IMMUNOHEMATOLOGY-LABORATORY (NOTES 1 OF 4)
Blood Groups
I. Basic Antigen-Antibody Testing
A. Basic Red Cell-Antibody Interactions
1. Agglutination
a.________ of red cells due to
antibody coating
b. Two stages:
1) Cell Coating (“______________”)
a) Affected by antibody specificity,
zeta potential,
pH, temperature, antigen and
antibody amounts
b) ____________________ (LISS)
decreases repulsive charges between
RBCs; tends to enhance cold
antibodies and autoantibodies
c) ________________ (PEG)
excludes H2O; tends to enhance
warm antibodies and autoantibodies.
2) ____________
a) Lattice structure formed by
antibodies and RBCs
b) IgG isn‟t great at this; too small to
bridge gap
c) IgM is better because of its
pentameric structure.
2. Hemolysis
a. Direct lysis of RBCs due to
antibody coating and
subsequent complement fixation
b. Uncommon, but equal to
agglutination.
1) IgM antibodies do this better than
IgG
B. Tube testing (Classic blood bank
testing)
1. Immediate spin (IS) phase
a. Mix serum, ____% RBC
suspension; spin ______ sec.
1) Most common: __ drops serum,__
drop RBCs.
b. Non-ABO antibodies reacting here
are most often ___
and insignificant
2. 37 C phase
a. Add potentiator if desired,
incubate at ____, spin.
b. Potentiators and incubation times:
1) ____ minutes for LISS
2) ____ minutes for PEG (do not
READ PEG at 37C!)
3) ____ minutes for no potentiation
(saline)
3. _________________ (AHG)
phase (aka “IAT” phase)
a. Wash above to remove unbound
globulins.
b. Immediately add AHG, spin.
c. Detects RBCs coated with IgG +/–
complement
d. Antibodies reacting at IAT are
more often significant
C. Modern alternatives to tube testing
1. Column agglutination technology
(“________________”)
a. Add RBCs/plasma to gel column
top, incubate, spin.
b. Tubes for IAT (see later) filled
with gel and anti-IgG
1) Gel particles separate RBC
clusters physically
(bigger agglutinates, less migration
through gel).
2) Anti-IgG grabs onto IgG-coated
RBCs and inhibits
their migration through gel
immunologically
 IMMUNOHEMATOLOGY-LABORATORY (NOTES 1 OF 4)

c. Results:
1) Negative: RBCs in button at e. Can be automated (Echo, NEO)
bottom of microtube. f. Similar sensitivity to PEG tube
2) Positive: RBCs stopped in areas testing and to gel
through the
microtube (more positive = higher D. The Antiglobulin Test
position in tube) (“___________”)
Image courtesy of Ortho
d. Also done without anti-IgG in gel 1. _________; demonstrates in-vitro
(ABO, other testing) RBC coating with antibody and/or
e. Can be automated (ProVue complement.
machine) 2. _________: red cells from patient
f. Similar sensitivity to PEG- washed, then mixed with antihuman
enhanced tube testing globulin; demonstrates in-vivo RBC
2. Solid-phase Red Cell Adherence coating with antibody and/or
(“Solid phase”) complement.
a. Antibody binds to lysed or intact
RBC antigens that
are bound by manufacturer to bottom
of microwells
b. Add patient plasma, incubate,
wash: If positive, IgG
binds to RBC antigens all over
bottom of the well.
c. Wash, add indicator RBCs coated
with anti-IgG,
centrifuge (RBCs bind diffusely to
bottom of well)
d. Interpretation:
1) Negative: RBCs in a button at
bottom of microwell,
(No bound IgG for indicator cells to
attach to).
2) Positive: RBCs spread in diffuse
“carpet” across The role of the Coombs test in the
bottom of well (attached to bound evaluationof hemolysis in adults.
IgG). 3. IAT variations
a. Unknown antibody check: Use
RBCs with a known
antigen profile to search for RBC
antibodies
b. Unknown RBC antigen check:
Use serum with known
antibody specificity to search for
RBC antigens
c. Can be used to check for an
unknown antigen OR
 IMMUNOHEMATOLOGY-LABORATORY (NOTES 1 OF 4)

unknown antibody, as in the b. Patient 2: Jk(a+b+) (less Jka

crossmatch procedure ; only 1 gene)
d. Can be done in tubes, gel, solid- c. Anti-Jka
phase, microwells has stronger rxn with “double dose”
4. Specificity possibilities for the RBCs
antiglobulin
a. Anti-IgG, –C3d (“polyspecific”); most
commonly used
1) Detect red cells coated with either
of the above
2) May also detect other
immunoglobulins (anti-IgG
detects shared kappa/lambda light F. Proteolytic enzymes (e.g., ficin, papain)
chains) 1. These enzymes cleave RBC surface
b. Anti-IgG and anti-IgG (heavy chain- proteins
specific) a. This may destroy certain RBC
1) Anti-IgG used for PEG, gel, and antigens
solid phase tests b. Strengthen others by allowing
c. Anti-C3b, –C3d antibodies to bind better
1) Detects either of the above to previously shielded antigens
complement components 2. Useful in antibody identification to
2) Useful in IgM-related hemolysis, confirm or refute a particular antigen as
cold agglutinin dz target of an antibody (see table)
5. IgG-sensitized RBCs (“Coombs
control”, “check cells”) 3. The “Enzyme Classification”
a. Use after negative DAT or IAT t
ube test (not gel or
solid-phase) to ensure functioning
AHG reagent
b. Add IgG-coated cells to AHG-cell
mixture
c. Negative = bad AHG or no AHG
added

E. Dosage
1. Some antibodies react more
strongly with RBCs that have
double-dose (“homozygous”) antigen
expression
2. Most common in Kidd, Duffy, Rh
and MNS systems
3. For example, imagine a patient
with anti-Jka
a. Patient 1: Jk(a+b-) (more Jka
because of 2 genes)
 IMMUNOHEMATOLOGY-LABORATORY (NOTES 1 OF 4)

G. Neutralization
1. Certain substances, when mixed with a
red cell antibody, inhibit the activity of that
antibody against test red cells.

H. Lectins
1. Seed/plant extracts react with certain
RBC antigens
2. Especially useful in polyagglutination
studies (T, Tn, etc)