Red Cell Agglutination Test Research Articles

Autoimmune hemolytic anemia in a preoperative patient: a case report and literature review

Background: In patients undergoing elective surgery, it is known that as many as one-third have preoperative anemia. Preoperative anemia is associated with an almost five-fold increase in the likelihood of postoperative mortality. So, it is essential to know the cause of anemia so that the management is appropriate so that surgery can be performed safely and prevent patients from dying on the operating table or postoperatively. This case report aims to describe the pathogenesis of the laboratory findings in a patient with autoimmune hemolytic anemia (AIHA) who was accidentally diagnosed during preoperative screening and the management performed. Case: A 65-year-old man with an enlarged prostate was scheduled for elective surgery. Preoperative examination revealed anemic right and left conjunctiva, decreased erythrocytes, hemoglobin, hematocrit, and increased MCV, MCH, and MCHC, peripheral blood smear dominated by red cell agglutination and positive direct Coomb's test. The patient was given methylprednisolone 62.5 mg intravenously before transfusion and given a transfusion of 2 units of slow drip packed red cells (PRC) under close supervision. There were no transfusion reactions during and after transfusion. There was an increase in Hb after transfusion so that the patient could be operated on. Conclusion: The patient in this case was diagnosed with AIHA based on preoperative laboratory findings. Laboratory results showed evidence of autoimmune hemolytic anemia with decreased erythrocytes, hemoglobin, hematocrits, spherocytes, and agglutination on peripheral blood smear and positive Coomb's test. Rapid identification and treatment of anemia help improve preoperative conditions.

The Cs a and Cs b Red Cell Antigens of the Cost Blood Group Collection Correspond to the HNA-3a and HNA-3b Neutrophil Antigens: Unexpected Twins with Implications for Sickle Cell Anemia

Recent genome-wide association and murine studies identified the human neutrophil antigen 3a/b polymorphism (HNA-3a/b) in SLC44A2 (rs2288904-G/A) as a risk factor in venous thromboembolism (VTE). We tested whether this polymorphism associates with clinical features in SCA patients in whose VTE and stroke are frequently encountered complications. Incidentally, we discovered that rare subjects with the homozygous HNA-3b/b genotype (genotype frequency < 2%) also carry the uncommon Cs(a-) red cell phenotype. Despite its discovery more than 50 years ago, the molecular basis of this red cell phenotype and the presumptive protein carrier for the antigens of the COST blood group collection (Cs a and Cs b) has remained unknown. Inspired by this fortuitous discovery, we hypothesized that rs2288904 and COST antigens are closely associated. Thus, we genotyped this SNP in a cohort of 35 Cs(a-) subjects and found that all of them showed a HNA-3b/b genotype, indicating that the high-frequency allele of rs2288904 (HNA-3a; 461G) encoding Arg154 might encode the Cs a antigen (COST1). Interestingly, anti-Cs a does not react with SLC44A2 null RBCs (exceptional VER- blood type) and SLC44A2 knockout K562 cell line, confirming that the COST antigens are carried on this protein. In addition, using a panel of K562 cells expressing HNA-3a or 3b antigens developed by CRISPR/Cas9 genome editing, we confirmed that anti-Cs a recognizes the HNA-3a antigen. Overall, our results confirmed that HNA-3 and COST antigens have the same molecular basis. As a result, the Cs a and Cs b red cell antigens can now be included in the CTL2 blood group system, encoded by the SLC44A2 gene. This new finding could be of medical and biological value because anti-HNA-3a may be responsible for severe cases of neonatal neutropenia and transfusion-related acute lung injury (TRALI). Current testing for HNA-3a antibodies includes the granulocyte agglutination test and granulocyteindirect immunofluorescence test, as recommended by the ISBT Working Party on Granulocyte Immunobiology. These classical methods, however, are cumbersome because they require fresh granulocytes that have to be prepared daily and used immediately. Consequently, screening for HNA-3a antibodies is restricted to a few expert laboratories. Therefore, we propose to use the simple indirect antiglobulin red cell agglutination test (IAT) and Cs(a-) RBCs to screen for this antibody in the plasma of donors and patients. Finally, given that the starting point for this project was the study of a potential association of the HNA-3 polymorphism with clinical and biological features in SCA patients, we found that the HNA-3b allele was significantly associated with a higher monocyte and platelet count (p<0.02) and the occurrence of leg ulcer (p=0.01), suggesting an important role of the SLC44A2 protein in the SCD physiopathology.

Rapid D-dimer assays to exclude deep venous thrombosis and pulmonary embolism: current status and new developments.

