ANTIGLOBULIN TEST and ANTIBODY DETECTION

1
ANTIGLOBULIN TESTandANTIBODY 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
• 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 gt3 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 lt 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 (gt90 of human population) or low
  (lt10) 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
• 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

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)