Heat Elution Research Articles

Adsorption and elution study detected misidentification of para-Bombay phenotype donor in an incompatible crossmatch with Bombay phenotype patient

Abstract Bombay and para-Bombay are rare phenotypes with H antigen-deficient red blood cells. A 54-year-old male patient suffering from suspected malignant pleural effusion was planned for thoracotomy. The patient’s blood group showed blood group discrepancy during pretransfusion testing due to ABO isoagglutinin. Further workup with anti-H lectin was negative with pan-reactive antibody screening and negative autocontrol, which suggested that the patient is Bombay RhD positive. A known Bombay phenotype donor was called for donation. That Bombay donor is a para-Bombay donor incorrectly labeled as Bombay in another hospital. During pretransfusion testing, there was an incompatible crossmatch. Hence, the adsorption of donor red cells with the patient’s serum was performed, followed by heat elution. The eluate was pan-reactive with an antibody screening panel indicating anti-H present. The Lewis phenotype of the donor is Leb positive, which reflects the secretor status of the individual. This case highlights the importance of reverse grouping, adsorption, Lewis phenotyping, and elution testing in detecting and differentiating Bombay and para-Bombay phenotypes to avoid fatal adverse transfusion events.

Resolving discrepancies in forward and reverse ABO blood group typing

Background/Aim: There is a need of investigation of high frequency of the cases of ABO discrepancy revealed by forward and reverse typing. The aim of the study was to reveal optimal methods for detection of weak subgroups of group A and B antigens on erythrocytes. Methods: Sixteen persons with A blood group and anti-B antibody adsorbing ability (weak Bel, Bm and B3 antigens), 5 persons with B blood group and anti-A antibody adsorbing ability (weak Ax antigen) and 2 persons with O blood group and anti-B antibody adsorbing ability (weak Bel antigen) were investigated as the cases of type II-III of ABO discrepancy and weak B antigen. Liquid chromatography, agglutination, inhibition of agglutination and adsorption reactions at 4 °C and 37 °C with prolonged incubation (12 hours) were used in the study. Results: The persons with A blood group revealed by forward typing at 4 °C and O blood group detected by reverse typing at 37 °C showed the ability of the erythrocytes to adsorb anti-B antibody. The persons with B blood group revealed by forward typing with presence of anti-B antibodies reactive at 37 °C showed the ability of erythrocytes to adsorb anti-A antibody. Prolonged incubation at reverse typing, adsorption-elution tests, inhibition of haemagglutination and liquid chromatography were used to define blood group specificity. Conclusion: Prolonged incubation during reverse typing at 4 °C and 37 °C, adsorption at 4 °C, heat elution and liquid chromatography might help to detect blood group type in cases of type II-III of ABO discrepancy.

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.

Hemolytic disease of newborn caused by multiple Rh antibodies

Hemolytic disease of Fetus and Newborn (HDFN) usually results due to natural occurring antibodies or alloimmunization in mother but the presence of multiple red cell antibodies increases the risk of development of significant HDFN. Here author reported a case of hemolytic disease of fetus and newborn in a preterm baby caused by multiple maternal antibodies. Direct Antiglobulin Test (DAT) on neonate blood sample was positive (3+) with monospecific DAT showed IgG type which was confirmed by heat elution. Antibody identification of eluate was done using commercial 11-cell panel by gel method showing specificity to anti-D and anti-C antibody which was differentiated from anti-G by sequential adsorption and elution studies. Neonate was treated with double volume exchange transfusion (DVET) using leucoreduced, irradiated O Rh D and C negative PRBC suspended in AB plasma and discharged 6th day in a stable condition. So, all pregnant women should be at least advised for ICT irrespective of Rh D negative status. If ICT is positive, they should be referred to higher center for proper Immunohematological work up, so that proper blood unit for DVET could be identified.

Heat elution: a modification of the Landsteiner-Miller method

Elution removes antibodies coating red blood cells and allows for serologic testing of the recovered antibodies. Heat elution may be used in the investigation of ABO hemolytic disease of the fetus and newborn, in the detection of weak A and B antigens in combination with adsorption-elution using polyclonal antibodies, and for the resolution of interfering IgM agglutinating antibodies.

