An enzyme-linked immunosorbent assay for platelet compatibility testing

The selection of platelet donors for patients who are refractory to random donor platelets often presents a difficult clinical problem. We describe an enzyme-linked immunosorbent assay (ELISA) for evaluating alloantibodies in refractory patients. Platelets from prospective donors are immobilized on microtiter plates and, after incubation with test serum and washing, platelet-bound IgG is detected with enzyme- linked anti-human IgG. Platelets from 46 prospective donors were tested. Twenty-two were judged compatible (reciprocal of the antibody titer less than 16) and, of these, 15 were used as platelet donors; each gave a measurable platelet increment after transfusion. The magnitude of the response was roughly proportional to the assay results. Platelets from donors giving antibody titers less than 4 resulted in platelet increments at 1 hr ranging from 4,890 to 22,200 (median 12,600), while platelets from donors giving titers of 8 or 16 resulted in lesser increments (550-4548). Conversely, 5 of the 24 patients found incompatible by the assay (titer greater than 16) gave no platelet increment, and in 3 instances, the recipient developed fever and chills after the transfusion. The assay is sensitive, simple, and adaptable to the clinical laboratory. Platelets from volunteer donor panels can be plated and stored for up to 6 mo.     

Determination of antiplatelet antibody activity in sera cytotoxic for human lymphocytes

Pooled serum aliquots obtained from sensitized potential renal allograft recipients on chronic hemodialysis were evaluated for their lymphocytotoxicity titers against the lymphocytes and then for alloantibodies against the platelets of 7 random donors by 5 methods. Platelet donor specific lymphocytotoxicity was present in 93% of 42 combinations. Of the positive combinations, 57% had a positive test for antiplatelet activity by the 14C serotonin release assay, 16% by the platelet aggregation method, and 19% as judged by acid phosphatase availability on the platelet membrane. No serum tested released beta-glucoronidase or lactic dehydrogenase. No correlation of the height of the titer of antiplatelet activity with that for lymphocytoxicity was detected. Thus, even in sera demonstrating significant activity against donor lymphocyte antigens, detection of associated platelet antibody activity is not uniform. Thus, a positive lymphocytoxic titer does not necessarily predict detectable antiplatelet activity. Therefore, additional tests for detection of antiplatelet activity should also be considered. This study shows that of the tests evaluated, the 14C serotonin release assay is the most sensitive for detection of antiplatelet antibodies.     

Occurrence of Bartonella henselae and Bartonella quintana in a healthy Greek population

The purpose of this study was to determine the prevalence of IgM and IgG antibodies against Bartonella henselae and B. quintana in a healthy Greek population using a commercially available immunofluorescent test (Focus test). Five hundred healthy individuals were divided by sex into four age groups and three groups according to contact with cats. IgM antibodies were not detected in any of the subjects examined, while 99 (19.8%) and 75 (15%) were IgG seropositive to B. henselae and to B. quintana, respectively. No statistical difference in the seropositivity was observed among these groups. The IgG antibody titers ranged from 1/64 to 1/256 for B. henselae and from 1/64 to 1/512 for B. quintana. A high percentage (12.4%) of cross-reactivity between the two species was observed. Our data show that the prevalence of both Bartonella species in Greece is high. However, low IgG antibody levels are not sufficient evidence of active infection.     

Prevalence and clinical significances of red cell alloimmunization and red cell bound immunoglobulin G in polytransfused patients with thalassemias

The study was to determine the prevalence and clinical significances of red blood cell (RBC)-bound IgG as detected by flow cytometry in polytransfused patients with thalassemias. Relationship of the presence of RBC-bound IgG with RBC alloimmunization was also evaluated. This study included 59 polytransfused patients with β-thalassemia disease. We studied the frequency of RBC autoantibodies and alloimmunization. Direct Coombs test and flow cytometry were performed to detect the presence of RBC autoantibodies while RBC alloantibodies were detected by antibody screening and identification assays.

