Anti-human Globulin Reagents Research Articles

Role of direct antiglobulin test in assessing the severity of extravascular hemolysis in autoimmune hemolytic anemia – analysis from a cross-sectional study

Background: Autoimmune hemolytic anemia (AIHA) is an immune hemolytic disease characterized by hemolysis and anemia which results from increased red cell destruction due to auto antibodies directed against self-antigens on red cells. Clinical characteristics and potential associated hemolysis in AIHA can greatly depend on the serological characteristics. Aims and Objectives: This study was done to find out the association of Grade of polyspecific Direct Antiglobulin test (DAT), complement involvement and immunoglobulin G (IgG) subclass with severity of hemolysis in an AIHA patients. Materials and Methods: This was a cross-sectional study conducted for a period of 18 months from March 1, 2012, to August 31, 2013. Fifty consecutive AIHA patients diagnosed by a positive DAT in EDTA anticoagulated blood samples using poly specific anti human globulin reagent were followed up and results of various immunohematological tests such as polyspecific and monospecific DAT, IgG subtyping, and thermal amplitude were performed. Hematological and biochemical parameters such as hemoglobin, percentage of reticulocyte, total serum bilirubin, and serum lactate dehydrogenase also were recorded. Data were analyzed using SPSS v17. Results: Majority of patients were belonging to 31–40 year age group and there was a remarkable female predilection. Secondary AIHA was more common (56%) than primary. SLE was identified as the most common underlying disorder in secondary AIHA. Majority (46%) were Grade 4 DAT positive. Predominant autoantibody identified was IgG (52%). Of the total IgG positive cases, IgG1 was the predominant subclass. C3d alone was detected in 18% of cases and C3d and IgG together in 30%. About 48% of our patients were of warm type, 32% mixed type, and 20% cold type. About 80% of the study population were treated with steroids alone. A small percentage of cases (16%) needed immunosuppressant in addition to steroids and still a smaller percentage (4%) needed splenectomy. Higher grades of DAT positivity were associated with more severe degree of anemia and hyperbilirubinemia. The study also demonstrated a positive correlation between DAT grade and severity of hemolysis. Conclusion: AIHA patients in this study were predominantly of Warm AIHA with more of IgG1 involvement. A significant association is found between higher grades of DAT results and severity of anemia and jaundice in this study group. A positive correlation exists between grades of DCT and hemolysis as well.

Red Cell Alloimmunization in Repeatedly Transfused Sudanese Patients with leukemia in Northern Sudan.

Leukemia frequently causes anemia; thus patients need products containing normal blood or RBCs for treatment. Anti-red blood cell alloantibody formation is still a critical challenge in transfusion. The current study aimed to investigate RBC alloantibodies in Sudanese leukemia patients who receive multiple blood transfusions at the cancer center in Dongola and Maroyee, Northern Sudan. At the Northern State oncology center in Dongola and Maroyee, Sudan, an across-sectional descriptive study design was used. In this study, 100 leukemic patients who had received blood transfusions three times or more were enrolled. From each participant, Peripheral blood was drawn in amounts of 3 ml in EDTA vacutainer tubes for ABO blood group and Rh factor testing and 3 ml in non-additive containers for antibody screening and Alloantibody identification. All individuals' ABO blood groups and Rh factors were determined using the slide method. Indirect Coombs test apply to detect alloantibodies by Polly Specific antihuman globulin reagents using tube method techniques. Alloantibody identification was performed by DiaMed-ID microtyping system. Incidence of alloimmunization was 11%, with 11 alloantibodies found in 11 patients. The most common alloantibody was kell (36.4%), followed by Lea (27.2%; 3/11), then P (18.2%; 2/11) and M (18.2%; 2/11). Anti kell antibody was the most prevalent alloantibody among leukemic patients with multiple transfusions.

A Study of Clinical and Serological Correlation of Positive Direct Antiglobulin Test in Blood Bank at a Tertiary Care Center.

