Antibody Information and Courses from MediaLab, Inc.

Reaction PhaseLook at the phase in which reactions are occuring and see if the results match the reaction patterns on your panel. Reactions occurring only at immediate spin phase could indicate a possible IgM antibody, cold agglutinin, or rouleaux. Reactions occurring in the AHG phase could indicate a possible IgG antibody. Reactions occurring in both reaction phases could indicate a combination of both IgM and IgG antibodies or a strong IgM antibody that carries through to AHG phase. If a tube method is used, reactions are usually read at immediate spin and AHG phase. If a gel method is used, reaction readings are done only at AHG phase. PatternComparing the pattern of reactivity of the sample reactions with that of the panel cells will help to determine which antibody or antibodies are present. If the reactions match, or closely match, a specific panel cell, that could be the antibody.The strength of the reaction does not correlate with the clinical significance of the antibody present. It does correlate to the amount of antibody that is available to react. If reactions are strong at immediate spin (IS) phase and get weaker at antihuman globulin (AHG) phase, it is possible that a strong IgM antibody is present that is carrying through to the AHG phase (e.g., 4+ at IS and 1+ at AHG).The presence of multiple antibodies should be considered if reactions vary in strength or there are two separate reaction patterns in the IS and AHG phases.

Most are IgM and not clinically significant May interfere with detection of clinically significant antibodies if they react at AHG phase. Screen cells and panel cells will have positive reactions in IS phase and strength will diminish or antibody will not be detected at AHG phase. Auto control will be positive if the cold antibody is an autoantibody. Binding of antibody to antigen occurs at room or colder temperatures and may start to disassociate from the red cell membrane at warmer temperatures. Reactions will appear weaker or be negative at warmer temperatures. (Example: 4+ at IS phase and W (weak)+ at AHG phase.) PrewarmingIf a non specific cold antibody or cold agglutinin is suspected, warm the sample and testing reagents, including saline, to 37° C. Only do reaction readings at AHG; bypassing the optimum reaction temperature prevents activation and binding of the cold antibody .

Cold antibody Immediate spin screen and panel cell reactions will be postive (W+ to 4+). The auto control may also be positive. AHG reactions may be weakly positive if the cold antibody is bound strongly to the red cells. Prewarming should prevent binding from occuring. So, prewarm panels and tests should have negative reactions.Warm antibody Immediate spin screen, panel cell and auto control usually not positive. AHG reactions will be positive including auto control (W+ to 4+). Prewarming of sample and reagents will not change positive reactions since they react best at 37°C and AHG phase. So, reactions will still be positive. Elution and autoadsorption techniques may be used to help further identify the antibody or to help identify other clinically significant antibodies that may be present.AutoadsorptionAutoadsorption is a technique that involves adsorbing unbound autoantibody from the patient's serum using the patient's own red cells. Once the autoantibody is removed, then testing can be performed to determine if any clinically significant antibodies are present.

Possible antibody is anti-C based on matching reaction pattern of sample at AHG. At least 3 positive reactions are present to rule in this antibody.Pink: negative reactions to use for rule-outsTurquoise: homozygous reactions used for rule-out (exceptions to homozygous rule are Rh group and Kk) Antibodies that can be ruled-out using "3 to rule out" rule: D, c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, M, N, S, s, P, LubAntibodies that cannot be ruled out: Cw, Kpa, Jsa, LuaPoints to remember: The pattern of positives and negatives on an antibody panel cell indicates whether that particular antigen is present on the testing cells The phase in which the reactions are occurring will help determine if it is an IgG clinically significant antibody or IgM antibody (usually not considered clinically significant). Stronger reactions seen if antibody exhibiting dosage. Think multiple antibodies if reactions occurring at different reaction phases.

