Alinity hq platelet count is not impacted by severe microcytosis.

BACKGROUND: Impedance technology has been shown to overestimate platelet (PLT) count in samples with microcytes, while the optical-fluorescence PLT count (PLT-F) by Sysmex has been suggested to be unaffected by microcytosis. The Abbott Alinity hq analyzer employs multi-dimensional optical PLT counting. Our goal was to assess the accuracy of this technology in microcytic samples. METHODS: Platelet measurements were performed by Alinity hq and the impedance (PLT-I) and PLT-F methods on a Sysmex XN-3000 analyzer on 464 samples. PLT concentration range was 6.56-947 × 109 /L and mean cell volume (MCV) 40.9-123.0 fL. Samples were categorized into normocytic (MCV > 80 fL), microcytic (MCV 65-80 fL), and severely microcytic (MCV < 65 fL) groups. RESULTS: Alinity hq PLT count showed excellent agreement with PLT-F (r = 1.00). Sysmex PLT-I data showed somewhat weaker correlation with both PLT-F and Alinity hq (r = 0.98). Increasing bias between Sysmex PLT-I and PLT-F was seen with decreasing MCV values, with mean bias of 35.2 × 109 /L in severe microcytosis. An inverse relationship was demonstrated between the PLT-I versus PLT-F bias and MCV (p < 0.0001). Consistent mean bias was observed between Alinity hq and PLT-F across all MCV ranges. Mean platelet volume was suppressed or flagged by Sysmex XN in 50% of the samples in the severely microcytic group, and markedly higher red cell distribution width (RDW) was reported compared to Alinity hq (18.1% vs 13.7%, p < 0.0001). CONCLUSION: The Sysmex PLT-I method overestimated the PLT count in samples with severe microcytosis. Alinity hq provided PLT counts and PLT and RBC indices that were not impacted by microcytosis.

Multicenter performance evaluation of the Abbott Alinity hq hematology analyzer.

Background Alinity hq (Abbott) is a new high-throughput hematology analyzer that exclusively employs optical principles for detecting and enumerating blood cells. It reports 29 parameters, including a six-part white blood cell (WBC) differential. The aim of this multicenter study was to evaluate the analytical and clinical performance of the Alinity hq. Methods Complete blood count (CBC) results and morphological flagging were compared to that of CELL-DYN Sapphire (Abbott) and 2 × 200-cell manual differential results, on 1473 whole-blood samples from a well-defined patient population from three different clinical laboratories in the Netherlands. In addition, within-run and within-laboratory precision, linearity, limit of quantitation, carryover and sample stability were assessed. External quality assessment samples were also evaluated. Results Data analysis demonstrated strong concordance of Alinity hq results with those of CELL-DYN Sapphire for all CBC parameters, except for basophil granulocytes. Alinity hq WBC differential showed high level of agreement with manual differential results and exhibited a better agreement with manual basophil results than CELL-DYN Sapphire. The sensitivity of the Alinity hq Blast flag was 57.6%, equal to the 57.6% sensitivity of the CELL-DYN Sapphire's Blast Alert. When considering samples with ≥5% blasts, the sensitivity of the Alinity hq Blast flag was 70.0%. Analytical performance of Alinity hq was shown to be consistent with state-of-the-art (SOTA) performance characteristics. Conclusions Alinity hq CBC measurands demonstrated good overall agreement with results obtained with CELL-DYN Sapphire, as well as manual WBC differential. The analytical and clinical performance characteristics of Alinity hq make it well suited for clinical laboratories.

Standardization of antiphospholipid antibody testing--historical perspectives and ongoing initiatives.

