PDGF enhances orbital fibroblast responses to TSHR stimulating autoantibodies in Graves' ophthalmopathy patients.

PURPOSE: Thyroid-stimulating hormone receptor (TSHR) stimulating autoantibodies are associated with Graves' ophthalmopathy (GO), the orbital manifestation of Graves' disease (GD). TSHR autoantibody levels and orbital TSHR expression levels correlate positively with GO disease activity. Platelet-derived growth factors (PDGF) are increased in GO and potently activate orbital fibroblast effector functions. We investigated the possible relationship between PDGF and TSHR expression on orbital fibroblasts and how that influences the immunopathological effects of TSHR autoantibodies on orbital fibroblast activity. METHODS: Orbital fibroblasts were stimulated with PDGF-AA, PDGF-AB, and PDGF-BB, and TSHR expression was determined by flow cytometry. Stimulatory effects of bovine TSH and GD immunoglobulins on orbital fibroblasts (with or without PDGF-BB preincubation) were determined by IL-6, IL-8, chemokine (C-C motif) ligand (CCL)-2, CCL5, CCL7, and hyaluronan ELISA. The TSHR blocking antibody K1-70 and the cAMP inhibitor H89 were used to determine involvement of TSHR signaling. RESULTS: PDGF-AB and PDGF-BB stimulation increased TSHR expression on orbital fibroblasts, whereas PDGF-AA did not. Furthermore, stimulation with bovine TSH and immunoglobulins from GD patients induced IL-6, IL-8, CCL2, and hyaluronan production by orbital fibroblasts, and PDGF-BB preincubation enhanced this response of orbital fibroblasts. Blocking studies with a TSHR blocking antibody and a cAMP inhibitor inhibited these effects, indicating the involvement of TSHR signaling and thus of TSHR stimulating autoantibodies herein. CONCLUSIONS: These findings indicate that PDGF-B containing PDGF isoforms amplify the immunopathological effects of TSHR-stimulating autoantibodies in GO patients by stimulating TSHR expression on orbital fibroblasts.

Orbit-infiltrating mast cells, monocytes, and macrophages produce PDGF isoforms that orchestrate orbital fibroblast activation in Graves' ophthalmopathy.

PURPOSE: Platelet-derived growth factors (PDGF) are regulators of fibroblast activity that may be involved in the pathophysiology of Graves' ophthalmopathy (GO). We unraveled the expression and origin of PDGF family members in GO orbital tissue and investigated the effect of PDGF isoforms on IL-6 and hyaluronan production and proliferation by orbital fibroblasts. METHODS: PDGF-A, PDGF-B, PDGF-C, PDGF-D, PDGF-Rα, and PDGF-Rß expression was determined by real-time quantitative PCR and PDGF-A and PDGF-B protein expression was determined by Western blot in orbital tissues. Orbital tissues were immunohistochemically stained for PDGF-A and PDGF-B expression, together with stainings for T cells, monocytes, B cells, macrophages, and mast cells. Effects of PDGF-AA, PDGF-AB, and PDGF-BB on orbital fibroblast proliferation and IL-6 and hyaluronan production were examined. Finally, effects of PDGF-BB- and PDGF-AA-neutralizing antibodies on IL-6 and hyaluronan production in GO whole orbital tissue cultures were tested. RESULTS: GO orbital tissue showed increased PDGF-A and PDGF-B mRNA and protein levels. Increased numbers of PDGF-A- and PDGF-B-positive monocytes, macrophages, and mast cells were present in GO orbital tissue. PDGF-BB stimulated proliferation and hyaluronan and IL-6 production by orbital fibroblasts the most, followed by PDGF-AB and PDGF-AA. Finally, in particular imatinib mesylate and PDGF-BB-neutralizing antibodies reduced IL-6 and hyaluronan production by whole orbital tissue cultures from GO patients. CONCLUSIONS: In GO, mast cells, monocytes, and macrophages may activate orbital fibroblasts via secretion of especially PDGF-AB and PDGF-BB. Preclinical studies with whole orbital tissue cultures show that blocking PDGF-B chain containing isoforms can be a promising treatment for GO.