In this example the patient's plasma tests positive with both screening cells at a strength of 4+. In the panel below, reaction patterns show varying strengths, 2+ to 4+ (highlighted in green).4+ could indicate one strong antibody or a combination of several antibodies that increases the strength of the reaction.3+ could indicate one strong antibody.2+ could indicate one the reaction between one weak antibody and the corresponding antigen that is present on with the other target antigen not present on that testing cell. If the panel cell is in the heterozygous state, the reaction of the antibodies present may be weaker if they commonly exhibit dosage. Since Cw, Kpa, Jsa, Lua are not present on the testing cells they are probably not causing these reactions. Perform rule outs using panel cells 5 and 7 (sample had no reaction in any phase with these panel cells) Cells that have at least 1 out of the 3 rule outs needed: C, c, e, K,k, Kpb, Jsb, Fya, Jkb, Lea, M, N, s, P1, Lub Antibodies that could not be ruled out with this panel: D,E, K, Fyb, Jka, Leb, S Predominant pattern of 4+ in panel cells 1,2,3,4,10 matches D Varying strengths in reactions indicates a possible second antibody so selected cells should be picked to aid in identification Find a panel cell negative for D (antibody you suspect) and homozygous positive for the antibody you are trying to rule out. For example: D E e K k Fya Fyb Jka Jkb Lea Leb S s Donor cell 1 0 0 + 0 + 0 + + + 0 + 0 + Donor cell 1 could be used as a rule out test for e, k, Fyb and Leb. Reactions should be negative if these antibodies are not present.You should have a total of 3 negative reactions with panel or screen cells to rule out potential antibodies. If reactions with this panel cell are negative, then e and k can be ruled out with a total of 3 to rule out reactions. Selected cells should be picked for each antibody that needs to be ruled out in order to determine the identity of the other antibody

Reactions are occurring strongly in IS phase but are weaker in AHG phase which could be due to some disassociation of the cold antibody occurring at the warmer testing phase.Prewarming of all reagents and sample will prevent binding of cold antibody. If the W+ reactions at AHG are due to residual cold antibody, the reactions should be negative with a prewarmed panel. No IS phase reading is performed. Prewarmed Panel

List panel cells to test for ruling-in or ruling-out remaining antibodies in Case Study Three. These would be your selected cells. For rule-out, selected cells should be negative for the antigens that correspond to the antibodies you have possibly identified. In this case, the selected cells for rule-out should be antigen-negative for K and Fya. If you are trying to rule in a possible antibody like K, then the panel cell should be positive for that corresponding antigen so that reactions will occur if the antibody is present.Panel cells 1 and 7 could be used for rule-in of K.Panel cells 2, 4, 5, 6, and 9 can be used for rule-outsPanel cell 2: to rule out C, e, Fyb, Jka, N, s Panel cell 4: to rule out Jka, Lea, N, SPanel cell 5: to rule out C, e, Jkb,MPanel cell 6: to rule out E, Jkb, Lea, N, and sPanel cell 9: to rule out M, S

Antibodies ruled out with 3 reactions: D, c, k, Kpb, Jsb, Leb, P1, and Lub (panel cells used for rule out are in green). Antibodies still needing selected cells for rule outs: C, Lea, E, M, Jkb, S, s (need 2 reactions)Fya,Jka, N, K (need 3 reactions)e, Fyb (needs 1 reaction) Jsa, Kpa, Cw, and Lua all need three reactions for rule-out but these are all low-frequency antigens. It is difficult to find panel cells with these antigens present to allow testing. They will fall in the "unable to rule out" category.Reactions are occurring in the AHG phase only and there is varying strengths of reactivity, which could indicate dosage and/or multiple antibodies.The pattern of reactivity closely matches Fya (cells 2,5,7,8,9 are positive). Of the remaining antibodies that have no rule-out reactions, anti-K is the possible second antibody (present on cell 2 and 10 and screen cell I). Explanation for the varying strengths in reactions: Panel cell 2: Fya (heterozygous) and K present so stronger reaction of 4+. Panel cell 5 and 8: Fya is heterozygous, so weaker reaction of 2+. Panel cell 7 and 9: Fya is homozygous, so stronger reaction of 3+. Panel cell 10: K is (homozygous, so stronger reaction of 3+.

Sometimes with Rh or K antibodies present, it may be difficult to find enough homozygous cells to use for rule out. In these cases, you can use heterozygous cells for rule out as long as you have at least 1 homozygous rule out reaction for that antibody. Only do this if you have checked all other available panels and your screen cell anagram reactions for possible homozygous cell reactions to use for rule out.If potential clinically significant antibodies cannot be ruled out completely with the first panel tested, then cells from other panels will need to be selected for testing. These are known as selected cell panels.

