Quantification of ß-region IgA monoclonal proteins - should we include immunochemical Hevylite® measurements? Point.

Accurate measurement of IgA monoclonal proteins presents a significant challenge to laboratory staff. IgA heavy/light chain (Hevylite, HLC) analysis is an alternative methodology for monoclonal protein assessment, giving an independent measure of IgAκ and IgAλ concentrations. Clonality is assessed by calculating the ratio of involved immunoglobulin to background uninvolved immunoglobulin concentrations (e.g. IgAκ/IgAλ in an IgAκ patient). Here we discuss the challenges faced by the laboratory in IgA monoclonal protein assessment, and compare the performance of Hevylite assays with electrophoresis and total IgA results. We present data which validates the use of Hevylite for response assessment: in most cases, Hevylite provides comparable response assignment to that provided by serum protein electrophoresis (SPE) and total IgA; in other cases Hevylite provides additional information, such as detection of residual disease or relapse.

The change in Ig regulation from children to adults disconnects the correlation with the 3'RR hs1.2 polymorphism.

BACKGROUND: In the immune system, the serum levels of immunoglobulin (Ig) increase gradually during ageing. Through B cell development, the Ig heavy chain expression is modulated by a regulatory region at the 3' of the constant alpha gene (3'RR), in single copy in rodents and, due to a large duplication, in two copies in apes. The human 3'RR1 and 3'RR2 are both characterized by three enhancers, the central of which, namely hs1.2, is highly polymorphic. Human hs1.2 has four different variants with unique binding sites for transcription factors (e.g. NF-kB and SP1) and shows variable allelic frequencies in populations with immune disorders. In previous works, we have reported that in several autoimmune diseases the *2 allele of hs1.2 is genetically associated to high level of IgM in peripheral blood. In subjects with altered levels of circulating Ig, an increased level was associated to *2 allele of hs1.2 and low levels corresponded to high frequency of *1 allele. RESULTS: We have correlated the allelic frequencies of hs1.2 with IgM, IgG and IgA serum concentrations in two cohorts of healthy people of different age and after three years follow-up in children homozygous for the allele. Here we show that when the expression levels of Ig in children are low and medium, the frequencies of *1 and *2 alleles are the same. Instead, when the Ig expression levels are high, there is a significantly higher frequency of the allele *2. The follow-up of children homozygous for *1 and *2 alleles showed that the increase or decrease of circulating Ig was not dependent on the number of circulating mature B cells. CONCLUSIONS: These data support the idea that under physiologic condition there is a switch of regulative pathways involved in the maturation of Ig during ageing. This mechanism is evidenced by hs1.2 variants that in children but not in adults participate to Ig production, coordinating the three class levels.

Identification of reduced circulating haptoglobin concentration as a biomarker of the severity of pulmonary embolism: a nontargeted proteomic study.

Risk stratification of patients with pulmonary embolism (PE) may identify patients at high risk of early death who may benefit from more intensive surveillance or aggressive therapy. Nontargeted proteomics may identify biomarkers useful for the risk stratification of patients with acute symptomatic pulmonary embolism (PE). We studied 6 patients presenting with low-risk PE and 6 patients presenting with intermediate (n = 3) or high-risk (n = 3) PE. Two-dimensional difference gel electrophoresis was used to compare their plasma protein abundances. Candidate protein markers were identified by matrix assisted laser desorption ionization time-of-flight mass spectrometry. A panel of four biomarkers (haptoglobin, hemopexin, α2-macroglobulin, and Ig α1-chain C region) showed differences in plasma abundance among patients with acute symptomatic PE of different severity. Haptoglobin and hemopexin were decreased, whereas α2-macroglobulin and Ig α1-chain C region were increased, in patients with high or intermediate-risk PE compared with low-risk PE patient. In a separate clinical population consisting of 104 adults with acute PE, serum haptoglobin concentrations had an 85% chance of correctly identifying patients with high-risk PE according to receiving operating characteristics curve analysis. Moreover, serum haptoglobin concentrations ≤1 g/l showed an 80% sensitivity and a 96% specificity for the diagnosis of high-risk PE. Nontargeted proteomics identified protein biomarkers for the severity of PE that are involved in iron metabolism pathways and acute-phase response. Among them, reduced serum haptoglobin concentrations show a high accuracy for the biochemical detection of high-risk PE.