The measurement of antiphospholipid antibodies (aPL) has been an important aspect of antiphospholipid syndrome (APS) characterization since the disease was first described in the 1980s. Despite significant efforts geared toward the standardization of immunoassays that measure anticardiolipin antibodies and anti-ß2-glycoprotein I spanning three decades, there are still reports of significant interassay and interlaboratory variation in the results of these assays. At the recent 13th International Congress on Antiphospholipid Antibodies (APLA 2010, April 13-16, 2010, Galveston, TX), a task force composed of internationally recognized experts in the field of APS was formed to address these issues. In this review, we discuss approaches that have been used in the past to achieve harmonization among aPL immunoassays as well as the ongoing efforts of the APLA task force. Our review also highlights the importance of cutoff determination in aPL assays and the clinical significance of positive aPL results of varying magnitudes.

Interference in antiphospholipid antibody assays.

Antiphospholipid antibodies (aPL) are detected with two types of laboratory tests: first, antigen-specific immunoassays for the determination of antibodies against cardiolipin, ß2-glycoprotein I and other phospholipids, and phospholipid-protein complexes; second, functional (coagulation) assays for the detection of lupus anticoagulants. Both aPL immunoassays and coagulation assays are prone to interferences, and clinicians need to be aware of the limitations of these assays. Interference is a clinically significant bias in the measured analyte concentration due to the effect of another component or property of the sample. Besides immune-mediated interferences (such as heterophile or human anti-animal antibodies, rheumatoid factor, high immunoglobulin levels, or factor inhibitors), aPL assays are uniquely affected by anticoagulants and the presence of residual platelets in test plasma. Interferences are usually analyte- and assay-specific and may go unrecognized in routine laboratory practice. Despite advances in our knowledge on the mechanisms of interferences in aPL assays, it is unlikely that total elimination will be possible.

Accurate white blood cell differential by Alinity hq: A comparison with flow cytometry and manual differential.

INTRODUCTION: White blood cell (WBC) differential by flow cytometry can report a six-part WBC differential and enumerate blasts. Some modern hematology analyzers are also able to provide a six-part WBC differential (including immature granulocytes). Our goal was to compare the WBC differential obtained by the Abbott Alinity hq hematology analyzer to an 8-color single-tube flow cytometry method and to manual WBC differential. METHODS: Samples from 144 patients were tested with Alinity hq, flow cytometry, and microscopic WBC differential. The WBC count ranged from 1.22 to 359 × 109 /L, and 34 samples were flagged by the analyzer for abnormal WBC morphology. RESULTS: Strong concordance was demonstrated between Alinity hq and flow cytometry for all six components of the differential, with correlation coefficients ranging from 0.86 (basophils) to 1.00 (lymphocytes). Small, clinically insignificant positive difference was observed between Alinity hq and flow cytometry for mature and total neutrophils (2.51% and 1.85%) and eosinophils (0.14%), and small negative difference for immature granulocytes (-0.65%), lymphocytes (-0.61%), and basophils (-0.21%). No bias was detected between the Alinity hq and flow cytometry monocyte counts. Alinity hq and flow cytometry results agreed with the manual differential, apart from small, clinically insignificant differences. Alinity hq nucleated red blood cell concentrations were equivalent with the manual results (r = 0.95, slope = 1.16). The percentage of blasts by flow cytometry demonstrated good correlation and agreement with the manual count (r = 0.99, slope = 1.35). CONCLUSION: Alinity hq has produced accurate six-part WBC differential in this three-way comparison, equivalent to flow cytometry and morphological classification.

Clinical utility of Abbott Alinity hq extended red blood cell parameters in differentiating ß-thalassemia trait and iron deficiency anemia.

