ANTIBODY DETECTION

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 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

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)