Objectives Detection of red cell bound immunoglobulins and/or complement by direct antiglobulin test (DAT) is a crucial serological assay in the diagnosis of autoimmune hemolytic anemia (AIHA). However, DAT may be positive in a variety of clinical conditions with or without hemolysis. We aimed at evaluating the clinical and serological correlation of positive DAT by categorizing the clinical conditions associated with positive DAT, estimating the presence of in vivo hemolysis in case of positive DAT with polyspecific and monospecific antisera and correlating the strength of positive DAT with the presence of hemolysis. Materials and Methods The prospective observational study was performed on 200 samples that were positive for DAT with polyspecific antiglobulin reagent as the baseline investigation. These samples were further tested with anti-immunoglobulin G and anti-C3 monospecific DAT reagents to evaluate the type of protein responsible for positive DAT. The antiglobulin tests were performed by tube technique. DAT positivity was graded (1+ to 4 + ) in each patient. Autocontrol test was included. The patients with positive polyspecific DAT were categorized into different clinical conditions. The presence or absence of in vivo hemolysis was evaluated in all clinical categories and also for each grade of positivity with polyspecific and monospecific antiglobulin reagents. Statistical Analysis Binomial logistic regression and Mann–Whitney U test were applied to between the group analyses. For categorical variables, Fisher's exact test and relative risk were used. The qualitative data were expressed in numbers and percentages. Results The highest number of patients (75/200, 37.5%) belonged to the autoimmune diseases group. Tuberculosis and hepatitis C were the main infectious diseases associated with positive DAT. Out of 200 DAT-positive patients, 98 (49%) had in vivo hemolysis and 102 (51%) did not have hemolysis. AIHA (22) and systemic lupus erythematosus (18) were the commonest clinical conditions associated with in vivo hemolysis. All the 11 samples that showed positivity with only anti-C3 reagent did not show any hemolysis. There was statistically significant increase in the incidence of in vivo hemolysis with increasing grades of DAT positivity with all the three antihuman globulin reagents. Conclusion There are different disease conditions which show positive DAT with or without hemolysis. So, it is important to clinically and serologically correlate positive DAT results.

Determination of C,c,E and e Antigens Section Prevalence in Rh D Negative Individuals: Is it Good Exercise with Utilities in Clinical Blood Transfusion Practices?

Introduction: Besides D antigen, C,c,E and e antigens of Rh blood group system are the most important antigens involved in alloimmunisation. The prevalence of C,c,E and e antigens in Rh D negative individuals in local population has many utilities in transfusion medicine practice. Aim: To determine the prevalence of C,c,E and e antigens in Rh D negative individuals. Materials and Methods: This prospective observational study was performed at Bhanumati clinical laboratory, Navsari, Gujarat, India, from January 2020 to January 2022. A total of 270 Rh D negative samples irrespective of ABO blood group status were typed for C,c,E and e antigen using monoclonal reagents from two different manufacturers using conventional tube technique. The most likely phenotype and genotype were determined using reference textbooks. Possibility of weak D in these 270 samples was ruled out using polyspecific Antihuman Globulin Reagent performing test for weak D antigens. Results were analysed using Excel spread sheet. Results: Prevalence of C,c,E, and e antigen in the 270 Rh D negative samples were, C (17, 6.29%), c (267, 98.88%), E (1, 0.37%) and e (270, 100%). Most probable genotypes were dce/ dce(rr), dCe/dce(r’r), dCe/dCe(r’r’) and dcE/dce(r’’r). Conclusion: Data obtained of C,c,E and e antigen prevalence in Rh D negative individuals is having varied utilities in blood transfusion services such as formulating rare blood group registry, preparing in house screening and panel red cells, and maintaining inventory of rare blood group units using freezing technology, if available.

A renewed understanding of anti-human globulin reagents: interference constraints using an optimization method in pretransfusion compatibility tests.