Human chorionic gonadotropin treatment of anti-Hu-associated paraneoplastic neurological syndromes.

OBJECTIVE: Paraneoplastic neurological syndromes associated with anti-Hu antibodies (Hu-PNS) are mediated by a T-cell immune response that is directed against the Hu antigens. In pregnancy, many Th1-mediated autoimmune diseases such as rheumatoid arthritis and multiple sclerosis regress. We hypothesised that this decreased disease activity during pregnancy may be related to high human chorionic gonadotropin (hCG) levels. METHODS: 15 Hu-PNS patients were treated in a prospective, uncontrolled and unblinded trial with 10,000 IU daily of hCG administered by intramuscular injection during 12 weeks. Primary outcome measures were functional improvement defined as a decrease of one or more points on the modified Rankin Scale (mRS) or stabilisation in patients with mRS score ≤3 and improvement of neurological impairment assessed with the Edinburgh Functional Impairment Tests (EFIT). Secondary end points included the change in activities of daily living as evaluated using the Barthel Index. RESULTS: Seven of 15 patients (47%) improved on the mRS or stabilised at mRS score ≤3. Four patients (27%) showed significant improvement of neurological impairment as indicated by an overall Edinburgh Functional Impairment Tests score of ≥1 point. Five patients improved on the Barthel Index (33%). CONCLUSION: Comparison with previous studies suggests that hCG may have immunomodulatory activity and may modify the course of Hu-PNS, although well-established confounding factors may have contributed in this uncontrolled trial.

Induction of antibodies by Staphylococcus aureus nasal colonization in young children.

In order to develop novel antistaphylococcal strategies, understanding the determinants of carriage and how humans respond to Staphylococcus aureus exposure is essential. Here, the primary S. aureus-specific humoral immune response and its association with nasal colonization was studied in young children. Sera from 57 colonized or non-colonized children, serially collected at birth and at 6, 14 and 24 months, were analysed for IgG, IgA and IgM binding to 19 staphylococcal proteins, using flow cytometry-based technology. The antibody responses showed extensive inter-individual variability. On average, the levels of antistaphylococcal IgA and IgM increased from birth until the age of 2 years (p <0.05), whereas the levels of IgG decreased (p <0.001). Placentally transferred maternal IgG did not protect against colonization. In colonized children, IgG and IgA levels for a number of proteins were higher than in non-colonized children. At both 14 and 24 months, the levels of IgG against chemotaxis inhibitory protein of S. aureus (at 24 months; median fluorescence intensity, 4928 vs. 24, p <0.05), extracellular fibrinogen-binding protein (987 vs. 604, p <0.05), and iron-responsive surface determinant H (62 vs. 5, p <0.05) were significantly higher in colonized children. The levels of IgA against CHIPS, IsdH and IsdA were higher (p <0.05). Therefore, CHIPS, Efb, IsdA and IsdH seem to play a role in nasal colonization of young children.

Women with rheumatoid arthritis negative for anti-cyclic citrullinated peptide and rheumatoid factor are more likely to improve during pregnancy, whereas in autoantibody-positive women autoantibody levels are not influenced by pregnancy.

OBJECTIVES: To determine whether changes in levels of anti-cyclic citrullinated peptide (anti-CCP) and rheumatoid factor (RF) are associated with the spontaneous improvement of rheumatoid arthritis (RA) during pregnancy and with the subsequent flare post partum. METHODS: Disease activity scores from the Pregnancy-induced Amelioration of Rheumatoid Arthritis (PARA) study of 118 patients were available for analysis. Before conception (if applicable), at each trimester and at 6, 12 and 26 weeks post partum, levels of the autoantibodies anti-CCP, IgM-RF, IgG-RF and IgA-RF were determined. Responses in disease activity were classified according to European League Against Rheumatism (EULAR) response criteria during pregnancy and post partum, and associated with the presence or absence of autoantibodies. RESULTS: The median levels of anti-CCP and all subclasses of RF during pregnancy were stable, whereas post partum the levels of anti-CCP, IgM-RF and IgA-RF declined. A significantly higher percentage of women without autoantibodies (negative for anti-CCP and RF) improved compared with women positive for either or both autoantibodies (75% vs 39%, p = 0.01). The occurrence of a flare post partum was comparable between these groups. CONCLUSIONS: Improvement of disease activity of RA during pregnancy was not associated with changes in levels of autoantibodies during pregnancy, however, improvement may occur more frequently in the absence of anti-CCP and RF.

