Performance evaluation of four dominant anti-hepatitis B core antigen (HBcAb) kits in Japan for preventing de novo hepatitis B virus (HBV) infection.

BACKGROUND: The determination of antibody to hepatitis B core antigen (HBcAb) has become an important means of evaluating the risk factors of de novo hepatitis B virus (HBV) infection before starting intensive immunosuppressive drug therapies. Four dominant HBcAb determination reagents used in Japan were evaluated with HBcIgM, HBsAg, HBsAb, HBeAb, and HBV DNA reagents in order to study their clinical utility. METHODS: Four kinds of HBcAb reagent kits (HBcAb Total and HBcAb-IgG reagent) were evaluated with 526 clinical specimens, including 344 negative specimens, at Osaka University Hospital. The dynamic range of each kit was evaluated by testing serially diluted serum from pooled sera with high HBcAb concentration. RESULTS: The reagent that showed the largest dynamic range was the Lumipulse HBcAb-N (HBcAb-IgG reagent). Regarding clinical sensitivity and specificity, Centaur HBcAb (HBcAb Total reagent) gave several "doubtful negative" results and ARCHITECT HBcII (HBcAb Total reagent) had the most discrepant positive results. By comparing the cut-off-index distribution of negative specimens using a parameter of "distance from the mean to the cut-off divided by the SD", Centaur was determined to be the best (distance/SD = 12.65), with Lumipulse and Elecsys Anti-HBc (HBcAb Total reagent) in the second group (8.13 and 7.00, respectively), and ARCHITECT rated as the worst (3.25). CONCLUSIONS: In this evaluation, Elecsys and Lumipulse HBcAb kits showed good clinical sensitivity and specificity and were considered to be suitable for evaluating the risk factors of de novo HBV infection.

Latex-enhanced turbidimetric immunoassay for everolimus in whole blood using the Nanopia TDM everolimus assay with the JCA-BM6010 automatic analyzer.

BACKGROUND: Everolimus is a novel proliferation signal inhibitor used in immunosuppressive therapies for the prevention of acute and chronic rejection. It is an immunosuppressant requiring routine monitoring in whole blood. We evaluated analytical performance of the Nanopia TDM Everolimus assay kit for the measurement of everolimus in whole blood. METHODS: Whole blood samples from patients receiving immunosuppressive therapy with everolimus after heart transplantation were used, and everolimus concentrations were measured by liquid chromatography combined with mass spectrometric detection and fluorescence polarization immunoassay and Nanopia TDM Everolimus assay. RESULTS: The within-assay coefficient of variation was 4.0%-6.8% (n = 20) at everolimus concentrations of 4.4-15.7 ng/mL, whereas the day-to-day coefficient of variation ranged from 3.6% to 5.4% at everolimus concentrations of 4.8-15.9 ng/mL. The limit of quantitation was 1.5 ng/mL. Calibration curves were stable for at least 14 days. The presence of conjugated bilirubin, unconjugated bilirubin, lipemic material, rheumatoid factor, and variation of the hematocrit did not affect this assay system. There was a strong positive correlation between values obtained with the Nanopia TDM Everolimus assay kit and the results of liquid chromatography combined with mass spectrometric detection (y = 0.960x + 0.312 ng/mL; r = 0.946; n = 91), as well as with data from the fluorescence polarization immunoassay (y = 0.966x - 0.440 ng/mL; r = 0.942; n = 91). CONCLUSIONS: The Nanopia TDM Everolimus assay showed good analytical performance. These results indicate that the Nanopia TDM Everolimus assay may be suitable for routine clinical use.

[Evaluation of the Creatine Kinase MB Activity Assay Kit].