INTRODUCTION: The objective of the study was to evaluate the performance of the Abbott Alinity hq advanced multi angle polarized scatter separation (MAPSSTM )-based optical RBC technology, for the differentiation between iron deficiency anemia (IDA) and ß-thalassemia carrier status. METHODS: Four hundred and sixty-four samples were analyzed. 228 were healthy controls, 30 were ß-thalassemia carriers, and 40 were IDA. Receiver operating characteristics analysis evaluated the performance of red cell parameters and mathematical formulas. RESULTS: RBC concentration was the most efficient discriminant (area under the curve; AUC of 0.963, Youden Index of 0.88) followed by red blood cell distribution width in size distribution (AUC of 0.960 and YI of 0.86), and red blood cell distribution width coefficient of variation (AUC of 0.924, and YI of 0.74). The absolute reticulocyte concentration showed good diagnostic efficiency, with AUC of 0.808. Hemoglobin distribution width, the %CV of directly measured cellular hemoglobin concentration, and CHCr, the average hemoglobin concentration of reticulocytes have emerged as novel discriminating parameters, with AUC of 0.749 and 0.785, respectively. The England and Fraser index was the best discriminating mathematical formula based on Youden Index of 0.91. The Ricerca, red blood cell distribution width index, Green and King, and Mentzner Index formulas also showed strong discriminative power. The Shine and Lal index, together with the recent mathematical formula M/H, (ratio of percent microcytic and hypochromic red blood cells) demonstrated moderate performance with AUC of 0.796 and 0.740, respectively. CONCLUSION: Extended red cell analysis delivered by the advanced optical technology on the Alinity hq hematology analyzer has efficient diagnostic utility in the initial discrimination of the two most common microcytic anemias: IDA and ß-thalassemia trait.

Alinity hq platelet results are equivalent with the international reference method in thrombocytopenic samples.

INTRODUCTION: Accurate and precise platelet (PLT) count is critical for the appropriate management of patients with thrombocytopenia. This study evaluated the performance of PLT counting with the Abbott Alinity hq hematology analyzer, which utilizes multi-dimensional optical technology. METHODS: Imprecision, linearity, and accuracy were assessed per CLSI guidelines. Alinity hq PLT results were compared to the international flow cytometry reference method (IRM) in the concentration range of 6.3 to 103.0 × 109 /L. Additional comparisons were made with Sysmex XN-3000 PLT counts: impedance (PLT-I), optical (PLT-O), and optical fluorescent (PLT-F) methods. RESULTS: The average within-run %CV was 4.7% on patient samples with PLT concentrations ranging from 13.1 to 41.7 × 109 /L, and the within-laboratory %CV was 3.6% at the level of 68.2 × 109 /L. Linearity evaluation indicated a maximum deviation of 3.1% from the linear fit in the range of 0.1 to 316.8 × 109 /L. Comparison between Alinity hq and the IRM PLT counts yielded a correlation coefficient of 0.99 and predicted bias of 0.0 and -0.5 × 109 /L at 10.0 and 20.0 × 109 /L transfusion thresholds, respectively. Alinity hq PLT counts also correlated well with Sysmex PLT counts, with strongest correlation obtained with PLT-F and PLT-O (r = .99) methods. CONCLUSION: This study demonstrated excellent analytical performance of Alinity hq PLT counting in thrombocytopenic samples, equivalency with the IRM and strong agreement with Sysmex PLT-F and PLT-O methods. The Alinity hq multi-dimensional optical PLT count is available with every CBC without additional reagents and may help promote efficiency in clinical laboratories.

New classification of the shared epitope in rheumatoid arthritis: impact on the production of various anti-citrullinated protein antibodies.