[Expression of major histocompatibility complex class I-related chain A antibody in patients with end-stage renal disease and its clinical implications].

OBJECTIVE: To study the frequency of major histocompatibility complex class I-related chain A (MICA) antibody in patients with end-stage renal disease (ESRD). METHODS: Luminex flow cytometry and beads loaded with 11 MICA antigens were used to identify the MICA antibody and evaluate the antibody specificity in 110 patients with ESRD. RESULTS: The positivity rate of MICA antibody was 40% (12/30) in PRA-positive patients, significantly higher than the rate of 17.5% (14/80) in PRA-negative patients (chi(2)=6.120, P=0.013). MICA-specific antibodies against 10 of the 11 MICA antigens were detected in 26 MICA antibody-positive patients, and 26.92% of the MICA antibody-positive patients had antibodies with single-specificity and 73.08% had polyspecific antibodies. Three MICA antibody-positive patients with cadaveric kidney transplantation showed good function of the graft without acute rejection 2 months after the operation. CONCLUSION: The positivity rate of MICA antibody is significantly higher in PRA-positive patients, suggesting a strong correlation between MICA and PRA positivity. The MICA antibodies are polyspecific and probably consist of IgM and IgG. These data can be used as prospective data for these ESRD patients considering potential renal transplantation, and may facilitate further investigation of the association of MICA with renal transplantation.

Roles of syndecan-1, bcl6 and p53 in diagnosis and prognostication of immunoproliferative small intestinal disease.

AIM: To evaluate roles of syndecan-1, bcl6 and p53 in diagnosis and prognostication of immunoproliferative small intestinal disease (IPSID) and to study profiles of kappa (kappa) and lambda (lambda) light chains and IgA heavy chain. METHODS: The study consisted of 11 cases of IPSID and similar number of controls which included 11 of normal intestinal mucosa and 11 of high grade B cell lymphoma of ileum. The parameters analyzed included clinical profiles, biochemical and other laboratory investigations, radiologic and histological findings including immunohistochemistry. RESULTS: All IPSID cases had demonstrable serum IgA heavy chain and heavy mucosal plasma cell infiltration. According to Galian's histological staging, there were 4 patients with stage A and 7 with stage B. kappa and lambda light chains were over-expressed in 7 patients; 1 stage A patient had H pylori-positive active gastritis and eradication of H pylori led to disease remission. Stage A biopsies had higher expression for syndecan-1, while stage B had higher expression for bcl6 and p53. Syndecan-1, kappa and lambda light chains and IgA heavy chain showed inverse relationship with bcl6 and p53. All patients were treated with doxycycline. CHOP regime was added in 5 patients who developed frank lymphoma. Three died of the disease due to extensive organ infiltration. CONCLUSION: Certain immunomarkers like syndecan-1, kappa and lambda light chains and IgA heavy chain could be of much help in identifying early stage IPSID. Stage B IPSID showed higher expression for bcl6 and p53 than stage A IPSID. bcl6 and p53 expressions correlated with a more advanced disease stage and aggressive tumour behavior.

Immunofixation reveals an apparent alpha heavy chain caused by precipitation of fibrinogen with IgA antiserum.

BACKGROUND: Fibrinogen present in serum specimens can interfere with the interpretation of serum protein electrophoresis. We report here the unexpected precipitation of fibrinogen by an IgA antiserum used in immunofixation electrophoresis. METHODS: Immunofixation electrophoresis of plasma combined with ethanol precipitation, serial dilution of plasma, and fibrinogen adsorption of the antiserum were used to investigate the apparent immunoreactivity of a commercial source of IgA antiserum to fibrinogen. RESULTS: Fibrinogen immunoreactivity by IgA antiserum was observed at fibrinogen concentrations > or =0.93 g/l. Ethanol precipitation of plasma removed fibrinogen and prevented the immunofixation of the protein by the IgA antiserum. Adsorption of the IgA antiserum with human fibrinogen removed its ability to precipitate fibrinogen, demonstrating cross-reactivity between the IgA antiserum and fibrinogen. CONCLUSIONS: Commercial sources of antiserum used for immunofixation electrophoresis may contain antibodies with specificity towards proteins typically absent from serum such as fibrinogen and can produce clinically misleading results.

