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ANTIGLOBULIN TEST and ANTIBODY DETECTION

ANTIGLOBULIN TEST and ANTIBODY DETECTION. AHLS 311. ANTIGLOBULIN TEST. Detection of non-agglutinating Ab, especially IgG 1 and IgG 3 or complement components (C3d) affixed to RBCs in vivo or in vitro .

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ANTIGLOBULIN TEST and ANTIBODY DETECTION

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  1. ANTIGLOBULIN TESTandANTIBODY DETECTION AHLS 311

  2. ANTIGLOBULIN TEST • Detection of non-agglutinating Ab, especially IgG1 and IgG3 or complement components (C3d) affixed to RBCs in vivo orin vitro. • Direct antiglobulin test (DAT) - detection of sensitization of RBC s (coated with Abs and/or complement components) in vivo • Indirect antiglobulin test (IDAT) - detection of sensitization of RBCs in vitro

  3. ANTIGLOBULIN TEST • Principle - Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCs • Pentameric IgM Abs are so large that, when bound to RBC Ags, the RBCs agglutinate (usually at RT) • IgG Abs usually need a little help, a bridge molecule, to agglutinate RBCs • AHG acts as a bridge molecule

  4. REAGENT PREPARATION • Polyclonal or monoclonal sources (see Figure 4-1) • Polyclonal - Animals hyperimmunized with human globulins; bled for antisera to obtain high titered, high avidity specificity to human • Monoclonal - Hybridoma cells from mice; collection of culture or ascites fluid yields antisera • mice hyperimmunized with human globulins • prepare cell suspension from spleens; fuse with immortalized myeloma cells • screen hybridoma clones for desirable specificities and affinities • maintain cultures of clones in vivo or in vitro • Product is highly regulated for potency

  5. REAGENT PREPARATION • Polyspecific AHG • Abs to human IgG, and • Abs to human C3d (C3b breaks down to C3c and C3d) • Advantage is that polyspecific AHG may detect complement-dependent Abs on RBCs (anti-Jka) • Disadvantage - more nuisance positives • Monospecific AHG • Abs to human IgG only or human C3d only • Fewer nuisance positives; may miss an important Ab

  6. DIRECT ANTIGLOBULIN TEST • Detects in vivo sensitization of RBCs • Procedure (see Color Plate 1) • Wash unknown RBCs >3 X with saline (removes unbound globulins) • Add AHG (binds to IgG or C3d, if any, that is bound to RBCs; forms agglutinates) • Centrifuge (accelerates agglutination) • Grade and record agglutination as 0 to 4+ • Add Ab-coated RBCs (check cells) to all negatives, spin, read and record (checks that AHG was added and was functioning)

  7. DAT: CLINICAL CONDITIONS • Hemolytic disease of the newborn (HDN) (maternal IgG crosses placenta and binds to infant RBCs; may be up to a 4+ reaction) • Hemolytic transfusion reaction (recipient Ab coats donor RBCs; usually about a 1+ reaction) • Autoimmune hemolytic anemia (AIHA) (autoAbs coat own RBCs; reaction strength variable)

  8. DAT: INTERPRETATION • Usually do initial DAT using polyspecific AHG and then more detailed testing, if necessary, with monospecific AHG • With a positive DAT, consider: • Evidence of in vivo hemolysis? • Recently transfused? • Unexpected allo- or autoAbs? • Medications? • Positive DATs with no clinical significance may be detected in up to 2% of population

  9. INDIRECT ANTIGLOBULIN TEST (IDAT) • Detection of in vitro sensitization of RBCs • Procedure (see Color Plate 1) • Same as DAT, except: • Step 1 entails incubating RBCs (reagent or unknown) with antisera (reagent or unknown) allowing time for in vitro attachment of Abs to RBCs

  10. IDAT: APPLICATIONS • When the unknown is sera: • Detection of Abs in recipient sera that may be incompatible with donor RBC Ags (compatibility test - in this case, the RBCs may also be considered “unknown”) • Detection of unexpected allo- or autoAbs in unknown sera (antibody screen) • Identification of unexpected allo- or autoAbs in unknown sera (antibody identification) • Titration of Ab in unknown sera or amniotic fluid

  11. IDAT: APPLICATIONS • When the unknown is RBCs: • Determination of RBC phenotype (detection of Ags) using known antisera (weak D testing)

  12. TEST SENSITIVITY • DAT detects about 150 to 500 IgG or C3d molecules/cell • IDAT detects about 100 to 200 IgG or C3d molecules/cell

  13. FACTORS AFFECTING SENSITIVITY • See Table 4-6 • Ratio of serum to cells (increasing ratio by adding more serum may increase sensitivity) • Temperature (37oC is optimal) • Incubation time • in saline (30 to 60 min) • in LISS (10 to 15 min) • Washing must be thorough (else, neutralization of AHG) and rapid (else, elution of bound Abs) • Centrifugation (1000 RCF for 20 seconds)

  14. REACTION MEDIA • 22% albumin • decreases zeta potential by buffering • allows Ab-coated cells to come closer together • Low Ionic Strength Solution (LISS) • decreases zeta potential • serum/cells very important; increase amount of serum with caution • Polyethylene glycol (PEG) • removes water, concentrating Ab • use monospecific AHG with anti-IgG (else, false positives) • do not read aggl. microscopically (false positives)

  15. ANTIBODY SCREEN • Detection of broad range of unexpected (not ABO) allo- or autoAbs in sample sera • Involves testing patient serum against 2 or 3 reagent RBC samples (not pooled) • O cells • Between the 2 or 3 samples, these Ags will be represented: D,C,E,c,e,M,N,S,s,P1,Lea, Leb, K,k,Fya, Fyb, Jka, and Jkb • Homozygous expression of Ags is valued over heterozygous expression (Ags may show dosage effect; greater antigen density per cell increases sensitivity)

