September 2007 Dr Debra Lane Serology Update September 2007 Dr Debra Lane

Initial detection and Identification of Alloantibodies Alloantibdies can be found in 0.3- 38% of the population Allogenic antibodies react only with allogenic cells Immunization can be from: Pregnancy Transfusion Transplantation Injections with immunogenic material ( needle sharing)

AB ID Rare cases no specific immunising even is identified Antigens exposure may be: Environmental Bacterial Viral

Definition of a Clinically significant antibody An antibody that shortens the lifespan of a transfused red cell or has been associated with Hemolytic Disease of the newborn. May be detected in serum or plasma Whenever possible, patients with clinically significant alloantibodies should be investigated, the antibody determined and antigen negative red cells provided

Medical History Useful to know: Clinical diagnosis History of transfusion Pregnancy history In some cases the ethnic background

Screening Commercial group O cells are available for screening Usually a two or three cells screen Up to the Lab Director to determine which to use Reagent cells must contain: D,C,E,c,e,M,N,S,s,P1,Lea,Leb,K,k,,Fya,Fyb,Jka,Jkb

Dosage Antigens in the Rh, MNSs, Kidd show dosage Reagent cells should be refrigerated when not in use Should not be used past their expiration date Lot number must be documented If there is a reaction on the screen cells then the next step is a panel test

Panels Most blood banks now use commercial panels Some blood banks attached to donor centers still prepare there own panels from regular donors with known phenotypes whose cells have been frozen Panels are usually set up to give the same characteristic patterns for the most common antibodies

Secondary Panels These panels have more cells, and are much more expensive Used for exclusions and often have more homozygous cells Panels come every 2 to 4 weeks Back of the panels often have the extended phenotypes listed on the back

Antiglobulin Reagents Most antibody identifications include an antiglobulin phase Either a polyspecific anti-human globulin or a monospecific IgG anti-human globulin is used IgG anti-human globulin is used to avid detecting the in-vitro binding of complement

Enhancement Media Many enhancement media have been utilized and have included: Saline Albumin LISS( Low Ionic Strength Saline) PEG (Polyethylene Gycol)

Auto control Cells that react with the enhancement media alone are not the same as a positive Direct Antiglobulin Test If the autocontrol is positive a DAT should be done If the DAT is positive then further studies such as an elution may need to be performed

Most places use the same technique or method for antibody detection as identification and crossmatch We don’t because we implemented Galileo for our screening [solid phase] and maintained the method our tech’s were familiar with for manual testing [PEG] Some techs start with immediate centrifugation and reading before adding enhancing media This is useful to detect M,N,P,I Lea, Leb We omit these steps since we want to avoid finding antibodies that react at lower temperatures Some antibodies may even cause complete red cell lyses such as anti-Lea, anti-Jkb

Special notes Patients with known antibodies do not require a full panel Some exclusion cells are done to confirm the antibody

Interpretation Presence or absence of agglutination is important Positive reactions include the phase and the strength of the reaction Single alloantibodies tend to give clear reaction patterns Negative reactions allow for exclusions of antibodies to antigens expressed on the nonreactive cells

Exclusions “crossout”- is the widely used first approach where antibodies are excluded when there is no reaction to a positive antigen cell If the antigen is present on the cell and the serum or plasma did not react, that antigen is excluded This usually leaves a group of antibodies that have not been excluded Next the cells reactive for the presumed antibody are evaluated and if the pattern matches exactly, that is most likely the specificity of the antibody in the serum

Exclusions Additional testing may be required to rule out other specificities If it fits anti-E but K and S have not been excluded, then one might test cells such as: E neg, K pos, S neg E pos, K neg, S neg E neg, K neg, S pos

Probability Probability has been studied to ensure that the reactivity is not due to chance Fischer’s exact method is to require three antigen positive cells that react and three antigen negative cells that d not react Harris and Hoschman have done calculations for minimum requirements for a (p) value of 0.05 are met by having two positive and three negative cells or one positive and seven negative cells