Anti‐human globulin (AHG) reagents are widely applied in pretransfusion compatibility tests. The accuracy of detection with AHG reagents is mainly affected by irregular antibodies or cold agglutinins in blood samples, which are related to the human complement system. Although much has been written about various types and applications of AHG reagents, their characteristics, interference factors and optimal selection in pretransfusion compatibility tests still need to be further clarified. Here, we review clinical practice and basic studies that describe each AHG reagent, summarize the advantages and disadvantages of using different AHG reagents in the presence of cold agglutinins or complement‐fixing antibodies, explore the potential mechanisms by which the complement system influences detection with AHG reagents and address the question of how to optimally select AHG reagents for clinically significant antibody detection.

Serological Characterisation of Auto-antibodies in Patients with Direct Antiglobulin Test Positive Autoimmune Haemolytic Anaemia at a Tertiary Care Teaching Hospital in Tirupati, India

Introduction: Haemolysis in Autoimmune Haemolytic Anaemia (AIHA) is a result of Immunoglobulin G (IgG) or Immunoglobulin M (IgM) auto-antibodies with or without complement components binding to the Red Blood Cell (RBC) surface and initiating its destruction. Serologic evidence is provided by autocontrol or Direct Antiglobulin Test (DAT). Diagnostic work-up is essential as the management depends on the antibody type. Characteristics of the bound antibody and the target antigen determine the degree of haemolysis. Serological characterisation in AIHA helps to differentiate into its various types which help the clinician to decide on the treatment to be given. Aim: To serologically characterise the auto-antibodies in patients with DAT positive AIHA at a tertiary care teaching hospital. Materials and Methods: This cross-sectional study was carried out in the Department of Transfusion Medicine, Sri Venkateswara Institute of Medical Sciences, Tirupati, Andhra Pradesh, India, from March 2019 to February 2020. A 40 consecutive patient samples were included in the study. Characterisation of antibody was done using polyspecific Anti-Human Globulin (AHG) reagent followed by mono-specific AHG reagent by gel method. If antibody was of IgG type, then the subclass was determined by a mono specific anti-IgG1 and anti-IgG3 gel card. Association between antibody types, subtype, and strength of DAT with severity of haemolysis were compared using Chi-square/Fisher’s-exact test. A p-value of less than 0.05 was considered statistically significant. Results: The total study population was 40 patients. The mean age of the study population was 45 years (range 13-78). Out of 40 patients, males were 30 (75%) and females were 10 (25%). The primary and secondary causes for AIHA include 4 (10%) and 36 (90%) respectively. Among 40 patients, 22 (55%) patients had IgG antibody alone, 17 (42.5%) patients had IgG antibody with combination of other antibodies and 1 (2.5%) had only complement (C3d). IgG1 was identified in 7 (18%) of patients, combination of IgG1 and IgG3 in 3 (7.7%). There was a significant association with IgG+combination (p-value=0.03), IgG1+IgG3 (p-value=0.029) and strength of reaction (p-value=0.003) with respect to severity of haemolysis. Conclusion: Presence of multiple antibodies, presence of IgG1 and IgG3 and with complement combination and presence of higher grading of reaction in gel column were associated with severity of haemolysis. We recommend that serological characterisation of auto-antibody in AIHA would help the clinician in assessing the severity of haemolysis so that management can be done appropriately.

Donation testing and transfusion transmissible infections

Donation testing and transfusion transmissible infections

Weak D Testing is not Required for D? Patients With C?E? Phenotype

BackgroundAlthough testing to detect weak D antigens using the antihuman globulin reagent is not required for D− patients in many countries, it is routinely performed in Korea. However, weak D testing can be omitted in D− patients with a C−E− phenotype as this indicates complete deletion of the RHD gene, except in rare cases. We designed a new algorithm for weak D testing, which consisted of RhCE phenotyping followed by weak D testing in C+ or E+ samples, and compared it with the current algorithm with respect to time and cost-effectiveness.MethodsIn this retrospective study, 74,889 test results from January to July 2017 in a tertiary hospital in Korea were analyzed. Agreement between the current and proposed algorithms was evaluated, and total number of tests, time required for testing, and test costs were compared. With both algorithms, RHD genotyping was conducted for samples that were C+ or E+ and negative for weak D testing.ResultsThe algorithms showed perfect agreement (agreement=100%; κ=1.00). By applying the proposed algorithm, 29.56% (115/389 tests/yr) of tests could be omitted, time required for testing could be reduced by 36% (8,672/24,084 min/yr), and the test cost could be reduced by 16.53% (536.11/3,241.08 USD/yr).ConclusionsOur algorithm omitting weak D testing in D− patients with C−E− phenotype may be a cost-effective testing strategy in Korea.