Detection of anti-MAG antibodies in polyneuropathy associated with IgM monoclonal gammopathy.

BACKGROUND: Detection of serum antibodies to myelin-associated glycoprotein (MAG) by Western blot (WB) is a valuable assay to diagnose a distinct type of demyelinating polyneuropathy with immunoglobulin M (IgM) monoclonal gammopathy. In this study, the diagnostic accuracy of a new and more practical ELISA to detect these antibodies was validated. METHODS: Routine WBs from 2 independent laboratories and ELISA were used to detect anti-MAG IgM in serum from 207 patients with neuropathy and controls. The sensitivity and specificity of these assays were compared and related to the patient clinical and electrophysiologic characteristics. RESULTS: In ELISA, anti-MAG antibodies were found in serum from 49 (72%) of 68 patients with demyelinating polyneuropathy and IgM monoclonal gammopathy. However, in this subgroup of patients, only 30 (44%) and 37 (54%) were positive in the 2 WBs. All of the patients positive in the 2 WBs were also positive in ELISA. A high correlation was found for IgM activity in ELISA to MAG and sulfate-3-glucuronyl paragloboside (SGPG) (Spearman rho = 0.72, p < 0.0001), supporting the notion that the shared sulfated glucuronic acid moiety of MAG and SGPG is preserved. Most patients positive in anti-MAG ELISA had a slowly progressive sensory-motor demyelinating polyneuropathy, even if the WB was negative. In control groups, however, 4 WB-negative patients with a nondemyelinating monoclonal gammopathy-related polyneuropathy were positive in anti-MAG ELISA. The remaining samples were negative in ELISA. CONCLUSION: ELISA is more sensitive than Western blot to diagnose anti-myelin-associated glycoprotein related polyneuropathy, although a positive serology may be found in other forms of polyneuropathy as well.

Granulocytosis and thrombocytosis in renal cell carcinoma: a pro-inflammatory cytokine response originating in the tumour.

BACKGROUND: In up to 20% of patients with renal cell cancer (RCC) an inflammatory response consisting of low-grade fever, weight loss and an elevated ESR and CRP may occur with modest granulocytosis and thrombocytosis. Clinical and experimental data suggest a pathogenic role for tumour-derived cytokine production, especially interleukin-6. CASE REPORT: A 79-year-old female with RCC presented with low-grade fever, weight loss and overt granulocytosis and thrombocytosis. Radiological examination revealed a right-sided renal tumour. During nephrectomy a gradient between the IL-6 levels in the renal artery and vein was demonstrated, providing direct evidence for in vivo production of IL-6 by the tumour affected kidney, which was confirmed by the demonstration of IL -6 in the tumour cells by immunohistochemical staining and in the supernatant of the homogenised tumour. Cytogenetic examination revealed complex abnormalities including a gain of chromosome 7. In addition we demonstrated production of IL-1alpha, IL-1beta, IL-8 and ICAM-1 in the tumour with systemic elevated levels of IL-6 and IL-8 with secondary increased serum G-CSF and TPO levels. CONCLUSION: We have provided direct evidence for the production of pro-inflammatory cytokines by renal cancer cells in a patient with RCC and a profound inflammatory response, with a central role of IL-6, probably due to a gain of chromosome 7. The extreme granulocytosis and thrombocytosis may have resulted from the secondary systemic production of G-CSF and TPO.

Is imatinib mesylate a promising drug in systemic sclerosis?