A case report: isolated a heavy chain monoclonal gammopathy in a patient with polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin change syndrome.

A 45-year-old South-Korean man presented with abdominal distension, progressive paresthesia and motor weakness of both lower extremities. Our case was identified as polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy and skin change (POEMS) syndrome based on diagnostic criteria. Circulating M components of POEMS syndrome consist mainly of IgG or IgA-lambda and rarely IgM-lambda, IgG-kappa or isolated light chains. In this case, the M-band on serum protein electrophoresis and isolated IgA heavy chain on serum immunofixation electrophoresis were demonstrated, but there was no abnormal light chain. We suggest that this case may be associated with a pattern of abnormal secretion of monoclonal protein or a coincidence of a heavy chain disease in POEMS syndrome, even though the latter possibility may be very rare.

Deficiencia selectiva de inmunoglobulina y enfermedades reumáticas inflamatorias / Selective deficience of immunoglobulin and inflammatory rheumatic diseases

La deficiencia selectiva de inmunoglobulina A (IgA) es la más frecuente de las inmunodeficiencias primarias e incluye individuos que sufren de infecciones respiratorias recurrentes, enfermedad diarreica crónicas y síndromes autoinmunes. En este estudio describimos la patología reumática inflamatoria que en nuesto medio se asocia a esta inmunodeficiencia. Durante los años 1977-1995 se detectaron dos casos de insuficiencia total y cinco de insuficiencia parcial de IgA. Los pacientes con insuficiencia total padecían de artritis reumatoide seronegativa el 1er caso y de enfermedad de Still del adulto y osteoartrosis erosiva el 2do caso; aquellos con insuficiencia parcial presentaban: lupus eritomatoso sistémico, anemia hemolítica secundaria y exoftalmos unilateral (caso No.1); enfermedad mixta del tejido conjuntivo (caso No.2); síndrome de Sjogren y lepra (caso No.3) y artritis reumatoide juvenil de inicio oligoarticular (casos No.4 y 5)

Sera of IgA nephropathy patients contain a heterogeneous population of relatively cationic alpha-heavy chains.

Sera of IgA nephropathy (IgAN) patients and normal subjects were analysed by two-dimensional (2-D) gel electrophoresis. Densitometric analysis of the 2-D gels of IgAN patients and normal subjects revealed that their protein maps were comparable. There was no shift of pI values in the major alpha-heavy chain spots. However, the volume of the alpha-heavy chain bands were differently distributed. Distribution was significantly lower at the anionic region in IgAN patients (mean anionic:cationic ratio of 1.184 +/- 0.311) as compared to normal healthy controls (mean anionic:cationic ratio of 2.139 +/- 0.538). Our data are in support of the previously reported findings that IgA1 of IgAN patients were lacking in sialic acid residues.

Enfermedad celíaca: valor de los parámetros inmunoquímicos / Celiac disease: value of immunochemistry parameter

En celíacos durante las distintas fases evolutivas (fase aguda, remisión, desafío) y en controles no celíacos estudiamos en forma prospectiva la eficacia clínica de la detección sérica de anticuerpos antigliodina -IgA e IgG- anticuerpos- antirreticulina, endomisio y músculo liso- y test de d-xilosa. Se investigaron 44 pacientes de 1 a 22 años, promedio 8 años, agrupados según el diagnóstico en: celíacos: fase aguda 14 (31.8 por ciento); remisión 9 (20,5 por ciento); desafío 8 (18,2 por ciento). No celíacos 13 (29,5 por ciento). Se efectuó biopsia de intestino delgado en forma simultánea con las determinaciones inmunoquímicas. Durante la fase aguda la IgA antigliadina posee 100 por ciento de sensibilidad, especificidad, valor predictivo positivo, valor predictivo negativo y eficacia. Estos índices disminuyen significativamente en el período de remisión, y en el desafío recuperan el 100 por ciento de positividad. La determinación de IgG antigliadina arroja porcentajes menores en la evaluación estadística. Destacamos la diferencia significativa entre los promedios de anticuerpos antigliadina -IgA e IgG- en las diferentes etapas de la enfermedad. Los anticuerpos antiendomisio poseen especificidad importante (85 por ciento). El test de la d-xilosa es el método menos específico. En conclusión, señalamos que la determinación de IgA-antigliadina es el método complementario más eficaz y específico para el diagnóstico y seguimiento de niños celíacos. Los anticuerpos antiendomisio tienen una asociación significativa, en especial para detectar verdaderos negativos y orientar en el diagnóstico

