Detection Of Red Cell Antibodies Research Articles

Red cell antibody detection by serology

Red cell antibody detection by serology

Unexpected red cell antibody detection by conditional combination of LISS/Coombs and NaCl/Enzyme gel tests at a tertiary care hospital in Korea: A 5-year study

BackgroundA combination of the LISS/Coombs and enzyme methods is recommended for identifying unexpected antibodies. However, many laboratories in which tests are to be performed within the limits of medical fees covered by insurance, use only the LISS/Coombs method because the permissible medical fee is low as compared to the price of reagents required for both methods. The NaCl/Enzyme gel is used as a secondary assay when the LISS/Coombs gel test yields inconclusive results. We compared the frequency of unexpected antibody identified by LISS/Coombs gel with that obtained by the conditional combination of LISS/Coombs and NaCl/Enzyme gels. We aimed at establishing evidence-based guidelines for antibody testing.MethodsFrom June 2007 to June 2012, antibody screening was performed for 69,986 samples; subsequently, antibodies were identified in samples showing positive screening results. These initial screenings and identifications were performed using the LISS/Coombs gel. We considered the results "inconclusive" when specific antibodies were not identified or reactions were too weak for accurate interpretation. For the inconclusive samples, we subsequently used NaCl/Enzyme gels.ResultsThe overall detection rate of unexpected antibodies was 1.23%. Among the samples analyzed using NaCl/Enzyme gels, 40.2% showed results different from those obtained using LISS/Coombs gels. Moreover, 41.9% of samples with nonspecific reactions in LISS/Coombs gels showed clinically significant Rh or Kidd antibodies with NaCl/Enzyme gels.ConclusionConsidering both patient safety and cost effectiveness, we recommend the use of conditional combination of LISS/Coombs and NaCl/Enzyme gels for antibody detection, especially in laboratories that must perform tests within an established budget.

Rhesus Alloimmunisation: Forgotten but not Gone

The prophylactic use of rhesus immunoglobulin has led to a significant reduction in the incidence of haemolytic disease of the newborn secondary to rhesus alloimmunisation, which now only occurs in 1 in 1000 live births. Despite prophylaxis, antibodies to the D antigen are still the commonest cause of rhesus isoimmunisation. Although now a rare pregnancy disorder rhesus isoimmunisation continues to be a challenging obstetric complication and while the face of this disease has undergone many changes over recent years, the principles of management remain unchanged. Pregnancies at risk of the disease are identified by past obstetric history and the antenatal detection of red cell antibodies with serial measurement of antibody levels. In cases of heterozygous paternity the real risk of fetal disease can be determined using new DNA techniques to determine the fetal blood cell type with tissue obtained from amniocentesis, and now by molecular analysis of circulating fetal DNA in the maternal plasma and serum. Sensitised pregnancies are monitored for the development of fetal anaemia. Since its introduction into clinical practise by Liley over 40 years ago, spectral analysis of amniotic fluid bilirubin as an indirect measure of fetal haemolysis has been the primary method of screening for fetal anaemia. Noninvasive evaluation is now possible with the use of doppler ultrasound to detect changes to fetal blood flow secondary to fetal anaemia, most commonly using the middle cerebral artery, and ultrasound evaluation of fetal haemopoetic organs and for the diagnosis of hydrops. Ultrasound guided intrauterine transfusion of red blood cells remains the treatment for the severely affected fetus. Perinatal survival rates in the non hydropic fetus are reported at over 90% and long term studies have revealed normal neurological outcomes for more than 90% cases.

P104 Red cell antibody screening with DiaMed card and with ACT 24 semi-automatic agglutination analyser based on the detection of red cell antibodies

P104 Red cell antibody screening with DiaMed card and with ACT 24 semi-automatic agglutination analyser based on the detection of red cell antibodies

The ScanGel System Shows at least Equal Sensitivity for the Detection of Red Cell Antibodies in Comparison to the ID Micro Typing System

Objective: The aim of this study was to compare 2 testsystems for red cell antibody screening. Materials andMethods: 30 fresh and 92 stored frozen samples withknown red cell antibodies (rbc-abs) and 356 consecutiveblood samples sent in for routine serology were investigatedin parallel using 2 different test systems: the IDMicro Typing System (test system A) and the ScanGelSystem (test system B). Results: Test system B demonstrateda significantly higher sensitivity (98.4 vs. 91.0%, p= 0.019) and equal specificity (99.7 vs. 100%) comparedto test system A. Conclusion: In this study, the ScanGelSystem was at least as good as the ID Micro Typing Systemin the detection of rbc-abs. It showed a significantlyhigher sensitivity that will have to be confirmed inprospective studies and across different batches of testcells.

