1. ANTIGLOBULIN TEST and ANTIBODY DETECTION
AHLS 311

2. 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

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 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

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)