CK-MB activity, which is measured by the immunoinhibition method, is an important marker for use in the diagnosis of acute coronary syndromes. In the present study, we evaluated the basal performance of a recently improved CK-MB activity kit, "L-system CK-MB," in which the activity of mitochondrial CK subunits is inhibited. Within-run and between-day precision were in the range of 1.9-2.3% and 3.2-6.0%, respectively. Diluted linearity and limit of detection were determined to be 600 U/L and 0.8 U/L, respectively. The use of L-system CK-MB allowed the inhibition of the activity of 98.1% of sarcomeric mitochondrial CK, 97.7% of ubiquitous mitochondrial CK, and 99.9% of CK-MM. The correlation coefficient (r) between CK-MB activity and CK-MB protein was 0.968. However, we found 4 cases showing CK-MB activity significantly higher than the protein concentration. Increased CK-BB activity was detected by electrophoresis in these cases. In some patients with malignant tumors, the presence of CK-immunoglobulin complex also lead to elevated CK-MB concentrations. Thus, the discrepancy in the CK-MB activity and the protein concentration may be caused by the presence of CK-BB and/or CK-immunoglobulin complex. More attention needs to be focused on samples with high CK-MB protein concentrations, especially when the CK-MB/CK ratio is high.

[Evaluation of the latex turbidimetric immunoassay for CK-MB protein].

CK-MB protein is an important marker for the diagnosis of acute coronary syndromes. In the present study, we evaluated the basal performance of recently developed CK-MB mass kit "L-type wako CK-MB mass". Within-run and between-day precision were obtained with 1.4-4.7% and 2.7-5.2%, respectively. Diluted linearity and lower limit of detection were obtained with 180.0 ng/mL and 2.1 ng/mL, respectively. Zone phenomenon was able to detect until 25,600.0 ng/mL. Analysis of interferent showed that only CK-BB positively influenced the assay results. CK-MB protein levels decreased to 82% at 72 hours in the room temperature, but it was stable at 4 degrees C and -20 degrees C. The correlation coefficient(r) between this assay and conventional assay was 0.999. However, discrepancy of the two cases was observed in the comparison between the two methods. In the case 1, CK isoenzyme analysis using electrophoresis indicated that CK-MB was not present and absorption test showed a 68% absorption effect of CK-MB protein values not by anti human IgG, anti human IgA, and animal serums, but anti human IgM. In the case 2, CK isoenzyme analysis indicated that there is not only CK-MB but CK-BB. CK-MB protein values between the two methods were fitted after decreasing CK-BB. Thus, Value discrepancy for CK-MB protein was resulting from IgM and CK-BB. We have to pay attention to such phenomenon when detecting an unlikely higher levels that could not be explained by clinical information.

[Effects of anticoagulants and storage temperature on immature platelet fraction % (IPF%) values in stored samples measured by the automated hematology analyzer, XE-5000--utility of CTAD-anticoagulation and room temperature storage].

Measurement of reticulated platelet percentage (RP%) is thought to be a useful marker for differential diagnosis and analysis of platelet kinetics in patients with thrombocytopenic disorders. Two methods are used to detect RP; flow cytometric method and immature platelet fraction (IPF) method using automated hematology analyzers. Although IPF% measured by the automated hematology analyzers is simple and convenient, we already reported that IPF% values were highly fluctuated in stored whole blood sample with EDTA-2K at 4 degrees C day by day. In this study we investigated the stability of IPF% in blood samples obtained from 11 patients with chronic immune thrombocytopenic purpura (ITP) and 19 healthy volunteers using the automated hematology analyzer, XE-5000 (Sysmex) under various storage conditions. EDTA-2K, 3.13% sodium citrate, acid-citrate dextrose solution (ACD), citrate-theophylline-adenosine-dipyridamole solution (CTAD), or sodium fluoride was used as an anticoagulant. When blood samples obtained from healthy subjects were stored at 4 degrees C, IPF% values markedly increased in a time-dependent manner by any anticoagulant examined. On the other hand, there was no significant or only slight difference in IPF% values at room temperature (RT) storage except sodium fluoride. However, in patients with ITP the elevated IPF% values fluctuated widely in EDTA-2K, sodium citrate and ACD-anticoagulated samples even at RT storage. In contrast, IPF% values in CTAD samples stored at RT were highly stable in all patients with ITP up to 4 day storage. These results suggest that the measurement of IPF% by XE-5000 provides quite stable data up to 4 day-storage in ITP patients as well as healthy subjects under CTAD-anticoagulation and RT storage conditions.

Development of a new modified Bethesda method for coagulation inhibitors: the Osaka modified Bethesda method.

