Antigen 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) .

If multiple antibodies are present and your selection of antibody panel cells is limited, performing an enzyme panel will give you more information as to which antibodies might be present. The benefit of using an enzyme panel is that enzymes will enhance some antigens and destroy other antigens. This makes it easier to narrow your choice of potential antibodies. However, you cannot use enzyme panel cells as the only rule-out cells because of the fact that some of the antigens are destroyed and you may not pick up an antibody that is present. Antigens enhanced: Rh, Kidd, Lewis, P1, I, and ABOAntigens destroyed: Duffy, MNS, XgaExample: You suspect an e and a Fyb and have already identified a D. By performing an enzyme panel, the e and D would be enhanced (strong reaction) and the Fyb would be destroyed (no reaction). Cell Number D e Fyb AHG 2 + 0 0 4+ 4 0 + 0 4+ 6 0 0 + 0

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

Start with the first panel cell where you have negatives in all the phases tested. Use the cells where patient reactions are negative in all phases tested for rule outs. Look at the screen cell antigram to see which cells the patient reacted with. You may use a screen cell as a rule out cell if there were no reactions in any of the phases tested and if it is homozygous for the antibodies you are trying to rule out; antigens should be in their homozygous state in order to rule out. Refer to the screen cell antigram example below to see homozygous screen cell that can be used for rule outs. Write down what you could not completely rule out with 3 homozygous cell reactions. If Jkb is the suspected antibody, then reactions with screen cell I should be negative. This screen cell may be used as a rule out cell.

Rule-out (also referred to as exclusion or cross-out) is a process by which antibodies are identified as being unlikely in a given sample because of the absence of an expected antigen-antibody reaction. In other words, the absence of a reaction is noted with a cell that is positive for the corresponding antigen. Non-reactive cells are selected for rule-out. To be classified as non-reactive, a cell must NOT have reacted in any phase of testing in a given panel or screen. In the case of cold antibodies: if reactions are only occurring at immediate spin and are negative in the AHG phase, then that panel cell can be used as a rule out cell for IgG reactive antibodies but not for antibodies that react at immediate spin (IgM).If there is no reaction with a panel cell then it is possible that antibodies to the antigens on that cell are not present in the sample being tested. Based on Fisher's statistical probability recommendation, the probability of having reliable results increases if you are able to have more rule-out and rule-in cells. By comparing the patterns of reactivity and non-reactivity, we can more safely assume that an observed pattern is not the result of chance alone. If a "3 (reactions) to rule in and 3 (reactions) to rule out" protocol is used, there is then a 95% probability that the reaction pattern is not due to chance alone. Homozygous cells are used so that weaker reacting antibodies which fail to react to the antigen present in the heterozygous state aren't accidentally ruled out. Examples of Homozygous and Heterozygous Antibodies Jka Jkb Patient IS Patient AHG Panel cell 10 + + 0 2+ Panel cell 11 0 + 0 4+ Panel cell 10 shows Jkb in the heterozygous state. The patient's reaction is weaker than the reaction with panel cell 11 which shows Jkb in the homozygous state.Reactions are weaker when antigens are present in the heterozygous state because there is less of the antigen present for the potential antibody to bind with.

Cells 5 and 8 can be used for rule-out cells. Jkb, Lea, M, N, and s still need more rule-out cells. P1, C, E and Fya have no cells for rule-out.Running an enzyme panel would help to enhance Jkb, Lea,P1 C and E if these antibodies are present.If M,N,s and Fya are present, no reaction would be seen because these antigens are destroyed by enzymes.

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.

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.

In electroimmunoassay electrophoresis, the antiserum is mixed in the gel during preparation. In the electrophoresis of the serum sample, the voltage drives the sample antigen into the antiserum creating a precipitin line in the shape of a rocket. This line is proportional to the concentration of the antigen, the protein to be detected. Each gel contains several serum samples, one antibody suspended in the gel, and standards of known concentration of antigen. Quantitation of the unknown antigen is derived from the height of the sample rockets compared to the height of the standard rockets. Electroimmunoassay electrophoresis is often referred to as rocket electrophoresis.

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

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.

In routine practice, specially prepared blood grouping sera containing anti-A, anti-B, (and optionally anti-A,B) are used to identify the four types of red cells. These sera will agglutinate cells with the corresponding antigen. This is called forward typing. ABO Blood Group Patient Red Cells Tested with Known Antisera Anti-A Anti-B Anti-A,B A 4+ 0 4+ B 0 4+ 4+ O 0 0 0 AB 4+ 4+ 4+ + = agglutination (graded 1+ to 4+)0 = no agglutination

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.

Specific sugars, attached to the red cell membrane in unvarying linkage conformations, determine ABO antigenic activity. Galactose resides at the end of this specific sugar chain. This configuration constitutes the ABO antigen precursor substance.

Without H substance (also known as H antigen or substance H), there is no way for additional sugar attachment to take place. Additional sugar attachment is necessary for the development of A and B antigenic activity. Therefore, without substance H there can be no A and B antigens developed. Once substance H is developed, the addition of the sugar N-acetylgalactosamine to the terminal position of the chain gives the molecule “A” antigenic activity.