Studies measuring the fibrin degradation product D-Dimer (DD) using enzyme-linked immunosorbent assays (ELISA) in patients suspected of deep venous thrombosis (DVT) or pulmonary embolism (PE) suggest that it is possible to exclude DVT/PE when the DD level is below a certain cut-off value. However, ELISA methods are time-consuming, bare high costs, and are only available in experienced laboratories. For this reason several rapid and less costly DD assays have been recently developed. This article reviews the current literature about rapid latex and ELISA DD assays in the diagnostic approach of DVT and PE. Two new latex assays seem suitable in clinical practice. The most extensively studied assay is the so-called SimpliRed DD, an autologous red cell agglutination test that can be performed on fresh whole blood. For DVT a sensitivity (Sens) and a negative predictive value (NPV) of 89-100% and 95-100%, respectively, have been reported, for PE 94-100% and 98-100%, respectively. The second test, Tinaquant, is a quantitative latex assay. Sens and NPV for DVT of 99% and 93% have been reported in one study. Two rapid ELISA assays have been investigated. The most extensively studied is the VIDAS DD assay, a fully automated quantitative ELISA method. Sens and NPV of 94-100% and 92-100% for DVT and both 100% for PE have been reported. For the other rapid ELISA, Instant IA DD, Sens and NPV of 92-93% and 76-77% have been reported for DVT. The last one is a qualitative assay giving only positive or negative results. These results show that low concentrations of plasma DD measured by especially SimpliRed or VIDAS DD, might be used to reliably rule out DVT or PE in clinically suspected patients. Tinaquant seems promising and has to be evaluated further. As for standard ELISA, increased DD concentrations are of no use because of the low specificity of the assays. Future studies should assess the clinical usefulness of both assays in management trials under routine conditions, in the frame of clinical decision-making diagnostic processes to prove that withholding further noninvasive testing and/or anticoagulants in patients with a low or negative DD is safe. Strategies to identify patients with false-negative results should be developed.

Evaluation of an autologous red cell agglutination test, VetRED FIV™, for the presence of FIV antibody in cats

A commercially available whole blood agglutination test, VetRED FIV ™, used for the detection of antibodies to feline immunodeficiency virus (FIV), was evaluated. The test is based on the use of a synthetic peptide conjugated to a non-agglutinating anti-feline red blood cell monoclonal antibody. The amino acid sequence of the synthetic peptide was derived from the predicted sequence of the transmembrane protein of FIV. The sensitivity and specificity of VetRED FIV™ was 100% when 34 known FIV-positive and 15 known FIV-negative cats were tested. These cats were part of studies on experimentally induced FIV infection, with their FIV status confirmed by virus isolation. Further, VetRED FIV™ was compared with another commercially available test for FIV antibody, PetChek ® in a field trial on 548 feline blood samples received by a diagnostic laboratory. Of the test results 94.2% ( 516 548 ) were in agreement: 112 were positive by VetRED FIV™ and PetChek ®; 404 were negative by both tests and 32 were discordant. These 5.8% discordant samples producing VetRED FIV™-positive/PetChek ®-negative or VetRED FIV™-negative/PetChek R-positive were further assessed by Western blot assay. In the field trial, the sensitivity and specificity of VetRED FIV™ was 97% and 97%, respectively, comparable to the 98% sensitivity and 99% specificity for PetChek ®. The results from the trial also confirm the relatively high overall prevalence of FIV in Australian cats predominantly among mature male cats in the 9–12 year age group. Given the simplicity of the VetRED FIV™ procedure, it is concluded that VetRED FIV™ is a useful addition to the available commercial tests for FIV infection.

Simplified conjugation chemistry for coupling peptides to F(ab′) fragments: autologous red cell agglutination assay for HIV-1 antibodies

The rapid whole blood test, developed for the detection of circulating antibodies to human immunodeficiency virus type 1 (HIV-1), is based on agglutination of autologous red blood cells using an anti-human glycophorin antibody conjugated to the HIV-1 immunodominant epitope of gp41 (579–613). A simplified procedure for preparing antibody-peptide conjugates for use in the autologous red cell agglutination test is described. F(ab′) 2 fragments of the anti-glycophorin antibody were prepared by pepsin digestion and reduced to F(ab′) fragments with the use of tri- n-butylphosphine (TBP). This permitted the simultaneous reduction of the F(ab′) fragments and coupling of a bromoacetyl derivative of the synthetic immunodominant peptide gp41 (579–613) [Cys-Acm 598, Lys-BrAc 604] containing ϵ-bromoacetyl-lysine at residue 604 to the resultant F(ab′) fragment. Conjugation to the F(ab′) fragment resulted in a stable thio-ether linkage between the peptide Lys-604 and the inter heavy chain cysteines of the F(ab′). The resultant F(ab′)-peptide conjugate was comparable to the previously described disulfide coupled conjugate when used in the autologous red cell agglutination test. This simplified conjugation chemistry may also be useful for the development of reagents for FACS analysis as well as targetted vaccines.

