Why identify???
- Pre-transfusion testing
- Hemolytic Disease of the Fetus and
Newborn (HDFN)
*important to know the specificity of
the disease - Transfusion Reactions
*what AB caused this reaction so that
it does not occur again - Hemolytic anemias
*warm autoantibody or an alloantibody
the cause
WHEN do we identify?
- Following a positive AB screen
- When testing suggests a NEW antibody
has developed - To confirm a previously identified AB
Differentiate the following sets of antibody terms:
a. Unexpected vs Expected
b. Immune Stimulated vs Naturally
Occurring vs Passively Acquired
c. Clinically Significant vs Clinically
Insignificant
A. Expected: ABO
Unexpected: all other ABs
B. Immune Stimulated: IgG
Naturally Occurring: IgM
Passively Acquired: not produced by the
patient’s immune system; administered
via transfusion; will eventually dissipate
from the body
Examples: IVIG, RHIG, ABO (through
incompatible plasma transfusions)
C. Clinically significant? Decreased survival
of RBCs
Clinical insignificant? No decreased
survival of RBCs
Compare and contrast the cells used for antibody screens and antibody panels to include:
a. ABO type
b. Required antigens to be present
c. Zygosity of the antigens present
d. Procedure for use of screening
cells vs panel cells
A. Group O: we don’t want to pick-up any
naturally occurring ABs
B. Screen: Rh, Kell, Duffy, Kidd, Lewis, P1,
MNSs
Panel: Some of the uncommon AGs as
well as the common AGs (the
AGs from the screen)
C. Homozygous: we want the AB to detect
the weakest expression of
the antigen
D. Screen: 2 or 3 cells used to detect for all
common ABs
Panel: 8 to 20 cells used to detect for
some of the uncommon ABs as
well as the common ABs
Describe how the following enhancement media aid in antibody identification
a. 22% Albumin
b. LISS
c. PEG
d. Enzymes (Ficin & Papain)
e. Sulfhydryl/ Thiol reagents (DTT, 2ME, AET)
f. P1 and Lewis Neutralization substances
A. Disperses charges around cells, thus
decreasing the zeta potential – requires
longest incubation
B. Decreases zeta potential and increases
the uptake of AB by the RBC in the
sensitization phase
C. Removes water in system, thus
concentrating AB; increasing RBC
sensitization (can cause nonspecific
aggregation of cells, must wash after
incubation)
D. Removes the sialic acid residues and
denatures/removes glycoproteins;
destroys some AGs, enhances others
E. Disrupt the disulfide bonds of IgMs;
destroys the Kell and Lutheran AGs
F. Use of Neutralizing substances; must use
a saline control to compare
State the four general steps of the antibody identification process.
- Antibody detection – IAT
- Antibody identification – panel, patient
AG typing - Determine clinical significance – IgG vs.
IgM - Appropriate transfusion considerations
are put in place – provide AG-negative
blood
State the antibody screen testing requirements according to AABB.
- 37 degrees C and use of AHG
- Pretransfusion testing
- Prenatal testing – HDN evaluation, RHIG
candidacy - Donor blood
Explain the “3+3 Rule” for antibody identification.
In order to identify an antibody with 95% confidence, you must:
1. Have 3 AG positive cells that react
positively
2. Have 3 AG negative cells that react
negatively
3. Exclude all other common alloantibodies
(prove they are NOT present)
4. Prove the patient is capable of making
the antibody
Describe the thought process (what questions one should consider) when starting an antibody
resolution.
- Recheck patient history for clues
*Recently transfused?
*Have they EVER been transfused?
*Have they received any RHIG or IVIG
recently?
*Diagnosis?
*Race - Is AC positive or negative?
- Is DAT positive?
- What phase(s) did the reaction occur?
- Does AB appear IgM or IgG?
- Do all positive cells react at the same
phase? - Are all reactions one strength or
variable? - Does there appear to be dosage?
- Are all common antibodies ruled out?
Explain the use of the autocontrol (AC) tube including:
a. procedure for testing
b. significance of positive and negative results
c. correlation of the AC to the DAT
A. The AC includes the patient’s RBCs and
serum along with the enhancements and
incubations that panel cells underwent
B. Positive AC could indicate the presence
of autoantibodies or antibodies to
medications
Negative AC indicates the presence of
an alloantibody
C. A positive AC should reflex to a DAT, that
should also be positive
List the 5 “uncommon” antigens that may or may not be present on panel cells but are not required to be ruled out on most panels, as presented in lecture.
V, C(w), Kp(a), Js(a), Lu(a)
State 3 antigen exceptions to homozygous rule-outs, as presented in lecture.
- “K” can be ruled out heterozygously
because it is nearly impossible to find
this antigen in the homozygous state