P058 Validation of anti-human antibodies used in flow cytometric crossmatching

Aim Flow cytometric crossmatches (FCXM) are widely used to detect recipient alloantibodies against donor tissue to assess compatibility for transplantation. Polyclonal anti-human globulin (AHG) is the reagent typically utilized for the detection of these alloantibodies. Humans produce four primary subtypes of IgG, designated as IgG1–4. Here, we describe a system to determine whether the AHG is able to detect all IgG subtypes. Methods Humanized monoclonal antibodies of each IgG subtype, specific for a red blood cell (RBC) antigen were produced. Briefly, the cDNA for the complimentarity-determining regions (CDR) of the heavy and light chains specific for the Kell RBC antigen were ligated into expression vectors for the separate IgG subtypes 1–4 and kappa light chains, co-transfected into CHO cells, and purified. These monoclonal IgG1–4 antibodies were then tested against RBCs, and three different lots of AHG were used to detect the antibodies bound to the RBCs in a standard FCXM assay. An anti-kappa light chain antibody that recognizes an epitope common in all IgG subtypes was used as a positive control. The AHG alone was used as a baseline control. Results As shown in Fig. 1, each lot of AHG was able to detect all four subtypes of IgG. In addition, titration experiments were performed to determine whether there was preferential detection of any IgG subtype(s). These experiments demonstrated that AHG does exhibit preferential binding to different IgG subtypes. In one specific lot of AHG, the hierarchy of binding/detection was IgG3 > IgG1 > IgG2 > IgG4. Download : Download high-res image (264KB) Download : Download full-size image Conclusion These data provide a proof-of-principle for a method to characterize and validate AHG reagents used in FCXM assays. This is important, because the inability of AHG reagents to detect IgG subtypes may lead to false-negative FCXM results. These animal-derived AHG reagents are polyclonal, and there may be lot-to-lot variability in their ability to detect different IgG subtypes. J. Zimring: Grant/Research Support; Company/Organization; Immucor, Inc.. Scientific/Medical Advisor; Company/Organization; Rubius Therapeutics. Employee; Company/Organization; Bloodworks NW.

Low incidence of D alloimmunization among patients with a serologic weak D phenotype after D+ transfusion.

By consensus definition, the red blood cells (RBCs) of patients that react weak to 2+ in the serologic D test are classified as having the serologic weak D phenotype (weak D). The risk of D alloimmunization in patients with weak D is not well studied. This study retrospectively determined the incidence of D alloimmunization in patients with weak D who received D+ blood products. Patient records at four institutions were reviewed. Eligible patients had at least 1 D typing result with agglutination strength that was weak to 2+ result in gel or tube (Institutions 1-3) or weak to 2+ result in solid phase or a positive tube weak D test using antihuman globulin reagent (Institution 4). All patients received at least 1 D+ allogeneic RBC or platelet transfusion and had a subsequent antibody detection test performed at least 30 days after the first D+ transfusion. The incidence of alloanti-D formation was 0.15% (6/4011, at Institutions 1-3) and 5.1% (3/59, at Institution 4; these incidences were significantly different [p=0.0002]). There were 0.15% (6/4011) patients who had autoanti-D detected; all were at Institutions 1 to 3. Until RHD genotyping is widely available, these data support the fact that patients with serologic weak D may be transfused with D+ RBCs if an urgent transfusion is required. At Institution 4, inclusion of a higher proportion of black patients compared to the other institutions may have affected the incidence of alloimmunization by perhaps including partial D recipients who are at risk of forming D antibodies.