Eight (13.6%) and 34 (57.6%) patients were found a positive direct Coombs test and flow cytometry, respectively. Twenty (33.9%) patients developed RBC alloantibodies. The four most frequent RBC alloantibodies were anti-E (55%), anti-Mia (40%), anti-Di(a) (25%) and anti-c (15%), respectively. There was no significant difference in the presence of RBC-bound IgG between polytransfused with thalassemia patients who developed RBC alloimmunization (13 of 20; 65%) and those without RBC alloantibodies (21 of 39; 53.8%), p?=?0.412. Splenectomy and increased transfusion requirement were significantly associated with the presence of RBC-bound IgG but not with RBC alloantibody formation.

The overall frequency of RBC alloantibody formation in polytransfused patients with thalassemias was 33.9%. The most common RBC alloantibody was anti-E. RBC autoantibody formation was more frequently detected by flow cytometry (57.6%) than by direct Coombs test (13.6%). Splenectomy was significantly associated with the development of autoreactive RBC-bound IgG antibodies in the polytransfused patients with thalassemias. The presence of the anti-RBC autoantibodies may cause an increase of transfusion requirement.     

Anti-sporozoite antibodies induced by natural infection

Serum samples from 120 individuals living in a malaria-endemic area, 31 patients with Plasmodium falciparum infection, and 58 healthy blood donors were tested for antibodies against P. falciparum and P. vivax sporozoites. Specific antibodies were determined by the circumsporozoite precipitation (CSP) reaction and indirect immunofluorescent (IFA) tests for IgG and IgM antibodies. It was found that a high proportion of adults living in the endemic area had IFA anti-sporozoite antibodies, usually IgG. Children and healthy donors were either negative or had low antibody titers. A positive correlation was found between IgG antibody titers against P. falciparum sporozoites and those against P. vivax sporozoites. CSP reactivity was demonstrated in 5 of 31 sera from patients with falciparum malaria, and was always associated with a high level of IFA antibodies. The anti-sporozoite antibodies were found to be stage- and species-specific.     

Screening of blood donors for erythrocyte alloantibodies

Abstract

Objective

To assess the prevalence of the anti-red blood cell antibodies in the donor population of Delhi.

Methods

This prospective study was conducted in Regional Blood Transfusion Centre (RBTC), Lady Hardinge Medical College (LHMC) and associated hospitals from March 2010 to March 2011. Antibody screening of all donor serum/plasma was performed as routine immunohaematological procedure. Any positive sera were further investigated to identify the specificity of irregular erythrocyte antibody by commercially available red cell panel (ID-Dia Panel, Diamed-ID Microtyping System). The titres and thermal amplitude of the identified antibodies were evaluated.

Results

A total of 7756 donors were screened, of which 7648 donors were males (98.6%) and 108 were females (1.4%). The maximum number of donors belonged to age group of 26–30 years. A total of four donors showed presence of alloantibodies in their serum (0.05%). On antibody identification, two of them were anti-C, one was anti-Lewis^a antibody and one was autoantibody.

Discussion

This study was conducted to highlight the significance of detecting irregular erythrocyte antibodies in healthy donors.     

SARS Coronavirus-2 Microneutralisation and Commercial Serological Assays Correlated Closely for Some but Not All Enzyme Immunoassays

Serological testing for SARS-CoV-2-specific antibodies provides important research and diagnostic information relating to COVID-19 prevalence, incidence and host immune response. A greater understanding of the relationship between functionally neutralising antibodies detected using microneutralisation assays and binding antibodies detected using scalable enzyme immunoassays (EIA) is needed in order to address protective immunity post-infection or vaccination, and assess EIA suitability as a surrogate test for screening of convalescent plasma donors. We assessed whether neutralising antibody titres correlated with signal cut-off ratios in five commercially available EIAs, and one in-house assay based on expressed spike protein targets. Sera from recovered patients or convalescent plasma donors who reported laboratory-confirmed SARS-CoV-2 infection (n = 200), and negative control sera collected prior to the COVID-19 pandemic (n = 100), were assessed in parallel. Performance was assessed by calculating EIA sensitivity and specificity with reference to microneutralisation. Neutralising antibodies were detected in 166 (83%) samples. Compared with this, the most sensitive EIAs were the Cobas Elecsys Anti-SARS-CoV-2 (98%) and Vitros Immunodiagnostic Anti-SARS-CoV-2 (100%), which detect total antibody targeting the N and S1 antigens, respectively. The assay with the best quantitative relationship with microneutralisation was the Euroimmun IgG. These results suggest the marker used (total Ab vs. IgG vs. IgA) and the target antigen are important determinants of assay performance. The strong correlation between microneutralisation and some commercially available assays demonstrates their potential for clinical and research use in assessing protection following infection or vaccination, and use as a surrogate test to assess donor suitability for convalescent plasma donation.     

