Direct Antiglobulin Test Strength Research Articles

Red cell serological characterization of autoimmune hemolytic anemia in systemic lupus erythematosus patients

Abstract: INTRODUCTION: Autoimmune hemolytic anemia (AIHA) is a clinical condition with increased red cell destruction or decreased red cell survival due to autoantibodies against self-antigens on red blood cells. AIHA may be primary or secondary to other underlying illness. Systemic lupus erythematosus (SLE) is the most common secondary cause of AIHA. Although the incidence of AIHA in SLE patients is low (5%–10%), sometimes, it causes severe anemia which requires blood transfusion. Characterization of these autoantibodies in AIHA is very important, because the clinical condition and treatment of the patient depend on the optimal temperature reactivity, class, and subclass of immunoglobulin responsible. AIM AND OBJECTIVES: The aim and objective of this study are to perform detailed characterization of red cell autoantibodies for accurate diagnosis of AIHA in SLE patients, to find out specific class (immunoglobulin G [IgG]/IgM/IgA) of immunoglobulin and/or complement (C3c/C3d) responsible for AIHA, and their association with severity of hemolysis. MATERIALS AND METHODS: This was a prospective analytical study, done at a tertiary care hospital in Hyderabad, conducted between February 2021 and May 2022. SLE patients with AIHA were included in the study. Monospecific direct antiglobulin test (DAT) was performed to identify the specificity of autoantibody or complement. Based on hematological laboratory values, the study population was divided into three groups, i.e., mild, moderate, and severe hemolysis. DAT strength, hemolytic parameters, and severity of hemolysis were compared between different types of AIHA in SLE patients. Correlation statistics were used to know the association of severe hemolysis with DAT strength and different combinations of autoantibodies. RESULTS: Among 150 patients enrolled, 16 (10.6%) patients had severe hemolysis, 51 (34%) patients had moderate hemolysis, and 83 (55.3%) patients had mild hemolysis. Only IgG was positive in 109 (72.6%) patients, both IgG and C3d were positive in 37 (24.6%) patients, C3d was positive in 4 patients, and IgM was positive in 19 patients. High DAT reaction strength (3+–4+) and presence of C3d or IgM along with IgG were moderately correlated with severe hemolysis. CONCLUSION: Warm AIHA is the most common type of AIHA in SLE patients mediated by IgG. Mild and moderate hemolysis was seen in most cases. Severe hemolysis was moderately correlated with the presence of C3d or IgM. Further studies are warranted to find the predisposing factors leading to severe hemolysis.

Role of direct antiglobulin test in anemia under evaluation: Its prevalence, laboratory workup, and significance in further patient management-A study from eastern India.

The direct antiglobulin test (DAT) is an important diagnostic tool for immune hemolytic anemia (IHA). The present study was primarily aimed to identify the prevalence of DAT positivity in anemia patients along with its specificity . A three months follow up of the DAT positive patients were performed for the response during course of illness in terms of transfusion requirement, hemoglobin level, persistence of DAT. This cross sectional study was performed at a government medical college on symptomatic anemia patients. At initial evaluation, complete blood count (CBC), blood grouping and DAT were performed in the EDTA blood. DAT positive blood samples were analyzed for their immunoglobulin specificity, auto or alloantibody type. Acid elution and red cell phenotyping were performed wherever applicable.Their clinical presentation, hematological and biochemical parameters of hemolysis were evaluated. Statistical analysis was performed on the results on SPSS (Version 23.0;.USA) and Graph pad Prism version 9. P value <0.05 was considered significant. DAT was present in 64 out of 501 patients with male female ratio 1: 4. Warm AIHA (WAIHA) was 93.7% with secondary WAIHA 60%. IgG was associated in 86% DAT positive samples, Only C3d was 14%. All the 4 cold AIHA (6.3%) had a higher antibody titre and thermal amplitude. DAT strength was directly proportional to the degree of hemolysis. During 3 months follow up , persistence of DAT and blood transfusion requirement was more in secondary WAIHA . Hemoglobin increment was more in primary WAIHA (75%). DAT played a significant role in the diagnosis as well as evaluation of AIHA.