A patient with therapy-resistant and progressive systemic sclerosis (SSc) with pulmonary involvement who was treated with imatinib mesylate is described herein. Prior to treatment, pulmonary fibroblasts obtained from the patient were cultured and incubated with imatinib mesylate. Preincubation of the fibroblasts for 16 hours with 2.5 microg/ml imatinib mesylate efficiently abrogated platelet-derived growth factor BB-induced fibroblast proliferation. Furthermore, transforming growth factor beta1-induced type I collagen gene transcription was blocked. During treatment, the patient's pulmonary involvement stabilized and her skin tightness improved. To our knowledge, this is the first report of a patient with therapy-refractory SSc responding to treatment with imatinib mesylate.

Imbalances in circulating lymphocyte subsets in Hu antibody associated paraneoplastic neurological syndromes.

In paraneoplastic neurological syndromes (PNS) associated with small cell lung cancer (SCLC) and Hu antibodies, neuron-specific Hu antigens expressed by the tumour hypothetically trigger an immune response that cross-reacts with Hu antigens in the nervous system, resulting in tumour suppression and neuronal damage. To gain more insight into the hypothesized cell-mediated immune pathogenesis of these syndromes, we analysed the circulating lymphocyte subsets in untreated patients with SCLC, PNS and Hu antibodies (n = 18), SCLC without PNS (n = 19) and controls (n = 29) using flow cytometry. SCLC patients with PNS had a variety of imbalances within their circulating lymphocyte subsets as compared with SCLC patients without PNS and healthy controls: (i) a lymphopenia of the major subsets (i.e. B, CD4+ and CD8+ T lymphocytes); (ii) increased proportions of activated CD4+ and CD8+ T cells; (iii) reduced numbers of terminally differentiated effector CD8+ T cells and cells with a cytotoxic T-cell phenotype (CD56+ and CD57+). Although indirect, our data provide further support for the involvement of T cells in the pathogenesis of Hu antibody associated PNS.

[Three patients with a paraneoplastic neurological syndrome: the significance of paraneoplastic antibodies]. / Drie patiënten met een paraneoplastisch neurologisch syndroom; de betekenis van paraneoplastische antistoffen.

Establishing the presence of paraneoplastic antibodies is important in identifying an often severe neurological syndrome as paraneoplastic and hence directing the search for an underlying neoplasm. A paraneoplastic neurological syndrome was diagnosed in 3 patients. The first was a 64-year-old woman in whom paraneoplastic encephalomyelitis was diagnosed. The diagnosis was strongly supported by a high titre of serum anti-Hu antibodies, despite three negative biopsies from a mediastinal mass. The patient died of a non-convulsive status epilepticus; autopsy revealed not only paraneoplastic encephalomyelitis but also small-cell lung cancer. The second patient was a 55-year-old woman with metastatic breast cancer. After a three-year period of progressive neurological deterioration, a high titre of anti-CV2/CRMP5 antibodies was detected, on the basis of which the clinical syndrome was diagnosed as paraneoplastic. She received immunotherapy and her condition stabilised. The third patient, a 41-year-old man, presented with severe limbic encephalitis. Biopsy from a paraaortic mass was positive for undifferentiated carcinoma. The patient had a high titre ofanti-Ma2 antibodies and was subsequently tested positive for serum alpha-foetoprotein (AFP) and beta-human-chorionic gonadotrophin (bta-HCG). During chemotherapy for a non seminoma testicular cancer, the limbic encephalitis improved both clinically and radiologically, but the patient died as a result of the toxicity of the treatment.

[A retrospective study on the role of antibodies against soluble liver antigen (anti-SLA antibodies) and other autoantibodies in the diagnostics of autoimmune hepatitis]. / Een retrospectieve studie naar de rol van antistoffen tegen oplosbaar ('soluble') leverantigeen (anti-sLA-antistoffen) en van andere autoantistoffen bij de diagnostiek van auto-immune hepatitis.