Sometimes identification is difficult We may resort to phenotyping the patient if they have not been transfused and give phenotypically matched blood Antibodies do not always react with all the positive cells Technical error Weak antigens Weak antibody reactivity

Zygosity Reaction strength may vary due to dosage React stronger with homozygous cells Or double dose of the antigen This is common in: Rh Duffy MNS Kidd

Antigens Present in Common Instead of excluding, one can look at what the reacting cells have in common cells reacting at room temp are P pos Except for one sometimes the panel will indicate it is a P weak cell

Variable Reactivity May indicate HLA antibodies Anti-Bga vary between individuals Perhaps the manufacturer has incorrectly labelled a reagent red cell May react with an antigen not routinely listed-Yta

Multiple Antibodies Observed pattern does not fit a single antibody Reactivity is present at different test phases Unexpected reactions are obtained when trying to confirm an antibody No pattern emerges

Other steps for Antibody Identification Exclusion method Test serum against homozygous cells Enhancement media Phenotype the patient (if not transfused) Methods to inactivate antigens Adsorption elution

Sneaky Antibodies All cells reactive with a positive DAT may not always be a warm auto Can be a transfusion reaction with an antibody to a high frequency antigen Like Diego The race of the individual can be helpful at this point

Antibodies to Ingredient in the Preservative solution Antibodies may be made to: Chloramphenicol Neomycin Tetracycline Hydrocortisone EDTA Sodium caprylate These may agglutinate cells suspended in that solution Can wash the reagent cells before testing

Antibodies to enhancement media Ingredients Antibodies to LISS or albumin; Sodium caprylate, thimersol

Cold Autoantibodies

Next step is usually to find antigen negative units for the patient

Cold autoantibodies Potent cold autoantibodies can create problems clinically if they react at room temperature May be benign or pathologic Thermal amplitude helps to determine if it is significant The freshly collected serum must be kept warm until the serum is separated

Determining the Specificities Requires ample to be warm until the serum is removed Autologous red cells Reagents: Pool O I adult red cells Group O I cells Pts own washed autologous cells ( 37o saline) Red cells same group of the patient Saline or phosphate buffered saline

Method Prepare serial two fold dilutions in saline or PBS( 12 tubes) Label 12 tubes with the dilution 2,4,8.. For each adult, I, autologous Dispense two drops into each tube Add I drop 3 to 5% red cells Incubate RT for 30 to 60 min Centrifuge for 15 to 20 sec 900 to 1000 g Examine for agglutination Grade and record Transfer tubes to 4 C and incubate for 1 to 2 hours centrifuge Place tubes in rack in ice water Grade results

Interpretation This method helps to determine the specificity and the titre Need to use separate pipette tips for dilutions Large volumes prepare better dilutions Potent examples do not show specificity until titration studies are performed This can be used to determine the titre and sensitivity If readings are taken sequentially after each incubation [37,30,4] the specificity, titre and thermal amplitude can be determined From AABB technical manual

Cold Agglutinin Titres High titred cold agglutinins may be pathologic May have overt hemolysis May be seen in B cell lymphomas

Cold Agglutinin Titres Specimen: serum separated at 37 from a sample that clotted at 37 or plasma from sample collected and maintained at 37 with periodic inversion Reagents Pool of t2 examples washed group O I PBS ph 7.3

Cold Agglutinin Titres Prepare serial dilutions in PBS ½ to 1/4096 Mix two drops dilution with 1 drop 3 to 5%red cells Mix and incubate at 4c for 1 to 2 hours Centrifuge 15 to 20 sec at 900 to 1000g Place in ice water bath Examine for agglutination Start with tube at highest grade

Cold Agglutinin Titres Titre is the r reciprocal of the highest serum dilution where agglutination occurs Titres above 64 are elevated Hemolytic anemia rarely occurs from colds unless the titre is greater than 1000 Titres may be lower with anti-I If the DAT is positive with complement only, and the patient has symptoms of hemolytic anemia Specificity and thermal amplitude studies should be performed.