mixing reference red blood cells with plasma from the patient in 3 different phases: room temperature, 37°c and ahg (with anti-human globulin, by the indirect antiglobulin test). step 1; annotated in blue: starting to exclude antigens without reaction in all 3 phases; looking at the first reference cell row with no reaction (0 in column at right, in this case cell donor 2), and excluding (here marked by x) each present antigen where the other pair is either practically non-existent (such as for d) or 0 (presence is homozygous, in this case homozygous c).when both pairs are + (heterozygous cases), they are both excluded (here marked by x), except for c/c, e/e, duffy, kidd and mns antigens (where antibodies of the patient may still react towards blood cells with homozygous antigen expression, because homozygous expression results in a higher dosage of the antigen). thus, in this case, e/e is not excluded in this row, while k/k is, as well as jsb (regardless of what jsa would have shown). step 2: annotated in brown: going to the next reference cell row with a negative reaction (in this case cell donor 4), and repeating for each antigen type that is not already excluded. step 3: annotated in purple. repeating the same for each reference cell row with negative reaction. step 4: discounting antigens that were absent in all or almost all reactive cases (here marked with \). these are often antigens with low prevalence, and while there is a possibility of such antibodies being produced, they are generally not the type that is responsible for the reactivity at hand. step 5: comparing the remaining possible antigens for a most likely culprit (in this case fya), and selectively ruling out significant differential antigens, such as with the shown additional donor cell type that is known to not contain fya but contains c and jka. in this case, the antibody panel shows that anti-fya antibodies are present. this indicates that donor blood typed to be negative for the fya antigen must be used. still, if a subsequent cross-matching shows reactivity, additional testing should be done against previously discounted antigens (in this case potentially e, k, kpa and/or lua). hemagglutination inhibition neutralizing substanceantigen cancelled hydatid cyst fluid pigeon eggs p1 salivah, lea breast milki guinea pig urinesda when multiple antibodies are present, or when an antibody is directed against a high-frequency antigen, the normal antibody panel procedure may not provide a conclusive identification. in these cases, hemagglutination inhibition can be used, wherein a neutralizing substance cancels out a specific antigen. alternatively, the plasma may be incubated with cells of known antigen profiles in order to remove a specific antibody (a process termed adsorption); or the cells can be treated with enzymes such as ficain or papain which inhibit the reactivity of some blood group antibodies and enhance others.: 723–9 the effect of ficain and papain on major blood group systems is as follows: enhanced: abo, rh, kidd, lewis, p1, ii destroyed: duffy (fya and fyb), lutheran, mns unaffected: kell people who have tested positive for an unexpected blood group antibody in the past may not exhibit a positive reaction on subsequent testing; however, if the antibody is clinically significant, they must be transfused with antigen-negative blood regardless. crossmatching main article: crossmatching crossmatching, which is routinely performed before a blood transfusion, involves adding the recipient's blood plasma to a sample of the donor's red blood cells. if the blood is incompatible, the antibodies in the recipient's plasma will bind to antigens on the donor red blood cells. this antibody-antigen reaction can be detected through visible clumping or destruction of the red blood cells, or by reaction with anti-human globulin, after centrifugation.: 600–3 if the transfusion recipient has a negative antibody screen and no history of antibodies, an "immediate spin" crossmatch is often performed: the red blood cells and plasma are centrifuged immediately after mixing as a final check for incompatibility between abo blood types. if a clinically significant antibody is detected (or was in the past), or if the immediate spin crossmatch demonstrates incompatibility, a "full" or "igg crossmatch" is performed, which uses the indirect antiglobulin test to detect blood group incompatibility caused by igg antibodies. the igg crossmatching procedure is more lengthy than the immediate spin crossmatch, and in some cases may take more than two hours. individuals who have a negative antibody screen and no history of antibodies may also undergo an "electronic crossmatch", provided that their abo and rh type has been determined from the current blood sample and that the results of another abo/rh type are on record. in this case, the recipient's blood type is simply compared against that of the donor blood, without any need for serologic testing.: 600–3 in emergencies, blood may be issued before crossmatching is complete.: 262–3 methods tube and slide methods blood typing by slide method: type a positive blood typing can be performed using test tubes, microplates, or blood typing slides. the tube method involves mixing a suspension of red blood cells with antisera (or plasma, for reverse grouping) in a test tube. the mixture is centrifuged to separate the cells from the reagent, and then resuspended by gently agitating the tube. if the antigen of interest is present, the red blood cells agglutinate, forming a solid clump in the tube. if it is absent, the red blood cells go back into suspension when mixed.: 611–12 : 214 the microplate method is similar to the tube method, except rather than using individual test tubes, blood typing is carried out in a plate containing dozens of wells, allowing multiple tests to be performed at the same time. the agglutination reactions are read after the plate is centrifuged.: 201 antibody screening and identification can also be carried out by the tube method. in this procedure, the plasma and red cells are mixed together in a tube containing a medium that enhances agglutination reactions, such as low ionic strength saline (liss). the tubes are incubated at body temperature for a defined period of time, then centrifuged and examined for agglutination or hemolysis; first immediately following the incubation period, and then after