OBJECTIVE: HLA-DR [shared epitope (SE)] alleles have recently been re-classified into S1, S2, S3P and S3D groups. S2 and S3P have been associated with increased risk for RA. We assessed the impact of S1, S2, S3P and S3D alleles on anti-citrullinated protein antibody (ACPA) production. Instead of comparing allele-carriers to non-carriers, we studied each allele group individually, using the X/X (non-SE) genotype as reference. METHODS: Serum and genomic DNA samples of 91 RA patients and 78 healthy controls were obtained. Various ACPAs and IgM RF were determined by ELISA. HLA-DRB1 genotyping and subtyping was performed by PCR. HLA-DRB1 alleles were re-classified as described above. Correlations between SE and ACPAs were determined. RESULTS: Not only S2 and S3P, but, to a lesser extent, S1 and S3D alleles also predisposed to anti-cyclic citrullinated peptide (CCP) production (P < 0.0001, P = 0.004, P = 0.01 and P = 0.027, respectively), with the following hierarchy of association: S2+S3P > S1+S3D > X/X. Similar associations were observed for anti-citrullinated vimentin. Anti-citrullinated fibrinogen (CF) exerted a different association pattern with the strongest correlation with S1 alleles [odds ratio (OR) 16.00; P = 0.05]. In addition, HLA-DRB1*15 alleles may represent a special predisposing effect for anti-CF antibody production. Finally, in this study, RF production was associated only with the HLA-DRB1*0401 SE allele (P = 0.04). CONCLUSIONS: Our approach of comparing individual S allele carriers with X/X genotype patients allowed us to perform unequivocal analyses and demonstrate new associations. Thus, novel subgroups of RA could be identified with potential relevance for prognosis and therapy.

Myth and reality: practical test system for the measurement of anti-DNA antibodies in the diagnosis of systemic lupus erythematosus (SLE).

The myth persists that only the labor intensive Farr radioimmunoassay and Crithidia luciliae immunofluorescence (CL-IFA) are systemic lupus erythematosus (SLE)-specific tests. We compared them to ELISA with bacteriophage lambda DNA (EL-dsDNA) and denatured calf thymus DNA (EL-ssDNA). By percentile ranking, the specificity cut-off level was set both out of clinical context (SOCC) on 100 blood bank donors, and in clinical context (SICC) on 100 patients with either rheumatoid arthritis or scleroderma (50/50). Clinical sensitivity was calculated on 100 random SLE patients. At 95% SICC, the sensitivity of Farr, CL-IFA, EL-dsDNA, and EL-ssDNA was similar (95%CI): 76% (66-84), 76% (66-84), 63% (53-72), and 75% (65-83), respectively; 87% of the patients were positive by at least one method and 55%by all methods. At 99% SICC, the sensitivity was also similar (95% CI): 57% (47-67), 47% (37-57), 58% (47-67), and 43% (33-53), respectively. The areas under ROC curve were similar (95% CI) when patients were used as controls for specificity. At 99% SOCC, EL-ssDNA identified 89% positive, 2 negative but positive by another method at 95% SICC, and 9 negative (i.e. 89/2/9), followed by CL-IFA (80/6/14), Farr (76/12/12), and EL-dsDNA (64/23/13). Thus, at relatively low cost and easy automation, under the same conditions of specificity, the two ELISA tests combined were at least as good, if not superior, to CL-IFA or Farr: they showed similar clinical sensitivity and also identified more patients with anti-DNA antibodies.

Smad-independent transforming growth factor-beta regulation of early growth response-1 and sustained expression in fibrosis: implications for scleroderma.

Transforming growth factor-beta (TGF-beta) plays a key role in scleroderma pathogenesis. The transcription factor early growth response-1 (Egr-1) mediates the stimulation of collagen transcription elicited by TGF-beta and is necessary for the development of pulmonary fibrosis in mice. Here, we report that TGF-beta causes a time- and dose-dependent increase in Egr-1 protein and mRNA levels and enhanced transcription of the Egr-1 gene via serum response elements in normal fibroblasts. The ability of TGF-beta to stimulate Egr-1 was preserved in Smad3-null mice and in explanted Smad3-null fibroblasts. The response was blocked by a specific mitogen-activated protein kinase kinase 1 (MEK1) inhibitor but not by an ALK5 kinase inhibitor. Furthermore, MEK1 was phosphorylated by TGF-beta, which was sufficient to drive Egr-1 transactivation. Stimulation by TGF-beta enhanced the transcriptional activity of Elk-1 via the MEK-extracellular signal-regulated kinase 1/2 pathway. Bleomycin-induced scleroderma in the mouse was accompanied by increased Egr-1 accumulation in lesional fibroblasts. Furthermore, biopsies of lesional skin and lung from patients with scleroderma showed increased Egr-1 levels, which were highest in early diffuse disease. Moreover, both Egr-1 mRNA and protein were elevated in explanted scleroderma skin fibroblasts in vitro. Together, these findings define a Smad-independent TGF-beta signal transduction mechanism that underlies the stimulation of Egr-1, demonstrate for the first time sustained Egr-1 up-regulation in fibrotic lesions and suggests that Egr-1 has a role in the induction and progression of fibrosis.