Detection of human monocyte-reactive alloantibodies by flow cytometry after selective downmodulation of the Fc receptor I

Monocyte-reactive human alloantibodies may be of importance in situations such as transfusion reactions and bone marrow and kidney transplantation. So far, only complement-binding monocyte-reactive antibodies can be detected with a cytotoxicity assay. No antiglobulin assays are yet available that also detect noncomplement-fixing monocyte-reactive antibodies. The binding of monomeric IgG with high affinity to the Fc receptor I (FcRI) on monocytes has severely hampered the development of such an assay until now. We report on the selective removal of the FcRI from monocytes to test human sera in a flow cytofluorometry assay for the presence of monocyte-reactive IgG alloantibodies. Selective downmodulation of FcRI was accomplished by incubating the cells with murine monoclonal antibodies against FcRI followed by a second incubation with goat-antimouse IgG polyclonal antibodies. With such modified cells, human complement-binding and noncomplement-binding IgG and IgM alloantibodies against polymorphic determinants of the HLA class I and II glycoproteins, the human monocyte antigen system and polymorphic antigenic determinants of the LFA complex, can be detected in a sensitive and reproducible manner.     

IgG and IgM autoantiidiotype antibodies against antibody to HBsAg in chronic hepatitis B

Antibody directed against HBsAg carries idiotypic determinants that may induce an autoantiidiotype antibody response. We describe a solid-phase radioimmunoassay which allows specific detection of either IgG or IgM antibody to antibody directed against HBsAg. Among 138 chronic hepatitis B virus carriers, IgG autoantiidiotype was detected in 98 (71%) and IgM autoantiidiotype in 10 (80%). The autoantiidiotype reaction was blocked with antibody directed against HBsAg after removal of immune complexes by polyethylene glycol precipitation. The prevalence and levels of both classes of autoantiidiotype antibodies were highest in patients with hepatitis B virus DNA or HBeAg in serum. During follow-up, patients who lost hepatitis B virus DNA and HBeAg from serum had lower titers of autoantiidiotype and were less likely to have autoantiidiotype than patients who persisted in having hepatitis B virus DNA and HBeAg in serum. Thus, the presence and titer of autoantiidiotype correlated with serologic evidence of active viral replication in chronic hepatitis B. These findings suggest that the antibody directed against HBsAg response may play a role in modulating viral replication in chronic hepatitis B.     

Avidity of specific IgG antibodies as markers of recent primary infection caused by Toxoplasma gondii]

For serologically characterizing a recent primary toxoplasma infection, the low avidity of IgG specific antibodies was studied. Avidity was evaluated as the decrease of IgG antibody titers in ELISA after treating plates with 6 M urea, as a dissociating solution of low avidity antigen-antibody complexes. Sixty nine serum samples were studied, presenting characteristic patterns of recent, transitional or chronic toxoplasmosis. Serological patterns were determined according to results of IgG and IgM immunofluorescence, IgM-capture, and hemagglutination tests. Twenty three serum samples from each of the referred patterns I, II and III were titrated. For chronic toxoplasmosis infections, which presented a serological pattern III, observed decrease of titers was 3% +/- 3%. For pattern I recent toxoplasmosis sera it was 34% +/- 12%, and for transition pattern II, 12% +/- 9%. Thus, a low avidity of IgG specific antibodies can be applicable for the diagnosis of a recent toxoplasmosis infection.     

IgG reactivity to phospholipid-bound beta(2)-glycoprotein I is the main determinant of the fraction of lupus anticoagulant activity quenched by addition of hexagonal (II) phase phospholipid in patients with the clinical suspicion of antiphospholipid-antibody syndrome.