Evaluation of the Use of Gel Card System for Assessment of Direct Coombs Test: Weighing the Pros and Cons

A positive direct Coombs test (DCT) is the hallmark of diagnosis of immune hemolytic anemias. The reagent used for the test is the Antihuman globulin (AHG), which may be either 'Polyspecific' or 'Monospecific'. The advent of the Gel card systems has made the procedure and interpretation of DCT simpler. Aim of this study is to evaluate three of the various techniques used for the performance and interpretation of DCT. A total of 96 EDTA samples were included in the study. DCT was performed by (i) polyspecific AHG manual tube method (ii) polyspecific AHG Gel card method and (iii) monospecific AHG (Anti IgG and Anti Complement) manual tube method. In our study we considered positivity by monospecific AHG as the standard for diagnosis. Of the total 96 samples evaluated, 44 cases positive by Gel card method, were also positive for either one or both the monospecific AHG reagents. 17 cases positive by Gel card were negative by all manual methods. These false positive cases were attributed to reasons such as increased ESR, macrocytosis and marked leucocytosis. Nine cases were negative by Gel card but were positive with the Monospecific AHG. The sensitivity of DCT done by the Gel card technique was 83.01% and the specificity was 60.46%. Use of Gel card technique to perform and interpret DCT is easier than manual tube methods, but positivity by Gel card needs to be correlated with clinical presentation of the patient and other laboratory findings. Monospecific antisera can be used to confirm cases that are positive by the Gel card systems.

Donation testing

Donation testing

Quality issues of laboratory reagents

Dear Sir, I send my sincere thanks to you and your team members for coming out with a very good Asian Journal of Transfusion Science. I have gone through each and every line of all the issues published till date and I have found them to be extremely useful. You and Your team members deserve special thanks because for Blood Bankers like me working in resource poor sectors, subscribing and reading costly international journals on transfusion medicine and blood banking is a dream to be fulfilled. AJTS has been a BOON to people like me working in charitable institutes which do not have funds for journals. I have few queries related to blood banking techniques and procedures. I would be highly obliged if you can answer them 1) We know that Monoclonal Anti D (IgG + IgM) is preferred over Monoclonal Anti D (only IgM) to pick up weaker variants of D antigen, then what is the role of Monoclonal Anti D (only IgM) and why do companies prepare and market them? 2) Acceptable quality of Anti Human Globulin reagent as per quality parameters detailed in NABH and DGHS publications have some technical issues to be used in routine practice a) Agglutination of red cells sensitized with anti D serum containing not more than 0.2 mg/ml antibody. How to measure this 0.2 mg/ml antibody activity in the anti D serum vial? b) Agglutination of red cells sensitized with a complement binding antibody (e.g. anti Le a) How to prepare or get such sensitized cells? c) Agglutination of red cells coated with C3b and C3d and no/weak agglutination with C4 coated cells How to prepare or get such red cells coated with C3b and C3d and also C4 coated cells?

Review: monoclonal reagents and detection of unusual or rare phenotypes or antibodies

Abstract Monoclonal antibodies have been used in the formulation of commercially available blood grouping reagents since the early 1990s. It became apparent early on that introducing them into routine use along with, or instead of, human- or animal-derived reagents could and did lead to discrepant reactions. These discrepancies most often came to light when confirming a blood type obtained previously with human- or animal-source reagents or when using two or more sources of a reagent from the same or another manufacturer to perform blood typing or antibody detection or identification testing. A number of factors contribute to differences in reactivity of reagents that are of the same specificity but are from more than one source. One factor is the use of different clones of the same specificity to manufacture blood bank reagents. Another is the effect of the various diluents used by different manufacturers to formulate reagents that contain the same clone(s). In addition, RBCs having unusual or rare phenotypes can cause discrepant reactions when performing phenotyping. Discrepant reactions can also occur because of patient or donor antibodies that react in an unusual manner when antiglobulin tests are performed with monoclonal antihuman globulin (AHG) versus rabbit AHG reagent. It is important to know the identity of the unusual or rare phenotypes and antibodies and to be able to recognize the different types of reactions that will be observed when using more than one reagent of the same specificity. Most importantly, one must be able to interpret reactions correctly and establish the true blood type of the RBCs or specificity of the antibodies. This review will describe situations in which the use of monoclonal reagents from more than one source or manufacturer, or comparison with results of human- and animal-source reagents, resulted in discrepancies with unusual or rare phenotypes or antibodies. Many of the samples described in this review were sent to the reference laboratory at Gamma Biologicals, Inc., in Houston, Texas, which later became ImmucorGamma with sites in Norcross, Georgia, and Houston, Texas. Immunohematology 2006;22: 52–63.