Molecular genetic analysis of Hungarian patients with the hyper-immunoglobulin M syndrome.

We have identified 9 disease-causing mutations in 18 hyper-immunoglobulin M (HIGM) syndrome patients from ten unrelated Hungarian families. CD40L mutation resulted in X-linked combined immunodeficiency in 11 patients (6 families) and AICDA mutation caused autosomal recessive HIGM characterized by B cell immunodeficiency in 5 patients (3 families). Two brothers with a genetically undefined form of HIGM and clinical manifestations of B cell deficiency were also included in this study. B cells from these two patients had defective CSR and skewed pattern of somatic hypermutation. Altogether, a novel CD40L truncation mutation (c.470 delA) and a new missense AICDA mutation (p.E58K) were identified. Carrier status was defined in 13 clinically healthy individuals allowing prenatal genetic testing that was performed in two affected families. This is the first comprehensive overview of molecular genetic features of Hungarian patients with HIGM syndrome.

Performance evaluation of the prototype Abbott Alinity hq hematology analyzer.

INTRODUCTION: The analytical and clinical performance as well as the workflow efficiency of the novel, prototype Alinity hq hematology analyzer was evaluated in the clinical laboratory of Universitair Ziekenhuis Brussel, Department of Hematology, Brussels, Belgium. METHODS: Within-run and within-laboratory imprecision, linearity, and carryover were assessed using clinical blood samples and commercial blood products. Four hundred and seventeen samples were selected for method comparison with Abbott CELL-DYN Sapphire, and for flagging performance analysis in comparison with smear review and manual microscopic white blood cell (WBC) differential. RESULTS: Within-run and within-laboratory imprecision verification demonstrated low %CV for complete blood count and WBC differential results within the normal ranges (0.1%-10.4%), except for basophil granulocytes. The linearity of the analytical measuring ranges was verified for WBCs, red blood cells, hemoglobin, and platelets. Alinity hq results showed strong agreement with those of CELL-DYN Sapphire. Good correlation was demonstrated with manual WBC differential results, with negative bias for neutrophil (NEU) granulocytes, and positive bias for lymphocytes and monocytes. Blasts were detected with 75% sensitivity and 96% specificity at 1% blast threshold, and 100% sensitivity at 5% blast threshold. Immature granulocyte detection was more sensitive (81% vs 76%, P = 0.086) and specific (88% vs 78%, P = 0.0002) than with CELL-DYN Sapphire. Nucleated red blood cell detection was more sensitive (89% vs 63%, P < 0.001) and just slightly less specific (96% vs 99%, P = 0.0067) than with CELL-DYN Sapphire. Re-run and reflex testing rates were lower with Alinity hq. CONCLUSION: The Alinity hq hematology analyzer is suitable for clinical use.

Association of HELLP syndrome with primary antiphospholipid syndrome--a case report.

Authors present the first Hungarian case of a young pregnant woman with the association of antiphospholipid syndrome (APS) and hemolysis, elevated liver enzymes, and low platelet count (HELLP) syndrome. After the onset of severe preeclampsia, the pregnancy was terminated but the patient's condition continued to worsen. New symptoms of APS, including deep vein thrombosis and ischemic nervus opticus lesion, developed in the patient followed by the onset of acute respiratory distress syndrome, which required respiratory therapy. Intensive treatment with plasmapheresis, high-dose intravenous immunoglobulin, high-dose corticosteroids, cyclophosphamide, and anticoagulants eventually led to full recovery. There have been only few scattered reports in the literature on the association of HELLP syndrome and APS, which was successfully managed with the combination of various immunosuppressive and immunomodulatory treatment modalities.

