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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)