Detection of red cell antibodies: comparison of two low ionic strength diluents

Various low ionic strength diluents are used routinely for red cell alloantibody detection in the antiglobulin test to increase the rate of antibody association to antigen, thereby allowing a reduction in the incubation time while achieving optimal agglutination. Two commercial low ionic strength diluents (DiaMed ID-CellStab and Inverclyde LISS) were assessed using the DiaMed-ID LISS Coombs’ microtube column system, to assess whether or not the choice of diluent influences red cell antibody detection. Effects of two low ionic strength diluents after 15-min incubation were assessed in 150 samples containing a wide range of typical red cell alloantibodies. Inverclyde LISS gave significantly higher reaction strengths in 25% of samples when compared with the same red cells suspended in ID-CellStab. Variation in reaction strengths ranged from 1+ to 2+, using Inverclyde LISS versus CellStab. Of 131 red cell alloantibodies directed against Rh, Kell, Kidd and Duffy antigens, Inverclyde LISS detected 90% after 15-min incubation, whereas 83% were detected with CellStab. This study suggests that Inverclyde LISS provides better red cell alloantibody detection than does ID-CellStab, and this may be due to the higher ionic strength of ID-CellStab.

The use of pooled red cells and column techniques for routine red cell antibody detection.

The object of antibody screening is to detect all clinically relevant antibodies. In order to do this effectively red cells are selected with an appropriate antigen profile. The introduction of column techniques for antibody screening by indirect antiglobulin testing (IAT) and two-stage enzyme testing (ETC) is perceived to lead to an increased sensitivity and an ability to detect red cell antibodies more easily than by traditional tube techniques because reactions in columns are more easily read and are stable. We evaluated the use of a column technology with pooled red cells for routine antenatal screening. The pooled cells used contained at least one cell with homozygous antigen expression for the majority of clinically significant antibodies known to be present, except for Kell. Pooled cell results were not as easy to read in gel columns when compared with single cell results due to weaker reactions which were often diffused throughout the gel in the column. We concluded that the use of pooled cells led to a decreased sensitivity which proved problematic for the interpretation of results. We used a two-cell and a three-cell pool and found that detection of known antibodies was reduced in IAT and ETC methods.

Drastische Verkürzung von Inkubationszeiten bei immunhämatologischen Untersuchungen durch Einsatz von Mikrowellengeräten

Many methods for the detection of red cell antibodies of the IgG type need incubation times between 15 and 20min at 37 °C. Microwaves induce a rapid movement of molecules resulting in heat delivery into the solution. A group from China shortened the incubation time of ELISA procedures from 1 -1.5 h to 15-20 s. To the best of our knowledge no attempts to use microwave irradiation in immunohematology have been published. We now report preliminary results for the gel technique (ID system), blood group typing on microtiter plates (Erytype), the SolidScreen test and the cross-match in plastic vials. Both the ICT and enzyme-dependent methods could significantly be shortened to incubation times of 60-75 s without a relevant loss of specificity and sensitivity for most of the antibodies tested. The commercially available microwave instruments that we used proved to be not ideal for our purposes. The choice of instruments capable of measuring the temperature within a defined solution should improve the results further, allowing microwave techniques to become routine methods of the immunohematological laboratory.

Serological and immunochemical assessment of anti-complement monoclonal antibodies

