Antibodies Information and Courses from MediaLab, Inc.

Look at the phase in which reactions are occurring. Reactions at immediate spin (IS) usually are not clinically significant. Reactions at AHG are clinically significant. Check for a match in the reactivity pattern by comparing sample reactions and individual antibody reactions Varying strength of reactions could indicate dosage. Dosage means that there are two "doses" of the same antigen present on the red cells . Antibodies that exhibit dosage react more strongly with homozygous cells (e.g., Jka Jka ) than with heterozygous cells (e.g., Jka Jkb) .

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.

1. Based on these reactions, which antibody or antibodies could be present?2. Based on these reactions, which antibodies can be ruled out?3. What further testing, if any, should be done to assist in the antibody identification?

Cells 4 and 9 may be used for rule outs due to negative sample reaction. Screen cell I may be used for rule outs due to negative sample reaction. Look at the antigens present on cells 4 and 9 that are in the homozygous state (highlighted in green). Remember the 3 to rule in and 3 to rule out procedure. Antibodies ruled out (with 3 reactions): e, k, Kpb, Jsb, Jka, Leb, P1, Lub. A selected panel should be set up to rule out (with 3 reactions) the remaining clinically significant antibodies (E, D, C,c, K, Fya, Fyb, Lea, M,N, S, and s).

Rule-outs can be done using screen cell I and panel cells 4 and 8 (highlighted in green) Antibodies ruled out using these panel and screen cells: C, e, Kpb, Jsb, Jka, Leb, M, P1 and Lub Performing an enzyme panel could help further identify the suspected antibodies. Antibodies needing rule out: D, c, E,K, k, Fya, Fyb, Jkb, Lea, N, S, s If these antibodies are present, a stronger reaction will be seen on the enzyme panel: D, c, E, Jkb, Lea. If these antibodies are present, there will be no reaction on the enzyme panel, since the antigens are destroyed by enzymes: Fya, Fyb, N, S, s.

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

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.

C is enhanced: reactions seen on cells 1,2,and 5 E is enhanced: reactions seen on cells 3 and 6 P1 is enhanced: pattern of reactivity at IS phase matches that of P1. Jkb not reacting: cells 7,8 and 10, so not present Lea not reacting: cell 7, so not present M, N, and s will not react with enzyme treated cells. The enzyme panel was used to remove the potential reactivity of these antibodies to help further identify other antibodies present.Another selected cell panel can be run to eliminate M,N and s.

Antibodies are immunoglobulin proteins secreted by B-lymphocytes after stimulation by a specific antigen. The antibody formed binds to the specific antigen in order to mark the antigen for destruction.The type of antigenic exposure occurring in the body determines if the antibody is a naturally occurring or immune antibody.Naturally occurring antibodies can be formed after exposure to environmental agents that are similar to red cell antigens, such as bacteria, dust or pollen. Sensitization through previous transfusions, pregnancy or injections is not necessary. These antibodies are usually IgM and react best at room temperature or lower. Most of these antibodies are not clinically significant with the exception of ABO antibodies. Examples of naturally occurring antibodies include anti-A, anti-B, anti-Cw, anti-M, and antibodies in the Lewis and P system.

Immune antibodies occur in the serum of individuals who become sensitized to foreign antigens through pregnancy or transfusion. IgM predominates in the primary response, IgG in the secondary response. Most react at 37°C and are considered clinically significant. Examples include antibodies in the Kell, Rh, Duffy, and Kidd systems. Immune antibodies can be classified as alloantibodies or autoantibodies.Alloantibodies Produced by exposure to foreign red cell antigens which are non-self antigens but are of the same species. They react only with allogenic cells. Exposure occurs through pregnancy or transfusion. Examples include anti-K and anti-E. Autoantibodies Produced in an autoimmune process and directed against one's own red cell antigens. React with patient's own cells and all cells tested. Can possibly mask the presence of other significant antibodies. It is very important to make sure that no underlying significant antibodies are present if an autoantibody is suspected. A positive direct antiglobulin test (DAT) or auto control could indicate the presence of an autoantibody. Examples include cold auto (P or I) or warm auto (Rh specificity).

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.

