Chapter 12: Detection and Identification PDF

Chapter 12 **Detection and Identification** Antibodies may be regular \"expected\" like the naturally occurring antibodies of the ABO system. Naturally occurring antibodies are stimulated by exposure to environmental sources such as pollen, fungus and bacteria. Antibodies may also be irregular \"u...

Chapter 12 **Detection and Identification** Antibodies may be regular \"expected\" like the naturally occurring antibodies of the ABO system. Naturally occurring antibodies are stimulated by exposure to environmental sources such as pollen, fungus and bacteria. Antibodies may also be irregular \"unexpected\" as a result of the exposure of the immune system to RBC antigen through transfusion, transplant or pregnancy. Other antibodies may also be passively acquired due to transfusion of plasma containing blood products or derivatives like intravenous immunoglobulin (IVIG). **Antibody Screen** AABB standards require that antibody screen be done on pre transfusion test for patients, prenatal test for the evaluation of the risk of HDFN and the evaluation of the mother\'s candidacy for prophylaxis (RhIG) and as part of donor testing. Antibody screen may be done using: 1. **Tube technique**: Serum or plasma is used. RBC reagents should be homozygous for Rh, Kidd, Duffy, MNSs and Lutheran antigens. The tube test is flexible, common available laboratory equipment can be used and it is also inexpensive. However it has an unstable reaction and the nature of grading is subjective. 2. **Gel technique**: Uses dextran acrylamide gel and screening cells suspended in LISS with the concentration of 0.8%. The washing step eliminated. The gel technique reaction is stable for up to 24 hours and is standardized. Mixed field reaction may be more readily detected. Moreover, automatic interpretation and interface with the hospital computer system is applicable. However, incubators and centrifuges are necessary. 3. **Solid Phase technique**: In this technique indicator cells are added rather AHG. It has the advantage of automation, it uses a smaller sample size and LISS that changes color when added to serum or plasma. However, positive and negative controls should be run with each batch. The following reagents may be used to enhance the reactions: 1. **22% albumin** works by reducing the zeta potential which allows the RBCs to approach each other that leads to increase the chances of agglutination. 2. **Low Ionic Strength Solution (LISS)** contains glycine in albumin solution. It increases the uptake of antibody onto the RBC during the sensitization phase which increases the possibility of agglutination. 3. **Polyethylene Glycol** **(PeG)** in a LISS solution removes water from the test system which increases the concentration of the antibody present. This increases the degree of RBC sensitization. This enhancement reagent is not suitable to patient with elevated levels of plasma proteins like in multiple myeloma. **Anti Human Globulin (AHG)** AABB standards require reagent contain anti-IgG for pre transfusion compatibility testing. Polyspecific AHG reagent called polyvalent or broad spectrum Coomb\'s serum is used. AHG should be checked by Coomb\'s control cells. **Antibody Screen Interpretation** Agglutination or hemolysis at any stage of testing is considered a positive test result. The investigator should consider the following questions: 1. In what phase(s) did the reaction occur? IgM react best at low temperature. IgG react best at 37°C. 2. Is the autologous control negative or positive? A positive \"AC\" autologous control indicates the presence of autoantibody or antibody to medication. If the patient received blood recently, the positive result may be due to alloantibodies coating the transfused cells. Negative \"AC\" with positive antibodies indicates alloantibodies. 3. Did more than one screening cell sample react; if so, did they react at the same strength and phase? A single antibody specificity should be suspected when all cells react at the same phase and strength. Multiple antibodies are most likely apparent when cells react at different phases and strengths. 4. Is hemolysis or mixed-field agglutination present? Anti-Le^a^, Anti-Le^b^, Anti-PP^1^P^k^ and Anti-Vel can cause in vitro hemolysis. Mixed-field reaction is associated with Anti-Sd^a^ and Lutheran antibodies. 5. Are the cells truly agglutinated or is rouleaux present? Patients with multiple myeloma or who have received high-molecular-weight plasma expanders like dextran may cause non specific aggregation of RBCs. Cells in rouleaux have a \"stacked coin\" appearance under microscopy. Rouleaux is observed in all tests containing the patients serum. Rouleaux does not interfere with the AHG phase and unlike agglutination, it is dispersed by the addition of one to three drops of saline. Screening reagents and methods are designed to detect clinically significant antibodies, where a negative result gives us 95% confidence that no clinically significant antibodies are present. Factors affecting the sensitivity of the antibody screen includes: 1. **Cell-to-serum ratio.** Prozone is present when antibody is in excess and postzone when antigen is in excess. Pro and post zones give false-negative results. 2. **Temperature.** IgG is clinically significant and reacts at the optimal temperature of 37°C. The optimal temperature of IgM is 21 to 25°C. 3. **Length of incubation.