The antiglobulin test for the detection of red cell antibodies is included in almost all regimes for pretransfusion testing. The polyspecific reagents produced for this purpose usually contain anti-complement antibodies to enable the detection of certain complement-binding antibodies of clinical significance. In addition the diagnosis of Cold Auto-immune Haemolytic Anaemia (CHAD) is supported by a positive direct antiglobulin test (DAT) using a reagent containing anti-C3d. Anti-C3d is therefore a necessary component of a polyspecific reagent. Unfortunately, if the strength of anti-C3d from a polyclonal reagent is optimal, it tends to be associated with unwanted reactions with red cells that have been stored in serum or plasma [9]. Even when apparently pure antigen is used for immunization, animals may make large amounts of antibody against minor contaminants. The specificity and potency varies from one animal and even one breed to the next, making reagent production both time consuming and uncertain. In recent years several monoclonal antibodies have been produced with a specificity for complement components or for certain fragments. These include an antibody to C3 which reacts with a C3d epitope which appears to be exposed on all complement-coated red cells encountered in blood transfusion serology, NBTS-BRIC 8 ( Holt et al., 1985) [10] and an anti-C3c, WM1 [17]. Chaplin et al., 1980) [2, 3] has reported a comparative study of four monoclonal anti-C3d and a series of 32 antibodies to C3c and C3d have recently been described and detailed serological and immunochemical investigations reported [5]. Some antibodies react with fragments such as C3g ( Lachmann et al., 1980) [11] which are not always present or exposed on complement-coated red cells. In the present study the serological and immunochemical properties of antibodies 12 W 1, 12 W 2, 12 W 3, 12 W 4, 12 W 5 and 9 W 14 were investigated.

Automated detection of red cell antibodies in donor sera using an automated technology.

The evaluation of two automated (AutoGrouper-16C) antibody detection techniques, designed for routine use is reported. Three channels on the instrument were modified for low-ionic-strength or enzyme methods. The incidence of antibody detection was 90.5 percent, and all antibodies that were not detected had titers of 4 or less and would be unlikely to cause adverse clinical reactions if transfused. The adaptation of this automated instrument for donor antibody screening resulted in substantial time and cost savings.

Red cell alloantibodies in thalassemia major. Results of an Italian cooperative study.

Clinical and serological data on 1435 Italian thalassemia major patients were collected during a cooperative study involving 19 centers in 10 regions. The main findings were as follows: 18 percent of the patients were under 6 years of age, 63 percent between 6 and 15, and 19 percent over 15. Forty-one percent had undergone splenectomy. Sixty-two percent of the patients were maintained at pretransfusion hemoglobin levels higher than 10 g per dl, 36 percent between 8 and 10 g per dl, and 2 percent below 8 g per dl. Overall, 5.2 percent of the patients had clinically significant red cell alloantibodies (136 alloantibodies in 74 patients). One-half of the immunized patients had more than one and one-fourth had more than two alloantibodies. The specificities of the 136 alloantibodies were almost exclusively confined to the common antigens of the Rh, Kell, Kidd, and Duffy systems, in that decreasing order of frequency. The antibody screening procedure, using a low-ionic-strength solution antiglobulin test against a three-red-cell panel and the patient's own red cells (autocontrol) with a serum to cell ratio of 100 to 1 was shown to be an adequate technique for red cell antibody detection.

A reappraisal of pre-transfusion testing procedures in a hospital blood bank

In recent years, experience with screening protocols for the detection of red cell antibodies has led to a reappraisal of pre-transfusion testing procedures. In the U.S.A. in particular, it has become accepted practice to perform only an antiglobulin crossmatch when an antibody screen has been negative. There is now debate in that country regarding the necessity of retaining even the antiglobulin crossmatch. In this laboratory, as is usual in Australia, room temperature, enzyme and antiglobulin tests are used for both antibody screening and crossmatching. A review was therefore conducted of the results of screening 58,227 samples from approximately 40,000 patients, involving 126,771 crossmatched blood units over a 53 mth period. Eight hundred and seventy-two red cell antibodies were detected in 718 patients. Forty-four of these antibodies were detected only in the crossmatch, and of these 23 were of potential clinical significance. Fourteen of the 23 were detected by the antiglobulin test, 8 by an enzyme test and only 1 by the saline method. Of 107 antibodies detected only in the room temperature phase, none were of clinical significance. The results indicate that exclusion of room temperature tests from all pre-transfusion testing, and deletion of enzyme tests from the crossmatch will not compromise patient safety.

A LISS spin enzyme method for the detection of red cell antibodies and its use in routine antibody screen procedures.

A technique for performing the enzyme phase of the antibody screen on red cells suspended in low-ionic-strength-salt solution (LISS) is described. The reliability of this LISS spin-enzyme (LSE) technique was compared with the two-stage papain-tile method, in the detection of 62 previously identified enzyme reacting antibodies. All 62 antibodies examined were detected by the LSE method, and no false-positive reactions were found. Using LSE and papain-tile methods in parallel, further assessment was obtained by screening 2000 sequential blood samples under routine service conditions. Fifty-six blood samples contained alloantibodies, of which 43 reacted by both methods, eight by the LSE method only, and five by the papain-tile method only. It was concluded that the LSE method was comparable to the papain-tile method.