Associations between serum anti-CCP antibody, rheumatoid factor levels and HLA-DR4 expression in Hungarian patients with rheumatoid arthritis.

BACKGROUND: The presence of anti-cyclic citrullinated peptide autoantibody is highly specific for rheumatoid arthritis. Certain HLA-DR4 (HLA-DRB1*04) alleles, also known as the "shared epitope," are associated with increased susceptibility to RA. In addition, these alleles may also have relevance for disease outcome. Anti-CCP antibody positivity has been associated with the presence of HLA-DR4 alleles in patients with RA. However, there is little information regarding a relationship between quantitative anti-CCP production (serum anti-CCP concentrations) and the shared epitope. OBJECTIVES: To determine the association between anti-CCP antibody production and various HLA-DRB1 alleles. METHODS: Serum anti-CCP, rheumatoid factor and C-reactive protein levels were assessed in 53 RA patients. All these patients underwent HLA-DRB1 genotyping. RESULTS: Of the 53 patients 33 (62%) were positive for anti-CCP antibody. We found significant correlations between anti-CCP and RF positivity (chi-square = 6.717, P < 0.01), as well as between anti-CCP and HLA-DRB1*04 positivity (chi-square = 5.828, P < 0.01). There was no correlation between RF positivity and serum levels, CRP serum levels and HLA-DRB1*04 positivity. When quantitatively comparing serum anti-CCP levels with shared epitope positivity, patients carrying one or two copies of HLA-DRB1*04 alleles had significantly higher anti-CCP concentrations (530.0 +/- 182.6 U/ml) compared to DRB1*04-negative patients (56.8 +/- 27.4 U/ml) (P < 0.01). There was no difference in serum anti-CCP antibody concentrations between patients carrying only one HLA-DRB1*01 allele but no HLA-DRB1*04 allele (12.0 +/- 8.6 U/ml) compared to SE-negative patients (76.8 +/- 56.2 U/ml). Regarding non-SE HLA-DRB1 genotypes, all 6 patients (100%) carrying DRB1*15 alleles and 6 of 7 (85%) patients carrying DRB1*13 were anti-CCP positive. In addition, patients with HLA-DRB1*13 (282.5 +/- 23.8 U/ml) and DRB1*15 (398.7 +/- 76.2 U/ml) produced significantly more anti-CCP than did any other non-SE HLA-DRB1 subtypes (P < 0.01). CONCLUSIONS: There is significant association between anti-CCP and RF, as well as between anti-CCP and SE positivity in RA. In addition, the presence of one or two copies of HLA-DRB1*04 alleles has been associated with higher serum anti-CCP antibody levels. Thus, patients carrying HLA-DRB1*04 alleles exhibited an overall tenfold increase in serum anti-CCP antibody levels in comparison to HLA-DRB1*04-negative subjects. Increased anti-CCP production may also be associated with other non-SE HLA-DRB1 genotypes, such as DRB1*13 or DRB1*15. In reports by other investigators, both anti-CCP concentrations and SE positivity were related to more rapid disease progression and unfavorable outcome.

Seroreactivity to microbial components in Crohn's disease is associated with ileal involvement, noninflammatory disease behavior and NOD2/CARD15 genotype, but not with risk for surgery in a Hungarian cohort of IBD patients.