Clinical profile and serological correlation with haemolysis in DAT-positive autoimmune haemolytic anaemia patients in Bangladesh

Discerning the type of autoimmune haemolytic anaemia (AIHA) is crucial for transfusion support and initiation of treatment. This study aimed to establish the clinical profile and serological character of red cell autoantibodies and to investigate the relationship with haemolysis in AIHA patients who were direct antiglobulin test (DAT)-positive. A total of 59 DAT-positive AIHA patients were included in this study. Clinical, laboratory and serological findings were evaluated to find the gradation of haemolysis and to investigate its correlation with age, sex, type of autoantibody and level of autoantibody. Study findings revealed that most patients (89.8%) had haemolysis, wherein moderate haemolysis (67.8%) was predominant. Weakness, palpitations, fever, pallor, tachycardia and splenomegaly were common among patients with severe and moderate haemolysis. The majority (66.1%) had an associated disorder. Warm autoantibody was the most common, followed by cold and mixed cases. The severity of haemolysis correlated strongly with the strength of the DAT reaction (Cramer V 0.636, p<0.001). These findings may be useful to clinicians while determining a treatment plan. The direct relationship between severity of haemolysis and strength of DAT needs further exploration in a large population to establish whether it can be used as a tool to formulate a treatment plan when assessing AIHA patients in low resourced countries.

Does strengths of a positive direct antiglobulin test predicts the need for phototherapy and duration of phototherapy? – a single center, retrospective study

Background Use of Direct Antiglobulin test (DAT) in management of neonatal hyperbilirubinemia is conflicting. Objective whether strength of positive DAT predicts the need for phototherapy, duration of phototherapy and need for major interventions. Methods We retrospectively collected data on all DAT positive neonates with birth gestational age ≥32 weeks over six years (2014–2019). Data regarding blood group, DAT and clinical details were obtained from a hospital database. We also collected data on serial hemoglobin and other relevant laboratory parameters. We also collected data on infants receiving major interventions such as exchange transfusion, in-utero transfusion, immunoglobulins, and postnatal transfusion for the duration of the study period. All of these infants were electronically followed up for a period of 6 weeks. This study was approved by institutional audit authority. All the statistics were performed using SPSS software. Results Out of 1285 DAT tests performed, only 91 infants were positive (7%), and 78 DAT positive infants were available for analysis. There were 54 infants with DAT (1+), 15 infants with DAT (2+), 7 infants with DAT (3+) and 2 infants with DAT (4+). There was no significant statistical difference in terms of need for phototherapy, duration of phototherapy, need for major interventions and hemoglobin levels at different time points between the groups (DAT 1+ Vs DAT ≥2+; DAT ≤2+ Vs DAT >2). A Total of 10 infants received major intervention, with one infant receiving all three interventions (DAT 3+ with significant maternal antibodies), 2 additional infants (both DAT1+) received exchange transfusion, 6 additional infants received immunoglobulin (2 infants: DAT 2+; 4 infants: DAT 1+) and one additional infant (DAT 1+) with significant maternal antibodies received a postnatal transfusion. Conclusion Strength of a DAT did not predict the need for phototherapy, duration of phototherapy, and the need for major hemolysis related intervention in the first 6 weeks of life.

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.

The Spectrum of Hemolytic Disease of the Newborn: Evaluating the Etiology of Unconjugated Hyperbilirubinemia Among Neonates Pertinent to Immunohematological Workup.

Background and objectiveThe exact burden of hemolytic disease of the newborn (HDN) attributed to neonatal unconjugated hyperbilirubinemia (NUH) in developing nations is still unclear. Still, anti-D is reported to be the most common cause of HDN in India. ABO incompatibility has emerged as a leading cause of exchange transfusion (ET) in many countries. But many centers in our country rely on direct antiglobulin test (DAT) as a screening tool to evaluate immunological causes, whereas advanced immunohematological workup like antibody screening, identification, and elution tests are also required. Early identification of implicated antibodies resulting in HDN can aid in the proper selection of blood units when ET is indicated, and hence also in managing the subsequent pregnancy. This study focused on determining the causes of neonatal hyperbilirubinemia (NH), especially with respect to immunohematological evaluation. This cross-sectional study was conducted on 240 neonates requiring neonatal intensive care unit (NICU) support for NUH at a tertiary care hospital.Materials and methodsDemographic data, along with detailed history pertaining to the cause of hyperbilirubinemia, was collected. Clinical and laboratory evaluation and complete immunohematological work including DAT, heat elution, antibody screening, antibody identification, and Rh Kell phenotyping were performed from neonate blood samples. Data were analyzed using SPSS Statistics version 19 (IBM Corp., Armonk, NY).ResultsPathological jaundice was more common (62.1%) than physiological jaundice (37.9%). The various pathological causes identified were HDN (42.6%), sepsis (12%), cephalohematoma (5.4%), and idiopathic (1.7%). Among HDN cases, ABO incompatibility (39.2%) was the most prevalent cause, followed by Rh HDN and G6PD deficiency (1.7% each). DAT was positive in only 14 cases out of 94 ABO-incompatible cases. Elution revealed antibodies in four DAT-negative neonates with ABO incompatibility and more specificity to the OA setting. DAT was positive with 100% sensitivity in Rh HDN cases (n=4). Elution demonstrated the presence of anti-D (n=2), anti-D + anti-C (n=1) and anti-E (n=1), confirming Rh HDN. DAT strength was found to be significantly associated with hemoglobin (Hb) level (p=0.048). The majority of cases were treated with phototherapy only (94.1%), and 10 cases received both ET and phototherapy. Four neonates' condition improved without any intervention.ConclusionThis study highlighted the shift in the trend from Rh HDN to ABO incompatibility as the cause of hemolytic jaundice in NICU neonates. Elution tests can aid in the diagnosis of DAT-negative ABO-incompatible hemolytic anemia. Early diagnosis, along with timely intervention and appropriate measures, can prevent neonatal morbidity and mortality. Negative DAT does not rule out HDN. Sensitive techniques like elution must be used in the presence of clinical suspicion.