OBJECTIVE: To determine the diagnostic value of antibodies against soluble liver antigen (anti-SLA antibodies) and a number of other antibodies for the diagnosis ofautoimmune hepatitis (AIH). DESIGN: Retrospective. METHOD: Anti-SLA, antinuclear antibodies (ANA), antibodies against smooth muscle (anti-SMA), anti-neutrophil cytoplasm antibodies (peri-nuclear pattern; pANCA) and antibodies against liver-kidney microsomal antigen type 1 (anti-LKM-1) were determined in the sera of 97 patients with AIH and 121 patients with other liver disorders including viral, drug-related and alcoholic liver disease. The sensitivity and specificity of each of the antibodies, or a combination ofantibodies, were calculated for the diagnosis 'AIH'. RESULTS: Anti-SLA antibodies were found only in AIH patients (specificity: 100%); 1 in 7 AIH patients (14%) had these antibodies and in 2% they were the only detectable antibodies. Anti-LKM-1 antibodies also showed a 100% specificity for AIH although the sensitivity was much lower (2%). Whilst the sensitivity of ANA (53%), pANCA (39%) and SMA (39%) was higher, the specificity of these antibodies for AIH was lower. 20% of AIH patients tested negative for all autoantibodies. The simultaneous presence of more than one antibody increased the probability of AIH diagnosis. CONCLUSION: When AIH is suspected, the presence of one or a combination ofanti-SLA, ANA, anti-SMA, anti-LKM-1 and pANCA antibodies is helpful for the often difficult differential diagnosis between AIH and other liver disorders. Anti-SLA antibodies are specific for AIH and appear to be a useful diagnostic parameter.

A hampered chemoattractant-induced cytoskeletal rearrangement in granulocytes of patients with unexplained severe chronic and relapsing infections of the upper and lower airways. In vitro restoration by G-CSF exposure.

Granulocytes play a major role in host defense against bacterial infections. Severe inborn defects in granulocyte function are associated with fulminant bacterial infections in early childhood. Subtle disturbances in granulocyte function might contribute to an enhanced susceptibility to bacterial infections in adulthood. We investigated chemoattractant (N-formyl-methionyl-leucyl-phenylalanine, fMLP and casein) induced cytoskeletal rearrangements (polarization) of blood granulocytes in 77 adults with chronic and recurrent therapy-resistant infections of the upper and lower airways. These infections could not be explained by B- and/or T-cell defects or local anatomic abnormalities. Besides polarization, chemotaxis of blood granulocytes was measured in 33 patients, as well as granulocyte superoxide production in eight patients. The chemoattractant-induced cytoskeletal rearrangement in patient blood granulocytes was significantly lower as compared to healthy control values with both fMLP and casein as stimuli. About two-thirds of the patients showed a defective polarization response to fMLP. Granulocyte colony-stimulating factor (G-CSF) when added in vitro corrected the defective polarization responses; responses in the normal range were not enhanced. The chemotactic motility of patient blood granulocytes was also slightly, but significantly lowered. However, it did not correlate to the lowered polarization. Granulocyte superoxide production was comparable in patients and in healthy controls. Our data thus show that subtle abnormalities in chemoattractant-induced cytoskeletal and motile function of blood granulocytes are frequent in patients with severe therapy-refractory bacterial infections of the upper and lower airways.

Therapeutic immune reconstitution in HIV-1-infected children is independent of their age and pretreatment immune status.

OBJECTIVE: To evaluate long-term immune reconstitution of children treated with highly active antiretroviral therapy (HAART). METHODS: The long-term immunological response to HAART was studied in 71 HIV-1-infected children (aged 1 month to 18 years) in two prospective, open, uncontrolled national multicentre studies. Blood samples were taken before and after HAART was initiated, with a follow-up of 96 weeks, and peripheral CD4 and CD8 T cells plus naive and memory subsets were identified in whole blood samples. Relative cell counts were calculated in relation to the median of the age-specific reference. RESULTS: The absolute CD4 cell count and percentage and the CD4 cell count as a percentage of normal increased significantly (P < 0.001) to medians of 939 x 106 cells/l (range, 10-3520), 32% (range, 1-50) and 84% (range, 1-161), respectively, after 48 weeks. This increase was predominantly owing to naive CD4 T cells. There was a correlation between the increase of absolute naive CD4 T cell counts and age. However, when CD4 T cell restoration was studied as percentage of normal values, the inverse correlation between the increase of naive CD4 T cell count and age was not observed. In addition, no difference in immunological reconstitution was observed at any time point between virological responders and non-responders. CONCLUSIONS: Normalization of the CD4 cell counts in children treated with HAART is independent of age, indicating that children of all age groups can meet their CD4 T cell production demands. In general, it appears that children restore their CD4 T cell counts better and more rapidly than adults, even in a late stage of HIV-1 infection.