The Nijmegen assay for the factor VIII (F-VIII) inhibitor is recommended by the International Society on Thrombosis and Haemostasis/Scientific and Standardization Committee. However, due to cumbersome and complicated preprocessing, it is presently difficult to introduce this assay into hospital laboratories. We used buffered plasma that was made by addition of 1 volume of 1 mol/l HEPES buffer at pH 7.35 to 9 volumes of plasma to form the test samples. The inhibitor titer was calculated by the remaining rate of F-VIII coagulation activity (F-VIII:C), using the ratio of actual value to the theoretical value. Five hundred microliters of the buffered test plasma and the control (30 mmol/l HEPES buffered saline at pH 7.35) were each mixed with equal volumes (500 µl) of normal pooled plasma in a test tube (11 mm internal diameter and 6.5 ml volume capacity), and incubated at 37°C for 2 h. In our modified Bethesda method, there were no significant changes in pH and F-VIII:C of control and test mixtures after incubation tests for stability. With the modified method, the inhibitor titers (mean, SD) from examining three hemophilia A plasma samples (F-VIII:C, <1-3%) and 40 normal samples (F-VIII:C, 34.5-168.3%) were 0.032, 0.057 and -0.009, 0.057, respectively. By our method, the F-VIII inhibitor titer of type I inhibitor-positive samples was higher than the Nijmegen method, and for type II inhibitor-positive samples, the titer was similar. We believe that our method can be applied to not only the type I inhibitor, but also to assays of type II inhibitor, without cumbersome and complicated preprocessing.

Low level of factor V is associated with development of deep-vein thrombosis in Japanese patients.

BACKGROUND: Factor V, having two functions (procoagulant and anticoagulant), is a key factor in blood coagulation, and low plasma levels of factor V may be a risk factor for thrombosis. OBJECTIVE: The levels of plasma factor V antigen (FV:Ag), and the phospholipid binding capability of Factor V (FV:PL-bound) were evaluated in patients with deep-vein thrombosis (DVT). METHODS: Levels of FV:Ag, and FV:PL-bound were expressed as a percentage of the normal level found in pooled plasma from control subjects. One hundred and twenty-three Japanese patients with deep-vein thrombosis (DVT) were included, with 100 age and sex-matched healthy control subjects. RESULTS: The FV:Ag, and FV:PL-bound values were significantly lower in DVT patients than in healthy subjects (p<0.05 and p<0.005, respectively). Among the 123 patients, 30 for FV:Ag (24.4%), and 32 for FV:PL (26%) had less than the arbitrary cutoff point (set at the 5th percentile of the value for FV:Ag and FV:PL-bound from healthy subjects), and the odds ratios (ORs) were 6.1 (95% confidence interval [CI], 2.3-16.5) and 6.7 (95%CI, 2.5-17.9), respectively. When patients with a deficiency of natural anticoagulants (antithrombin, protein C, and protein S) were excluded from the analysis, the ORs increased for all patients (6.6 for FV:Ag (95%CI, 2.4-18.3) and 7.4 for FV:PL-bound (95%CI, 2.7-20.3). Moreover, twenty-one (17%) of the 123 DVT patients, and 1 (1%) of 100 control subjects had values below the cutoff points for both FV:Ag and FV:PL-bound, and the OR was 21.6 (95%CI, 2.85-163.1). CONCLUSIONS: These results suggest that low levels of factor V are associated with development of DVT, and may be a predictor for DVT.

[Comparison between two methods for the measurement reticulated platelet and their clinical significance--flow cytometry (FCM) method and IPF method Using automated hematology analyzer (XE-2000)].

Reticulated platelet (RP) is thought to be a useful marker for differential diagnosis and analysis of platelet kinetics in patients with thrombocytopenia. In this study, we compared two methods for the measurement of RP: flow cytometric (FCM) method and immature platelet fraction (IPF) method using automated hematology analyzer (XE-2000). There was a relatively good correlation between RP% measured by FCM method and IPF% measured by IPF method in patients with idiopathic thrombocytopenic purpura (ITP) (Y = 0.806X-0.050, r = 0.634, p < 0.001). We then measured RP% and IPF% in 61 patients with ITP and 27 patients with aplastic anemia (AA). For the differential diagnosis for ITP, the sensitivity (82%) and specificity (93%) of FCM method were better than those of IPF method (sensitivity 67% and specificity 63%). Our data demonstrate the significant difference between two methods by analyzing clinical samples in parallel.