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.

Study on the effect of direct anti-human globulin test positive strength on patients' red blood cell transfusion

Objective: To investigate the effect of direct antiglobulin test (DAT) positive strength on the clinical effect of red blood cell transfusion. Methods: The clinical data was collected on blood transfusion compatibility test and clinical transfusion of patients who underwent DAT testing from January 2015 to March 2019 in the Department of Blood Transfusion, Beijing Shijitan Hospital affiliated to Capital Medical University. A retrospective cohort study method was used to analyze the correlation between the DAT positive strength and the blood match results, irregular antibody test results, bilirubin value after blood transfusion, and red blood cell transfusion effect. Results: According to the blood matching test results, the minor cross-matching results were significantly correlated with the DAT strength (r=0.58, P<0.01). The DAT strength of the antibody-screening positive patients was greater (χ(2)=126.810, P<0.01). DAT-positive strength influences the direct bilirubin, the higher the DAT-positive strength, the higher the direct bilirubin concentration of the patients (χ(2)=32.069, P<0.01). DAT-positive strength also had an effect on patients' total bilirubin value (χ(2)=17.981, P=0.001). From the effect of red blood cell transfusion, patients with a DAT positive strength of 0.5 had a lower effective rate of transfusion (28.6%). The remaining DAT strength had no relation to the effect of red blood cell transfusion, and the efficiency of blood transfusion in patients with different DAT-positive strengths (1, 2, 3, 4) was 57.0%, 60.1%, 65.6% and 60.0%, respectively. With the same DAT strength, the patient's transfusion efficiency was higher (χ(2)=41.071, P=0.009). Conclusion: Different DAT-positive strengths are correlated with the patient's minor cross-match results and bilirubin content, while the effect of red blood cell transfusion has no significant correlation with DAT-positive strength. However, with the increase of DAT positive strength, patients are more likely to have irregular antibodies.

Clinical Implication of Immunohaematological Tests in ABO haemolytic disease of newborn: Revisiting an old disease.

We aimed to assess the frequency distribution of of ABO haemolytic disease of newborn (ABO-HDN) and to know the predictive value of immunohaematological tests in identifying at risk neonates. ABO incompatibility, although a common cause of haemolytic disease of newborn, has several unaddressed issues related to it. A prospective study over 20 months was carried out in a tertiary care centre in South India. Blood grouping, Direct Antiglobulin test (DAT) and elution studies were performed on neonatal samples, whereas blood grouping, antibody screening and antibody titration were performed on maternal samples. In suspected cases, ABO-HDN was diagnosed after excluding other possible causes for haemolysis. The laboratory results were correlated with the clinical details to assess the predictive value of the tests. Of the total 2856 pregnancies, 34% had ABO incompatibility. On testing with columnagglutination test (CAT), the overall DAT positivity and that among ABO-incompatible cases were 3.8% and 11.2%, respectively,) whereas by conventinal tube technique (CTT) it was 0.6% and 2.4% respectively. CAT was found to have higher sensitivity, and the predictive value was higher for CTT. Maternal IgG titre showed a positive linear relationship with the DAT strength and the rise in indirect bilirubin levels. The positive predictive value of combination of tests such as DAT, elution and titation was 94.12%, which was much higher than that of the individual tests. DAT positivity is a predictor of early rise in serum bilirubin level, and a combination of tests has a better predictive value than individual tests towards development of clinically significant hyperbilirubinemia in ABO-HDN.

