[Screening for alpha1-antitrypsin deficiency using dried blood spot: Assessment of the first 20 months]. / Dépistage du déficit en alpha1-antitrypsine sur sang capillaire recueilli sur papier-filtre : bilan des 20 premiers mois.

INTRODUCTION: Alpha1-antitrypsin deficiency is a predisposing factor for pulmonary disease and under-diagnosis is a significant problem. The results of a targeted screening in patients with respiratory symptoms possibly indicative of severe deficiency are reported here. METHODS: Data were collected from March 2016 to October 2017 on patients who had a capillary blood sample collected during a consultation with a pulmonologist and sent to the laboratory for processing to determine alpha1-antitrypsin concentration, phenotype and possibly genotype. RESULTS: In 20 months, 3728 test kits were requested by 566 pulmonologists and 718 (19 %) specimens sent: among these, 708 were analyzable and 613 were accompanied by clinical information. Of the 708 samples, 70 % had no phenotype associated with quantitative alpha1- antitrypsin deficiency, 7 % had a phenotype associated with a severe deficiency and 23 % had a phenotype associated with an intermediate deficiency. One hundred and eight patients carried at least one PI*Z allele which is considered to be a risk factor for liver disease. CONCLUSIONS: The results of this targeted screening program for alpha1- antitrypsin deficiency using a dried capillary blood sample reflect improvement in early diagnosis of this deficiency in lung disease with good adherence of the pulmonologists to this awareness campaign.

Heavy+light chain monitoring correlates with clinical outcome in multiple myeloma patients.

Novel anti-myeloma agents have improved patient response rates, which are historically based on reductions of the M-protein. These methods can be inaccurate for quantifying M-proteins at low concentrations. We compared the consistency and clinical impact of response assignment by electrophoretic and heavy+light chain (HLC) immunoassays post-consolidation in 463 newly diagnosed patients. The two methods gave similar assignments in patients with partial (PR; 79% agreement) or complete response (⩾CR; 92%). However, in patients achieving very good PR (VGPR) there was poor concordance between methods (45%). Median progression-free survival (PFS) for standard VGPR patients was 34.5 months; HLC responses stratified these patients further into PR, VGPR and ⩾CR, with median PFS of 21.3, 28.9 months and not reached, respectively; P<0.001. At this time, abnormal HLC ratios had better concordance with multiparametric flow cytometry (sensitivity 10-4) (37 and 34% positive, respectively), compared to immunofixation (62% positive). In addition, HLC-pair suppression was identified in 38% of patients and associated with shorter PFS (30.6 months vs not reached; P<0.001). We conclude that HLC monitoring could augment electrophoretic assessments in patients achieving VGPR. The prognostic significance of HLC responses might partly depend on the patients' ability to recover their immune system, as determined by normalisation of HLC measurements.

[DEFI-ALPHA cohort and POLYGEN DEFI-ALPHA clinical research hospital programme. A study about clinical, biological and genetics factors associated with the occurrence and the evolution of hepatic complications in children with alpha-1 antitrypsin deficiency]. / Cohorte DEFI-ALPHA et projet hospitalier de recherche clinique POLYGEN DEFI-ALPHA. Étude des facteurs cliniques, biologiques et génétiques associés à l'apparition et à l'évolution de complications hépatiques chez les enfants présentant un déficit en alpha-1 antitrypsine.

INTRODUCTION: The alpha-1 antitrypsin (α1-AT) deficiency, most frequently caused by homozygosity for the Z variant (SERPINA1: c.1096 G>A; Glu342Lys), can give rise to two clinical patterns: (i) respiratory impairment with emphysema (mainly in adulthood) because of a pulmonary quantitative defect in anti-elastase activity; (ii) hepatic impairment (mainly in childhood) due to the misfolding of the PiZ protein which accumulates in hepatocytes thus providing cytotoxicity. CURRENT KNOWLEDGE: To date, the clinical and genetic factors responsible for the development of major hepatic injuries (fibrosis and portal hypertension) during childhood in PiZ patients are not known. METHODS: The DEFI-ALPHA cohort, created in 2008, aims to inventory and prospectively study all α1-AT deficient children diagnosed and included after occurrence of a hepatic sign. The POLYGEN DEFI-ALPHA PHRC has recently (2013) been added to the project to identify modifiers genes by two complementary approaches: (i) the candidate genes strategy with the SERPINA1, CFTR (cystic fibrosis gene), MAN1B1 and SORL1 genes, these two latter being implied in the degradation of misfolding proteins; (ii) the whole exome sequencing (WES) strategy in families in which the PiZ proband has a PiZ brother or sister free of any hepatic sign. EXPECTED RESULTS: The clinical parameter we want to explain is the apparition of a portal hypertension in PiZ children. In the DEFI-ALPHA project, three criteria will be tested: (i) age of inclusion in the cohort, (ii) the way of inclusion (neo-natal icterus or later hepatic impairment) and (iii) treatment or not with ursodesoxycholic acid and, if so, its duration. Genetically, polymorphisms on the SERPINA1 and MAN1B1 genes have already been associated in the literature with different clinical evolutions of the A1ATD but very inconstantly. Our study thus aims to confirm or not this association. The CFTR and SORL1 genes have never been studied in the α1-AT deficiency. Finally, the whole exome sequencing strategy could allow the discovery of new unexpected modifiers genes in this disease.