Choose cells that can help rule out more than one antibody at a time in order to help decrease supply usage and tech prep time. Example: Ruling out C, Fyb, and M if you have a suspected Jka c C Fya Fyb Jka Jkb M N Panel cell 9 0 + 0 + 0 + + 0 Panel cell 10 0 + + + 0 + 0 + Panel cell 11 + + 0 + 0 + + 0 Panel cell 12 + + + + 0 + + 0 Instead of running 3 separate cells to rule out the antibodies, you can choose one that is homozygous positive for M, C, Fyb and negative for Jka. Panel cell 9 works in this case.If the only antibody that is present is Jka, then your test results should be negative. If the results are positive then further rule outs will be needed to determine what is present.

Suspect dosage if varying strengths in reactivity are seen and reactions are in the same phase. Weaker reactions will be seen if suspected antibody is reacting with antigens in the heterozygous state. Stronger reactions are seen if the antigen is present on the testing cells in the homozygous state. This allows more corresponding antibody to bind with the antigen. Remember the antibodies known for showing dosage are: Rh, Kidd, Duffy, MNSs, and Lutheran. Dosage may be seen if cells are R2R2 (DcE/DcE). These red cells have more D antigen sites so reaction with anti-D may be stronger.Refer to Example 5 on the following page.

Antibody panel resultsResults of the antibody panel show reactions at immediate spin and AHG with varying strengths. The pattern at IS matches P1. Remember that varied strengths can mean multiple antibodies, dosage or both.There are not enough rule-out cells to rule anything out with 3 negative reactions. You can use panel cells that reacted at IS and are negative at AHG for rule-out. Use cells 4 and 10 for rule-outs. Antibodies that have no rule-outs from this panel are: C, E, Cw, Kpa, Jsa, Fya, Lea, M,s, P1, and Lua. Cw, Kpa, Jsa, and Lua are usually not present on panels and fall under the "unable to rule out" catagory. C,E, Fya and s are clinically siginificant and should have further testing done to rule-out or rule-in these antibodies. Lea , P1 and M tend to react at IS, so if the pattern of reactivity is compared to the reaction pattern at IS, there is a match for P1.A selected cell panel was then performed.

In this example, anti-M can be identified as the possible antibody by looking at the patterns of reactivity. Reactions are only occurring at immediate spin, so this would not be considered a clinically significant antibody. Clinically significant antibodies are usually IgG and react at 37°C and at the AHG phase.IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control

Monospecific anti-human globulin (IgG) enables sensitized red cells to cross-link so that agglutination is visible.Enhancement media are sometimes used to further promote agglutination and reduce incubation time. Low ionic strength saline (LISS) is the most common enhancement media. LISS reduces the ionic strength in the testing sample and causes reduction of the zeta potential. It increases antibody uptake and decreases incubation time. Polyethylene Glycol (PEG): brings red blood cells (RBCs) closer together and concentrates antibodies by removing water molecules from the testing sample. It is the most sensitive of the enhancement media; strengthening almost all clinically significant antibodies. However, it will also enhance some clinically insignificant antibodies as well. Centrifugation should be avoided when PEG is used. PEG can cause aggregates to form if the sample (red cell - serum mixture) with PEG added is centrifuged. Reaction readings should only be done at the AHG phase. 22% Albumin: reduces zeta potential, bringing the RBCs closer together and enhancing agglutination. Albumin does not contribute much to antibody uptake. Longer incubation time is needed with this media than with the previously discussed media. Detection of some IgG antibodies can be enhanced with enzyme test methods. Proteolytic enzymes (papain and ficin) denature some RBC antigens and remove negative charges from the RBC membranes. This reduces the zeta potential, bringing the cells closer together. Enzyme techniques are particularly useful in the identification of Rh antibodies and antibodies in the Kidd, Lewis, P and I systems. However, enzymes destroy some antigens including Fya, Fyb, M, and N. The effect of proteolytic enzymes on the S and s antigens are variable.

Antibody screening and antibody identification are critical components in blood bank testing. Clinically significant antibodies must be identified so that appropriate blood products are selected for transfusion and the risk of adverse reaction is minimized. Clinically significant antibodies are capable of causing transfusion reactions, hemolytic disease of the newborn and in severe cases, death.This course will discuss the techniques that are used by blood bank technologists to detect and identify various types of antibodies.