Blocked D in RhD hemolytic disease of fetus and newborn

A 3-day-old term neonate was admitted to our hospital for the evaluation of neonatal jaundice. The neonate was born to an RhD-negative multiparous mother who had never received anti-D prophylaxis. The neonate's direct antiglobulin test was 4+. Blood group was B, and Rh D typing was negative using tube technique and positive (2+) using gel card. The mother had anti-D, and anti-D titers were 1:1024. After heat elution, RhD typing of the neonate was positive using tube (2+) and gel technique (4+). The eluate was shown to have anti-D. As the red cells of the neonate were saturated with maternal anti-D, commercial monoclonal anti-D could not bind to D antigen which resulted in false-negative D typing in the neonate. Such blocking phenomenon is rare. The discrepancy was identified while using two different techniques (tube and gel) which aided in early diagnosis and tailored an appropriate treatment. The neonate improved after the initiation of a combination of intravenous immunoglobulin and phototherapy.

Frequency of DEL phenotype in RhD negative donor population of north India

BackgroundThere is a paucity of data for DEL phenotype in the Indian population. Ours is a tertiary care Regional Blood Transfusion Centre in north India collecting more than 50,000 blood units out of which 8–9% are RhD negative donors. AimTo determine the frequency of DEL phenotype in RhD negative blood donor population at our centre. Material and MethodsA total of 200 RhD-negative blood donor samples were included in this study which was conducted over a period of 4 months (October 2013 to January 2014). All these blood samples were tested for extended Rh typing including C, E, c and e antigens and also for adsorption elution testing. The heat elution method at 56 °C in water bath for 10 minutes was utilized. The eluate and the last wash supernatant were used for indirect antiglobulin test against O RhD positive and O RhD negative cells by gel technique using the LISS Coombs' AHG gel cards (Biorad, Morat, Switzerland). Those donor samples which were found positive on adsorption elution testing were also further investigated by gel technique for Direct Antiglobulin test (DAT), Weak D testing and Auto Control test. ResultsOut of the total 200 Rh D negative donor samples tested, 3 (1.5%) samples were found to give positive result and, thus, were the DEL phenotypes. It was found that 20 (10%) donor samples were positive for C antigen, 5 (2.5%) for E antigen, 200 (100%) for c antigen and 200 (100%) for e antigen. Among the DEL negative (= 197), 193 (98%) were E antigen negative and only 4 (2%) were E antigen positive, whereas among the DEL positive samples (= 3), 2 (66.6%) were E antigen negative and 1 (33.3%) was E antigen positive. The C antigen positivity was only in 2 (66.6%) individuals in the DEL positive group and 178 (90.3%) in the DEL negative samples. All the three samples which were found to be positive as DEL phenotype also gave negative result for DAT, Weak D testing and Auto Control. ConclusionThe frequency of DEL phenotype in north Indian RhD negative donor population is 1.5%.

Blocked D phenomenon and relevance of maternal serologic testing

Abstract A blood requisition for double-volume exchange transfusion was received for a 2-day-old male child born to a 29-year-old multiparous female (P2002) referred to our institute having neonatal jaundice with encephalopathy; no maternal sample was received. The neonatal blood sample was typed as group A, D–, and the direct antiglobulin test (DAT) was strongly positive (4+) using the gel method. Mono-specific DAT showed the presence of IgG antibodies on neonatal red blood cells (RBCs). Acid elution and gentle heat elution (at 56°C) confirmed the presence of anti-D on neonatal RBCs. The baby received two exchange transfusions with group O, D–, packed RBCs compatible with his own serum. Later, on day 3, the neonate’s mother was typed as group AB, D–, and her serum revealed the presence of alloanti-D, -C, and -S reactive in the anti-human globulin phase. The anti-D titer was 1024. This report highlights the “blocking” phenomenon caused by maternal anti-D in a case of hemolytic disease of fetus and newborn with a positive DAT. Immunohematology 2015;31:116–118.