BACKGROUND AND OBJECTIVE: Autoantibodies to beta(2)-glycoprotein I (beta(2)-GPI) and/or prothrombin (FII) have been involved in the expression of lupus anticoagulant (LA) activity, an in vitro phenomenon associated with an increased risk of arterial and/or venous thromboembolic events. However, LA activity sustained by anti-FII antibodies has a much weaker association with thrombosis than LA activity sustained by anti-beta(2)-GPI antibodies. Because assays aimed at detecting LA activity are now commercially available, we evaluated the relative sensitivity to anti-FII and anti-beta(2)-GPI antibodies of a commercial LA assay in a consecutive series of patients with the clinical suspicion of anti-phospholipid antibody (APA) syndrome. DESIGN AND METHODS: One hundred and ten consecutive patients with the clinical suspicion of APA syndrome (primary in 39) and 36 healthy controls were evaluated for the presence of LA activity (LA, Staclot, Stago), anticardiolipin antibodies (Quanta Lite aCL IgG, IgM, Inova Diagnostics), and IgG binding to solid-phase and/or phospholipid (PL)-bound beta(2)-GPI and FII by ELISA assays developed an optimized in our laboratory. Odds ratios for the association of IgG binding activity with LA and the aCL IgG status were calculated. In LA patients, dependency of LA potency (as assessed by clotting time prolongation in absence or presence of hexagonal phospholipid) on autoantibody titers was analyzed by the generalized linear model. Total IgG fractions were purified from selected patients to evaluate their ability to inhibit prothrombin activation at low FII concentration. RESULTS: Anticardiolipin antibodies (aCL) of the IgG or IgM type were found in 64 and 23 patients and LA activity in 49 patients. Anti-beta(2)-GPI and anti-FII (solid-phase and PL-bound) IgG titers exceeding by more than 3 standard deviations the mean values observed in control subjects were found in 46 and 47 patients and in 56 and 30 patients respectively, with the highest titers detected in the subgroup of patients with both LA and aCL IgG. The relative risk of LA for patients free of anti-FII and/or anti-beta(2)-GPI IgG was 0.03 after stratification for the aCL IgG status. Anti-beta(2)-GPI (solid-phase and PL-bound) IgG (RR 34.4 and 12.6) and anti-FII (solid-phase) IgG (RR 6.33) were all associated with LA activity. However, when taking into account co-existence of anti-FII and anti-beta(2)-GPI IgG in the same patients, the relative risk of LA for patients with isolated anti-FII IgG (solid-phase and/or PL-bound) was 0.50, whereas it ranged from 4.24 to 8.70 for all the antibody combinations including anti-beta(2)-GPI IgG. Anti-beta(2)-GPI (PL-bound) and aCL IgG titers were the only significant predictors of LA potency determined in absence phospholipid (anti-beta(2)-GPI IgG) or in presence of hexagonal phospholipid (aCL IgG). Total IgG fractions purified from 12 patients (6 with anti-FII IgG) did not significantly inhibit factor II activity up to a 150-fold molar excess. INTERPRETATION AND CONCLUSIONS: These results highlight the high prevalence of anti-FII and anti-beta(2)-GPI IgG in patients with the clinical suspicion of APA syndrome and particularly in the subgroup of patients with LA activity. The fraction of LA activity which can be quenched by addition of hexagonal phospholipid is, however, only dependent on IgG directed to PL-bound beta(2)-GPI. Other antibodies associated with anticardiolipin IgG may explain residual clotting time prolongation observed in the presence of hexagonal phospholipid.     

Age-related changes in auto- and natural antibody in the werner syndrome

To assess the immunologic disturbance in Werner syndrome, antibodies to “intrinsic” (auto)antigens (anti-DNA antibodies and rheumatoid factors) and “natural” antibodies to “extrinsic” antigens (hemagglutinins for sheep red cells and antibodies against ABO blood type antigens) were measured in serum samples from 16 patients with Werner syndrome and compared with those from 150 healthy persons ranging in age from less than a year to 98. Employing a sensitive solid-phase radioimmunoassay, we found that the levels of both anti-double-stranded and anti-single-stranded DNA antibodies in the IgG class gradually increased with age in normal donors; a more abrupt increase with age was observed In those with Werner syndrome, although they lacked any complication of renal disease and hypocomplementemia. The titers of rheumatoid factor detected by sensitized sheep cell agglutination also gradually rose in normal persons and patients with Werner syndrome. In contrast, the titers of natural antibodies declined with age in both groups. These disturbances in antibody production suggested that Werner syndrome expresses an accelerated form of aging in immunologic aspect.     

