Multicenter evaluation of a new 4th generation HIV screening assay Elecsys HIV combi.

Fourth-generation screening assays which permit a simultaneous detection of human immunodeficiency virus (HIV) antigen and antibody reduce the diagnostic window on average by four days in comparison to third-generation antibody assays. Recently, the new automated Elecsys HIV combi was compared in a multicenter study to alternative fourth- and third-generation assays, p24 antigen test and HIV-1 RNA RT-PCR. A total of 104 serocon-version panels, samples of the acute phase of infection after seroconversion (n = 33), anti-HIV-1 positive specimens (n = 572) from patients in different stages of the disease, 535 subtyped samples from different geographical locations, including group M (subtypes A-J) and group O, anti-HIV-2 positive sera (n = 364), dilutions of cell culture supernatants (n = 60) infected with different HIV-1 subtypes, selected performance panels, 8406 unselected samples from blood donors originating from different blood transfusion centers, 3810 unselected sera from daily routine and from hospitalized patients, 9927 unselected samples from South Africa and 1943 potentially interfering samples were tested with the Elecsys HIV combi. Elecsys HIV combi showed a comparable sensitivity to HIV-1 Ag stand-alone assays for early detection of HIV infection in seroconversion panels. The mean time delay of Elecsys HIV combi (last negative sample + 1 day) in comparison to HIV-1 RT-PCR for 92 panels tested with both methods was 3.23 days. The diagnostic window was reduced with Elecsys HIV combi between 1.56 and 5.32 days in comparison to third-generation assays. The specificity of Elecsys HIV combi in blood donors was 99.80% after repeated testing. Our results show that a fourth-generation assay with improved specificity and sensitivity like the Elecsys HIV combi is suitable for blood donor screening due to its low number of false positives and since it detects HIV p24 antigen with a comparable sensitivity to single antigen assays.

Multicenter performance evaluation of a transcription-mediated amplification assay for screening of human immunodeficiency virus-1 RNA, hepatitis C virus RNA, and hepatitis B virus DNA in blood donations.

BACKGROUND: The performance of the recently launched Procleix Ultrio (Chiron/Gen-Probe) human immunodeficiency virus-1 (HIV-1), hepatitis C virus (HCV), and hepatitis B virus (HBV) blood screening assay was evaluated in a European multicenter study. STUDY DESIGN AND METHODS: Serial dilutions of reference materials were tested to determine the detection limits. Robustness and specificity were assessed by testing alternating high-load HCV RNA-positive and -negative samples, and 2912 test pools of eight donations. The added value of minipool and single-donation HBV nucleic acid testing protocols was compared to the currently used Prism (Abbott GmbH & Co. KG) hepatitis B surface antigen (HBsAg) and Auszyme (Abbott GmbH & Co. KG) dynamic HBsAg tests in 15 HBV seroconversion panels. RESULTS: The 95 percent detection limits (and 95% confidence interval [CI]) on the WHO International Standards was 26 (16-58) IU per mL for HIV-1 RNA, 4.6 (3.7-6.5) IU per mL for HCV RNA, and 11 (7.3-22) IU per mL for HBV DNA. No cross-contamination was observed. Testing 2912 pools of eight donations revealed 16 initial reactive samples; 11 were confirmed. The specificity after initial testing and percentage of invalid results were 99.83 and 0.48 percent, respectively. The HBV window-period (WP) reductions relative to HBsAg seroconversion in Prism and Auszyme dynamic HBsAg were, respectively, 6 days (95% CI, 3-8) and 9 days (95% CI, 7-12) in 1:8 minipool (MP) testing. CONCLUSION: The performance characteristics of Procleix Ultrio assay and the Procleix HIV-1 and HCV assay are comparable. The sensitivity for HIV-1 and HCV met the directives of the Paul-Ehrlich Institute and the FDA. The assay can reduce the WP for HBV by 6 days to 2 weeks when used in small MP (<1:8) or single-donation screening protocols.

Sequencing of two new HLA class II alleles: DRB3*0218 and DQB1*030202.

Two novel human leukocyte antigen (HLA) class II alleles for DRB3 and DQB1 genes detected in Caucasoid Spanish individuals are described: DRB3*0218 and DQB1*030202. Both alleles have been found during routine high-resolution typing by sequencing. DRB3*0218 shows a novel DRB3 gene polymorphic position, located at amino acid residue 58, alanine to glutamic acid. This residue is shared by several DRB1 alleles, including all described DRB1*11 subtypes. DQB1*030202 differs from DQB1*030201 by a point mutation at position 319 (T to C). This nucleotide change generates a new codon at amino acid position 75 that is not shared by any other DQB1 allele.

Collection, processing and cryopreservation of umbilical cord blood for unrelated transplantation.