Molecular and flow cytometric analysis of the Vbeta repertoire for clonality assessment in mature TCRalphabeta T-cell proliferations.

Clonality assessment through Southern blot (SB) analysis of TCRB genes or polymerase chain reaction (PCR) analysis of TCRG genes is important for diagnosing suspect mature T-cell proliferations. Clonality assessment through reverse transcription (RT)-PCR analysis of Vbeta-Cbeta transcripts and flow cytometry with a Vbeta antibody panel covering more than 65% of Vbeta domains was validated using 28 SB-defined clonal T-cell receptor (TCR)alphabeta(+) T-ALL samples and T-cell lines. Next, the diagnostic applicability of the V(beta) RT-PCR and flow cytometric clonality assays was studied in 47 mature T-cell proliferations. Clonal Vbeta-Cbeta RT-PCR products were detected in all 47 samples, whereas single Vbeta domain usage was found in 31 (66%) of 47 patients. The suspect leukemic cell populations in the other 16 patients showed a complete lack of Vbeta monoclonal antibody reactivity that was confirmed by molecular data showing the usage of Vbeta gene segments not covered by the applied Vbeta monoclonal antibodies. Nevertheless, this could be considered indirect evidence for the "clonal" character of these cells. Remarkably, RT-PCR revealed an oligoclonal pattern in addition to dominant Vbeta-Cbeta products and single Vbeta domain expression in many T-LGL proliferations, providing further evidence for the hypothesis raised earlier that T-LGL derive from polyclonal and oligoclonal proliferations of antigen-activated cytotoxic T cells. It is concluded that molecular Vbeta analysis serves to assess clonality in suspect T-cell proliferations. However, the faster and cheaper Vbeta antibody studies can be used as a powerful screening method for the detection of single Vbeta domain expression, followed by molecular studies in patients with more than 20% single Vbeta domain expression or large suspect T-cell populations (more than 50%-60%) without Vbeta reactivity.

Effect of an inhaled glucocorticoid on reactive oxygen species production by bronchoalveolar lavage cells from smoking COPD patients.

Oxidative stress in the lung is important in the pathogenesis of COPD. Published data indicate that glucocorticoids inhibit blood cells in their capacity to produce reactive oxygen species (ROS). We investigated the effect of Fluticasone propionate (FP) on the ROS production capabilities of pulmonary cells. Bronchoalveolar lavage (BAL) was performed in smoking COPD patients, before and after a six month, placebo-controlled treatment with FP. BAL cells were stimulated with phorbol myristrate acetate (PMA) alone, and together with superoxide dismutase (SOD). From kinetic plots of ferricytochrome-c conversion we calculated the maximal rate of superoxide production: V(max). We also examined BAL cell subsets and performed correlation analyses on ROS production and relevant clinical determinants. Paired results were obtained from 6 FP- and 9 placebo-treated patients. No significant change of V(max) was found in both patient groups. Also BAL cellularity was unchanged. Correlation analyses showed a significant (inverse) association of V(max) with the number of cigarettes smoked per day. We concluded that a potent inhaled glucocorticoid had no effect on the ROS production capability of BAL cells from smoking COPD patients. Apparently, heavy smoking impaired the ability of alveolar macrophages to produce ROS, which was not further decreased by FP.

Flow cytometric analysis of the Vbeta repertoire in healthy controls.