BACKGROUND: Antibodies directed against Saccharomyces cerevisiae (ASCA), perinuclear components of neutrophils (pANCA), and porin protein C of Escherichia coli (anti-OmpC) are reported to be associated with disease phenotype and may be of diagnostic importance in inflammatory bowel disease (IBD). Since limited data are available from Eastern Europe, we assessed the above antibodies in Hungarian IBD patients. METHODS: In all, 653 well-characterized, unrelated consecutive IBD patients (Crohn's disease [CD]: 558, m/f: 263/295, duration: 8.1 +/- 10.7 years; ulcerative colitis [UC]: 95, m/f: 44/51, duration: 8.9 +/- 9.8 years) and 100 healthy subjects were investigated. Sera were assayed for anti-Omp and ASCA by enzyme-linked immunosorbent assay (ELISA) and ANCA by indirect immunofluorescence assay (IIF). TLR4 and NOD2/CARD15 variants were tested by polymerase chain reaction/restriction fragment length polymorphism (PCR-RFLP). Detailed clinical phenotypes were determined by reviewing the medical charts. RESULTS: Anti-Omp, ASCA, and atypical pANCA antibodies were present in 31.2%, 59.3%, and 13.8% of CD, 24.2%, 13.7%, and 48.5% of UC patients, and in 20%, 16%, and 5.6% of controls, respectively. ASCA and anti-Omp positivity were associated with increased risk for CD (odds ratio [OR](ASCA) = 7.65, 95% confidence interval [CI]: 4.37-13.4; OR(Omp) = 1.81, 95% CI: 1.08-3.05). In a logistic regression analysis, anti-Omp and ASCA were independently associated with ileal and noninflammatory disease, but not with a risk for surgery or response to steroids or infliximab. A serology dosage effect was also observed. ASCA and anti-Omp antibodies were associated with NOD2/CARD15, in addition to a gene dosage effect. No associations were found in UC. CONCLUSIONS: Serological markers were useful in the differentiation between CD and UC in an Eastern European IBD cohort. Reactivity to microbial components was associated with disease phenotype and NOD2/CARD15 genotype, further supporting the role of altered microbial sensing in the pathogenesis of CD.

Increased production of the soluble tumor-associated antigens CA19-9, CA125, and CA15-3 in rheumatoid arthritis: potential adhesion molecules in synovial inflammation?

Some tumor-associated antigens (TAAs) are expressed on inflammatory cells. We previously detected carcinoembryonic antigen (CEA; CD66) in the rheumatoid (RA) synovium. The production of CEA, CA19-9, CA125, and CA15.3, may be increased in patients with RA, scleroderma, lupus, and Sjögren's syndrome (SS). Some of these TAAs contain sialylated carbohydrate motifs and they are involved in tumor-associated cell adhesion and metastasis. We assessed levels of TAAs in the sera of RA patients and healthy subjects. Serum TAA levels were correlated with disease markers including serum rheumatoid factor (RF), C-reactive protein (CRP), and anti-CCP antibody levels, DAS28, age disease duration. TAAs including CEA, CA15-3, CA72-4, CA125, and CA19-9, and neuron-specific enolase (NSE) were assessed by immunoassay in the sera of 75 patients with RA and 50 age- and sex-matched healthy controls. Normal upper limits for these TAAs were 3.4 microg/L, 25 kU/L, 6.9 kU/L, 35 kU/L, 34 kU/L, and 16.3 microg/L, respectively. There were significantly more RA patients showing abnormally high levels of CA125 (10.8% versus 7.1%), CA19-9 (8.1% versus 0%), and CA15-3 (17.6% versus 14.3%) in comparison to controls (P < 0.05). The mean absolute serum levels of CA125 (23.9 +/- 1.8 versus 16.8 +/- 2.2 kU/L) and CA19-9 (14.2 +/- 1.2 versus 10.5 +/- 1.6 kU/L) were also significantly higher in RA compared to controls (P < 0.05). Among RA patients, serum CEA showed significant correlation with RF (r = 0.270; P < 0.05). None of the assessed TAAs showed any correlation with CRP, anti-CCP, DAS28, age or disease duration. The concentration of some TAAs may be elevated in the sera of patients with established RA in comparison to healthy subjects. CEA, CA19-9, CA125, and CA15-3 contain carbohydrate motifs and thus they may be involved in synovitis-associated adhesive events. Furthermore, some TAAs, such as CEA, may also correlate with prognostic factors, such as serum RF levels.