** Saline environment requires 45 minutes to 1 hour of incubation. Potentiators may require 10 minutes. 4. **pH.** Most antibodies react between 6.8 to 7.2. **Antibody Identification** Once an antibody has been detected, the antibody should be identified and its clinical significance determined. To detect and identify an antibody, it is necessary to get the patient\'s history including the age, sex, race, diagnosis, transfusion, history of pregnancy and medication. The panel reagent used for identification is a collection of 11 to 20 \"O\" cells with various antigen expressions including homozygous expression of Rh, Duffy, Kidd and MNSs antigen system, positive for low-frequency antigen and negative for high-frequency antigen. Rule out or exclude all negative results with all homozygous positive antigen. Evaluation of panel results should be carried out in a logical step-by-step method: 1. In what phase(s) and what strength(s) did the positive reaction occurred? Homozygous cells reacts more strongly than heterozygous cells. Different reaction strength could indicate the presence of more than one antibody. Antigens with variable expression on the cell like I, P1, Le^a^, Vel, Ch/Rg and Sd^a^. 2. Do all of the positive cells react at the same phase or do any react at different or multiple phases? Reactions of certain cells at one phase and different cells at another phase may indicate the presence of multiple antibodies. Homozygous cells react at an earlier phase than the heterozygous cells. IgM antibodies most often react at the immediate spin phase and are usually not significant. IgG antibodies are clinically significant and most detected by AHG phase. 3. Does the serum reactivity match any of the remaining specificities? When a single alloantibody is present the pattern of reactivity usually matches a pattern exactly. 4. Are all commonly encountered RBC antibodies ruled out? Antibodies to low-frequency antigens are uncommon and it may not be necessary to rule them out. For a commonly encountered antibody which is not ruled out, it is important to test selected cells that will rule out the presence of the antibody. 5. Is the autologous control positive or negative? A negative auto control indicates that the positive reactions are caused by an alloantibody while a positive auto control is caused by an autoantibody or mix between allo and autoantibodies. 6. Is there sufficient evidence to prove the suspected antibody? Conclusive antibody identification requires at least three antigen positive and three antigen negative cells \"3 and 3 rule\". A probability (P) value of 0.05 or less is required for identification results to be considered valid. 7. Is the patient lacking the antigen corresponding to the antibody? A negative result is expected and indicates that the identification results are correct. For DAT positive patients which could not be phenotyped by antisera detected by AHG and to remove the antibody coating the RBCs for phenotyping, use gentle heat elution, chloroquine diphosphate, glycine/EDTA reagent (suitable for Kell antigen). Absorption method has been described for antibodies which resist elution. For recently transfused patient, reticulocyte typing can be performed. Flow cytometry has been used to detect small quantities of antigens. Polymerase chain reaction (PCR) may be used for fetal antigen typing. Positive control should have a heterozygous expression of the antigen. 1. Use of **extra cells** where the cells should have minimal overlap in the antigens they posses. Multiple antibodies can also be identified using extended cell panels. 2. **Enzymes** such as ficin, papain, trypsin and bromelin may be used. The enzyme destroys certain antigens and enhance the expression of others. 3. **Neutralization** of antibodies with the use of soluble substances similar to antigen can inhibit agglutination. 4. **Adsorption** Antibodies may be removed from the serum by adding the target antigen and allowing the antibody to bind to the antigen. a. Commercial reagents for adsorption includes **human platelet concentrate,** used to adsorb Bg-like antibodies from the serum while leaving other specificities in the serum, antibody identification can then be performed on the adsorbed serum; **rabbit erythrocyte stroma (REST),** used to adsorb I, H, IH-like antigen and leave other antibody specificities unaffected by REST adsorption. Cross matching with REST-adsorbed serum is not recommended. b. **Autoadsorption** uses the patient\'s own cells. It removes the auto antibody leaving the alloantibody. The temperature of the incubation will depend on the thermal range of the autoantibody being removed. The patient should not be transfused with blood during the last 3 months. c. **Homologous adsorption** is performed when there is not enough RBCs available to perform autoadsorption or if the patient has been recently transfused. Use exact match or focus on finding cells that lack the antigens to which the patient may form antibodies. d. **Differential adsorption** is performed when phenotyping the patient is difficult because of a positive DAT or recent transfusion or if the patient has multiple antibodies. Use three different cells R1R1, R2R2 and rr in which one must be negative for Jk^a^, K, Jk^b^. **DAT and Elution techniques** DAT is performed for the