Autologous red cell agglutination test for antibodies to feline immunodeficiency virus

The T-lymphotropic lentivirus, feline immunodeficiency virus (FIV) is now recognised as a major viral pathogen affecting domestic cat populations worldwide. A rapid, autologous red cell agglutination test for antibodies to FIV has been developed. A synthetic peptide analog corresponding to the immunodominant epitope within the FIV transmembrane glycoprotein gp40 residues (680–715) KVEAMEKFLYTAFAMQELGC (Acm)NQNQFFK(BrAc)KIPLELWTR was conjugated to an anti-feline erythrocyte antibody using a thio-ether linkage. Within 3 min of adding this reagent to 20 μl of whole blood, circulating antibody to the peptide epitope caused agglutination of the red blood cells. The performance of this simple test is comparable with the two commercially available enzyme immunoassay (EIA) kits and an EIA based on this peptide. A variant of the gp40 (680–715) peptide corresponding to the FIV, PPR strain gp40 (678–716) sequence was also synthesised and no difference in reactivity was observed in an EIA on 211 seropositive samples, indicating that the peptide-based test may be applicable to other known strains of the virus.

Kinetics of the autologous red cell agglutination test

The kinetics of the autologous red cell agglutination test for detecting circulating antibodies to HIV-1 were studied. Two monoclonal anti-red blood cell antibodies (1C3/86 and 10F7MN) were used to construct Fab-peptide conjugates for the test. Both antibodies recognize glycophorin α on the surface of erythrocytes by immunoprecipitation or immunoblotting techniques. The number of binding sites, association and dissociation constants of 1C3/86 Fab and 10F7MN Fab′ fragments were determined ( n = 4.80 × 10 5 sites/erythrocyte, K a = 0.43 × 10 7 M −1, K d = 23 × 10 −8 M for 1C3/86, n = 4.66 × 10 5 sites/erythrocyte, K a = 1.05 × 10 7 M −1, K d = 9.5 × 10 −8 M for 10F7MN. The binding studies were performed under the same conditions as the autologous red blood cell agglutination test. When 0.9 μg of anti-glycophorin Fab was added to 10 μl of blood 0.25 μg of 1C3/86 Fab was bound whereas 0.29 μg for 10F7MN Fab′ was bound. Antibody binding reached a plateau after 2 min and once bound did not exchange with unbound Fab over the time scale of the test. The binding of the anti-peptide antibody (cross-linking antibody) was also complete within 2 min. Addition of approximately 0.1 μg of anti-peptide antibody gave half a maximal agglutination score. This is equivalent to 10 μg/ml circulating antibody. Under the agglutination test conditions, Fab-peptide conjugate was bound to 14% of available glycophorin molecules. Half maximal agglutination occurred when approximately 1.1% of the bound Fab-peptide conjugates were cross-linked. A maximum agglutination score of four occurred in the presence of 1 μg of anti-peptide antibody equivalent to 100 μg/ml circulating antibody whereas an agglutination score of 1+ was elicited by only 0.032 μg anti-peptide antibody and involved the cross-linking of approximately 160 glycophorin per red cell.

Autologous red cell agglutination assay for HIV-1 antibodies: simplified test with whole blood.

An antibody detection procedure based on agglutination of autologous red cells has been developed for samples of whole blood. A nonagglutinating monoclonal antibody to human red blood cells conjugated to a synthetic peptide antigen (in this case residues 579 to 601 of the HIV-1 envelope precursor, Arg-Ile-Leu-Ala-Val-Glu-Arg-Tyr-Leu-Lys-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys - Ser-Gly-Lys) permitted the detection of antibodies to the human immunodeficiency virus type 1 (HIV-1) in 10 microliters of whole blood within 2 minutes. Agglutination was specifically inhibited by addition of synthetic peptide antigen but not by unrelated peptides. The frequency of false positive results was 0.1% with HIV-1 seronegative blood donors (n = 874). The false negative results were approximately 1% (n = 81). The autologous red cell agglutination test is potentially suitable for simple, rapid, qualitative screening for antibodies to a variety of antigens of medical and veterinary diagnostic significance.