Low-incidence antigens are antigens that occur in less than 1% of the population.Antibodies to low-incidence antigens Low-incidence antigens are not usually found on screen cell and antibody panels. Antibodies are hard to test for, but it is usually not difficult to find compatible blood. Suspect this antibody if an AHG crossmatch is incompatible and other causes have been ruled out, such as a positive donor DAT or ABO incompatibility. Examples of low-incidence antigens include: Cw, V, Kpa, Jsa. When going through the process of Ruling Out, antibodies like anti-V, anti-Cw, anti-Lua, anti-Kpa, and anti-Jsa usually fall into the "unable to rule out" category. High-incidence antigens are antigens that occur in greater than 99% of the population. Antibodies to high-incidence antigens Antibodies are rare and may be difficult to identify due to lack of negative panel cells for other high-incidence antigens (difficult to rule out). Reactions with screen and panel cells will all be positive (same strength and same phase). Auto control will be negative. Difficult to find antigen-negative compatible blood. Examples of antibodies to high-incidence antigens are: anti-k, anti-Kpb, anti-Jsb, and anti-Lub. If an antibody to either a high- or low-incidence antigen is present, it may be difficult to identify and may require further testing in a reference blood bank.

Antibodies to low-incidence antigens will be difficult to test for since most screen and panel cells do not have these antigens on the testing cells. Further testing may be needed at a reference laboratory where a larger selection of antibody panels are available to locate cells positive for these antigens.Suspect an antibody to a low-incidence antigen if: AHG crossmatch is incompatible and Other causes have been ruled out (positive donor DAT, ABO incompatibility) Examples of antibodies to low-incidence antigens are: anti-V, anti-Cw, anti-Kpa, anti-Jsa, and anti-Lua.

Smith KR, Fisher HC III, Hook, EW III: Prevalence of fluorescent monoclonal antibody-nonreactive Neisseria gonorrhoeae in five North American sexually transmitted disease clinics.J Clin Microbiol 34:1551-1552, 1996We compared a direct fluorescent monoclonal antibody (DFA) test with alternative enzymatic and fermention tests for identifying presumptive gonococcal isolates in a systematic sample from patients attending five sexually transmitted disease clinics in five cities.Fourteen (2.5%) of 556 isolates from three clinics were nonreactive with the DFA confirmatory reagent and reactive by both the Quad-Ferm and Rapid NH tests. The prevalence of DFA-nonreactive Neisseria gonorrhoeae isolates varies geographically and is independent of local methods for the identification of possible gonococci.On the basis of our findings, we recommend that for use in medicolegal and other instances in which a diagnosis of gonorrhea has the potential to have far-reaching effects, it is appropriate to test DFA reagent-nonreactive, oxidase-positive, gram-negative diplococci by alternative methods of gonococcal confirmation.Although the prevalence of such isolates could change, the fluorescent monoclonal antibody confirmation reagents remain useful for many clinical situations. Their ease of use and ready applicability for screening large numbers of isolates make them useful for many laboratories.

Blotting techniques were developed to discriminate fragments of nucleic acids. These techniques involve several processes; electrophoresis is one of the processes and is used to separate fragments of DNA and RNA. In Southern blotting (named after Edward Southern) restriction enzymes cut fragments of DNA are separated by AGE or PAGE, transferred to a membrane or blot, and visualized by hybridization with labeled probes.Northern blotting (not named after an inventor but by analogy to Southern blotting) separates RNA. RNA molecules are shorter and have defined lengths; cutting by restriction enzymes is not required. Denaturing conditions are required because of RNA secondary structures. After membrane blotting, the separated types of RNA are visualized with staining or labeled probes.Western blotting (again not named after an inventor but by analogy to Southern blotting)does not separate nucleic acids; it separates proteins in a mixture. The proteins are usually separated with PAGE, transferred to the membrane and visualized with a labeled antibody against the proteins of interest.

Informed consent must be obtained first before a HIV antigen or antibody test can be ordered.Informed consent must be preceded by: Explanation of the test subject's right of confidentiality. Notification that a positive HIV test result will be reported to county health department with enough information to possibly identify the test subject. Availability and location of sites where anonymous testing is performedInformed consent can be given by a legal guardian or other person authorized by law when the test subject is: not competent incapacitated a minor

While the predictability of ABO antibodies in persons lacking the corresponding antigen makes the ABO blood group system an easy one for testing purposes, it can be treacherous as far as transfusion is concerned. If a patient receives cells containing A or B antigens and his/her serum contains the corresponding antibody, the donor cells will be destroyed almost immediately with severe and sometimes fatal transfusion reaction. It is, therefore, of utmost importance to thoroughly understand the ABO blood group system. Compatibility of the ABO system is essential for all other pre-transfusion testing.

