of 100 A Xa ICU/kg/bwt of Nadroparin Calcium subcutaneously 4 hours prior to the operation and also once a day until the 3rd postoperative day; between the 4th and 10th postoperative days doses of 150 A Xa ICU/kg/bwt Nadroparin Calcium were given. The 10 control animals (Group II.) did not receive anticoagulant treatment. In both groups platelet count, activated partial thromboplastin times (APTT), prothrombin and fibrinogen levels as well as activated factor X inhibition (F Xa) were measured prior to surgery and also until the 14th postoperative day. No changes in APTT and prothrombin levels were detected during the experiment, however platelet count and fibrinogen levels as well as the extent of F Xa inhibition showed significant and different changes in groups I. and II. The Group I. which had received thromboembolic prophylaxis did not develop deep venous thrombosis or pulmonary embolism, but the control group did. Based on their investigations, the authors concluded that they had been able to achieve F Xa inhibition by giving the above mentioned doses of Nadroparin Calcium which was enough to prevent thromboembolic complications in their model experiment of implanting hip endoprosthesis.