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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
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
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?
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
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)
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: 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 Patients 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 patients auto control was positive; patient has an autoAb
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)