Clinical Profile and Severity of Hemolysis in Adult Patients of Primary Autoimmune Hemolytic Anemia and Their Response to Steroid: A Prospective Cohort Study from Single Institution.

Autoimmune hemolytic anaemia (AIHA) has traditionally been classified based on the temperature sensitivity of the autoagglutinins as warm (WAIHA), cold (CAIHA) and mixed type. Autoagglutinin may be of IgG or IgM type. The present prospective study was conducted to evaluate the profile of clinical picture, severity of haemolysis, treatment response of steroid. This study on patients of adult primary AIHA was conducted by taking complete history followed by detail physical examination. Laboratory investigations were performed to establish haemolytic anaemia and to assess severity of haemolysis. Immunehematological work up including blood grouping, direct antiglobulin test (DAT), IAT, antibody screening, adsorption elution was performed to diagnose type of AIHA. All cases were followed up to assess the response to prednisolone. All the data were collected and analysed by SPSS 19. Out of 62 primary AIHA cases, female were affected more than male (41:21). WAIHA is most common type (42, 67.8%) followed by mixed (20.9%) and cold AIHA (11.3%). Severity of haemolysis showed significant correlation with the DAT strength and not with type of AIHA. (P < 0.05) On oral prednisolone, 22 cases attended complete remission, while relapse, drug dependency and partial remission was achieved in 13, 9, 3 cases respectively. Severity of haemolysis in AIHA is directly related with DAT strength. WAIHA is most common type and can be managed with oral prednisolone (cr 45.2%), without red cell transfusion in most of cases. Mixed type AIHA cases were presented mostly with severe haemolysis, with minimum therapeutic response to prednisolone and maximum relapse/drug dependency.

Correlation of Direct Antiglobulin Test Strength in Umbilical Cord Blood and Hyperbilirubinemia in ABO Incompatible Neonates with Two Methods

Introduction: The direct antiglobulin test (DAT) detects the presence of IgG and/or C3 on the red blood cell (RBC) membrane and is used to diagnose autoimmune hemolytic anemias as well as hemolytic disease of the fetus and newborn (HDFN). A positive DAT in a neonate occurs when maternal IgG crosses the placenta and binds to fetal RBCs, which may lead to hemolysis and clinical manifestations of HDFN such as hyperbilirubinemia. Historically, DATs in umbilical cord blood samples were performed manually. We have recently switched to using the Ortho Vision Analyzer (gel technology) for this purpose in our institution, and noticed a trend toward stronger positive reactions. The purpose of this study was to compare the DAT results obtained with the conventional tube method and the gel method, and their association with hyperbilirubinemia and/or the need for phototherapy in the neonates tested.Methods: We retrospectively reviewed all cord DAT results of infants born between January 2016 and June 2018. We included all tests of neonates of blood group A or B born to blood group O mothers who had a positive DAT and performed a retrospective chart review to collect the following data: DAT strength, gestational age, birth weight, hematocrit, blood type, initial and peak serum total bilirubin levels, use of phototherapy, antibody screening results, and transfusion history. Neonates whose mothers had a positive antibody screen were excluded from the analysis.Results: During the study-period, 100 ABO-incompatible neonates with a positive DAT were tested by the tube and gel methods (50 with each methodology) and met our inclusion criteria. Their demographic information and laboratory results, including the strength of the positive DAT are in Table 1. There was a clear trend toward stronger DAT results since the gel methodology was introduced; the most common result in the Ortho Vision Analyzer was 1+ positive (52%), and 40% of neonates had a 2+ positive result. The latter was only seen in 2% with the tube method. Furthermore, although there were no significant differences in any demographic or laboratory parameters between the two groups of neonates, an increased percentage of those tested with the new methodology (23/50 or 46%) received phototherapy compared with 14/50 (28%) of those tested manually (Figure 1).Conclusions: We found that the Ortho Vision Analyzer produced stronger DAT results compared to the tube method without evidence of increased hemolysis. It is essential that Transfusion Medicine physicians communicate the change in methodology to neonatologists to make them aware of the reason for the stronger DAT results in order to avoid unnecessary phototherapy. DisclosuresNo relevant conflicts of interest to declare.