Prolonged hemodialysis for acute kidney injury in myeloma patients.

OBJECTIVE: Cast nephropathy, due to free light chain (FLC) toxicity, is the main cause of acute kidney injury in multiple myeloma, with about 10% of patients requiring dialysis. In these patients, in addition to chemotherapy that prevents FLC production, daily hemodialysis using high cutoff or adsorptive membranes, showed promising results by decreasing quickly toxic serum FLC concentrations. CASE HISTORY: We report here the case of 2 patients presenting with acute kidney injury and high FLC serum concentration and M-components one with IgG Kappa and the other with IgD lambda. Both were treated with bortezomib and dexamethasone and received a 24-h continuous hemodialysis using a high and sharp cutoff (around 35,000 Daltons) polysulfone membrane (ultraflux® HD 1000, Fresenius Medical Care GmbH, Bad Homburg, Germany) with citrate regional anticoagulation using a safe and dedicated device (multi filtrate Ci-Ca®). CONCLUSION: Despite similar range of depuration, serum plasma FLC decreased importantly in the patient with the kappa type who recovered but was unchanged in the lambda type patient who remained under maintenance dialysis. Further studies are needed to confirm this new approach therapy.

Oligo-monoclonal immunoglobulins frequently develop during concurrent cytomegalovirus (CMV) and Epstein-Barr virus (EBV) infections in patients after renal transplantation.

In the present study we report that the appearance of oligo-monoclonal immunoglobulins (oligoM-Igs) in the sera of transplanted individuals is concurrent with the detection of coincident active CMV infection and EBV replication. Eighty-four renal allograft patients were monitored with respect to CMV isolation, to CMV conventional serology and humoral response against the EBV trans-activator ZEBRA (an immediate-early antigen also called BZLF1). Titration of anti-ZEBRA antibodies (IgG and IgM) and amount of EBV DNA in serum were evaluated. Using the combination of four techniques (agarose gel electrophoresis, analytical isoelectric focusing, high resolution immunoelectrophoresis, immunofixation electrophoresis), oligoM-Igs were found in 25% of patients after allografting and significantly associated with rejection episodes (P < 0.001). Twenty out of 23 (86%) concurrent CMV/EBV infections were associated with serum oligoM-Igs (P < 0.001). One can thus reasonably assume that a sustained EBV replication following iatrogenic immunosuppression can promote the immunoglobulin heavy chain expression in EBV-infected B lymphocytes. The proliferation of immunoglobulin-secreting clones might occur after active CMV infection, through a transient over-immunosuppression or via immune subversion.

[Immunonephelometric analysis of immunoglobulin light chains: agreement between the theoretical and experimental model. Assessment of the reference values of the kappa/lambda ratio and the heavy to light chain ratio]. / Dosage immunonéphélémétrique des chaînes légères d'immunoglobulines: concordance du modèle théorique et du modèle expérimental. Détermination des valeurs de référence des rapports kappa/lambda et chaînes lourdes/chaînes légères.

Since the introduction of fully automated nephelometric systems simultaneous measurements of immunoglobulin light chains kappa (kappa) and lambda (lambda) and IgG, IgA and IgM have become increasingly used for the routine assessment of humoral immunity. From these data two ratios were calculated, the kappa/lambda ratio and the heavy chains to light chains ratio. As changes in these ratios might have some predictive clinical value besides reflecting a monoclonal component, it is necessary to know mean and reference limits of these ratios. On account of differences in the calibration method of the light chains measurements (either free light chains or light chains bound to a complete molecule) and of differences in the calculation method of the heavy chains to light chains ratio we were led to conduct our own investigation. IgG, IgA and IgM and kappa and lambda light chains were immunonephelometrically measured in the sera of 84 blood donors. For each sample theoretical values for kappa + lambda, kappa and lambda, and kappa/lambda were calculated using the existing relation between the concentration of a given immunoglobulin and the concentration of bound light chains. Using the Valtec Protocole and the t test we were able to evidence highly significant differences (p < 10(-4) between theoretical and experimental values of kappa, lambda and kappa + lambda; those differences could be proved to be directly linked to the nephelometric technique itself. However the experimental kappa/lambda ratio did not appear to differ from the theoretical one nor the standardization method to have an effect on the reference values of this ratio, our values (mean and reference limits, 1.81, 1.29-2.53) being very similar to previously published results. Concerning the so called heavy chains to light chains ratio two methods were used to express it, one consisting in the ratio of the theoretical kappa + lambda value to the experimental one with the following results, 1.05 and 0.93-1.18 for the mean and reference limits and the other one using the raw data. The results were as follows: mean 3.50, reference limits 3.11-3.94.

