Lack of rivaroxaban influence on a prothrombinase-based assay for the detection of activated C protein resistance: an Italian ex vivo and in vitro study in normal subjects and factor V Leiden carriers.

INTRODUCTION: Activated protein C resistance (APCr) leads to hypercoagulability and is due, often but not exclusively, to Factor V Leiden (FVL). The aim of this study was to assess the ex vivo and in vitro interference of the direct factor Xa inhibitor rivaroxaban (RIV) on a prothrombinase-based assay for APCr detection. METHODS: An ex vivo study was performed on fresh plasma samples obtained from 44 subjects with FV wild-type and seven with FVL heterozygous, all treated with RIV. An in vitro study was performed on 15 plasma samples (six from normal subjects, six from heterozygous, and three from homozygous FVL carriers, all frozen specimens) spiked with RIV. RIV concentration was evaluated using a chromogenic assay, and APCr was evaluated by a prothrombinase-based assay. RESULTS: No significant interference of RIV on APCr results obtained by a prothrombinase-based assay was observed for drug concentrations up to 400 ng/mL in FV wild-type and FVL carriers (homozygous and heterozygous). These results were confirmed both ex vivo and in vitro. CONCLUSIONS: RIV did not significantly interfere with the prothrombinase-based assay used for the assessment of APCr, and this was observed to occur independently of FV status. However, only concentrations up to 400 ng/mL were tested and, therefore, what occurs in the presence of higher doses remains to be investigated.

Polyendocrine syndrome type 3C in a family from Pakistan.

In type 3 polyendocrine syndrome (PAS3), autoimmune thyroiditis occurs with other organ-specific autoimmune disease, but not with autoimmune adrenalitis. In this report we described a family from Pakistan in which mother and three daughters were affected by a PAS3. We studied a family from Pakistan: Father MMu age 44, mother KN aged 44, three daughters MM age 20, MH age 16 and MA age 14 and a son MU age 18. These subjects were tested for thyroids function, metabolic function, adrenal function, autoimmune disease. In this family the four females were shown hypothyroidism with presence of anti thyroid autoantibodies (AA) and high TSH serum concentration in association with the presence of anti transglutaminase AA. Moreover KN, MM and MH were positive for anti nuclear AA (granular pattern) and for antibodies against Saccaromyces cerevisiae. MM was positive for AA against nuclear extractable antigens (SSA and SSB) too. No diabetes or pernicious anemia were observed. Adrenal and Pituitary function were normal. PAS 3C is an uncommon disease. In this family from Pakistan we observed a PAS3C in the four female members: mother and three daughters while father and son were unaffected.

Evaluation of the GeneXpert assay in the detection of Factor V Leiden and prothrombin 20210 in stored, previously classified samples.

BACKGROUND: In this study, we evaluated the GeneXpert HemosIL Factor II and Factor V assay, an innovative assay for the detection of Factor V Leiden (FVL) and prothrombin G20210A mutation (GPRO). PATIENTS AND METHODS: We evaluated 132 patients that were previously classified (with a concordant result) using two commercial real-time PCR assays supplied, by Applied Biosystems and Roche Molecular Biochemicals. The cohort comprised 75 normal subjects, 10 FVL homozygous, 35 FVL heterozygous, 7 GPRO heterozygous, 2 GPRO homozygous and 3 double heterozygous FVL and GPRO subjects. All of the samples were evaluated using the GeneXpert HemosIL Factor II and Factor V assay. RESULTS: All of the samples were correctly identified using the GeneXpert HemosIL Factor II and Factor V assay; therefore, in this patient series, the specificity and sensitivity of the test under evaluation was 1.00. DISCUSSION: We have shown that the GeneXpert HemosIL Factor II and Factor V assay, a rapid fully automated assay, can accurately characterise the presence of FV G1691A and FII G20210A polymorphisms with specificity and sensitivity that are comparable to other current real-time PCR-based methods. The theoretical advantages of such an assay include improved standardisation across varying healthcare environments, more thorough sample manipulation and reduced human error.

