Transcript ANTIGLOBULIN TEST - Austin Community College District

ANTIGLOBULIN TEST
and
ANTIBODY DETECTION
AHLS 311
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
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
• Product is highly regulated for potency
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
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)
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)
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
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
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
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)
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)
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
– 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
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)