Plasma cell proliferation in monoclonal gammopathy: relations with other biologic variables--diagnostic and prognostic significance.

PURPOSE: We investigated the place of direct plasma cell proliferation analysis beside other biologic data in monoclonal gammopathy, particularly the serum level of C-reactive protein (C-RP) PATIENTS: Eighty patients were studied at the time of their diagnosis. Patients with a serum creatinine level greater than 200 mumol/L were excluded. METHODS: Plasma cell proliferation analysis was performed after bromodeoxyuridine incorporation and double immunoenzymatic labeling on cytological smears, making determination of the plasma cell labeling index (LI) possible. The other biologic variables studied were related to tumor burden (plasmacytosis, hemoglobin, serum levels of monoclonal immunoglobulin, albumin) or to kidney function (creatinine). beta 2-microglobulin (beta 2-M), C-RP, lactic dehydrogenase (LDH), and calcemia were also assessed. RESULTS: No correlation was found between LI and serum C-RP. LDH was the sole variable significantly correlated to C-RP (P < 0.01). Besides the biologic parameters used for the staging according to the Durie and Salmon classification, beta 2-M, albumin and LI were significantly different between stages (P < 0.0002, < 0.0004, < 0.00001). LDH and C-RP showed no significant difference. Results were similar when patients with and without bone lesions were analyzed separately. Multivariate analysis ranked these variables as follows with respect to prognostic value: beta 2-M, LI, and age. CONCLUSION: Among the variables analyzed, LI is the sole true reflector of cell proliferation. We confirm its diagnostic and prognostic value.

Tobacco smoking and other factors in relation to serum alpha-1-antitrypsin.

Serum levels of alpha-1-antitrypsin were measured by radial immunodiffusion, and phenotypes were determined by electrofocusing in acrylamide gel in 160 subjects who were used as controls in a case-control study of hepatocellular carcinoma (HCC). The results were studied in relation to age, sex, diagnostic category, tobacco smoking, consumption of alcoholic beverages, presence of hepatitis B surface antigen (HBsAg), and serum levels of alphafetoprotein (AFP) by modeling the data through multiple regression. There was no relation of serum alpha-1-antitrypsin values with sex, HBsAg, AFP, consumption of alcoholic beverages, and diagnostic category (p > 0.25). By contrast, there were statistically significant dose-dependent positive associations of serum alpha-1-antitrypsin with age and tobacco smoking (p < 0.01 in both instances). The positive association of serum alpha-1-antitrypsin with tobacco smoking and the previously reported excessive elevation of serum alpha-1-antitrypsin in hepatitis B-negative tobacco-related cases of HCC suggest that alpha-1-antitrypsin is intimately related to the pathogenetic process linking tobacco smoking to HCC.

Epstein-Barr virus associated lymphoproliferative diseases (B cell lymphoma) after transplantation.

We report 12 cases of lymphomas which occurred among 1670 patients with kidney or combined renal and pancreatic transplantation. Group 1 comprised nine patients presenting with the diffuse form of the disease where immunoblasts or mature plasma cells massively infiltrated all organs. The first symptom was a viral syndrome, associated with a restriction of heterogeneity of immunoglobulins; oligoclonal to monoclonal peaks of immunoglobulins appeared about 50 days after transplantation. All patients received antilymphocyte globulins (ALG), and seven were treated with cyclosporin. EBV infection could be demonstrated in almost all patients; three EBV lymphoblastoid cell lines were established, their HLA phenotype being the same as the recipient of the graft. All patients finally died with renal and hepatic failure. Group 2 comprises three patients who presented solid B cell tumours of tonsils, lungs, and spleen at onset, extending to liver, kidney graft, lymph nodes, and brain. All received cyclosporin; two patients were treated with ALG, and one with OKT3. Immunoglobulins were polyclonal, oligoclonal, or decreased. Cell surface immunoglobulins were monoclonal on two tumours. EBV-DNA was positive within two tumours. Two patients presented EBV and CMV primary infection. CD4+T lymphocytes subsets were diminished at onset, and increased after cessation of immunosuppressive therapy. One patient died because of brain involvement; the two others are alive, one with perfect graft function. Therapy consisted of stopping immunosuppressive treatment, Acyclovir, and in two patients of group 2, monoclonal antibodies to pan-B and EBV receptor antigens.