Prevalence of genetic mutations in protein S, protein C and antithrombin genes in Japanese patients with deep vein thrombosis.

INTRODUCTION: Genetic deficiencies of PROS1, PROC, and SERPINC1 (antithrombin) are risk factors for deep vein thrombosis (DVT). Diagnosis of the inherited deficiencies of these three genes is sometimes difficult because of the phenotypic variability. This study was undertaken to reveal the frequency of nonsynonymous mutations of these three genes in Japanese DVT patients. PATIENTS/METHODS: One hundred seventy-three DVT patients were registered by the Sub-group of Blood Coagulation Abnormality, from the Study Group of Research on Measures for Intractable Diseases. We sequenced the entire coding regions of the three genes in all DNA samples and identified the nonsynonymous mutations. RESULTS AND CONCLUSIONS: For PROS1 we identified 15 nonsynonymous mutations in 28 DVT patients; for PROC, 10 nonsynonymous mutations in 17 patients; and for SERPINC1, 13 nonsynonymous mutations in 14 patients. Five patients had two mutations in PROS1 and PROC, and all of them had PROS1 K196E mutation. We previously identified one patient with a large PROS1 gene deletion. Thus, 55 out of 173 patients (32%) carried at least one genetic defect in the three genes. The PROS1 K196E mutation found in 15 Japanese DVT patients was the most prevalent. Mutations of PROC K193del and V339M were the second, each found in four patients. Our data suggested that the PROC K193del mutation caused the loss of the anticoagulant activity but not the amidolytic activity. Our effort is the first DNA resequencing study to identify the genetic variations in DVT patients without any consideration of their plasma activities and antigens. To minimize selection bias in a future evaluation of the contribution of genetic deficiency to DVT, we must recruit patients consecutively.

Association of Asn221Ser mutation in tissue factor pathway inhibitor-beta with plasma total tissue factor pathway inhibitor level.

Tissue factor pathway inhibitor (TFPI) is an anticoagulant protease inhibitor that inhibits the tissue factor-initiated blood coagulation cascade reactions. Based on these anticoagulant functions of TFPI, we hypothesized that genetic variations in TFPI may alter the TFPI expression or impair the anticoagulant function and could predispose persons to deep vein thrombosis (DVT). This study was undertaken to examine whether the genetic polymorphisms in TFPI are associated with the plasma TFPI levels and risk for DVT. We sequenced the entire coding regions of TFPI in 175 Japanese DVT patients and identified 12 genetic variants, including one missense mutation, Asn221Ser. The missense mutation occurred at the site presumably attached to the glycosylphosphatidylinositol anchor in the TFPI-beta form. The allele frequency of the mutant Ser-coding allele of the Asn221Ser mutation was 8% in the Japanese general population consisting of 1684 individuals. The Asn221Ser mutation was significantly associated with the total TFPI levels (Asn/Asn, n = 108, total TFPI = 56.57 +/- 0.88 ng/ml (mean +/- SD) vs. Asn/Ser + Ser/Ser, n = 16, total TFPI = 63.44 +/- 2.28 ng/ml, P = 0.0058). The genotype was not associated with the free TFPI level. This Asn221Ser mutation was not associated with DVT. Thus, the Asn221Ser mutation occurring in the TFPI-beta form was associated with the total TFPI level, but not a risk for DVT. The absence of the putative glycosylphosphatidylinositol anchor in TFPI-beta under pathological conditions remains to be studied.

[Progression from a routine examination to a study. Having come this far, what should be done from now? A thrombosis risk factor study].