Clinical thrombotic manifestations in SLE patients with and without antiphospholipid antibodies: a 5-year follow-up.

OBJECTIVE: To analyze the association of antiphospholipid antibodies (aPL) with the development of clinical thrombotic manifestations and to characterize the efficacy of anti-thrombotic therapies used. METHODS: 272 systemic lupus erythematosus (SLE) patients participated in the study. Patient files and a cumulative database were used to collect patients' medical histories. Anti-cardiolipin (aCL), anti-beta2-glycoprotein I (abeta2GPI) antibodies, and lupus anticoagulant (LAC) were measured according to international recommendations. New thrombotic events were registered during follow-up. RESULTS: The patients were prospectively studied for 5 years, of whom 107 were aPL negative (aPL- group). Criteria for antiphospholipid syndrome (APS) were fulfilled by 84 of 165 aPL-positive patients (APS+ group) indicating that SLE patients with aPL have around 50% risk to develop thrombotic complications. The aPL+ group (n = 81) consisted of aPL+ but APS- patients. LAC was the most common aPL (n = 27, 32.1%) in patients with APS. The cumulative presence of aPL further increased the prevalence of thrombotic events. During the follow-up period, aPL developed in 8 of 107 patients (7.5%) from the aPL- group, of whom 3 (2.8%) presented with thrombotic complications. Other types of aPL developed in 7 of 165 (4.2%) aPL+ patients within 5 years. New thrombotic events occurred in 3.7% of aPL+ (n = 3) and 8.3% (n = 7) of the APS group. During follow-up, 52 of 81 aPL+ patients received primary prophylaxis, and 1 (1.9%) had transient ischemic attack (TIA). In the non-treatment group, 2 (6.9%) had stroke. Seventy-nine of 84 of the APS patients received secondary prophylaxis, and myocardial infarction occurred in 2 patients (on cumarine therapy maintaining an international normalized ratio around 2.5-3.0), and 5 suffered a stroke/TIA (1 on aspirin and 4 on aspirin + cumarine). CONCLUSION: The findings emphasize the importance of determining both aCL and abeta2GPI antibodies and LAC in SLE patients and the need for adequate anticoagulant therapy.

Intracellular TGF-beta receptor blockade abrogates Smad-dependent fibroblast activation in vitro and in vivo.

Fibrosis, the hallmark of scleroderma, is characterized by excessive synthesis of collagen and extracellular matrix proteins and accumulation of myofibroblasts. Transforming growth factor-beta (TGF-beta), a potent inducer of collagen synthesis, cytokine production, and myofibroblast transdifferentiation, is implicated in fibrosis. Profibrotic TGF-beta responses are induced primarily via the type I activin-like receptor kinase 5 (ALK5) TGF-beta receptor coupled to Smad signal transducers. Here, we investigated the effect of blocking ALK5 function with SM305, a novel small-molecule kinase inhibitor, on fibrotic TGF-beta responses. In normal dermal fibroblasts, SM305 abrogated the ligand-induced phosphorylation, nuclear import, and DNA-binding activity of Smad2/3 and Smad4, and inhibited Smad2/3-dependent transcriptional responses. Furthermore, SM305 blocked TGF-beta-induced extracellular matrix gene expression, cytokine production, and myofibroblast transdifferentiation. In unstimulated scleroderma fibroblasts, SM305 caused a variable and modest reduction in type I collagen levels, and failed to abrogate constitutive nuclear accumulation of Smad2/3, or alter the proportion of smooth muscle actin stress fiber-positive fibroblasts. In vivo, SM305 prevented TGF-beta-induced Smad2/3 phosphorylation type I collagen (COL1)A2 promoter activation in dermal fibroblasts. Taken together, these results indicate that SM305 inhibits intracellular TGF-beta signaling through selective interference with ALK5-mediated Smad activation, resulting in marked suppression of profibrotic responses induced by TGF-beta in vivo and in vitro.