Umbilical cord blood (UCB) transplantation is being used as an alternative source of hematopoietic stem cells for bone marrow reconstitution. Separation and processing of UCB samples in large numbers for storage in cord blood banks ideally needs to be partially automated. The aim of this study was to establish and standardize a method for unrelated cord blood banking as well as the biological characterization of the samples. Up to October 1999, a total of 938 UCB units (mean volume 84.6 +/- 23.6 ml, nucleated cell (NC) count 0.90 +/- 0.37 x 109, total CFU-GM 79 +/- 72 x 104, CD34+ cell count 2.46 +/- 2.72 x 106) had been collected. Twenty-three per cent of all UCB samples had a NC count below 0.4 x 109 and were discarded. The initial bacterial contamination rate was reduced to less than 5% as a result of extensive training in collection procedures. Using a modification of a triple bag system and adding a solution of 6% hydroxyethyl starch, the UCB was separated by two centrifugation steps into three components: buffy coat, red cell and plasma fractions. The overall recoveries for NC, CFU-GM and CD34+ cells were 87.4 +/- 8.5%, 88.8 +/- 6.6% and 90.3 +/- 12.4%, respectively, in a mean final volume of 27 +/- 4.2 ml.

Human immunoglobulin isotype profiles produced in response to antigens recognized by monoclonal antibodies specific to Anisakis simplex.

BACKGROUND: Anisakis simplex is a medically important pathogen which not only causes anisakiasis but may provoke allergy reactions, ranging from mild urticaria to anaphylactic shock. OBJECTIVE: To investigate anti-Anisakis isotype profiles in anisakiasis and Anisakis allergy patients. METHODS: Capture ELISA techniques were used to investigate the isotype profiles of antibodies specific for two defined Anisakis simplex antigens, in serum from Japanese patients with confirmed anisakiasis and from Spanish patients with allergy to Anisakis. The antigens were 'UA2R antigens' (two proteins with MW of 48 and 67 kDa, recognized by our monoclonal antibody UA2) and 'UA3R antigens' (two proteins with MW of 139 and 154 kDa, recognized by our monoclonal antibody UA3). RESULTS: Considering IgG, the two most frequent isotypes in the response to the UA2R antigens were IgG1 and IgG2, with IgG4 detected in only one case; in response to the UA3R antigens, by contrast, the two most frequent isotypes were IgG1 and IgG4 (though IgG2 remained reasonably frequent). As regards potential utility for serodiagnosis, 95% of the Japanese anisakiasis patients and 84% of the allergy patients showed detectable IgG1 antibodies to the UA3R antigens. Furthermore, all allergy patients showed IgE antibodies to these antigens. CONCLUSION: Anisakis simplex contains antigens that induce responses which are differentially regulated. Because of their immunogenicity, immunodominance and allergenic nature, we consider that the 139/154-kDa antigens recognized by our MoAb UA3 are good candidates for use in tests for the diagnosis of anisakiasis and of the allergy caused by this parasite.

[Properties of stored platelet concentrates: effect of the suspension media and type of pouch used]. / Propiedades de los concentrados de plaquetas almacenados: influencia del medio de suspensión y del tipo de bolsas utilizadas.

PURPOSE: We have evaluated in vitro the effects of using plasma (group A) or PAS-2 (group B) in storing platelets, regarding as well the type of container (conventional polyolefin--PL732--or new oxygen permeable platelet containers--PL2410, Compoflex--), on the metabolism of platelet concentrates--PC--from pools of five buffy coats. MATERIAL AND METHODS: 87, A and B pools of PCs collected in two types of bags were studied. The samples were taken on days 2, 5 and 7, and cell counts, pH, glucose, platelet activation (% CD62) and aggregation response were measured. RESULTS: In group A, when we compared the two types of bags, we observed a difference (p < 0.01) in values of pH and glucose on day 5. These values were more advantageous in new oxygen permeable platelet containers. The rest of the parameters analysed didn't show significant differences. When we compared the PL-2410 containers from groups A and B, we found a lower level of glucose (p < 0.01) in group B, although the levels of glucose in this group on days 5 and 7 of storage were sufficient to support the platelet metabolism. CONCLUSIONS: The use of new oxygen permeable polyolefin containers and additive solutions, PAS-2, allows us to obtain pools of PC with suitable metabolic parameters during storage.

[Antineutrophil cytoplasmic antibodies in patients with sickle cell anemia]. / Anticuerpos anti citoplasma de neutrófilos en pacientes con anemia drepanocítica.

PURPOSE: To determine whether anti neutrophil cytoplasmic antibodies (ANCA): anti myeloperoxidase (anti MPO) and anti proteinase 3 (anti PR3), are present in patients with sickle cell anaemia (SCA) during painful crisis. PATIENTS AND METHODS: 55 patients with SCA were included in this study, 35 of them had painful crisis and the rest were in steady state. We determined levels of anti MPO and anti PR3 by two enzyme immunoassays commercially available (Progen Biotechnik GMBH). RESULTS: A significant increase of anti MPO in the group of patients with painful crisis was found in comparison with steady state patients and controls. CONCLUSIONS: Our results suggest the possibility that anti MPO interacting with MPO on primed neutrophils lead to neutrophil activation, enhanced neutrophil adhesion and amplification of endothelial cell damage.

Reduction of the diagnostic window with a new combined p24 antigen and human immunodeficiency virus antibody screening assay.