A person exposed to a specific immunizing event may produce “immune” ABO antibodies of the same specificity as the “naturally” occurring antibody, but with different biological behavior. Such immunizing events include pregnancy with an ABO incompatible fetus or transfusion of ABO incompatible red cells. After immunization, the subject’s antibody may increase in titer and/or avidity, develop powerful hemolyzing properties, or become more active at 37ºC.

Other rare subgroups of A exist. These comprise less than 1% of the total pool of A genes. Of these rare types, A3 is the most common, but Ax, Aend, and Ael have also been identified. In subgroup A3, the red blood cells characteristically give a “mixed field” agglutination pattern when tested with anti-A and anti-A,B. Small clumps of agglutinated cells are present among large numbers of cells which absorb the antibody to their surface but are not agglutinated by it.

An increasing number of transfusion services are using automated blood banking systems. These systems may employ either solid phase or gel techniques. Use of automation may increase productivity, reduce costs, and, by decreasing the number of manual steps in the testing process, potentially reduce errors.

Antibody - A modified type of serum globulin synthesized by lymphoid tissue in response to antigenic stimulus. By virtue of specific combining sites each antibody reacts with only one antigen. Anucleate - Having no nucleus. Azurophilic granules - The well-defined large reddish granules (lysosomes) which may be present in large lymphocytes. They are called "azurophilic granules" because they stain blue with the azure stains which were originally used. Basophilic granules - Specific granules present in the cytoplasm of basophils. These granules are large and stain purple-black due to their strong affinity for basic stain. B-cell - Bone marrow derived lymphocytes which produce humoral antibodies. Biconcave - Having two concave surfaces. Cellular Immunity - The capacity of a small proportion of lymphoid population to exhibit response to a specific antigen. Chromomere - The centrally located granular portion of the platelet. Clone - A population of cells descended from a single cell. Delayed Hypersensitivity - (part of cellular immunity) that develops slowly over a period of 24-72 hours after an antigenic stimulus. It consists of an accumulation of cells around small vessels and/or nerves. Example: Tuberculin skin test reaction. Digestive Enzyme - A substance that catalyzes or accelerates the process of digestion. Eosinophilic Granules - Specific granules present in the cytoplasm of eosinophils. These granules are large, refractile spheres which stain reddish-orange due to their strong affinity for acid stain. Erythrocyte (red blood cell, RBC) - One of the elements found in peripheral blood. Normally the mature form is a non-nucleated, circular, biconcave disk adapted to transport respiratory gases. Fixed Macrophage - A phagocyte that is non-motile. Free Macrophage - An ameboid phagocyte present at the site of inflammation. Graft Rejection - A transplanted tissue that is rejected by the body's antibodies. Graft vs. Host Reaction - A complication that occurs when an implanted piece of tissue, which contains antibodies, rejects the host's tissue. Granulocyte - A leukocyte which contains granules in its cytoplasm, i.e., neutrophilic, eosinophilic, or basophilic granules. Half-life - is the length of time it takes for half of the cells circulating at a given time to leave the blood for the tissues. Hemocyte - Any blood cell or formed element of the blood. Hemostasis - A mechanism of the vascular system to arrest an escape of blood. It involves an interaction between blood vessels, platelets, and coagulation. Heparin - A mucopolysaccharide acid which, when present in sufficient amounts, functions as an anticoagulant by inhibiting thrombin. Histamine - A powerful dilator of capillaries and a stimulator of gastric secretions. Humoral Immunity - Acquired immunity produced after response to an antigenic stimulus in which B cells produce circulating antibodies. Hyalomere - the clear, blue non-granular zone surrounding the chromomere of a platelet. Immune Response - The interaction of a cell and an antigen that results in a proliferation of the cell and a capacity to produce antibodies. Isotonic Fluid - A fluid whose elements have an equal osmotic pressure. Leukocyte (white blood cell, WBC) - One of the formed elements of the blood; involved primarily with the body's defense. Lysosome - A microscopic body within cell cytoplasm; contains various enzymes, mainly hydrolytic, which are released upon injury to the cell. Megakaryocyte - A giant cell of the bone marrow from which platelets are derived. Mononuclear - A cell having a single nucleus.