A Low Complexity Rapid Molecular Method for Detection of Clostridium difficile in Stool

Here we describe a method for the detection of Clostridium difficile from stool using a novel low-complexity and rapid extraction process called Heat Elution (HE). The HE method is two-step and takes just 10 minutes, no specialist instruments are required and there is minimal hands-on time. A test method using HE was developed in conjunction with Loop-mediated Isothermal Amplification (LAMP) combined with the real-time bioluminescent reporter system known as BART targeting the toxin B gene (tcdB). The HE-LAMP-BART method was evaluated in a pilot study on clinical fecal samples (tcdB +, n = 111; tcdB −, n = 107). The HE-LAMP-BART method showed 95.5% sensitivity and 100% specificity against a gold standard reference method using cytotoxigenic culture and also a silica-based robotic extraction followed by tcdB PCR to control for storage. From sample to result, the HE-LAMP-BART method typically took 50 minutes, whereas the PCR method took >2.5 hours.In a further study (tcdB +, n = 47; tcdB −, n = 28) HE-LAMP-BART was compared to an alternative commercially available LAMP-based method, Illumigene (Meridian Bioscience, OH), and yielded 87.2% sensitivity and 100% specificity for the HE-LAMP-BART method compared to 76.6% and 100%, respectively, for Illumigene against the reference method. A subset of 27 samples (tcdB +, n = 25; tcdB −, n = 2) were further compared between HE-LAMP-BART, Illumigene, GeneXpert (Cepheid, Sunnyvale, CA) and RIDA®QUICK C. difficile Toxin A/B lateral flow rapid test (R-Biopharm, Darmstadt, Germany) resulting in sensitivities of HE-LAMP-BART 92%, Illumigene 72% GeneXpert 96% and RIDAQuick 76% against the reference method. The HE-LAMP-BART method offers the advantages of molecular based approaches without the cost and complexity usually associated with molecular tests. Further, the rapid time-to-result and simple protocol means the method can be applied away from the centralized laboratory settings.

Removal of antibodies from red cells: Comparison of three elution methods

Background:Direct antiglobulin test (DAT) is the most common test done in immunohematology lab, which detects immunoglobulin and fragments of complement attached to the red blood cells. These coated red blood cells are difficult to accurately phenotype, which may be required for selection of appropriate unit of red blood cells for transfusion.Aims:We have studied the efficacy of various elution methods in removing the antibodies coating the red cells and their impact on different blood group antigen activity.Materials and Methods:Patient samples sent for serological evaluation of autoimmune hemolysis were included in the study. DAT and Indirect antiglobulin test (IAT) were performed using gel cards (ID system, DiaMed Switzerland). Antibody coated red cells, either by in-vivo or in-vitro sensitization, were used to assess the outcome of three elution methods.Results:Out of 93 DAT positive samples already sensitized in vivo, 28 (30 %) samples became DAT negative post elution using either of three methods, while 36 (38.8%) showed reduction in strength of reaction, whereas in 29 (31.2%) there was no change in strength of reaction. Similarly, out of the 17 samples prepared by in vitro sensitization, 12 samples became completely negative after glycine-HCl/EDTA elution, 9 and 5 samples became negative after heat elution and chloroquine diphosphate elution methods, respectively.Conclusion:On comparative analysis glycine-HCl/EDTA elution method was better than the other two methods and can be used for eluting immunoglobulins from intact red cells.

Detection of IgG anti-A/B must be essential for safe transfusion support in patients undergoing ABO incompatible allogeneic HSCT

Recipients of ABO incompatible allogeneic HSCT present with unusual ABO groups and crossmatch problems accompanied by change of the ABO group from that of the host to the donor. We report on a patient undergoing major ABO incompatible allogeneic HSCT (donor/recipient: A/O) whose blood group was wrongly established to have completely switched to blood group A when using a routine tube method and micro gel column blood grouping card on the 68th day post transplantation. The major crossmatch test with added antiglobulin and blood group A red cells was still positive. After further investigation, an explanation was found because we could not detect IgG anti-A in the serum. At the same time, anti-A coated the patient’s RBCs and could be identified using a heat elution method although the DAT was negative. We also found an obvious mixed field with the LISS-IAT gel card. Hence, routine methods of ABO grouping are unfit for ABO incompatible allogeneic HSCT patients and a micro column neutral gel card is recommended for forward typing especially to detect mixed fields and a LISS-IAT gel card or IAT tube method for detecting IgG anti-A/B in reverse typing.