Oligoclonal "fingerprint" of CSF IgG in multiple sclerosis patients is not modified following intrathecal administration of natural beta-interferon.

The IgG pattern in CSF was studied in 11 patients with multiple sclerosis who exhibited an oligoclonal banding upon thin-layer polyacrylamide gel isoelectric focusing followed by silver stain of unconcentrated CSF. Each patient received beta-interferon intrathecally during a 2 month period. No modification was observed over a 6 month period. In addition, the oligoclonal pattern was remarkably unique for each individual representing a typical "fingerprint" which allowed the identification of any single CSF.

Alpha 1-antitrypsin levels and phenotypes and hepatitis B serology in liver cancer.

Serum levels of alpha 1-antitrypsin (alpha 1 AT) were measured by radial immunodiffusion and phenotypes were determined by electrofocusing in acrylamide gel in 39 patients with hepatocellular carcinoma (HCC) positive for serum hepatitis B surface antigen (HBsAg), 41 patients with HCC negative for serum HBsAg, and 160 age- and sex-matched hospital controls. There was no difference between the control series and either of the two HCC groups with respect to alpha 1 AT phenotype pattern; also, there was no evidence of association between HCC and either the M2 allele or any of the alpha 1 AT deficiency phenotypes. However, HCC cases negative for HBsAg had significantly higher serum alpha 1 AT values (mean 665 +/- 26 mg 100 ml-1) than HCC cases positive for HBsAg (mean 571 +/- 23 mg 100 ml-1), who in turn, had significantly higher alpha 1 AT values than hospital controls (mean 434 +/- 13 mg 100 ml-1). These results indicate that in Greece, as in other high HCC incidence countries, genetically determined alpha 1 AT deficiency is not aetiologically important; the increase of serum alpha 1 AT is an important correlate of HCC with possible aetiologic significance and diagnostic potential and HBsAg-positive HCC and HBsAg-negative HCC are manifest differently as well as being aetiologically distinct.

Interaction of group-specific component (vitamin D-binding protein) with immobilized Cibacron blue F3-GA.

Group-specific component (vitamin D-binding protein) was purified to homogeneity from human plasma by a three-step procedure involving pseudo-ligand affinity chromatography on immobilized Cibacron blue F3-GA followed by gel filtration and ion-exchange chromatography. Upon pseudo-ligand chromatography, Gc globulin was separated into two peaks. The first, which represented approx. 4% of the total Gc globulin, was eluted together with other alpha-globulins of similar Mr and/or pI, and the second (96% of Gc globulin) was clearly retarded. Collection of the latter provided a fraction 10-fold enriched in Gc globulin, with yields higher than 90%. Incubation of plasma with trace amounts of radioactively-labeled 25-OH vitamin D3 showed that the radioactivity coeluted with the first peak. In addition, after saturation with 25-OH vitamin D3, all the Gc globulin was eluted in the first peak. This indicates that the two peaks correspond to the holo and the apo forms of the protein, respectively, and suggests that either the interaction of the apo form with the Cibacron blue dye involves the binding site for vitamin D metabolites, or that the holo-protein undergoes a conformational change as a consequence of formation of the complex.

Pi-Gm linkage: evidence for linkage in males but not in females and for an effect of the S allele of the Pi system.

Linkage between the Pi (alpha 1-antitrypsin) and Gm (immunoglobulin heavy chain) loci was studied in thirty-four families including forty-one informative parents and 142 children. In females, the results did not provide evidence for linkage (posterior probability of non-linkage 0.98). In contrast, in males, there was strong evidence for linkage (peak lod 3.9 at theta = 0.18, posterior probability of linkage 0.98). The two populations appeared to be significantly different (0.001 less than P less than 0.01) with respect to the heterogeneity criterion of Morton. In addition, the effect of the possession of the S allele (associated with significantly decreased serum alpha 1-antitrypsin levels) was studied in fifteen informative parents and fifty-three children of the same group. No evidence for or against linkage was found in females, but in males close linkage between Pi S and Gm was demonstrated (peak lod 7.7 at theta = 0.05, posterior probability of linkage 0.9999). These data indicate significant linkage between Pi and Gm in males but not females and close linkage between the Pi S and Gm markers in males.