In vitro immunoglobulin synthesis of the peripheral blood and synovial lymphocytes in patients with rheumatoid arthritis

The synthesis of immunoglobulins (IgG, IgA and IgM) by mononuclear cells (MNC) from peripheral blood and synovial fluid in patients with rheumatoid arthritis (RA) was studied emploing radioimmunoassay. The correlations between Ig synthesis rates and clinical data were also studied.The decreased synthesis of IgM by peripheral MNC, and of IgA by synovial fluid MNC, was characteristic of RA patients. A coincidence was found between the synthesis of IgM by peripheral MNC and the decreased serum IgM levels in RA patients. Furthermore, significant correlation was found between IgM synthesis rates by synovial fluid MNC and (a) blood sedimentation rate and (b) sensitized sheep red blood cell agglutination test. In contrast, Igsynthesis rates by peripheral blood MNC did not correlate with the clinical data. These data suggest the significant role of the synovial fluid MNC.

Rheumatoid factor in diabetes mellitus (author's transl)

This study was undertaken to try to determine the incidence of rheumatoid factor in patients with diabetes mellitus by the Hyland RA slide test and the RAHA test (the sensitized sheep red cell agglutination test). Sera from 507 patients with diabetes mellitus and 2073 controls were investigated for the presence of rheumatiod factors. RA test was positive in 8.7 per cent and RAHA test in 6.1 per cent in sera from patients with diabetes mellitus compared to 4.1 percent and 3.6 per cent respectively in healthy subjects.

Radioimmunologic assay for antithyroglobulin antibodies

Abstract A double-antibody radioimmunologic assay for antithyroglobulin antibodies (RATA) in serum is described. The assay employs human thyroglobulin labeled in vitro. The reaction product of antithyroglobulin antibodies and labeled human thyroglobulin is measured after precipitation with rabbit antihuman gamma-globulin serum. This test has a high degree of positivity in autoimmune thyroid disorders. In chronic lymphocytic thyroiditis, 100 per cent of sera from patients with surgically proved disease were positive and 83.8 per cent of those in whom this disease was clinically suspected were positive. In Graves' disease, 87.5 per cent of untreated patients' sera and 57.7 per cent of treated patients' sera were positive. In primary hypothyroidism, 94.4 per cent of patients' sera were positive. The sera from many patients with autoimmune thyroid disorders which were negative or had low titers to the commonly used tanned red cell agglutination test (TRCA) were positive with the RATA test. Four per cent of sera from normal control subjects, 15 per cent of sera from patients with other autoimmune disorders, and 8 per cent of sera from patients with thyroid disorders not considered to be of an autoimmune etiology were positive to the RATA. The RATA is simple and quantitative with sensitivity and specificity equal to or exceeding present tests for circulating antithyroglobulin antibodies.

Subtyping of hepatitis B antigen by use of monospecific antibody-coated cells.

A sensitive red cell agglutination (RCA) method for the detection of hepatitis B antigen (HB Ag) was adapted for subtyping. The technique used human type O, Rh-negative erythrocytes stabilized with pyruvaldehyde and formaldehyde and coated with IgG containing monospecific anti-d or anti-y hepatitis B antibody prepared from antiserum produced in guinea pigs. Optimal coating of stabilized erythrocytes occurred at 25 C and at pH 4.0 with a concentration of IgG of 0.25-0.5 mg/ml and a 60-min incubation period. The coated cells were stable for at least one month when stored at 4 C or for at least four months when frozen at -70 C or -196 C. The RCA test was conducted at 25 C and read after 3 hr. Nonspecific agglutination was not observed at serum dilutions of > 1:10. Results were verified by agar gel diffusion or by a more sensitive double antibody radioimmunoassay method that was modified to detect subspecificities of HB Ag. HB Ag endpoint titrations can be obtained within 3 hr, and the test is 100to 200-fold more sensitive than the agar gel diffusion technique.

Evaluation of a Red Cell Agglutination Test for Detection of Australia Antigen (HB Ag)

An investigational red cell agglutination (RCA) test was evaluated for sensitivity in detecting and titering hepatitis B antigen (HB Ag) in comparison with two counterelectrophoresis (CEP) systems and a solid-phase radioimmunoassay (RIA). The RCA procedure was found to be significantly more sensitive than the CEP methods and compares favorably in sensitivity with the solid-phase RIA, detecting even lower concentrations of the HB Ag. Since the RCA test can be completed in 2 to 3 h and requires relatively inexpensive equipment, it offers a highly sensitive and rapid procedure suitable for use in blood banks to screen donors or detect low levels of antigen in serum of patients.

A heat-labile inhibitor in human serum with the horse red blood cell agglutination test (Mono test) for infectious mononucleosis.