In order to reduce the window phase between time of human immunodeficiency virus (HIV) infection and laboratory diagnosis, new fourth generation screening assays which permit a simultaneous detection of HIV antigen and antibody have been developed. In a multicenter study, a new automated fourth generation assay, Enzymun-Test HIV Combi (Boehringer Mannheim GmbH) was compared to third generation assay, p24 antigen tests and Western blot. A total of 37 seroconversion panels, samples of the early infection (n = 42), HIV-1 antibody positive sera, including subtypes A E, and O (n = 1118), HIV-2 positive samples (n = 252) and cell culture supernatants infected with different HIV-1 subtypes and HIV-2 (n = 50), blood donors (n = 6649), hospitalized patients (n = 475), HIV neg. sera with indeterminate Western blot (n = 32), potentially cross reactive serum samples (n = 435) and HIV negative specimens from Cameroon (n = 68) were tested. A total of 16 of 29 seroconversions were detected on average 8.5 days earlier with Enzymun-Test HIV Combi than HIV-1/HIV-2 3rd generation EIA (Abbott Laboratories). Overall, in the 29 panels investigated comparatively with the two assays, the mean time delay between Enzymun-Test HIV Combi and HIV-1/HIV-2 3rd generation EIA was 4.7 days. HIV antigen was detected in three out of 35 seroconversions one bleed earlier with HIV-1 Ag Monoclonal than with Enzymun-Test HIV Combi. Enzymun-Test HIV Combi showed a sensitivity of 100% for HIV antibody detection for HIV-1 group M and O and HIV-2 positive specimens. While p24 antigen of different HIV-1 subtypes was detected with Enzymun-Test HIV Combi in all the 49 cell culture supernatants, HIV Ag was not detected in an HIV-2 virus lysate. A total of 66 false positive results out of 7659 HIV negative samples were obtained with the Enzymun-Test HIV Combi. The specificity for unselected blood donors was 99.6%. The Enzymun-Test HIV Combi permits an earlier diagnosis of HIV infection than third generation assays through the detection of p24 antigen, which may be present in serum samples from individuals with recent HIV infection prior to seroconversion and it shows an excellent sensitivity for antibodies to all known HIV-1 subtypes and HIV-2. The specificity in blood donors and hospitalized patients is comparable to that of other assays.

Circulating immune complexes in sickle cell anaemia.

Circulating immune complexes were investigated in sera of 100 patients with sickle cell anaemia (SCA) in different clinical stages, using inhibition of complement-dependent rosette formation (EAC rosette inhibition), anticomplementary activity, polyethylene glycol 6000 precipitation, immunoconglutinin titre, total haemolytic complement, C4 and C3d. A high positivity was observed in patients with hepatic crisis (75%) and cases with leg ulcers (43%) compared with painful crisis (12%) and asymptomatic (7%) patients. These results provide evidence for the presence of circulating immune complexes during leg ulcers and hepatic crisis in SCA.

Plasma exchange for acute cholestasis in homozygous sickle cell disease.

A child with sickle cell disease presented acute chest syndrome, acute cholestasis and a neurological syndrome. Bilirubin, LDH, alkaline phosphatase, urea and creatinines were markedly increased. Circulating immune complexes were slightly elevated. The patient improved dramatically after a plasma exchange.

Dengue haemorrhagic fever in Cuba. II. Clinical investigations.

Clinical and serological studies were carried out on 114 patients admitted to hospital in Havana, Cuba with Dengue Haemorrhagic Fever and Dengue Shock Syndrome (DHF/DSS). Serological confirmation of dengue was obtained in 90% of cases, with 5% of cases primary and 95% secondary. Fever, haemorrhagic manifestations, vomiting and headache were the most frequent signs and symptoms. Among haemorrhagic manifestations, petechiae and vaginal bleeding were reported in a larger number of patients. 21 patients presented shock and, of these, 20 were secondary infections. The disease appeared more frequently in white persons and in women. The aetiopathogenicity of the syndromes is discussed. 95% of the cases could be explained on the basis of the secondary infection hypothesis.

[Enzymatic changes in cancer]. / Alteraciones enzimáticas en cáncer.

Several enzymes were estimated in different tumors. Ceruloplasmin, lactate dehydrogenase (LDH), and phosphohexose isomerase (PHI) showed a direct correlation with tumor growth and remission. The ratio LDH/PHI was low in solid tumors and high in lymphomas, suggesting a different metabolism.

Circulating immune complexes in paroxysmal nocturnal hemoglobinuria.

Circulating immune complexes were investigated in sera of 13 patients with paroxysmal nocturnal hemoglobinuria. A significant inhibition was observed in the complement-dependent rosette formation (EAC) in 6 cases; 2 patients presented an increased anticomplementary activity; antilymphocyte antibodies were demonstrated in 2 patients. The levels of C3d and immunoconglutinins were also increased; there were no differences in the total hemolytic complement between patients and normal controls. There was a significant correlation between C3d and immunoconglutinins with the inhibition of the EAC rosette formation test. These results provide evidence for the presence of circulating immune complexes in paroxysmal nocturnal hemoglobinuria.