Laboratory diagnosis of renal failure: urine conductivity and tubular function.

AIM: Conductivity is a measure of a material's ability to conduct an electric current and it works thanks to movable charges. Conductivity in urine is directly proportional to ionic contents. The aim of this study was to evaluate the significance of urine conductivity by using the Sismex UF-100 analyser in correlations with other surrogate parameters of osmolality and renal diuresis, relative density, electrolytes and creatinine concentration. METHODS: For this study 140 urine samples were submitted for diagnostic urinalysis to the Clinical Pathology laboratory. Samples were collected from 70 healthy subjects, 42 diabetics with poor metabolic control and significant glicosuria, 28 patients with monoclonal gammopathy of uncertain significance, with significant proteinuria. All the samples were assessed for conductivity (UF-100 Sysmex), relative density (refract meter Zeiss), sodium, potassium, chlorine, creatinine, urea, glucose, protein (Olympus AU-2700). RESULTS: Urine conductivity appears to be related to ionic concentration but not to glucose and/or protein presence. CONCLUSIONS: This study results suggest that conductivity determination should be useful in diabetic patients to study the tubular function minimising interferences due to osmotic action of glucose.

High prevalence of HFE gene mutations in hemodialysis patients.

AIM: Hemochromatosis (HH) was a common inherited disease characterized by iron overload. This disease is usually the result of mutations in the HLA-linked hemochromatosis gene (HFE). The aim of this study was to evaluate the frequency of HFE mutations in a group of Venetian hemodialysis patients. METHODS: Sixty-one hemodialysis patients, 62 patients with laboratory findings suggestive for iron overload, 57 repeat blood donors were enrolled in the study. HFE mutations were detected by using a commercial strip assay. RESULTS: In this study only H63D and C282Y mutations were observed. The overall prevalence of HFE mutations was 40.9% among hemodialysis patients, 30.6% among patients with laboratory findings of iron overload and 15.8% among blood donors. CONCLUSION: A high prevalence of HFE mutation among hemodialysis patients was observed. Prevalence of HFE mutation in this group was 40.9%, significantly higher than results observed among blood donors (15.8%, P<0.005) or among patients with laboratory signs of iron overload (30.6%, P<0.01). These data are, at present inexplicable, and this results need further confirmation.

Laboratory assessment of hypercoagulable state. A study in a group of patients with venous thromboembolism born in Chioggia.

AIM: Authors performed a Laboratory assessment for thrombophilia risk factors in a group of patients with previous deep venous thrombosis. METHODS: 123 consecutive patients were considered. The following parameters were investigated by using commercially available methods: PT, aPTT, TT, Fibrinogen, D-Dimer, Anti thrombin 3 (AT), Protein C (PC), Protein S (PS), activated C protein (APC) resistance, Lupus anticoagulant (LA), FV Leiden (G1691a mutation), Prothrombin G20210A mutation, MTHFR mutation (G677T mutation), anti Prothrombin auto-antibodies (PR) IgG and IgM, anti Beta 2 glycoprotein 1 (B2GP1) auto-antibodies IgG and IgM, anti Cardiolipin (CL) auto-antibodies IgG and IgM, homocysteine. RESULTS: In the 123 patients considered we observed: two AT deficiency, one PC deficiencies, one PS deficiency, 60 FV Leiden mutation (six homozygous), 1 Prothrombin gene mutations (heterozygous), 71 MTHFR mutations (15 homozygous). Study of anti phospholipid auto antibodies showed 10 patients positive for LA, 9 for anti CL antibodies (6IgG and 3IgM), 10 for anti B2GP1 antibody (5 IgG and 5IgM), 3 for anti PR antibody (IgG). Thirty nine patients showed hyper homocysteinemia. CONCLUSION: In our study only 19 patients were free of demonstrable thrombophilia risk factor. In 51 subjects a single risk factor was identified and in 53 multiple (from 2 to 5) risk factors were identified. In our opinion a Laboratory assessment of thrombophilia risk factors after a previous episode of deep venous thrombosis is a diagnostic tool of great importance. As a matter of fact, in our experience, by using a standard analytical panel, it was possible to highlight one or more risk factors in about 85% of the patients considered.