detection of antibodies coating RBCs when investigating hemolytic transfusion reaction (HTRs), HDFN, autoimmune hemolytic anemia and drug induced hemolytic anemia. DAT is used to detect in vivo sensitization of RBCs. Elution technique is used to release, concentrate and purify antibodies by changing the attractive forces between antigen and antibody or change the structure of the RBC surface. Eluate is a solution containing released antibody from the RBCs. Total elution releases bound antibody and the RBC antigens are rendered useless, it is necessary when performing antibody identification. Partial elution removes the antibody but the RBC antigen remains intact, it is useful when performing phenotype or preparing cells for auto adsorption. There are different kinds of elution based on temperature, pH and the use of organic solvents. Gentle heat elution method is performed at 45°C for partial elution and at 56°C for total elution. Lui freeze method is performed at -18°C. Temperature dependent elution are best at detecting IgG antibodies directed against antigens of the ABO system. A common and relatively quick and easy method for total elution for the detection of non-ABO antibodies includes acid elution, a glycine acid solution at a pH of 3.0. Citric acid and digitonin acid may also be used. Several organic solvents have also been used in total elution methods like dichloromethane, xylene and ether. These solvents act on the lipids in the RBC membrane to reduce surface tension and lead to the reversal of the Van der Waal forces that hold the antigens and antibodies together. This method is best for detecting non-ABO antibodies but this procedure is time consuming. The last wash should be run in parallel with the eluate and should be negative. **Antibody Titration** Antibody titer can be evaluated by flow cytometry, radioimmunoassay and enzyme-linked immunoassay. Performing an antibody titration can help determine antibody concentration levels. The titer level is the reciprocal of the greatest dilution in which agglutination is observed. A score may be assigned based on the strength of reactivity. Each reaction is given a value and the score is determined by adding up the individual values. A change in titer level of 2 or more tubes or a change in score of 10 or more is considered to be significant. The cells selected should contain the target antigen, homozygous or heterozygous depending on the last titration or the hospital policy, lack the other target antigens if present to avoid interference. Titer level studies are useful in monitoring the obstetric patient who has an IgG antibody that may cause HDFN. Increasing titer levels may indicate the need for intrauterine exchange transfusion. The titer level in RhIg is rarely above 4. HTLA (High titer, low avidity) are high frequency antibodies and are observed with weakly positive reactions persisting through extensive dilution (as high as 2048) like Anti-Ch, Anti-Rg, Anti-Cs^a^, Anti-Yk^a^, Anti-Kn^a^, Anti-McC^a^ and Anti-JMH. These antibodies are usually not clinically significant but may mask significant antibodies. **Providing Compatible Blood Products** Providing compatible blood products is determined by the frequency of the antigen in the population and by the clinical significance of the antibody. If the antibody does not cause a decreased survival of antigen positive RBCs, then the use of random blood products that are crossmatch compatible is acceptable, such as Anti-M, -N, -P1, Le^a^ and Le^b^. If the patient\'s sample contains a clinically significant antibody or the patient has a past history of a clinically significant antibody, units for transfusion must be antigen negative. The crossmatch technique must demonstrate compatibility at AHG phase. If there is a sufficient sample, start with the random crossmatch then antigen-type the units that are crossmatch compatible. If the quantity of the sample is not sufficient or the antibody is no longer detectable in the serum, units should be antigen-typed first and then cross matched. The frequency of the antigen in the population is helpful to determine the number of units that must be antigen-typed to find a sufficient number to fill the crossmatch request. The number of units requested is divided by the frequency of antigen negative individuals. No. of units to be typed = [Units requested] Frequency of antigens For typing multiple antigens: No. of units to be typed = [Units requested] Frequencies of antigen negative are multiplied together Choosing ethnicity of the donor in selecting the units depends on the frequency in races population. **Resolving difficult antibody identification problems** 1. **Case One : Multiple antibodies**: Suspect multiple antibodies when all or most of the screening and panel cells are positive but reactions are at different strengths or in different phases and the auto control is negative. This can be resolved by performing a selected cell panel to exclude certain specificities, by using enzyme techniques that may allow for the separation of certain specificities and by neutralization and absorption techniques. 