Detection of IgG antibodies to type-specific Pseudomonas aeruginosa lipopolysaccharides by solid-phase radioimmunoassay.

Previous studies have suggested that IgG serotype-specific antibodies are protective against infections with pseudomonas aeruginosa. In the present study, type-specific IgG antibodies to P. aeruginosa were detected by solid-phase radioimmunoassay in sera from 15 volunteers before and after vaccination with lipopolysaccharides from P. aeruginosa and from four patients with endocarditis due to P. aeruginosa. Significant type-specific increases in IgG antibody occurred after both vaccination and infection. The correlation coefficients comparing net counts per minute by solid-phase radioimmunoassay with hemagglutination titers in the 15 vaccinees were 0.940, 0.874, 0.792, 0.903, 0.882, 0.869, and 0.704 for serotypes 1--7, respectively.     

Human antibodies to a Mr 155,000 Plasmodium falciparum antigen efficiently inhibit merozoite invasion.

IgG from a donor clinically immune to Plasmodium falciparum malaria strongly inhibited reinvasion in vitro of human erythrocytes by the parasite. When added to monolayers of glutaraldehyde-fixed and air-dried erythrocytes infected with the parasite, this IgG also displayed a characteristic immunofluorescence restricted to the surface of infected erythrocytes. Elution of the IgG adsorbed to such monolayers gave an antibody fraction that was 40 times more efficient in the reinvasion inhibition assay (50% inhibition titer, less than 1 microgram/ml) than the original IgG preparation. The major antibody in this eluate was directed against a parasite-derived antigen of Mr 155,000 (Pf 155) deposited by the parasite in the erythrocyte membrane in the course of invasion. A detailed study of IgG fractions from 11 donors with acute P. falciparum malaria or clinical immunity revealed the existence of an excellent correlation between their capacities to stain the surface of infected erythrocytes, their titers in reinvasion inhibition, and the presence of antibodies to Pf 155 as detected by immunoblotting. No such correlations were seen when the IgG fractions were analyzed for immunofluorescence of intracellular parasites or for the presence of antibodies to other parasite antigens as detected by immunoprecipitation of [35S]methionine-labeled and NaDodSO4/PAGE-separated parasite extracts. The results suggest that Pf 155 has an important role in the process of erythrocyte infection and that host antibodies to this antigen may efficiently interfere with this process.     

Detection of Platelet-Bound and Serum Antibodies in Autoimmune Thrombocytopenia by Enzyme-Linked Assay

Platelet antibodies were looked for in 47 patients with autoimmune thrombocytopenia using a modification of the enzyme-linked assay previously described. Surface-bound antibodies measured as increased platelet-associated IgG were found in 32 (68%) of the patients. After incubation in test sera, the platelet-associated IgG of normal donor platelets was significantly increased in 27 of the 47 patients (57%), thus demonstrating the presence of platelet antibodies free in their sera. 6 patients had antibodies only in the serum without any elevation of their platelet-associated IgG. When both tests are evaluated together no antibody was detected by either the direct or the indirect test in 9 of the 47 patients (19%) studied. The technique used is described and the interpretation of our results discussed.     

Screening of Tissue Transglutaminase Antibody in Healthy Blood Donors for Celiac Disease Screening in the Turkish Population

Celiac disease (CD) is a disease having the characteristic pathology of the mucosa of the small intestine. The prevalence of CD in the Turkish population has not been investigated previously. The present study was designed to determine the prevalence of CD in healthy blood donors. Serum samples of 2000 healthy blood donors presenting to Hacettepe University Faculty of Medicine Hospital Blood Bank were tested for tissue transglutaminase (tTG) IgA and IgG antibodies with enzyme-linked immunosorbent assay (ELISA; Euroimmune, Germany). The histopathological findings for the cases with positive serology were evaluated. The distribution of sex was 95.7% male, and 4.3% female. The mean age was 33 +/- 9. Among 2000 donors, 23 (1.15%) were positive for tTG IgA antibody and 3 (0.15%) were positive for tTG IgG antibody. None of the samples was positive for both antibodies. Serum total IgA was measured in two cases with only tTG IgG positivity and was found to be low in one case. Twelve subjects positive for tTG agreed to endoscopy and biopsy. Histopathological examination revealed changes classified as Marsh III-II in one, Marsh II in two, Marsh I in seven, and Marsh 0 in two donors. This was the first study conducted to determine the prevalence of tTG positivity in the Turkish population. The tTG antibody positivity prevalence in healthy blood donors was as high as 1.3%. This study shows that the prevalence of CD in the Turkish population is relatively high in comparison to that in the Western world.     