We were able to carry out a study of deep venous thrombosis (DVT) with the cooperation of clinicians. I herein describe a part of the study that I have conducted. Japanese patients with DVT were investigated regarding their incidences of antithrombin III (AT-III), protein C (PC), and protein S (PS) deficiencies, and the results were compared with those of normal subjects. We confirmed that the Japanese population has a high frequency of PC and PS deficiencies. Furthermore, we recommend that PS activity should be measured for the screening of thrombosis since type II deficiency accounted for approximately 50% of PS deficiency cases in both patients and the normal group in Japanese. We evaluated the reactivity of thrombin to TM by determining the TM-bound thrombin (TMBTh) to total thrombin generation (t-Th) ratio (TMBT ratio). We also examined whether a decreased TMBT ratio is associated with an increased risk of thrombosis. The TMBT ratio (TMBTh/t-Th) was significantly lower in patients with DVT than in control subjects. These results suggest that it is possible to evaluate the reactivity of thrombin to TM by determining the TMBT ratio, and this ratio may be a predictor of deep vein thrombosis. However, we have yet to investigate the causal mechanism.

Frequencies of mild factor V, VII and X deficiencies in a Japanese population.

We investigated the frequencies of coagulation factor deficiencies in a Japanese population. We measured factor II, V, VII and X activity in 100 healthy individuals. A specific factor deficiency was determined according to the factor activity and the ratio of the factor activity to that of other coagulation factors. Seven samples showed factor activity less than the mean -2SD of standardized factor activity (factor II: three; factor V: one; factor VII: one; factor X: one and factor V+factor VII: one). The samples with low factor II and factor VII activity were determined not to be due to deficiency because the ratios of these factor activities to other factor activities were within the range of the mean +/- 2SD. We measured activity ratios in the remaining 97 samples and identified one sample with factor V deficiency and two with factor VII deficiency. Thus, six samples with coagulation factor deficiency were identified (factor X: one; factor V: two; factor VII: two and factor V + factor VII: one). These results suggest that the Japanese population has relatively high frequencies of mild factor V, factor VII and factor X deficiencies, in which activity is reduced to approximately 50% (36-64%) of normal plasma.

Impaired binding of thrombin to thrombomodulin is associated with risk of deep vein thrombosis.

The complex of thrombin and thrombomodulin (TM) activates protein C, and impaired binding of thrombin to TM may be a risk factor for thrombosis. In this study, we evaluated the reactivity of thrombin to TM by determining the TM-bound thrombin (TMBTh) to total thrombin generation (t-Th) ratio (TMBT ratio). We also examined whether a decreased TMBT ratio is associated with increased risk of thrombosis. TMBTh was measured on TM-coated plates. Thrombin was generated by addition of prothrombin time reagent to plasma. Levels of t-Th and TMBTh were expressed as percentages of the levels in pooled normal plasma. The study included 124 patients with deep vein thrombosis and 150 age- and sex-matched healthy subjects. The TMBT ratio (TMBTh/t-Th) was significantly lower in patients than in control subjects (p<0.05). Among the 124 patients, 43 (34.7%) showed TMBT ratios below the 5th percentile value of control subjects, and the odds ratio (OR) for development of deep vein thrombosis was 9.4 (95% confidence interval [CI], 4.6-19.1). When patients with a deficiency of natural anticoagulant (antithrombin III, protein C, or protein S) were excluded from analysis, the TMBT ratio in 37 (42.5%) of the remaining 87 patients was below this cutoff point, and the OR (13.1; 95% CI, 6.4-26.9) was increased compared to that in the total patient group. These results suggest that it is possible to evaluate the reactivity of thrombin to TM by determining the TMBT ratio, and this ratio may be a predictor of deep vein thrombosis.

Tissue factor expression on monocytes induced by anti-phospholipid antibodies as a strong risk factor for thromboembolic complications in SLE patients.

Our aim was to clarify the role of anti-phospholipid antibodies in the pathogenesis of monocyte tissue factor (TF) expression and thromboembolic complications (TE) in patients with SLE. We examined cell surface expression of TF on monocytes in 93 SLE patients. Monocyte TF expression was significantly higher in SLE patients who had TE than in other SLE patients, and confirmed that the high expression of monocyte TF was a strong risk factor for TE. Furthermore, the presence of anti-cardiolipin/beta2-glycoprotein I antibodies (anti-CL/beta2-GPI) was strongly associated with the high expression of monocyte TF. We therefore studied the in vitro effect of IgG anti-CL/beta2-GPI on lipopolysaccharide (LPS)-induced expression of TF on monocytes in healthy peripheral blood and found that purified IgG containing anti-CL/beta2-GPI significantly enhanced LPS-induced monocyte TF expression. These results suggest that anti-CL/beta2-GPI cause persistently high TF expression on monocyte, which may contribute to the risk of thromboembolic events in SLE patients.

