people with cold agglutinin disease produce antibodies against their own red blood cells that cause them to spontaneously agglutinate at room temperature, leading to false positive reactions in forward grouping. cold agglutinins can usually be deactivated by warming the sample to 37 °c (99 °f) and washing the red blood cells with saline. if this is not effective, dithiothreitol can be used to destroy the antibodies.: 141 cord blood samples may be contaminated with wharton's jelly, a viscous substance that can cause red blood cells to stick together, mimicking agglutination. wharton's jelly can be removed by thoroughly washing the red blood cells.: 141 in a rare phenomenon known as "acquired b antigen", a patient whose true blood type is a may show a weak positive result for b in the forward grouping. this condition, which is associated with gastrointestinal diseases such as colon cancer: 139 and intestinal obstruction,: 126 results from conversion of the a antigen to a structure mimicking the b antigen by bacterial enzymes.: 477 unlike the true b antigen, acquired b antigen does not react with reagents within a certain ph range.: 139 reverse grouping infants under 3 to 6 months of age exhibit missing or weak reactions in reverse grouping because they produce very low levels of abo antibodies.: 136 therefore, reverse grouping is generally not performed for this age group.: 486 elderly people may also exhibit decreased antibody production, as may people with hypogammaglobulinemia. weak reactions can be strengthened by allowing the plasma and red cells to incubate at room temperature for 15 to 30 minutes, and if this is not effective, they can be incubated at 4 °c (39 °f).: 137–8 approximately 20% of individuals with the blood type a or ab belong to a subgroup of a, termed a2, while the more common subgroup, encompassing approximately 80% of individuals, is termed a1. because of small differences in the structure of the a1 and a2 antigens, some individuals in the a2 subgroup can produce an antibody against a1. therefore, these individuals will type as a or ab in the forward grouping, but will exhibit an unexpected positive reaction with the type a1 red cells in the reverse grouping. the discrepancy can be resolved by testing the person's red blood cells with an anti-a1 reagent, which will give a negative result if the patient belongs to the a2 subgroup. anti-a1 antibodies are considered clinically insignificant unless they react at 37 °c (99 °f). other subgroups of a exist, as well as subgroups of b, but they are rarely encountered.: 127–32 if high levels of protein are present in a person's plasma, a phenomenon known as rouleaux may occur when their plasma is added to the reagent cells. rouleaux causes red blood cells to stack together, which can mimic agglutination, causing a false positive result in the reverse grouping. this can be avoided by removing the plasma, replacing it with saline, and re-centrifuging the tube. rouleaux will disappear once the plasma is replaced with saline, but true agglutination will persist.: 140–1 antibodies to blood group antigens other than a and b may react with the reagent cells used in reverse grouping. if a cold-reacting autoantibody is present, the false positive result can be resolved by warming the sample to 37 °c (99 °f). if the result is caused by an alloantibody, an antibody screen can be performed to identify the antibody,: 141–2 and the reverse grouping can be performed using samples that lack the relevant antigen.: 477 weak d phenotype further information: weak d approximately 0.2 to 1% of people have a "weak d" phenotype, meaning that they are positive for the rhd antigen, but exhibit weak or negative reactions with some anti-rhd reagents due to decreased antigen expression or atypical variants of antigen structure. if routine serologic testing for rhd results in a score of 2+ or less, the antiglobulin test can be used to demonstrate the presence of rhd.: 159 weak d testing is also performed on blood donors who initially type as rhd negative. historically, blood donors with weak d were treated as rh positive and patients with weak d were treated as rh negative in order to avoid potential exposure to incompatible blood. genotyping is increasingly used to determine the molecular basis of weak d phenotypes, as this determines whether or not individuals with weak d can produce antibodies against rhd or sensitize others to the rhd antigen. red cell antibody sensitization the indirect antiglobulin test, which is used for weak d testing and typing of some red blood cell antigens, detects igg bound to red blood cells. if igg is bound to red blood cells in vivo, as may occur in autoimmune hemolytic anemia, hemolytic disease of the newborn and transfusion reactions,: 260 the indirect antiglobulin test will always give a positive result, regardless of the presence of the relevant antigen.: 477–8 a direct antiglobulin test can be performed to demonstrate that the positive reaction is due to sensitization of red cells.: 289 other pretransfusion testing further information: blood transfusion some groups of people have specialized transfusion requirements. fetuses, very low-birth-weight infants, and immunocompromised people are at risk for developing severe infection with cytomegalovirus (cmv)―an opportunistic pathogen for which approximately 50% of blood donors test positive―and may be transfused with cmv-negative blood to prevent infection.: 749 those who are at risk of developing graft-versus-host disease, such as bone marrow transplant recipients, receive blood that has been irradiated to inactivate the t lymphocytes that are responsible for this reaction. people who have had serious allergic reactions to blood transfusions in the past may be transfused with blood that has been "washed" to remove plasma.: 342–5 the history of the patient is also examined to see if they have previously identified antibodies and any other serological anomalies. a direct antiglobulin test (coombs test) is also performed as part of the antibody investigation. donor blood is generally screened for transfusion-transmitted infections such as hiv. as of 2018, the world health organization reported that nearly 100% of blood donations in high- and upper-middle-income countries underwent infectious disease screening, but the figures for lower-middle-income and low-income countries were 82% and 80.3% respectively. history karl landsteiner circa 1930 in 1901, karl landsteiner published the results of an experiment in which he mixed the serum and red blood cells of five different human donors. he observed that a person's serum never agglutinated their own red blood cells, but it could agglutinate others', and based on the agglutination reactions the red cells could be sorted into three groups: group a, group b, and group c. group c, which consisted of red blood cells that did not react with any person's plasma, would later be known as group o.: 5 a fourth group, now known as ab, was described by landsteiner's colleagues in 1902.: 7 this experiment was the first example of blood typing.: 120 in 1945, robin coombs, a.e. mourant and r.r. race published a description of the antiglobulin test (also known as the coombs test). previous research on blood group antibodies had documented the presence of so-called "blocking" or "incomplete" antibodies: antibodies that occupied antigen sites, preventing other antibodies from binding, but did not cause red blood cells to agglutinate. coombs and his colleagues devised a method to easily demonstrate the presence of these antibodies. they injected human immunoglobulins into rabbits, which caused them to produce an anti-human globulin antibody. the anti-human globulin could bind to antibodies already attached to red blood cells and cause them to agglutinate. the invention of the antiglobulin test led to the discovery of many more blood group antigens. by the early 1950s, companies had begun producing commercial antisera for special antigen testing.: 62–72 notes the rhd antigen is commonly known as the "rh factor", although there are several other antigens in the rh antigen system.: 150 : 26 besides from c/c, e/e, duffy, kidd and mns, clinically significant dosage effects is rare but not impossible for other antigens, which thus may still be considered if subsequent cross-matching is reactive.