A heat-labile inhibitor in human serum with the horse red blood cell agglutination test (Mono test) for infectious mononucleosis.

Endemic goiter in Misiones, Argentina: pathophysiology related to immunological phenomena.

ABSTRACT A group of 316 patients from an endemic goiter region, Apostoles, Misiones, Argentine Republic, was studied from the immunological point of view and the results obtained were compared with various tests assaying thyroid function. A tanned red cell agglutination test (TRA) and an agar gel immunodiffusion test (AGID) were used for assaying circulating antithyroglobulin antibodies. Eighty-four per cent of the sera were positive for TRA and 56% for AGID, this being the most outstanding aspect of the study. Coincidence of results in both tests was obtained in 66% of the cases. Patients with high TRA titers showed a statistically significant increase in total serum globulins and gamma globulins when compared with low TRA titers or with values obtained in the general population. No changes in albumin values were observed. No correlation was found between immunotests, age, sex, and PB127I. Sixty-eight per cent of the patients with high TRA titers had at the same time high thyroid 131I uptake; 63% had hig...

Distinction between idiopathic primary myxedema and secondary pituitary hypothyroidism by the presence of circulating thyroid antibodies.

Immunologic studies for the detection of circulating antibodies, including the tanned red cell agglutination test, the complement fixation reaction and the double-layer technique of immunofluorescence, were compared in idiopathic primary myxedema (29 cases) and in secondary pituitary hypothyroidism of various causes (41 cases). The immunofluorescence technique was also applied for the detection of antibodies directed against the gastric parietal cells. Thyroid antibodies were detected at a significant level in idiopathic primary myxedema in 55% of cases by the TRC, and in 51.7 and 65.5%, respectively, for the cytoplasmic and the colloidal antigens by the immunofluorescence method, in contrast to 0 % by the TRC and 4.9 and 9.8% for the cytoplasmic and the colloidal antigens in secondary pituitary hypothyroidism. The complement fixation test was positive in only 10 cases, all primary myxedema. Antibodies to stomach were found frankly positive in 45 % of cases of primary myxedema and in 2.4% of cases of secondary hypothyroidism. The difference was still significant if the women of both groups were compared. The theoretical implications of these results are discussed with respect to the unsettled question whether antibodies precede, accompany, or are the consequence of anatomical alteration of the thyroid gland. The usefulness of these immunologic tests in differentiating primary involvement of the thyroid from secondary hypothyroidism is pointed out.

QUANTITATIVE ASPECTS OF THE RED BLOOD CELL AGGLUTINATION TEST FOR INFLUENZA VIRUS

A detailed study has been made of the nature of the variables inherent in the chicken red cell agglutination test for influenza virus in an effort to obtain a method of measurement of biological activity of sufficient accuracy that it might be employed as a reliable index of chemical purity of preparations of the virus. It was found that the temperature at which the test is conducted has a marked effect on the titer, whereas within the range of pH 6-8 the pH has a negligible effect. It was also found that a variation in results may be encountered due to a variation in the specific behavior of red cells from different chickens and to an instability of the red cells themselves. Preparations of purified influenza virus held at 4 degrees C., on the other hand, were found to be stable with respect to chicken red cell agglutinating activity for several months. This fact, together with the fact that in duplicate measurements upon different samples the accuracy was such that the chances were 19 out of 20 that differences of 8.4 per cent in the mean end points were significant, made it possible to establish a reproducible standard of CCA activity based on a unit weight of purified virus material. As a result, it was possible to devise a standardized procedure for carrying out with high accuracy quantitative measurements of influenza virus.

The Chick Red Cell Agglutination Test with the Viruses of Newcastle Disease and Fowl Plague

Summary The high loads were found to have the following main causes, which occured together in many work situations: 1. Hirst's red cell agglutination test, has been shown to be applicable to the viruses of Newcastle disease of fowls and of fowl plague. 2. Serum-inhibition tests show that there is no serological relationship between the viruses. 3. The test provides a useful method for the rapid laboratory diagnosis of either disease.

A Method for the Titration of Influenza Hemagglutinins and Influenza Antibodies with the Aid of a Photoelectric Densitometer

Summary The construction and use of a simple photoelectric densitometer for agglutination studies have been described. This instrument facilitates the estimation of hemagglutinin and antibody titers when using the red cell agglutination test in influenza work. The use of this instrument makes the determination of end points more precise and objective than the technic previously described.

Identification of Mice in Genus Peromyscus by a Red Blood Cell Agglutination Test

Identification of Mice in Genus Peromyscus by a Red Blood Cell Agglutination Test