Determination of leukocyte counts in cerebrospinal fluid with a disposable plastic hemocytometer.

Manual microscopic cell counting in a Fuchs-Rosenthal (FR) chamber has been the gold standard for quantification of leukocytes in cerebrospinal fluid (CSF). However, for accurate determination of the number and differentiation of cells by chamber counting, hemocytometers must be prepared carefully and kept clean. Improper fitting of the chamber and coverslip changes the volume of sample introduced into the chamber well. Moreover, because conventional hemocytometers are used repeatedly and are breakable, there is a risk of exposure to potentially infectious material. To address these issues, disposable plastic hemocytometers have been developed. However, the accuracy, precision, and clinical usefulness of disposable chambers for CSF cells counting have not been determined. In the present study, we evaluated use of a disposable plastic counting chamber (C-Chip DHC-F01) by comparing its performance with that of an FR chamber for counting of CSF specimens and cell suspensions. Within-run precision of C-Chip counting was comparable or superior to that of FR chamber counting, and excellent correlation between cell counts obtained with the C-Chip chamber and FR chamber was observed. However, C-Chip chambers that were kept at 4 degrees C yielded significantly low cell counts. The disposable hemocytometer will reduce the risk of exposure to potentially infectious material. However, use of C-Chip chambers should be avoided in cold environments.

Haplotype of thrombomodulin gene associated with plasma thrombomodulin level and deep vein thrombosis in the Japanese population.

INTRODUCTION: Thrombomodulin (TM) is an essential cofactor in protein C activation by thrombin. Here, we evaluated the contribution of genetic variations in the TM gene to soluble TM (sTM) level and deep vein thrombosis (DVT) in Japanese. PATIENTS AND METHODS: We sequenced the TM putative promoter, exon, and 3'-untranslated region in DVT patients (n=118). Among 17 genetic variations we identified, two missense mutations (R385K, D468Y) and three common single nucleotide polymorphisms (-202G>A, 2487A>T, 2729A>C) were genotyped in a general population of 2247 subjects (1032 men and 1215 women) whose sTM levels were measured. We then compared the frequency of these mutations in DVT patients with that in the age, body mass index-adjusted population-based controls. RESULTS: We identified one neutral mutation (H381) and three missense mutations (R385K; n=2, A455V; n=53 heterozygous, n=14 homozygous, D468Y; n=2) of TM in the DVT patients. Age-adjusted mean values of sTM were lower in C-allele carriers of 2729A>C than in noncarriers in the Japanese general population (women: 16.7+/-0.3 U/ml vs. 17.9+/-0.2 U/ml, p<0.01, men: 19.4+/-0.3 U/ml vs. 20.4+/-0.3 U/ml, p=0.03). Additionally, the CC genotype of this mutation was more common in the male DVT patients than in the male individuals of the general population (odds ratio=2.76, 95% confidence interval=1.14-6.67; p=0.02). This mutation was in linkage disequilibrium (r-square>0.9) with A455V mutation. CONCLUSIONS: TM mutations, especially those with a haplotype consisting of 2729A>C and A455V missense mutation, affect sTM levels, and may be associated with DVT in Japanese.

Urine flow cytometer quantification of leukocytes in samples containing a large proportion of lymphocytes.

OBJECTIVE: We compared leukocyte counts obtained by cytometric analysis and Fuchs-Rosenthal (FR) chamber counting in different proportions of lymphocytes (Lym%) suspensions and cerebrospinal fluid (CSF). DESIGN AND METHODS: UF-100 (UF) was evaluated. For preparation of cell suspensions, gradient density centrifugation method was used. RESULTS: The regression equation for UF and FR chamber counting of the cell suspensions was y=0.88x+18.8 WBC/microL (r=0.832, n=106). For a few high Lym% samples, markedly underestimated WBC counts were obtained by UF. CONCLUSIONS: Underestimated WBC count is due not to systematic error but to random error. Counts of the "other" population by UF may be useful for detection of underestimated samples.