One of the problems with Lp(a) measurement is that the Apo(a) protein has a variable mass. It can have a molecular weight ranging from 275,000 to 800,000 daltons. This is due to variable amounts of repeating regions of the protein. Immunoassay antibodies which recognize these regions will thus give more signal for larger Apo(a) molecules compared to smaller Apo(a) molecules. This is not ideal since again, we would prefer to quantify the number of particles and Lp(a) containing large Apo(a) molecules will produce more signal, skewing the count. One assay system that tries to correct for this is the Lp(a) Cholesterol Electrophoresis Assay sold by Helena Laboratories. This assay uses electrophoresis followed by cholesterol staining and densitometry to calculate the concentration of cholesterol in Lp(a). Although this method still does not enumerate particles, it does appear to have less heterogeneity.Lp(a) is an acute phase reactant. This means that Lp(a) levels will rise in the context of general inflammation. Thus, Lp(a) should not be measured when there is extensive inflammation, such as immediately following a cardiovascular event. Concentrations of Lp(a) above 30 mg/dL are associated with increased cardiovascular risk. The risk of having a cardiovascular event increases 2 to 3 fold if Lp(a) cholesterol is > 30 mg/dL. Fifteen to 20% of the Caucasian population have Lp(a) levels >30 mg/dL. Africans, or people of Aftican descent, generally have levels higher than Caucasians and Asians, however, results must be evaluated in conjunction with clinical history.

Landsteiner, knowing that none of his subjects had been immunized, realized that “natural” antibodies must develop which are directed against antigens not present on the red cells. Individuals with “A” antigens on their red cells had sera containing “Anti-B” antibody. Individuals with “B” antigens had sera containing “Anti-A.” “AB” individuals had sera with no ABO antibodies present and “O” individuals’ sera contained “Anti-A” and “Anti-B.” Sera from group O individuals may contain a separate antibody, “Anti-A,B.” Anti-A,B possesses serologic activity not found in mixtures of Anti-A and Anti-B. Anti-A,B sera will agglutinate A, B, and AB cells. It is particularly useful in detecting weak A and B antigens. See the table on the next page.

It has been demonstrated that antibodies occur predictably in the sera of all normal adults in association with the ABO antigens. Demonstration of these antibodies is therefore necessary for definitive classification of an individual’s ABO cell type. The individual’s serum is therefore tested against reagent red cells containing known antigens. Patient ABO Blood Group Patient Serum Tested with Known Reagent Cells A Cells B Cells A 0 4+ B 4+ 0 O 4+ 4+ AB 0 0 + = agglutination (graded 1+ to 4+)0 = no agglutination or hemolysis

In most other blood group systems, antibody may be formed after an individual has been immunized by an antigen that is missing from his or her red cells; perhaps as the result of pregnancy or transfusion. In the ABO system, when the antigen is missing from the cells, the corresponding antibody will predictably be found in the serum and must be found before determining the ABO type. There are few exceptions to this rule and any exception must be explained before the true ABO blood type can be determined.

ABO antibodies are not usually produced by an infant until 3 to 6 months of age. Antibodies found in the sera of newborns are almost always IgG, passively acquired from the mother. Thus, serum testing of newborns is not performed. Anti-A and anti-B titers are highest at ages 5-10 years and then they gradually decrease. Thus, in elderly patients, ABO antibodies may be difficult to detect. In patients with hypogammaglobulinemia, some leukemias, lymphomas or patients who are taking immunosuppressive drugs, the expected antibodies may be weak or even absent, reflecting the low levels of gamma globulin in the patient’s serum. As previously mentioned, these and other ABO typing discrepancies must be resolved before true ABO type can be determined.

The predominant immunoglobulin class for the B antibodies produced by individuals with group A phenotype and the A antibodies produced by individuals with group B phenotype is IgM. Small quantities of IgG may also be present. IgG is the predominant immunoglobulin for the anti-A and anti-B antibodies found in individuals with group O phenotype. Infants of group O mothers are at higher risk for hemolytic disease of the newborn (HDN) than those born to mothers with group A or B because IgG immunoglobulins readily cross the placenta. IgM molecules do not readily cross the placenta because of their larger size. It is important to note that immune antibodies are usually IgG. Both naturally occurring and immune ABO antibodies are critically important in transfusion since both sensitize and usually hemolyze red cells with the corresponding antigen.