Detection of IgM autoantibodies in eluates from red blood cells

Forty-two patients with autoimmune haemolysis, whose red cells were sensitized with IgM, were studied in order to determine suitable techniques for investigating the form of erythrocyte-bound IgM, particular emphasis being placed on identifying forms of low molecular weight. Four elution techniques were assessed for their efficiency in removing cell-bound IgM; these were the heat elution, acid stromal, organic solvent and freeze-thaw methods. Eluted IgM was detected by polyacrylamide gel electrophoresis followed by immunoblotting; the heat elution method proved to be the best. Although oligomeric forms of IgM were found in 64% of patients' sera tested concurrently, only pentameric IgM was eluted; from this it was concluded that low molecular weight IgM was not causing haemolysis in any of the cases studied but that the methods would readily detect its presence.

Heat elution chromatography of immunoglobulins

A tremendous increase has taken place over the last decades in the biochemical and clinical use of antibodies. Unfortunately, the constantly growing demand has not been matched by a corresponding easy access to pure immunoglobulin, as purification procedures tend to be either laborious, expensive, or inefficient. We present a new and simplified method to obtain pure antibody based on the special thermal properties of the streptococcal M proteins, a family of cell–surface exposed coiled-coil molecules which bind different sets of host plasma proteins. The coiled-coil structure is already destabilized at low temperatures and the M proteins unfold reversibly, usually below 40 °C. We demonstrate the use of this property to purify immunoglobulin G from rabbit serum with protein H from the AP1 strain of Streptococcus pyogenes. Recombinant protein H is linked to nickel–agarose via a C-terminal histidine tag. After mixing with rabbit serum and washing at room temperature, pure IgG can be eluted from the gel with a moderately heated buffer. In this case, protein H has been used to purify rabbit IgG, but the principle should be applicable to other M protein–ligand pairs.

解離試験法とＥＬＩＳＡとを組み合わせた高感度ＡＢＯ式血液型検査法の開発と自動化

A highly sensitive method for forensic ABO blood grouping using monoclonal antibodies was developed in combination with an absorption-elution test and an enzyme-linked immunosorbent assay (ELISA). In this method, monoclonal antibodies eluted from bloodstains with the absorption-elution, were determined by ELISA, for detection of ABO blood group substances in bloodstains. This method enabled us to determine blood group substances in bloodstains with high sensitivity about 10 times greater than a usual absorption-elution test and also to handle many samples (32 samples per a ELISA plate) at the same time. Furthermore, changing the heat elution of antibodies to the acid elution permitted automated analysis for ABO blood grouping using BIOMEK 1000 apparatus (Beckman Instrument, California, U. S. A.).

Conversion of the human blood group H antigen to A antigen in vitro

A-transferase (N-acetylgalactosaminyl transferase) was purified from human group A plasma using Sepharose 4B affinity chromatography. Human anti-A antibodies were purified from human serum by adsorption to an immunosorbent column and heat elution in order to detect the A antigen. Conditions appropriate for the development of the A antigen on O red cells were examined and several buffer systems were found to be equally effective. Expression of the developed A antigen was found to be similar to that on group A red cells, indicating that the system in vitro has similar activity to the system in vivo. The H antigen from human saliva was coupled to Sepharose 4B or adsorbed to a nitrocellulose membrane. The A antigen was able to be developed on these materials by the action of group A-transferase. The procedures enabled the identification in vitro of sugar-transferase activities which can be useful in studies within the A,B,H antigen system or other carbohydrate antigen system.

IgG subclasses of anti-A and anti-B antibodies bound to the cord red cells in ABO incompatible pregnancies.

IgG subclasses were determined in 138 A or B infants weighing over 2,500g, born to O mothers. Direct antiglobulin test (DAT) was positive in 43 infants and negative in 95 with anti-A and/or anti-B antibodies detected by heat elution test. In 59 out of 131 infants without ABO hemolytic disease (ABO-HDN), no IgG subclass was detectable. In the 72 others, IgG1 was found in 29/72, IgG2 in 63/72, and IgG3 was not detected. In 7 infants with ABO-HDN, DAT was positive in 4 and negative in 3. In conclusion, in DAT-positive infants without HDN, IgG1 or IgG2 may be bound to erythrocytes, but the amount of IgG1 is too small to cause hemolysis. In DAT-positive ABO-HDN the amount of IgG1 is sufficient to cause hemolysis. In DAT-negative ABO-HDN, IgG3 is responsible for hemolysis, even though undetectable by DAT.