The presence of anti-phosphatidylserine/prothrombin antibodies as risk factor for both arterial and venous thrombosis in patients with systemic lupus erythematosus.

In an effort to clarify the clinical significance of anti-phospholipid antibodies (aPL) detected by enzyme-linked immunosorbent assay (ELISA), we examined the prevalence of anti-cardiolipin antibodies (aCL), anti-beta2-glycoprotein I antibodies (anti-beta2-GPI), antiprothrombin antibodies (anti-PT), and anti-phosphatidylserine/prothrombin antibodies (anti-PS/PT) in 175 patients with systemic lupus erythematosus (SLE) comprising 67 patients with thrombotic complications. The present study showed that positive results of anti-beta2-GPI-ELISA and anti-PS/PT-ELISA could serve as markers of thrombotic complications in patients with SLE, whereas aCL and anti-PT are less reliable as markers of these complications. Furthermore, results of the anti-PS/PT-ELISA correlate best with the occurrence of both arterial and venous thrombosis in patients with SLE.

[Fragmented red cells are the major cause of spuriously high platelet counts in patients with fragmented red cells when counts are obtained by automated blood cell counter].

Platelet counts measured by automated blood cell counter often show spuriously high values when measuring samples contain particles of equal size to platelets. The major cause of spuriously high platelet counts in samples with fragmented red cells (FRC) is thought to be the FRC themselves. We studied the correlation between FRC and spuriously high platelet counts in 40 patients demonstrating FRC on blood smears. FRC were measured by manual hemocytometry and by flow cytometry using a monoclonal antibody against glycophorin A (GPA method). There was a significant correlation between spuriously high platelet counts and FRC by manual hemocytometry (r=0.60, p<0.001) or FRC by the GPA method (r=0.45, p<0.005). These data suggest that FRC are the major cause of spuriously high platelet counts in samples with FRC.

Acquired activated protein C resistance associated with IgG antibodies against beta2-glycoprotein I and prothrombin as a strong risk factor for venous thromboembolism.

BACKGROUND: Venous thromboembolic events such as deep vein thrombosis and pulmonary embolism are common manifestations of antiphospholipid syndrome. Our aim was to clarify the roles of anti-phospholipid (aPL) antibodies in the pathogenesis of venous thromboembolism (VTE) in patients with systemic lupus erythematosus (SLE). METHODS AND RESULTS: We examined anti-cardiolipin/beta2-glycoprotein I (anti-CL/beta2-GPI) antibody concentrations, anti-phosphatidylserine/prothrombin (anti-PS/PT) antibody concentrations, and lupus anticoagulant (LA) activity in 87 patients with SLE (21 with VTE and 66 without thrombosis). Both anti-CL/beta2-GPI and anti-PS/PT antibodies strongly correlated with LA activity. Multivariate logistic analysis confirmed that both anti-CL/beta2-GPI and anti-PS/PT antibodies were significant independent risk factors for VTE (odds ratios = 4.98 and 7.54, respectively; 95% confidence intervals, 1.51-16.4 and 2.30-24.7, respectively). We therefore studied the in vitro effects of IgG fractions containing anti-CL/beta2-GPI or anti-PS/PT antibodies on the anticoagulant activity of activated protein C (APC) and found that purified IgG containing anti-CL/beta2-GPI or anti-PS/PT antibodies significantly hampered the anticoagulant activity of APC. We also studied the ability of IgG fractions to impede the anticoagulant activity of APC before and after complete removal of anti-CL/beta2-GPI or anti-PS/PT antibodies by adsorption. Removal of anti-CL/beta2-GPI or anti-PS/PT antibodies from all positive IgG samples clearly decreased the inhibitory effect of those samples on APC anticoagulant activity. CONCLUSIONS: Anti-CL/beta2-GPI and anti-PS/PT antibodies independently cause APC resistance, which may contribute to risk of VTE in patients with SLE.