BACKGROUND: Analysis of the T-cell receptor (TCR)-Vbeta repertoire has been used for studying selective T-cell responses in autoimmune disease, alloreactivity in transplantation, and protective immunity against microbial and tumor antigens. For the interpretation of these studies, we need information about the Vbeta repertoire usage in healthy individuals. METHODS: We analyzed blood T-lymphocyte (sub)populations of 36 healthy controls (age range: from neonates to 86 years) with a carefully selected most complete panel of 22 Vbeta monoclonal antibodies, which together recognized 70-75% of all blood TCRalphabeta(+) T lymphocytes. Subsequently, we developed a six-tube test kit with selected Vbeta antibody combinations for easy and rapid detection of single ("clonal") Vbeta domain usage in large T-cell expansions. RESULTS: The mean values of the Vbeta repertoire usage were stable during aging in blood TCRalphabeta(+) T lymphocytes as well as in the CD4(+) and CD8(+) T-cell subsets, although the standard deviations increased in the elderly. The increased standard deviations were caused by the occurrence of oligoclonal T-cell expansions in the elderly, mainly consisting of CD8(+) T lymphocytes. The 15 detected T-cell expansions did not reach 40% of total TCRalphabeta(+) T lymphocytes and represented less than 0.4 x 10(9) cells per liter in our study. Vbeta usage of the CD4(+) and CD8(+) subsets was comparable for most tested Vbeta domains, but significant differences (P < 0.01) between the two subsets were found for Vbeta2, Vbeta5.1, Vbeta6.7, Vbeta9.1, and Vbeta22 (higher in CD4(+)), as well as for Vbeta1, Vbeta7.1, Vbeta14, and Vbeta23 (higher in CD8(+)). Finally, single Vbeta domain expression in large T-cell expansions can indeed be detected by the six-tube test kit. CONCLUSIONS: The results of our study can now be used as reference values in studies on distortions of the Vbeta repertoire in disease states. The six-tube test kit can be used for detection of single Vbeta domain expression in large T-cell expansions (>2.0 x 10(9)/l), which are clinically suspicious of T-cell leukemia.

External quality assessment of flow cytometric HLA-B27 typing.

A biannual external quality assurance (EQA) scheme for flow cytometric typing of the HLA-B27 antigen is operational in The Netherlands and Belgium since 1995. We report here on the results of the first seven send-outs to which 36 to 47 laboratories participated. With the send-out, four specimens from blood bank donors, who had been typed for HLA Class I antigens by complement-dependent cytotoxicity, were distributed. Subtyping of the HLA-B27 allele was performed by PCR-SSP. Ten samples were HLA-B27(pos) (all HLA-B*2705) and 18 were HLA-B27(neg). For flow cytometry, the most widely monoclonal antibody (MoAb) used was FD705, followed by GS145.2 and ABC-m3. The majority of laboratories used more than 1 anti-HLA-B27 MoAb for typing. The HLA-B27(pos) samples were correctly classified as positive by the large majority of participants (median 95%; range 85% to 100% per send out); some participants considered further typing necessary and misclassification as negative was only sporadically seen. The classification of HLA-B27(neg) samples as negative was less straightforward. Ten samples were correctly classified as such by 97% (82% to 100%) of the participants, whereas 64% (range 53% to 70%) of the participants classified the remaining eight samples as HLA-B27(neg). There was no significant prevalence of a particular HLA-B allele among these eight "poor concordancy" samples as compared to the ten "good concordancy" samples. Inspection of the reactivity patterns of the individual MoAb with HLA-B27(neg) samples revealed that ABC-m3 showed very little cross-reactivity apart from its well-known cross-reactivity with HLA-B7, whereas the cross-reactivity patterns of GS145.2 and FD705 were more extensive. The small sample size (n = 18) and the distribution of HLA-B alleles other than HLA-B27 did not allow assignment of specificities to these cross-reactions. Finally, we showed that standardized interpretation of the combined results of two anti-HLA-B27 MoAb reduced the frequency of false-positive conclusions on HLA-B27(neg) samples. In this series, the lowest frequency of false-positive assignments was observed with the combination of the FD705 and ABC-m3 MoAb.