Haemoglobin E in pregnancy. A case report. Diagnosis, familiar study and counselling, follow up until delivery and new-born observation.

Haemoglobin E is a beta chain variant quite common in Southeastern Asia. The case of a gravid Thai woman with a microcytic anaemia is reported. The diagnosis of homozygous haemoglobin E was suspected on the basis of ethnic considerations when analysis of her haemoglobin showed the absence of normal HbA1 and about 100% of a variant Hb with electrophoretic mobility with HbC and HbA2. Identification of the haemoglobin variant was performed by using an association of alkaline electrophoresis on agarose gel, acid electrophoresis on agarose gel, haemoglobin isoelectrofocusing, high performance liquid chromatography. A study of haemoglobin pattern in the partner, parents and siblings was also performed. Pregnancy continued without any problems until the 40th week when a caesarean section was performed due to a difficult labour with foetal distress. The haemoglobin pattern of the new-born was studied at birth and after 1 year; as expected, it was quite normal at birth and a heterozygous condition for HbE was observed after 1 year. HbE, in even heterozygous and homozygous states, gives a mild clinical picture but its association with other haemoglobinopathies, such as a double heterozygous state (i.e. HbE/beta Thalassaemia) gives rise to a severe transfusion dependent thalassaemia syndrome. It is the authors' opinion that only a strict interaction between obstetricians and pathologists is the possible correct answer to the new diagnostic question proposed by a rapidly evolving inter-ethnic society.

Biological qualification of blood units: considerations about the effects of sample's handling and storage on stability of nucleic acids.

BACKGROUND: In transfusional setting introduction of nucleic amplification technique (NAT) for HBV-DNA, HCV-RNA and HIV-RNA in biological qualification of blood units suggest some problems. At first the opportunity to operate on mini-pool, at second the need to store the samples at +4 degrees C. The authors therefore have tried to estimate the impact of these conditions on the operativity of NAT testing in the transfusional setting. METHODS: The following parameters has been estimated: distribution of viral-load in untreated subjects, stability of nucleic acids during storage at +4 degrees C, stability of nucleic acids after repeated cycles of freezing and defrosting, robustness of the test to the cross-contamination, definition of the detection-limit (95%). Quantitative tests has been performed by using the following kits: Cobas Amplicor HBV Monitor, Cobas Amplicor HCV Monitor, Cobas Amplicor HIV Monitor; the qualitative tests has been performed by using the following kits: Ampliscreen HBV, Ampliscreen HCV 2,0, Ampliscreen HIV 1,5 all supplied by Roche Molecular System (Brancburg, NJ). RESULTS: Viral load in untreated subjects showed wide variation for HBV, HCV and HIV. HBV has been demonstrated much stable to the conservation +4 degrees C also until 168 h while for HCV and HIV a greater decrease of the viral-load was observed. For all and three virus the conservation to +4 degrees C until 72 h does not seem to involve meaningful fall in the viral-load. A remarkable reduction of the viral-load has been observed after five cycles of freezing and defrosting. All the tests showed a good robustness to cross-contamination. The detection-limit (95%) was 8 U/ml for HBV, 21 U/ml for HCV and 27 copy/ml for HIV. CONCLUSIONS: Samples for NAT testing, can be stored until 72 h to +4 degrees C without appreciable lowering of the viral-load. Repeated cycles of changes of state should be avoided. The tests showed a good robustness to cross-contamination. NAT tests for biological qualification of blood units had a minimal sensibility around 50 (copy/unit/ml). In our experience the detection-limit (95%) was 21 U/ml for HCV, 27 copies/ml for HIV, 8 U/ml for HBV. The availability of NAT test for HBV-DNA, HCV-RNA e HIV-RNA, sensitive and reliable, together with epidemiological data, suggest the opportunity to place side by side, in the biological qualification of the blood units, to add the tests for HBV-DNA and HIV-RNA to the test for HCV-RNA mandatory by low, in Italy in the biological qualification of blood units.