石家庄市大学生献血者弓形虫感染调查

目的 了解石家庄市大学生献血者弓形虫感染情况。 方法 2012年3-10月采集石家庄市4所大学大学生献血者和健康体检成人血清标本, 采用酶联免疫吸附试验 （ELISA） 检测弓形虫感染情况。 结果 共检测石家庄市4所大学864 例大学生献血者和95例健康成人, 弓形虫IgG阳性率分别为5.1%和7.4%, 差异有统计学意义 （P ＜ 0.05）。男性和女性大学生献血者弓形虫IgG阳性率分别为5.0%和5.2%, 差异无统计学意义 （P ＞ 0.05）; 不同大学的大学生献血者弓形虫IgG阳性率差异无统计学意义 （P＞0.05）。结论 大学生献血人群中部分感染弓形虫, 有必要加强弓形虫病防控工作。     

Immunity against Pseudomonas aeruginosa adoptively transferred to bone marrow transplant recipients

Infection is a common problem for bone marrow transplant (BMT) recipients during the period of neutropenia that immediately follows the procedure. Gram-negative infections present a particular hazard in these immunocompromised hosts. To augment host defenses against one such pathogen, Pseudomonas aeruginosa, we immunized bone marrow transplant donors and/or recipients with a polyvalent O-polysaccharide-toxin A conjugate vaccine. When either donor or recipient alone was vaccinated before transplant, no increase in specific antibody titers to any of the vaccine components was observed in the recipient. However, when both donor and recipient were vaccinated before transplant, increases in antibody titers to all polysaccharide components occurred to levels shown to be protective in animal models of gram-negative sepsis. Specific antibodies were primarily of the IgG1 and IgG2 subclass even though IgG2 subclass deficiency is common after BMT. The requirement for both donor and recipient immunization reflects the need for primed donor B lymphocytes in the marrow inoculum to be transferred into an antigen-containing environment so that maximum B-cell proliferation and antibody secretion can occur. Adoptive transfer of antibody responses to Pseudomonas aeruginosa and other common bacterial pathogens has the potential to reduce infection-related morbidity and mortality after allogeneic bone marrow transplantation.     

Antibody elutions in Thai patients with a positive direct antiglobulin test

Background

The direct antiglobulin test is performed to determine whether an anaemic patient with evidence of haemolysis has autoimmune or alloimmune haemolytic anaemia.

Materials and methods

We determined the antibody specificity of eluted IgG antibodies from patients’ blood samples with a positive direct antiglobulin test. Overall, 134 Thai patients were included in this study. EDTA blood samples were obtained from recently transfused patients, patients with unexplained anaemia and patients who had serum antibodies detected during routine pre-transfusion tests from different hospital blood banks. These complicated samples were sent to the National Blood Centre of the Thai Red Cross Society for investigation and to find compatible blood components. Each blood sample underwent a direct antiglobulin test with the gel technique using polyspecific antihuman globulin and mononospecific anti-IgG and anti-C3d. Acid eluates were prepared from the samples for which the direct antiglobulin test was positive and the specificities of the eluted antibodies were determined by the gel technique.

Results

Of the samples tested, 101 showed a positive direct antiglobulin test result (75.4%) using polyspecific antihuman globulin sera whereas only 95 samples (70.9%) were positive with anti-IgG or anti-IgG and anti-C3d. Moreover, 54 of 95 eluates (56.8%) were positive for antibody screening and tested with the reagent panel cells. Twenty-one eluates had specific alloantibodies, which were concordant with the findings in the patients’ sera and all patients had a history of blood transfusion. Additionally, 33 eluates contained pan-agglutinins. Interestingly, alloantibodies could be determined using titration studies in 5 of 26 eluates with pan-agglutinins.

Conclusion

Although the direct antiglobulin test is not routinely performed in pre-transfusion screening, this test and elution studies would be useful in patients with a history of previous transfusions, and in those for whom compatible blood cannot be found.