Fluoresence polarization immunoassay (FPIA) is also a homogenous competitive immunoassay. In this system, fluorescein-labeled drug competes with unlabeled drug from the patient's serum sample for binding sites on an antibody reagent. The patient's sample, presumably containing the therapeutic drug that is being monitored, and the fluorescein-labeled drug are added to a chamber containing antibody for that drug. The labeled and unlabeled drug will compete for binding sites on the antibody. The greater the amount of drug in the sample, the fewer the number of binding sites that are available for the labeled analyte, leaving a greater number of small, free fluorescein-labeled molecules in the solution.When the chamber is excited with plane polarized light, fluorescein will absorb the light and emit it at a higher wavelength as fluorescent light. A small, free fluorescein-labeled drug rotates randomly and faster than it would if it were bound to antibody, interrupting the light and leading to less emission of light. The larger antibody-drug-fluorescein complexes rotate slower and emit more light in the measured plane. A lower level of drug in the patient's sample results in greater emission of polarized light because there are more antibody-drug-fluorescein complexes present to produce light in the measured plane. A higher level of drug in the patient's sample results in a lower emission of polarized light. This inverse relationship between the concentration of the drug and the polarization units (signal) is illustrated in the image below.

Acute hepatitis panel: Hepatitis A antibody (IgM) Hepatitis B core antibody, IgM (HBcAb) Hepatitis B surface antigen (HBsAg) Hepatitis C antibody

3. Do the results of the initial antibody screen support the presence of the identified antibody?No: All 3 screen cells reacted in the initial screen. Upon review, however, only Screen Cells 1 and 3 were Jk(a+); Screen Cell 2 reacted but was Jk(a-).This anomalous result was investigated by a reference laboratory. It was discovered that the patient had anti-Rd, an antibody to the low frequency antigen Radin (Rd). By chance, Screen Cell 2 was Rd-positive. Radin has a frequency of less than 0.5% in several populations tested. The screen cell manufacturer was notified. They would likely confirm that the cell was Rd-positive, make their clients aware of it, and document it in future antigrams.

The p value is a statistical tool that increases the confidence that an antibody has been identified with a scientifically acceptable level of uncertainty (0.05). As applied to antibody identification, it is computed using Fisher's exact test. Tidbit: This is the same Fisher who helped developed the Fisher-Race theory of Rh inheritance.The p value is calculated using the number of cells that are positive and negative with the patient's plasma. Calculating p values is beyond the scope of this case study but basic understanding of p values at the conceptual level is covered.

In immunohematology textbooks, the "rule of three" is sometimes presented as follows:1. If a patient plasma or serum gives positive results with a minimum of three antigen-positive cells and negative results with a minimum of three antigen-negative cells, concluding that the serum contains an antibody directed against the antigen has a p value of 0.05.2. Therefore, a p value of 0.05 requires at least three positives and three negatives.The first statement is correct but second statement is a misinterpretation of the p value.Three positives and three negatives are required to identify an antibody with a p value of 0.05 ONLY if you have only a 6-cell panel. It does not mean that you always need three positive cells and three negative cells to get p=0.05.For example: A 10-cell panel with eight Jk(a+) cells and two Jk(a-) cells gives a probability of 0.02 if all the positive cells and none of the negative cells react. A 10-cell panel with eight K- cells and two K+ cells gives a probability of 0.02 if all the positive cells and none of the negative cells react. Learning point: You do not need three positive cells and three negative cells to get an acceptable p value of 0.05.

This case study presents a scenario in which a patient had an unexpected antibody that disappeared after he was transfused with 2 units of unmatched group O Rh negative RBC. The patient developed a positive DAT with MFA but an antibody identification using the post-transfusion red cell eluate was inconclusive, making the antibody unidentifiable. Fortunately, the patient improved and further transfusion was not required. Ultimately, the patient's antibody was identified as anti-Jka, with a second antibody to a low frequency antigen (Radin) also unexpectedly present.The case illustrates the risks involved in using unmatched blood.

