ANTIGLOBULIN TEST and ANTIBODY DETECTION AHLS 311

ANTIGLOBULIN TEST Detection of non-agglutinating Ab, especially IgG1 and IgG3 or complement components (C3d) affixed to RBCs in vivo or in 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

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

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

REAGENT PREPARATION Polyspecific AHG Monospecific 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

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)

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)

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

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

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

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

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

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)

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)

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)

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)

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)

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)

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.)

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)

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

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?

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

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

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

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

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

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)

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)