The manual polybrene test: a simple and rapid procedure for detection of red cell antibodies.

A rapid manual Polybrene test for detection of red blood cell antibodies have been devised which uses standard laboratory equipment. Red blood cells are incubated with the test sera in a low ionic medium at room temperature for one minute. Polybrene, a quaternary ammonium polymer, is then introduced to cause nonspecific red blood cell aggregation. The test tubes are centrifuged, the cell free supernatant fluid decanted, and the Polybrene effect on the cells is neutralized by adding a dilute sodium citrate-glucose solution. The hemagglutination results are evaluated macroscopically and microscopically. The entire procedure is completed in less than three minutes. In the Rh system, the test is 10--160-fold more sensitive than the antiglobulin reaction. In other systems tested, except for the Kell, a high sensitivity is achieved. The sensitivity for the Kell system is markedly increased, however, by performing a supplementary antiglobulin reaction on the sensitized, Polybrene-treated, red blood cells. The antiglobulin reagent used for this purpose should lack anti-C4 and anti-C3 activities. Sensitivity for cold reactive antibodies is augmented by cooling the cells for 30 seconds before citrate-glucose reagent is added.

The Influence of Ionic Strength and Incubation Time on the Sensitivity of Methods Using Papainized Red Cells

The sensitivity of a combination of various papain methods with normal and low ionic strength (lis) of the serum-cell mixture for the detection of red cell antibodies was tested after varying periods of incubation of the mixture. The mixture was centrifuged before reading when the period of incubation was short. The maximum sensitivity without centrifugation was always reached after a period of 60 min. With a longer incubation the sensitivity decreased. A sensitivity that equalled the maximum sensitivity without centrifugation could be obtained after only 10 min of incubation, if it was followed by a centrifugation before reading. Lis consistently increased the sensitivity of the papain methods, especially after 45--60 min of incubation.

Automated red cell antibody detection. An improved method.

A single-channel AutoAnalyzer was used to screen potential blood-grouping sera to exclude the presence of unwanted antibodies. In order to detect the widest possible range of antibodies, a low-ionic strength Polybrene method was employed, but difficulty was encountered because the aggregated cells adhered to the glass surfaces of the reaction coils, resulting in instability of the system. Modifications were made to the apparatus, and the improvements in the demonstration of antibodies that resulted are described.

Automated Red Cell Antibody Detection

Automated Red Cell Antibody Detection

Kinetic Studies of a Doubly Bound Red Cell Antigen-Antibody System

The Polybrene method for detection of red cell antibodies which utilizes continuous flow equipment was modified so that kinetic studies could be performed on red cell antibodies doubly bound between adjacent red cells. In the anti-Rh(o)-Rh(o) erythrocyte system, deaggregation by temperature was studied over an antibody concentration range of from approximately 1 to 500 antibody molecules per erythrocyte, a residence time range of approximately eightfold, and a temperature range of from 10 to 55 degrees C. The rate of dissociation of antigen-antibody complex, as determined from deaggregation of antibody-dependent red cell aggregates, was found to be of apparent zero order. The apparent activation energy for the antigen-antibody reaction under the experimental conditions was determined and found to be higher than would be expected for singly bound antigen-antibody systems. Possible explanations are considered for these findings in terms of an antigen-antibody bond-breaking model.

Temperature gradient dissociation of red cell antigen-antibody complexes in the polybrene technique.

Summary. The automated Polybrene technique for the detection of red cell antibodies has been adapted for the study of the thermal properties of various red cell antigen‐antibody reactions. A continuous flow of antibody‐induced red cell aggregates was subjected to gradually increasing temperature. The results produced ‘temperature gradient dissociation curves’ (TGDC) which represented not the influence of temperature on antibody uptake but the dissociation of already established immunological bonds. Temperature of dissociation was influenced by the thermal properties of the antibodies and by their concentration. Antibodies could be classified into ‘cold’, ‘intermediate’ and ‘warm’ reacting groups. The technique was also applicable to the study of antibodies that occur on sensitized red cells and, particularly, to patients with acquired haemolytic anaemia. Determination of TGDC has provided a new approach to the study of autoimmune diseases, and may have significant clinical implications.