The ABO locus is on chromosome number 9. There are three major allelic genes and numerous rare genes. The three principle genes are A, B, and O. The A gene determines the product N-acetylgalactosaminyltranferase activity. The B gene determines galactosyltransferase activity. The O gene does not produce a functional enzyme. The enzyme products of the A and/or B genes act on H substance to convert it to A and/or B antigens. Not all H substance is converted; thus, all cells normally contain some H substance along with the A and/or B antigens. If both the A and B genes are present, some H antigen sites are converted to A antigen and other H antigen sites are converted to B antigen. (A single antigen site does not have both A and B antigens.) The O gene is an amorph and doesn’t act on H substance, therefore group O cells contain only H substance. See the diagram on the next page.

The A, B, and H antigens, like many other blood group antigens, are the expression of genes inherited from the previous generation. If the antigen is demonstrated, the gene controlling it must have been inherited from one or both of the parents.  As previously mentioned, the genes A, B, and O are allelic genes. Assuming the production of H substance, these three genes, in various possible combinations of two, account for the four recognized ABO groups: A, B, AB, and O. Each individual inherits two ABO genes, one from each parent, and these genes determine which ABO antigen will be present on that individual’s red cells. These genes exhibit co-dominance, meaning that if both A and B genes are present, both will be expressed.

Those who type as group O must have two O genes present (since both the A and B genes would have produces recognizable antigens, neither of which is present on group O cells). Therefore, in the case of an AB individual or an O individual, we can tell exactly which genes are present, or a genotype. Typing that show persons to be group A or group B reveal only one gene product and thus only a phenotype can be determined. Persons of phenotype A can be genotype AA or AO , while those of phenotype B can be genotypically BB or BO. Family studies may be done to determine the genotype of an A or B individual. Fore example, if the mating of one A and one O parent produced a group O child, the second gene present in the A parent must have been O since the child has inherited one O gene from each parent.

It is possible that since anti-A and anti-B develop so predictably, without a recognizable immunizing event, that they are “naturally” occurring. Their production is thought to be stimulated by bacteria which have been shown to contain substances that are chemically similar to human A and B antigens. (Anti-A and anti-B are absent in germ-free animals.) Except for the rare hh individuals who lack H substance, everyone has some H in their cellular makeup.

The most common classifications are A1 and A2. These account for over 99% of group A bloods. Of this 99%, A1 comprises approximately 80%. Commercial anti-A typing serum does not differentiate between A1 and A2 cells. A1 cells contain “A” antigen and “A1” antigen. A2 is not really a unique antigen. It is thought to be simply “A” antigen with no “A1” antigen. Several preparations are available that will react with A1 cells, but not other subgroups of A. An extract of the seeds of the plant, Dolichos biflorus has specific anti-A1 activity. “Absorbed anti-A” serum can also be prepared. To do this, the anti-A from group B people is absorbed with A2 cells. Anti-A is removed and a second antibody that reacts only with A1 cells remains. Anti-A1 can also be found as a separate antibody in the sera of A2 and A2B individuals.

For the most part, subgroups are merely of academic interest, but occasionally they present clinical problems. The antigen may be so weak that it is not detected and the red cells are mistyped as group O. This is especially dangerous if the cells are those of a donor. Problems may arise because the serum of an A2 or A2B, A3 or Ax individual might contain anti-A1. This antibody may be detected in serum typing and cause confusion. You would not expect to find a person with A antigen on his red cells and anti-A in his serum. Anti-A1 is produced by about 1-2% of group A2 persons and about 25% of group A2B persons. Subgroups may be determined by reactions with antisera as seen in the table on the next page.

Forward typing is done using known antisera to detect ABO antigens present on the patient’s red cells. In the tube test, known antisera and patient cells are placed in labeled test tubes, centrifuged, and observed for agglutination. Each manufacturer has specific instructions for its own antisera, detailing the percent of cell suspension, number of drops of cell suspension versus number of drops of antisera, and the rate and length of centrifugation. Though the details differ, the theory behind the tests is the same.

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.

CBC Hepatitis B surface antigen Antibody, rubellaSyphilis test (RPR) Antibody screen Blood type, Rh and ABO

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.

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.

If the physician had decided to continue transfusing the patient at this stage, the following information should be communicated: Although all donors appear to be compatible in the post-transfusion crossmatch, they are not. The results are false negatives - the patient's antibody has been "mopped up" by adsorbing to the incompatible transfused O Rh-negative RBC. Given that 6 donors were positive using the pretransfusion plasma, the antigen is a higher frequency antigen and most donors would likely be antigen-positive and incompatible. The patient's physician should consult the TS medical director before any decision to transfuse is made. Transfusing RBC before tests are complete requires physicians to sign an emergency release form in which they assume full responsibility.

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.

There are two potential problems in typing a recently transfused patient who develops a positive DAT: There will be two cell populations, patient and donor red blood cells. If the typing sera reacts by IAT, the positive DAT will cause false positives. In the case presented, the DAT has become negative. This also suggests that most (if not all) transfused donor red cells have been removed from the patient's circulation.Regardless, to be on the safe side, the patient's initial pretransfusion specimen, which was DAT negative and consisted of only the patient's red blood cells, should be used for antigen phenotyping.