Neonatal Alloimmune Neutropenia Due to Anti-HNA-1b

Neonatal Alloimmune Neutropenia Due to Anti-HNA-1b

International Reference Reagents: Antihuman Globulin

AbstractAn international working party has conducted a study designed to select a suitable reference reagent for antihuman globulin, to replace those first made available in 1987. The chosen preparation contains levels of anti‐lgG and anti‐C3 (anti‐C3c and anti‐C3d) potency that are considered suitable to serve for reference when evaluating either polyspecific antihuman globulin reagents or those containing their separate monospecific components. The reference material is available in 2‐ml freezedried aliquots from seven assigned distribution centres.

Survey on pretransfusion testing.

Hospitals and blood centers throughout the United States use a variety of reagents and methods to perform pretransfusion testing. A survey was developed to determine the reagents and methods in use and their relative prevalence in different work settings. A national survey on pretransfusion testing was conducted. Surveys were distributed to state and regional blood bank associations, which then distributed them to hospitals and blood centers within their region. In most instances, the blood centers distributed the survey to the local hospitals. Completed surveys were returned to the authors for review, and all information was entered into a database for analysis. Analysis of the data shows that the majority of blood banks use monoclonal reagents for ABO testing and monoclonal-polyclonal blended reagents for Rh testing. The data show that anti-IgG and polyclonal antihuman globulin reagents are used almost equally for antibody screening (detection) tests and that most blood banks use a three-cell antibody-screening test. Slightly more than 50 percent of hospitals use an immediate-spin crossmatch in the absence of unexpected antibodies. A number of approved reagents and methods are used by blood bank laboratories for pretransfusion testing. Facility size (number of beds) and type tend to influence the choice of methods and reagents employed. This survey provides an opportunity for blood bank laboratories to compare their current practices with those of their peers.

A new microplate red blood cell monolayer technique for screening and identifying red blood cell antibodies.

A new method has been developed to immobilize red blood cells in wells of microplates using a cell fixation buffer. This method has been employed for detecting and identifying red blood cell antibodies with greater sensitivity than haemagglutination antiglobulin tests, without loss of specificity. This method decreases the amount of test erythrocytes and anti-human globulin reagents employed per test, consequently lowering the cost.

A New Microplate Red Blood Cell Monolayer Technique for Screening and Identifying Red Blood Cell Antibodies

A new method has been developed to immobilize red blood cells in wells of microplates using a cell fixation buffer. This method has been employed for detecting and identifying red blood cell antibodies with greater sensitivity than haemagglutination antiglobulin tests, without loss of specificity. This method decreases the amount of test erythrocytes and anti-human globulin reagents employed per test, consequently lowering the cost.

Laboratory medicine parameter. Utilizing monospecific antihuman globulin to test blood-group compatibility.

When using polyspecific antihuman globulin (AHG) reagents to detect unexpected antibodies or for crossmatching, reactivity in the AHG phase may be due exclusively to the AHG anticomplement component. A lengthy evaluation may be needed to prove that the reactivity is caused by a "nuisance" antibody, one that is clinically insignificant. Clinically significant transfusion intolerance is rare when caused by blood group alloantibodies, which are detected only with AHG sera that contain anticomplement activity. Using monospecific anti-IgG AHG reagents to detect unexpected antibodies offers reliability while avoiding interference from some common and clinically insignificant IgM complement-fixing antibodies, and thereby saves time and expense.