Diagnosis and treatment of autoimmune hemolytic anemia: classic approach and recent advances.

Diagnosis and treatment of autoimmune hemolytic anemia: classic approach and recent advances.

Autoimmune hemolytic anemia: From lab to bedside

Autoimmune hemolytic anemia (AIHA) is not an uncommon clinical disorder and requires advanced, efficient immunohematological and transfusion support. Many AIHA patients have underlying disorder and therefore, it is incumbent upon the clinician to investigate these patients in detail, as the underlying condition can be of a serious nature such as lymphoproliferative disorder or connective tissue disorder. Despite advances in transfusion medicine, simple immunohematological test such as direct antiglobulin test (DAT) still remains the diagnostic hallmark of AIHA. The sensitive gel technology has enabled the immunohematologist not only to diagnose serologically such patients, but also to characterize red cell bound autoantibodies with regard to their class, subclass and titer in a rapid and simplified way. Detailed characterization of autoantibodies is important, as there is a relationship between in vivo hemolysis and strength of DAT; red cell bound multiple immunoglobulins, immunoglobulin G subclass and titer. Transfusing AIHA patient is a challenge to the immunohematologist as it is encountered with difficulties in ABO grouping and cross matching requiring specialized serological tests such as alloadsorption or autoadsorption. At times, it may be almost impossible to find a fully matched unit to transfuse these patients. However, transfusion should not be withheld in a critically ill patient even in the absence of compatible blood. The “best match” or “least incompatible units” can be transfused to such patients under close supervision without any serious side-effects. All blood banks should have the facilities to perform the necessary investigations required to issue “best match” packed red blood cells in AIHA. Specialized techniques such as elution and adsorption, which at times are helpful in enhancing blood safety in AIHA should be established in all transfusion services.

Autoimmune Hemolytic Anemia With Gel-Based Immunohematology Tests

We used gel centrifugation tests (GCTs) to analyze the relationship between the diagnosis and immunohematology tests used for autoimmune hemolytic anemia (AIHA). The study included 588 samples positive for the direct antiglobulin test (DAT). Of these, 52 were from patients diagnosed with AIHA. Immunoglobulin (Ig) class, IgG1, IgG3, and complement were measured. DAT strength had the strongest correlation with AIHA diagnosis (odds ratio [OR], 23), followed by anti-IgG titer 300 (OR, 8.4), anti-IgG titer 1,000 (OR, 10.5), and C3d agglutination strength (OR, 1.7). Decision tree analysis revealed that DAT strength and anti-IgG titer higher than 100 were the best predictors of AIHA. Multidimensional scanning analysis found a high grade of similarity among DAT strength, anti-IgG titer, and IgG strength in the AIHA samples. This observation was not detected in DAT-positive samples from patients without AIHA. DAT strength remained the best diagnostic indicator for AIHA and had the strongest association with AIHA compared with other commercially available immunohematology tests. The other tests, despite good correlation with AIHA diagnosis, did not add useful information.

Significance of Quantitation of Autoantibodies in the Eluate of Sensitized Red Cells in Warm Autoimmune Hemolytic Anemia

Background Concentrated autoantibodies obtained by elution can be subjected for total serological characterization including their quantitation. The present study was performed to correlate the quantity of immunoglobulin (Ig) G (IgG) autoantibodies in eluate with direct antiglobulin test (DAT) reactivity and markers of in vivo hemolysis in warm autoimmune hemolytic anemia (WAIHA). Methods Detailed autoantibody characterization was performed on 34 samples with regards to antibody class, IgG subclass, DAT dilution and quantification study on eluate. Cold acid elution was performed on the DAT positive red cells and quantitation of IgG in eluates was done using ELISA. Number of IgG molecules eluted per RBC was calculated using Avogadro’s number. Results We observed a significant correlation between the concentration of IgG in eluate and DAT strength (r = 0.53, P = 0.014), hemoglobin (Hb)(r = −0.41, P = 0.04), serum bilirubin (r = 0.67, P = 0.000) and serum lactate dehydrogenase (LDH) (r = 0.50, P = 0.02). The number of IgG molecules/RBC also correlated significantly with the DAT strength (r = 0.69, P = 0.001) as well as DAT dilutions (r = 0.53, P = 0.004). Conclusions Quantification of autoantibodies in eluate in WAIHA helps to assess the severity of the disease and plan appropriate management.