Assessment of in vitro platelet activation by Advia 120 platelet parameters.

The Advia 120 Hematology System provides new platelet parameters that have been proposed as useful markers of platelet activation. The aim of this study was to investigate platelet parameter variations after adenosine diphosphate (ADP) activation of platelet-rich plasma (PRP) in vitro, with particular interest in the mean platelet component (MPC), which was compared with two well-known degranulation antigens. Changes in platelet parameters that were induced by the activation of PRP with different concentrations of ADP were examined first. The time course of parameter values up to 60 minutes after maximal ADP activation and the relationships between the MPC and P-selectin and granulophysin expression as determined by flow cytometry were then investigated. After 10 minutes of ADP stimulation, the MPC presented a dose-dependent increase. At the maximal ADP concentration, the initial increase of the MPC was followed by a progressive decrease, leading the MPC to become significantly lower with respect to the baseline after 60 minutes of incubation. Significant variations in other parameters are also described. Finally, a negative correlation was found between the MPC change with respect to time 0 and both P-selectin and granulophysin expression. The present study suggests that platelet parameter variation, particular in the MPC, may be used to assess platelet activation in PRPs stimulated by ADP.

Field evaluation of a second-generation cytometer UF-100 in diagnosis of acute urinary tract infections in adult patients.

AIMS: The authors evaluated the analytical performance of the Sysmex UF-100 cytometer vs. the diagnosis of urinary tract infections (UTI). METHODS: We considered 2010 subjects, aged between 18 and 78, 870 males and 1140 females. The majority (90.2%) of the samples were voided urine specimens collected by using the midstream technique. Each sample was subjected to microbiological evaluation (culture + residual antibacterial activity), dipstick tests, UF-100 examination and microscopic observation. In order to obtain a final diagnosis of UTI these laboratory results were taken into consideration together with clinical data and patients' characteristics. The analytical performance of the laboratory tests was obtained by adopting this diagnosis as standard practice. RESULTS: Out of the total 2010 subjects considered a clinical diagnosis of UTI was obtained in 529 cases (26.32%). The UF-100-based screening had sensitivity, 0.94; specificity, 0.93; positive predictive value, 0.83; negative predictive value, 0.98; and correctly classified incidence, 0.93. CONCLUSIONS: In our experience the results of the UF-100-based screening show a very good correlation with the diagnosis of acute UTI in adults patients.

[Foetal-maternal alloimmunizations in the South-East area of the Venice province]. / Alloimmunizzazioni feto-materne nel Sud-Est del Veneto.

BACKGROUND: This study report the results obtained in a retrospective analysis of the foetal-maternal alloimmunizations observed from 1993 to 1999 in the South-East area of the Venice province. METHODS: The data concerning 17,000 pregnancy observed in this area from 1993-1999 have been collected. For each pregnancy data concerning maternal ABO, Rh, Kk and IAT as well as foetal ABO, Rh, Kk and DAT were available. Further data (mainly antibodies concentration and specificity) were available if a foetal-maternal alloimmunization was detected and if transfusional support was given after the birth. RESULTS: The authors observed 465 alloimmunizations (prevalence 2.7%): 381 (82%) of these were due to an ABO foetal-maternal incompatibility, 23 due to D incompatibility and the other 61 due to other blood groups antigens. Only 6 cases needed transfusional support: 5 exchange transfusion (a patient needed 2 exchanges) and a delayed transfusion. CONCLUSIONS: Foetal-maternal alloimmunizations are today a rare but not exceptional event (about 3% of pregnancy), the great majority of these alloimmunizations are due to an ABO incompatibility. Despite the prevention of alloimmunization in D negative women by using Rh immune globulin, anti-D alloimmunization is still observed. A great number of other blood groups antigens are involved in foetal-maternal alloimmunization mainly within the Rh system (CcEe, etc.). In the authors' experience the great majority of foetal-maternal alloimmunizations were clinically silent, only 6 cases (1.3% of patients with a positive DAT) needed transfusional therapy.