Antibodies of the ABO system cause agglutination of saline-suspended red cells at 4°C to 20°C. Heating to 37° weakens the reaction. “Naturally” occurring ABO antibodies may not be strong enough to agglutinate cells without centrifugation. Thus, testing serum for the presence of anti-A or anti-B has classically been performed using the tube system in which serum and cells added to a test tube are centrifuged and then evaluated for agglutination. A slide test has also been performed for forward reactions. Although tube tests are still in wide use, newer systems utilizing other technology such as gel agglutination are becoming more prevalent. The image on this page illustrates agglutination reactions observed with the tube system, from 4+ in the topmost image, to 0 in the lowest image. ABO reactions should be strong. Weak or missing reactions occur, but must be "resolved" before blood products can be released.4+ agglutination: Red blood cell button is a solid agglutinate; clear background.3+ agglutination: Red blood cell button breaks into several large agglutinates; clear background.2+ agglutination: Red blood cell button breaks into many medium-sized agglutinates; clear background; no free red blood cells.1+ agglutination: Red blood cell button breaks into many small clumps barely visible macroscopically; background is turbid; many free red blood cells.Negative: No agglutinated red blood cells present; red cells are observed flowing off the red blood cell button during the process of grading.Other reaction which may occur are the mixed-field reaction, in which mixtures of agglutinated and unagglutinated red blood are present; and hemolysis, in which red cells are hemolyzed by the antibody. Both of these patterns are considered positive reactions.

Patient Serum Tested With Known Reagent Red Cells Antibodies Present in Serum A1 Cells B Cells 0 4+ Anti-B 4+ 0 Anti-A 4+ 4+ Anti-A and Anti-B 0 0 No ABO antibodies present + = agglutination (graded 1+ to 4+) 0 = no agglutination or hemolysis

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.

Chemiluminescent assays use antibodies that are conjugated to enzymes, such as peroxidase or alkaline phosphatase. These enzymes, mixed with chemiluminescent substrates, produce light in the visible spectrum. A direct relationship exists between the amount of drug that is present in the sample and the light units that are produced and measured by the luminometer in the instrument. Assays that use chemiluminescence are more sensitive than immunoassays that rely on the generation of a colored product.

Similar to evaluating inconsistencies, one of the post-analytic tools for confirming that the serological data fit the solution is to consider the "big picture." For example: Is there a likely red cell stimulus (prior transfusion or pregnancy) for IgG antibodies such as anti-Jka? Can different reaction strengths with panel cells be explained by the identified antibody (e.g., dosage) or by the presence of more than one antibody? Is the antibody unusual for a patient of a particular race? For example, anti-Dib is more likely to occur in Native Americans than in Caucasians.

When p values are calculated for antibody identifications, we think of the null hypothesis as meaning, "the relative proportions of one variable (panel cell being antigen-positive) are independent of the second variable (patient's plasma reacting with the cell). In other words, the results could be due to another cause (different antibody, combination of antibodies, or spurious reactions), not the antibody that we have identified as being probable.Therefore, a p value of 0.05 can be interpreted as meaning that the same results produced by another antibody or cause would be expected to occur by chance alone only one in 20 times (5% of the time), given the number of cells tested. By scientific tradition, this is an acceptable level of uncertainty.A p value of 0.05 does not mean that we have identified the correct antibody.

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.

The following signs and symptoms are associated with acute HTR due to ABO incompatibility but can be associated with other blood group incompatibilities. ABO incompatibility typically results from patient misidentification.The more serious symptoms result from intravascular hemolysis (IVH) caused by antibodies such as anti-A and anti-B that can bind complement to C9.Signs and symptoms typically appear within minutes of the transfusion but can occur anytime during the transfusion. They may include: 1. Burning sensation along the vein being transfused (IVH due to complement activation to C9)*2. Lower back pain in the area of the kidneys (renal failure with subsequent oliguria/anuria) *3. Unexplained bleeding/oozing from a surgical site (fibrinolysis following DIC)*4. Hypotension leading to hypovolemic shock (release of vasoactive substances caused by C3a and C5a)5. Tightness in substernal area of the chest (bronchial constriction due to release of vasoactive substances caused by C3a and C5a fragments)6. Other symptoms: fever, chills, skin flushing, dyspnea, wheezing, anxiety, malaise, nausea, headache. * If untreated, these complications may lead to patient death.

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