Application of flow cytometry in detection of red cell bound IgG in Coombs negative AIHA

Coombs negative autoimmune hemolytic anemia (AIHA) is characterized by laboratory evidence of in vivo hemolysis along with a negative direct antiglobulin test (DAT) performed by conventional tube technique (CTT) in clinically suspected AIHA patients. The sensitive gel test (GT) and flow cytometry (FC) can effectively diagnose such patients where CTT does not detect low level of red cell autoantibodies. We investigated the use of FC in the serological evaluation of CTT DAT negative AIHA and its comparison with GT DAT. Of the 50 patients with suspected AIHA, CTT DAT was negative in 5 patients (Coombs negative AIHA). GT DAT could detect red cell autoantibodies in 4 of these 5 patients. Monospecific GT DAT showed IgG and/or C3d as the responsible autoantibody. FC was considered as reactive when MFI was > 3.6 (mean of 20 healthy negative volunteers +2SD). FC was reactive in all five Coombs negative AIHA patients. The mean MFI in five known CTT DAT positive samples taken for comparison was significantly higher compared to 5 DAT negative AIHA (18.3 ± 7.78 vs. 7.88 ± 1.35, p < 0.05). There was poor correlation between strength of GT DAT and MFI by FC. We conclude that FC is more sensitive test than the CTT and helps in the serological diagnosis of Coombs negative AIHA. However, in resource poor settings, GT DAT can be a good alternative to FC.

Lower hemoglobin levels in human immunodeficiency virus–infected patients with a positive direct antiglobulin test (DAT): relationship with DAT strength and clinical stages

There are conflicting opinions regarding the effect of positive direct antiglobulin test (DAT) on hemoglobin (Hb) levels in human immunodeficiency virus-infected (HIV+) patients. A total of 166 samples from HIV+ outpatients were studied. The DAT was performed with the tube test and column agglutination technology (CAT). The DAT was positive in 18.67 percent with the tube method and 33.73 percent with the CAT. Patients with DAT-positive results showed lower Hb levels than DAT-negative patients, 12.3 g per dL versus 14.3 g per dL (p = 0.0002). The univariate logistic regression enabled us to study the phenomenon better and fit the probability of having a DAT-positive result on the basis of the Hb levels. The relationship between the CAT and the tube test when washing the red blood cells (RBC) at 4 degrees C was stronger than when washing these at room temperature (phi = 0.8156; p = 0.000). The Hb levels were significantly lower in the positive DATs of Stage C (acquired immune deficiency syndrome [AIDS]) and Stage B (symptomatic non-AIDS patients), which showed decreasing Hb values for increasing agglutination strengths (p = 0.000). Anemia was related with the DAT results (odds ratio [OR], 8.005; p = 0.000) but not to the AIDS condition (OR, 1.741; p = 0.221). Our study indicates that the DAT-positive results may be specifically related to lower Hb levels in HIV+ patients. The immunologic RBC clearance could be part of the anemic multifactorial condition in HIV+ patients.

Spontaneous agglutination of red cells with a positive direct antiglobulin test in various media.

Red cells from 475 individuals (donors and patients) with a positive direct antiglobulin test (DAT) (66% were 1/2+ to 1+, 19% were 1 1/2+ to 2+, and 15% were 2 1/2+ or greater) were examined for spontaneous agglutination following incubation in various media. Twenty-three percent of the samples showed spontaneous agglutination in commercial Rh control solutions, 6 percent in 30 percent albumin, 3 percent in 10 percent albumin, 2 percent in 6 percent albumin, and 1 percent in saline. Cells suspended in serum were more prone to spontaneous agglutination. The strength of spontaneous agglutination varied; more than 50 percent of the samples reacted only 1+ or less, approximately 10 percent reacted 2+ or stronger. There was not a complete correlation with spontaneous agglutination and quantity of IgG on the cells, as determined by the antiglobulin test. Of those samples showing spontaneous agglutination, 50 percent were associated with a DAT strength of more than 2+, 27 percent with a DAT of 1 1/2+ to 2+, and 23 percent with a DAT of 1/2+ to 1+. Spontaneous agglutination occurred with the same frequency whether the cells were sensitized with both IgG and C3 or with IgG alone. Surprisingly, 7 percent of the samples sensitized with C3 but no detectable IgG also demonstrated spontaneous agglutination. Marked differences in reaction strength were seen with Rh control solutions from different manufacturers, and the degree of spontaneous agglutination was inconsistent with individual products.