Life-Threatening HemorrhageDespite potential risk, sometimes immediate transfusion is necessary, even for patients with red cell antibodies. In such cases transfusion service staff should alert the medical director, who can discuss options with clinical staff.The medical director will generally talk to the staff attending the patient and indicate that, if possible, they should hold off transfusion. But if it is a case of massive bleeding where exsanguinating hemorrhage is likely, it is better to give some blood and monitor for a delayed hemolytic transfusion reaction than to let the patient bleed to death.Transfusing when bleeding is brisk will result in much of the autologous and incompatible blood bleeding out, with the possibility of a delayed hemolytic reaction once the patient's antibody rebounds and destroys still present antigen-positive donor red cells.Some transfusion services also try to minimize the risk of unmatched blood by typing their emergency supply of O Rh negative RBCs for the K antigen, since anti-K is a relatively common clinically significant antibody. See Resources for two papers that discuss the risks of transfusing un-crossmatched emergency blood.

These ABO, Rh, and antibody screen results were obtained by the TS using the blood specimen that was collected prior to starting the emergency transfusion with O Rh-negative RBCs. ABO and Rh typing ABO Forward Group ABO Reverse Group Rh anti-A anti-B A1 cells B cells anti-D 0 0 4+ 4+ 3+ Antibody screen Cells Gel IAT* Screen Cell I 3+ Screen Cell II 2+ Screen Cell III 2+ * IAT = indirect antiglobulin test

Immediate post-transfusion antibody identification results Cell Rh Rhesus Kell Duffy Kidd MNSs P Lewis Lu Results1 Results2 C D E c e Cw K k Kpa Fya Fyb Jka Jkb M N S s P1 Lea Leb Lua Gel IAT Gel IAT 1 rr 0 0 0 + + 0 0 + 0 + 0 + 0 0 + + + +S + 0 0 0 w+ 2 rr 0 0 0 + + 0 0 + 0 0 + + + 0 + + + +S + 0 0 0 0 3 rr 0 0 0 + + 0 0 + 0 + 0 0 + 0 + + 0 + 0 + 0 0 0 4 r"r 0 0 + + + 0 0 + 0 + + 0 + 0 + 0 + + + 0 0 0 0 5 R2R2 0 + + + 0 0 + 0 0 + 0 + + + 0 + 0 + 0

The patient's post-transfusion plasma was also retested with the 6 RBC that tested positive initially. Like the antibody panel done on the post-transfusion plasma, they are now all negative by gel IAT.Unfortunately, the panel results with the patient's post-transfusion eluate do not give clear results (only cells #1 and #9 react) and the antibody remains unidentifiable. Suppose that the physician had decided to continue transfusing the patient at this stage. Take a moment to think about what you would advise regarding the compatibility of such transfusions, all of which appear to be compatible in the crossmatch. When you have considered the options, continue to the next page.

The patient's physician was notified that compatible blood was unavailable and that the patient's antibody was still being investigated.When asked whether or not the patient was experiencing a transfusion reaction due to the transfusion of the two unmatched and incompatible O Rh negative RBC, the nurse in the OR stated that the patient was undergoing surgery and completely sedated. A transfusion reaction was not apparent but they would investigate and closely monitor.Hemolytic Transfusion Reactions (HTR)Before proceeding to the next page, make a short list of signs and symptoms associated with immediate hemolytic transfusions reaction and another list associated with delayed hemolytic transfusion reactions.

In this case the patient's antibody has disappeared from the plasma by adsorbing to transfused donor red cells. It is detectable but unidentifiable in the post-transfusion red cell eluate. Several trial and error procedures exist to enhance weak antibodies. Which methods will enhance the reactivity of a given antibody depend on its characteristics. Methods to investigate weak antibodies include: Use a higher plasma to red cell ratio (add more antibody-containing plasma or eluate) Increase incubation time (if consistent with manufacturer instructions, if applicable) Use enzyme-treated panel red cells (enzymes enhance IgG antibodies in Rh and Kidd blood systems but denature some antigens, e.g., Fya, Fyb, S) Try alternative antibody detection methods, e.g., if using LISS routinely, try polyethylene glycol (PEG) or column agglutination methods such as gel, providing they have been validated for use in the TS laboratory.

Use this antibody exclusion protocol to identify the antibody or antibodies present.

A standard follow-up to antibody identification is to antigen phenotype: Patient's red cells (expecting them to lack the corresponding antigen) Donor red cells (in this case, those transfused before an antibody was identified, or, more typically, to find suitable antigen-negative donors to crossmatch prior to transfusion).If you had wanted to type the patient for any antigens at this point in the investigation (2-weeks post-transfusion), which specimen would you have used? Think about any antigen typing problems and how to overcome them before proceeding to the next page.