washing and addition of anti-human globulin reagent.: 722 crossmatching, likewise, may be performed by the tube method; the reactions are read immediately after centrifugation in the immediate spin crossmatch, or after incubation and addition of ahg in the full crossmatching procedure.: 725 the slide method for blood typing involves mixing a drop of blood with a drop of antisera on a slide. the slide is tilted to mix the cells and reagents together and then observed for agglutination, which indicates a positive result. this method is typically used in under-resourced areas or emergency situations; otherwise, alternative methods are preferred.: 214 : 476 column agglutination blood typing by column agglutination method: type o positive column agglutination techniques for blood compatibility testing (sometimes called the "gel test") use cards containing columns of dextran-polyacrylamide gel. cards designed for blood typing contain pre-dispensed blood typing reagents for forward grouping, and wells containing only a buffer solution, to which reagent red blood cells and plasma are added, for reverse grouping. antibody screening and crossmatching can also be carried out by column agglutination, in which case cards containing anti-human globulin reagent are used. the gel cards are centrifuged (sometimes after incubation, depending on the test), during which red blood cell agglutinates become trapped at the top of the column because they are too large to migrate through the gel. cells that have not agglutinated collect on the bottom. therefore, a line of red blood cells at the top of the column indicates a positive result. the strength of positive reactions is scored from 1+ to 4+ depending on how far the cells have travelled through the gel. the gel test has advantages over manual methods in that it eliminates the variability associated with manually re-suspending the cells and that the cards can be kept as a record of the test.: 269–71 the column agglutination method is used by some automated analyzers to perform blood typing automatically.: 590 these analyzers pipette red blood cells and plasma onto gel cards, centrifuge them, and scan and read the agglutination reactions to determine the blood type.: 270–1 solid-phase assay solid-phase assays (sometimes called the "antigen capture" method) use reagent antigens or antibodies affixed to a surface (usually a microplate).: 214 microplate wells coated with anti-a, -b and -d reagents are used for forward grouping. the test sample is added and the microplate is centrifuged; in a positive reaction, the red blood cells adhere to the surface of the well.: 590 : 477 some automated analyzers use solid phase assays for blood typing.: 275–6 genotyping genetic testing can be used to determine a person's blood type in certain situations where serologic testing is insufficient. for example, if a person has been transfused with large volumes of donor blood, the results of serologic testing will reflect the antigens on the donor cells and not the person's actual blood type. individuals who produce antibodies against their own red blood cells: 202 or who are treated with certain drugs may show spurious agglutination reactions in serologic testing, so genotyping may be necessary to determine their blood type accurately.: 261 genetic testing is required for typing red blood cell antigens for which no commercial antisera are available. the aabb recommends rhd antigen genotyping for women with serologic weak d phenotypes who have the potential to bear children. this is because some people with weak d phenotypes can produce antibodies against the rhd antigen, which can cause hemolytic disease of the newborn, while others cannot. genotyping can identify the specific type of weak d antigen, which determines the potential for the person to produce antibodies, thus avoiding unnecessary treatment with rho(d) immune globulin. genotyping is preferred to serologic testing for people with sickle cell disease, because it is more accurate for certain antigens and can identify antigens that cannot be detected by serologic methods. genotyping is also used in prenatal testing for hemolytic disease of the newborn. when a pregnant woman has a blood group antibody that can cause hdn, the fetus can be typed for the relevant antigen to determine if it is at risk of developing the disease. because it is impractical to draw blood from the fetus, the blood type is determined using an amniocentesis sample or cell-free fetal dna isolated from the mother's blood.: 202 the father may also be genotyped to predict the risk of hemolytic disease of the newborn, because if the father is homozygous for the relevant antigen (meaning having two copies of the gene) the baby will be positive for the antigen and thus at risk of developing the disease. if the father is heterozygous (having only one copy), the baby only has a 50% chance of being positive for the antigen. limitations abo discrepancies in abo typing, the results of the forward and reverse grouping should always correspond with each other. an unexpected difference between the two results is termed an abo discrepancy, and must be resolved before the person's blood type is reported.: 136 forward grouping weak reactions in the forward grouping may occur in people who belong to certain abo subgroups—variant blood types characterized by decreased expression of the a or b antigens or changes in their structure. weakened expression of abo antigens may also occur in leukemia and hodgkin's lymphoma. weak reactions in forward grouping can be strengthened by incubating the blood and reagent mixture at room temperature or 4 °c (39 °f), or by using certain enzymes to enhance the antigen-antibody reactions.: 139 occasionally, two populations of red blood cells are apparent after reaction with the blood typing antisera. some of the red blood cells are agglutinated, while others are not, making it difficult to interpret the result. this is called a mixed field reaction, and it can occur if someone has recently received a blood transfusion with a different blood type (as in a type a patient receiving type o blood), if they have received a bone marrow or stem cell transplant from someone with a different blood type, or in patients with certain abo subgroups, such as a3. investigation of the person's medical history can clarify the cause of the mixed field reaction.: 138 : 314