Enfermedad celíaca: valor de los parámetros inmunoquímicos / Celiac disease: value of immunochemistry parameter

En celíacos durante las distintas fases evolutivas (fase aguda, remisión, desafío) y en controles no celíacos estudiamos en forma prospectiva la eficacia clínica de la detección sérica de anticuerpos antigliodina -IgA e IgG- anticuerpos- antirreticulina, endomisio y músculo liso- y test de d-xilosa. Se investigaron 44 pacientes de 1 a 22 años, promedio 8 años, agrupados según el diagnóstico en: celíacos: fase aguda 14 (31.8 por ciento); remisión 9 (20,5 por ciento); desafío 8 (18,2 por ciento). No celíacos 13 (29,5 por ciento). Se efectuó biopsia de intestino delgado en forma simultánea con las determinaciones inmunoquímicas. Durante la fase aguda la IgA antigliadina posee 100 por ciento de sensibilidad, especificidad, valor predictivo positivo, valor predictivo negativo y eficacia. Estos índices disminuyen significativamente en el período de remisión, y en el desafío recuperan el 100 por ciento de positividad. La determinación de IgG antigliadina arroja porcentajes menores en la evaluación estadística. Destacamos la diferencia significativa entre los promedios de anticuerpos antigliadina -IgA e IgG- en las diferentes etapas de la enfermedad. Los anticuerpos antiendomisio poseen especificidad importante (85 por ciento). El test de la d-xilosa es el método menos específico. En conclusión, señalamos que la determinación de IgA-antigliadina es el método complementario más eficaz y específico para el diagnóstico y seguimiento de niños celíacos. Los anticuerpos antiendomisio tienen una asociación significativa, en especial para detectar verdaderos negativos y orientar en el diagnóstico

Alpha-chain disease: analysis of alpha-chain protein and secretory component in jejunal fluid.

BACKGROUND: It is unclear why different forms of alpha-chain disease protein appear in intestinal fluid. This was studied in a 23-year-old Mauritanian man in whom alpha-chain disease was diagnosed localized to the duodenum and jejunum, nasopharynx, and bone marrow. METHODS: The duodenal infiltrate was studied by immunohistochemistry. Forms of alpha chain-containing proteins in serum and jejunal fluid were analyzed by ultracentrifugation and radioimmunoassays. RESULTS: The infiltrating cells contained alpha-1 chain but no light chains, and approximately 66% showed variable expression of J chain. Serum contained a large fraction of monomeric alpha-chain disease protein, whereas both monomeric and heavier forms appeared in jejunal fluid. Some of the latter were bound to secretory component, and the fluid contained virtually no free component. CONCLUSIONS: Linkage of polymeric alpha-chain disease protein to secretory component depends on balanced synthesis of alpha chains and J chain in the proliferating B cells, giving rise to polymers with binding site for secretory component expressed as an epithelial receptor. Insufficient receptor-mediated transport capacity (either relative and/or because of intestinal crypt reduction) results in passive external transfer of polymers without bound secretory component along with leakage of serum-derived or locally produced monomeric alpha-chain disease protein, the latter presumably originating from immunocytes with little or no J-chain synthesis.

Haemoglobin-H disease due to a unique haemoglobin variant with an elongated O-chain

The clinical and genetic properties of an unusual O-chain variant of human haemoglobin are described. It constitutes less than 1 percent of the total haemoglobin in heterozygotes and, when inherited together with an O-thalassaemia gene, produces the clinical picture of haemoglobin-H disease. Preliminary structural studies indicatge that, in addition to the 141 aminoacid residues which constitute the normal O-chain, this variant has about 31 extra residues attached to the C-terminal end.(SUMMARY)