ABO Antigens in Red Cells and Digestive Organs of Non-human Primates

Blood group A, B and H antigens on red cells of non-human primates were examined by direct hemagglutination reaction, antibody absorption and heat elution tests. ABH substances on red cells from prosimians, New World and Old World monkeys were detected only by elution test with human anti-A, anti-B and chicken immune anti-H antibodies and their activities were equivalent to those from variants of human group A and B. Red cells of apes except gorilla were agglutinated with anti-A, -B and -H from human antisera, monoclonal antibodies and lectins, and their activities corresponded to the activities of human intermediate types of blood group A and B. Red cells of gorilla reacted weakly with human antisera, but failed to react with monoclonal antibodies, and such reactivities of red cells were corresponded to those of human subgroups.In various species of non-human primates, as in human, blood group specific anti-A and/or -B antibodies were present in the serum according to Landsteiner's rule. Water soluble and chloroform-methanol soluble extracts from digestive tissues of prosimians, New World and Old World monkeys showed the ABH-activities demonstrated on red cell. Extracts from submaxillary glands, stomach and upper part of small intestine showed stronger activities than lower part of intestine, and were almost equivalent to those of tissues from human secretors.

IgG Subclasses of Anti-A and Anti-B Antibodies Bound to the Cord Red Cells in ABO Incompatible Pregnancies

IgG subclasses were determined in 138 A or B infants weighing over 2,500g, born to O mothers. Direct antiglobulin test (DAT) was positive in 43 infants and negative in 95 with anti-A and/or anti-B antibodies detected by heat elution test. In 59 out of 131 infants without ABO hemolytic disease (ABO-HDN), no IgG subclass was detectable. In the 72 others, IgG1 was found in 29/72, IgG2 in 63/72, and IgG3 was not detected. In 7 infants with ABO-HDN, DAT was positive in 4 and negative in 3. In conclusion, in DAT-positive infants without HDN, IgG1 or IgG2 may be bound to erythrocytes, but the amount of IgG1 is too small to cause hemolysis. In DAT-positive ABO-HDN the amount of IgGl is sufficient to cause hemolysis. In DAT-negative ABO-HDN, IgG3 is responsible for hemolysis, even though undetectable by DAT.

ＡＢＯ式血液型不適合妊娠における新生児赤血球の直接グロブリン試験陽性の意義について ＩｇＧサブクラスの検討

Direct antiglobulin test (DAT) and heat elution test were examined on the cord red cells of 257 A or B infants weighing over 2, 500g born to O mothers. DAT was positive in 43 infants (group I), while was negative in 95 infants which were detected anti-A and/or anti-B antibodies by elution test (group II), and remaining 119 infants were not detected any antibody in their eluates. IgG subclasses were determined in 138 infants of group I and group II. In 59 out of 131 infants without ABO hemolytic disease (ABO-HDN), all IgG subclasses were undetectable in their eluates. In the eluates of 72 others, IgG1 was detected in 29/72, IgG2 in 63/72, and IgG3 was not detected in any infant. In 7 infants with ABO-HDN, DAT was positive in 4 cases and negative in 3. In conclusion, in DAT positive infants without HDN the only IgG subclasses bound to erythrocytes are IgG2 or IgG2 with a small amount of IgG1 which cannot cause hemolysis, and in DAT-positive ABO-HDN the amount of IgG1 is enough to cause hemolysis. In DAT-negative ABO-HDN, IgG3 is responsible for hemolysis even though in amounts undetectable by DAT.

Quantitative Evaluation of Elution Methods for Red Cell Antibodies

Two recent techniques using xylene and chloroform for antibody elution from red blood cells (RBC) were compared with the most widely used ether and heat elution by means of a quantitative 125 I-antiglobulin test. Free serum alloantibodies and cell-bound alloantibodies from in vivo sensitization of delayed hemolytic transfusion reactions and of hemolytic disease of the newborn as well as autoantibodies in autoimmune hemolytic disease were studied. With alloantibodies, xylene proved more efficient than chloroform (p<0.025), while both methods were superior to heat or ether elution (p<0.005). Autoantibody elution was equally effective with xylene and chloroform (p>0.1) but again better than heat or ether elution (p<0.02). However, no single method produced the strongest eluate with all antibodies studied. We conclude that xylene and/or chloroform elution are valuable implementations in RBC serology, but, in selected instances, the classical elution methods will retain their importance.