Platelet count and parameters determined by the Bayer ADVIA 120 in reference subjects and patients.

In order to study the behaviour of traditional and new platelet parameters determined by the ADVIA 120 Hematology System, five hundred samples from reference subjects, divided for sex and age, were processed. Significant variations on the basis of sex and age were found. Reference ranges as 95% confidence limits were therefore calculated for each age class, and platelet parameters proved to have specific variations during lifetime. Moreover, one hundred samples from thrombocytopenic patients were processed by the ADVIA 120 System. When compared with those of reference subjects matched for sex and age, all platelet parameters, except mean platelet component (MPC), showed significant differences.

The stability of hepatitis C virus RNA after storage at +4 degrees C.

One of the major issues in nucleic acid testing is how to store blood samples to obtain reliable results. We therefore studied hepatitis C virus (HCV) RNA concentration in samples after storage at +4 degrees C or freezing and thawing. Six HCV RNA-positive, untreated subjects were studied. Blood samples were collected from these subjects in plasma preparation tubes. The HCV RNA concentration was analysed after storage at +4 degrees C for 168 h or after five freeze-thaw cycles. For HCV RNA quantification we used a qualitative and a quantitative commercial test. After 168 h of storage at +4 degrees C, the HCV RNA concentration was similar to that observed at time-point 0 (5.025 log vs 5.492 log). In one sample we observed a significant fall in HCV RNA concentration. After five freeze-thaw cycles, the HCV RNA concentration was lower than that observed at time-point 0 (4.454 log vs 5.492 log) and in four samples we observed a significant fall in HCV RNA concentration. Our data suggest that HCV RNA is stable in whole blood samples stored at +4 degrees C for 168 h. Based on our results, we conclude that the standard procedures for transport of blood samples (at room temperature for a maximum of 5 h) and storage schedules (+4 degrees C for a maximum of 48 h) can be maintained without compromising the quality of results.

In subjects with antibody to hepatitis C virus a high serum level of interleukin-2 soluble receptor suggests activity of liver disease.

The hepatitis C virus (HCV) is an RNA virus without apparent cytopathic effects, and hepatocellular damage in chronic infection is generally believed to be immune-mediated by cytotoxic T lymphocytes. Activated T cells release the soluble form of the interleukin-2 (IL-2) receptor (sIL-2R) and its concentration is correlated with the degree of lymphocyte activation. We measured sIL-2R in 69 subjects: 24 healthy repeat blood donors (group I), 17 HCV carriers without liver damage (group II) and 28 patients with HCV-related chronic active hepatitis (group III). There was no significant difference between sIL-2R levels in patients of group I (36.5 +/- 14.6 U ml-1) and group II (46.8 +/- 17.4 U ml-1), and the levels for both of these groups were significantly lower than those observed in the patients with active HCV, group III (176.9 +/- 59.5 U ml-1). Hence, among HCV-infected subjects (HCV RNA positive) with persistently normal alanine aminotransferase (ALT) levels, the plasma levels of sIL-2R are normal, but, in patients (HCV RNA positive) with HCV-related chronic active hepatitis there are increased plasma levels of sIL-2R. We conclude that in HCV infection high levels of sIL-2R are related to activity of the disease rather than to virus replication. In patients with HCV-related chronic liver disease, the sIL-2R concentration may be a useful marker of disease activity.