2. **Case Two: Antibody to a high-frequency antigen :** Suspect an antibody to a high-frequency antigen (\>98%) when all or most screening and panel cells are positive with reaction in the same phase and same strength and the auto control is negative. This can be resolved by testing additional cells that lack the antigen, consulting a reference laboratory, phenotyping the patient and performing absorption studies to confirm the presence or absence of the antibody. Autologous donation and blood from family members should be chosen for transfusion. 3. **Case Three:** **Antibody to a Low-frequency antigen** : Most cases of antibody to a low-frequency antigen gives a negative screening and panel cell result. It is detected when only one cell becomes positive, cross match is incompatible or the DAT of the newborn is positive even if the blood group is compatible with the mother. The extended panel may be used to detect rare antibody. 4. **Case Four**: **Cold Reacting Autoantibodies**: Most adult sera contain low titers of cold-reacting autoantibodies, Anti-I, Anti-H and Anti-IH. Cold antibody interferes with antibody screen, panel and results are all positive at immediate spin phase and gets weaker at 37°C and AHG phase. This can be resolved by using monospecific Anti-IgG Coomb\'s, pre-warm technique and autoadsorption at 4°C. Rabbit erythrocyte stroma (REST) adsorption can be applied if the patient has a strong reacting antibody or if the patient has been recently transfused. REST adsorbed serum is not suitable for crossmatch as anti-B may be removed. The use of DTT and 2-Mercaptoethanol to break the disulfide bonds and denature IgM may also be useful. 5. **Case Five**: **Warm Reacting Autoantibodies**: Warm autoantibody may occur secondary to chronic lymphocytic leukemia, lymphoma, systemic lupus erythematosus and other autoimmune diseases. If the patient has not been recently transfused the problem can be resolved by partial elution to prepare cells for autoadsorption using gentle heat elution and chemical methods by ZZAP, autoadsorption \"autologous adsorption\", phenotyping for the corresponding target antigen. For cross matching select least incompatible blood that is negative for the antigen corresponding to the alloantibody. If the patient has been recently transfused, resolve the problem by allogeneic adsorption and homologous or differential adsorption. Choose negative antigen for the clinically significant antibody for transfusion. **Important Points** - The purpose of the antibody screen is to detect unexpected antibodies, which make up approximately 0.2-2% of the general population. The antibodies may be classified as immune (the result of RBC stimulation in the patient), passive (transferred to the patient through blood products or derivatives), or naturally occurring (the result of environmental factors). Antibodies may also be classified as alloantibodies, directed at foreign antigens, or autoantibodies, directed at one's own antigens. - A clinically significant antibody is one that results in the shortened survival of RBCs possessing the target antigen. Clinically significant antibodies are IgG antibodies that react best at 37^0^C and/or in the AHG phase. They are known to cause haemolytic transfusion reactions and HDN. - Screening cells are commercially prepared group O cell suspensions obtained from individual donors who are phenotyped for the most commonly encountered and clinically important RBC antigens. - RBCs from a homozygous individual have a double dose of a single antigen, which results from the inheritance of two genes that code for the same antigen, whereas heterozygous individuals carry only a single dose each of two different antigens. (Each gene codes for a different antigen). - Antibodies in the Kidd, Duffy, Lutheran, Rh, and MNSs blood group systems show dosage and yield stronger reactions against RBCs with homozygous expression of their corresponding antigen. - Enhancement reagents, such as LISS and PEG, are solutions added to serum and cell mixtures in the IAT to promote antigen-antibody binding or agglutination. - Coombs' control cells are RBCs coated with human IgG antibody, which are added to all AHG-negative tube tests to ensure that there was an adequate washing step performed and that the AHG reagent is present and functional in the test system. - Gel and solid phase testing are alternatives to tube testing. These methods may be automated to increase efficiency. - The antibody exclusion method rules out possible antibodies based on antigens that are present on negatively reacting cells. - Conclusive antibody identification is achieved when the serum containing the antibody is reactive with at least three antigen-positive cells (i.e., reagent cells that express the corresponding antigen), negative with at least three antigen-negative cells (i.e., reagent cells that do not express the corresponding antigen), and the patient's RBCs phenotype negative for the corresponding antigen. - The DAT detects cells that were sensitized with antibody in vivo. Elution methods are used to free antibody from the cell surface to allow for identification. - The calculation for determining the number of random donate units screened for patients with an antibody is described as dividing the number of antigen-negative units desired for transfusion by the incidence of antigen-negative individuals in the donor population. - The relative quantity of an RBC antibody can be determined by testing serial twofold dilutions of serum against antigen-positive RBCs; the reciprocal of the highest serum dilution showing agglutination is the antibody titre.