  16. Ab SCREEN: SAMPLES & REAGENTS • Plasma or serum samples (no C’ in most plasma samples because of Ca++ chelation) • Monospecific AHG with Anti IgG • less interference from nuisance cold agglutinins • minimal risk of missing C’ dependent Abs • Enhancement reagents (help reduce zeta potential and allow sensitized RBCs to come closer together) - 22% albumin, LISS, PEG • Coombs control (check) cells (IgG coated RBCs)

  17. Ab SCREEN: PROTOCOL • Add 2 drops unknown serum to 2 or 3 appropriately labelled tubes • Add 1 drop screening cells to appropriate tubes • Centrifuge, read for agglutination (0 - 4+), record • Add 2 drops LISS or PEG; incubate for 15” at 37oC • Centrifuge, read and record • Wash 3X with saline • Add AHG, centrifuge, read and record • Add check cells to tubes negative for aggl., centrifuge, read and record (geared for 2+ rxn)

  18. Ab SCREEN: AUTO CONTROL • Auto control consists of 2 drops unknown serum and 1 drop unknown RBC suspension • May or may not be included in Ab screen • May provide a negative to observe • When positive, indicates that unknown RBCs have: • An unexpected autoAb (eg., AIHA) • A positive DAT (eg., recently transfused with incompatible blood)

  19. Ab SCREEN: INTERPRETATION • Phase of agglutination or hemolysis? • Pentameric IgM Abs usually react in immediate spin phase at RT (Abs to N, I, P1) • IgG Abs typically react in AHG phase (Abs to Rh, Kidd and Duffy Ags) • Abs to Kell, Lewis and M Ags are variable • Result of auto control? • Did more that 1 screen cell react and, if so, did they react at same strength and phase? (If not, consider multiple Abs or dosage.)

  20. Ab SCREEN: LIMITATIONS • Low frequency Ags (Ags found on < 10% of all human RBCs) may not be detected • Ab titers that are low may not be detected • ABO Abs will not be detected (of interest in HDN)

  21. Ab IDENTIFICATION • Uses a panel of RBCs (type O) of known Ag content to determine unknown Ab specificity • Applications • Providing information for donor unit selection for recipients with unexpected Abs • Working up a case of HDN • Working up a case of AIHA • Samples, most reagents (except cells) and protocols usually same as those for Ab screen

  22. Ab ID CONSIDERATIONS • Patient medical history (race; previous transfusion, pregnancies, transplantations; medications/IV fluids; diagnosis) • Antigen profile of panel cells • Result of auto control? • What phase(s) and at what strength(s) was agglutination or hemolysis seen? • Crossing out procedure • Only tubes negative in all phases except check cells phase) • Only antigens with homozygous expression • Do Abs left match the reaction pattern?

  23. Ab ID CONSIDERATIONS • If remaining Abs do not fit the reaction pattern: • Multiple Abs • Dosage effect (heterozygous vs. homozygous) • Abs to high (>90% of human population) or low (<10%) frequency Ags • Cold reacting Abs • Confirming the Ab specificity • Testing serum against 3 known Ag-negative RBCs and 3 known Ag-positive RBCs gives a 95% confidence level • Usually need to use RBCs from multiple panels

  24. Ab ID CONSIDERATIONS • If auto control was negative and Ab screen and ID were positive, patient has an alloAb • Patient’s RBCs should be lacking the Ag to which the alloAb has specificity • Final confirmation of Ab specificity requires demonstrating that patient lacks that Ag • Test patient RBCs with known Ab of same specificity as suspected alloAb; should be negative (Ag phenotyping) • This relationship does not hold true if patient’s auto control was positive; patient has an autoAb

  25. SELECTING COMPATIBLE UNITS FOR PATIENT WITH Ab • If patient has an alloAb, he/she needs units that are negative for that Ag • Select random ABO/Rh compatible units, perform compatibility test and, if negative, check units for Ag of interest using known antiserum • How many random donor units will it take to find needed units? Use known Ag frequencies to determine

  26. SELECTING COMPATIBLE UNITS FOR PATIENT WITH Ab • Divide number of units needed by the frequency of population that is negative for the Ag (see text); screen that many random units • What if the patient has multiple (clinically significant) alloAbs? • Multiply the population frequency of those negative for one with the frequency of those negative for the other • Divide # units needed by that number

  27. SPECIAL TECHNIQUES • Use of enzyme treated panel cells (enzymes remove Abs to Fya, Fyb, M, N, and S) - see Table 11-3 • Elution of Ab from the surface of RBCs • Material (Ab?) recovered from cell membranes is called the eluate • Perform Ab screen/ID on eluate as if it was serum • Use of heat, freeze/thaw, organic solvents, and acidic solutions provide methods for disassociating Abs from RBC membranes

  28. SPECIAL TECHNIQUES • Adsorption • Removal of Ab from serum by combining serum with appropriate RBCs • Following incubation, cells and serum are separated; absorbed serum may be used for further studies • Especially helpful when patient has both autoAb (adsorbed by patient cells) and alloAg (not adsorbed)

  29. SPECIAL TECHNIQUES • Neutralization • Uses soluble Ag to inhibit reactivity of some Abs in testing • Add soluble Ag to serum, incubate, then use serum to do testing • Especially helpful when a patient has a nuisance cold Ab (neutralized) and a clinically significant Ab (not neutralized)

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