BLOOD BANK (2ND WEEK)

ANTIBODY IDENTIFICATION
Antibody Screens
- Test donor/patient serum (unknown) with
reagent RBCs (known)
- Use 2 or 3 screening cells to detect if
antibodies are present in the serum
- If antibodies are detected, they must be
identified.

Reagent RBCs
- Screening cells - An autocontrol should also be run with ALL
o Antibody detection panels (patient RBCs + patient serum)
o Sets of 2 or 3 vials
- Panel cells
o Antibody identification
o At least 10 vials per set

- The same phases used in an antibody screen

are used in a panel.

Panel
- Group O red cells

Antibody ID Testing
- A tube is labeled for each of the panel cells
plus one tube for AC:
- NOTES:
o 10 tubes, each contains RBC antigen
then 1 autocontrol; 1 drop of patient
RBC in AC and 2 drops of patient
serum in all tubes (11 tubes) 
- Each of the panel cells has been antigen proceed to centrifugation (immediate
typed (shown on antigram) spin) then read if there’s
o + refers to the presence of the agglutination
antigen
o 0 refers to the absence of the antigen
IS Phase
- Perform immediate spin (IS) and grade
agglutination; inspect for hemolysis.
o Hemolysis is also a positive (+) sign of
reaction
- Record the results in the appropriate space as
shown:
(LISS – Low Ionic Strength Solution) 37°C
Phase
- 2 drops of LISS are added, mixed and
incubated for 10-15 minutes.
o Checks for agglutination
- Centrifuge and check for agglutination.
- Record results.
- NOTES:
o LISS – it potentiates the attachment
of your antibody with your RBC via
electroneutrality. If there’s a lot of
negative outside the cell membrane it
will convert/modify the external
electroneutrality to weakly positive
 makes the RBC outside membrane
more electroattractive.
Indirect Antiglobulin Test (IAT) Phase (or
AHG)
- Anti-Human Globulin reagent (AHG)
o Polyspecific
o Anti-IgG
o Anti-complement
- Wash cells 3 times with saline after LISS stage
- Add 2 drops of AHG and gently mix.
o Centrifuge
o Read
o Record reactions
AHG Phase
- Every after antibody identification you should
always add check cells
o Check cells
 use to consume antibodies
that did not react to serum
 use to check if you really did
drop a serum because if not
restart from the beginning
 should always have check
cells reaction in panels where
reaction did not happen (yung
mga panels na puro 000 sa
dulo)
Interpretation of Results
- need to identify what antibody circulating is
present to patient
o How to detect? Rationale is the
patient has circulating antibody
against a minor antigen, you assume
that its reaction is positive (+) to any
of these 3 panels (IS, 37, AHG)
1st – highlight those that have reaction
(cell 1, 4, 7, 9)
2nd – check for the presence of your
autoantibody  patient or AC, if
positive for AC automatically rule out
that autoantibody is present, report
it as positive. If the AC is negative it
 means that alloantibody is present
against the minor blood group.

Done only when delayed or acute

hemolytic transfusion reaction na
hindi justifiable by ABO or RH alone.

3rd – rule out possible antigens to

know what causes the reaction. Rule
out everything that is negative.

Compare the dosage effects (C, c; E,

e; M, N; S, s; Lea, Leb; K, k; Fya,
Fyb; Jka, Jkb) If dosage effect is both
positive then they’re not the one that
causes the reaction.

4th – the previously cross out cell 

mental barrier to cross out the whole Possible Results and Interpretations
column
- Autocontrol
If the dosage effect is repeated for o Negative – alloantibody
2x, this is the time that you can cross o Positive – autoantibody or DTR (i.e.,
it out. (sa pang 3rd time actually, sa alloantibodies)
cell #7 na cross out  look for M, N) - Phases
o IS – cold (IgM)
o 37° – cold (some have higher thermal
range) or warm reacting
o AHG – warm (IgG)
- Reaction strength
o Consistent strength – one antibody
o Different strengths – multiple
antibodies or dosage

Rule of Three
- The rule of three must be met to confirm the
presence of the antibody:
o Positive with 3 cells with the antigen
Interpretation:
o Negative with 3 cells without the
- Most prominent: anti-f, anti-Lea, anti-k antigen
- cells that can’t rule out: anti-c, anti-Fyb - If there are not enough cells in the panel to
- look for the most prominent, if its consistent fulfill the rule, then additional cells from
positive for all columns then proceed to rule another panel could be used OR antigen
of 3s (3 positive on positive panel and 3 typing on red cells can be performed,
negative on negative panel) provided no recent transfusion occurred.
o anti-Lea is the circulating
alloantibody Multiple antibodies
- Reaction strengths can vary
- Matching the pattern is difficult
o Report all antibodies that cannot be
rule out

o If the reaction is like this (example

above)  it indicates two different
 antibodies present; the cold phase  Bromelin (pineapple)
and warm phase.  Papain (papaya)**
o Enzyme procedures may be:
Multiple Antibody Identification  One-step
- Selected cells  Two-step
- Neutralization o Enzymes remove the sialic acid from
- Chemical treatment the RBC membrane, thus “destroying”
o Proteolytic enzymes it and allowing other antigens to be
o Sulfhydryl reagents “enhanced”.
o ZZAP o Antigens destroyed: M, N, S, s, Duffy
o Antigens enhanced: Rh, Kidd, Lewis,
Selected Cells I, and P
o If there is no agglutination after
- Selected cells are chosen from other panel or
treatment, then it is assumed the
screening cells to confirm or eliminate the
enzymes destroyed the antigen.
antibody.
o Enzyme Techniques
- The number of selected cells needed depends
 One-stage
on how many antibodies are identified.
 Enzyme is added
- Every cell should be positive for each of the
directly to the
antibodies and negative for the remaining
serum/cell mixture.
antibodies
 Two-stage
- NOTES:
o Need to rule out cell/s that cannot be  Panel cells are pre-
treated with enzyme,
rule out
incubated and
Let’s say you ran a panel and identified 3 different washed.
antibodies: anti-S, anti-Jka, and anti-P1  Patient serum is
added to panel cells
and tested.
- Sulfhydryl Reagents
o Cleave the disulfide bonds of IgM
molecules and help differentiate
between IgM and IgG antibodies.
o Good to use when you have both IgG
and IgM antibodies (warm/cold).
 Dithiothreitol (DTT) is a thiol
and will denature Kell
antigens
Neutralization  2-mercaptoethanol (2-ME)
- Some antibodies may be neutralized as a way - ZZAP
of confirmation o A combination of proteolytic enzymes
- Commercial “substances” bind to the and DTT
antibodies in the patient serum, causing them o Denatures Kell, M, N, S, Duffy and
to show no reaction when tested with the other less frequent blood group
corresponding antigen (in panel). antigens
- Common substances: o Does not denature the Kx antigen
o P1 substance (sometimes derived o Good for adsorption techniques
from hydatid cyst fluid)  “Frees” autoantibody off
o Lea and Leb substance (soluble patient’s cell, so that
antigen found in plasma and saliva) autoantibody can then be
o I substance can be found in breast adsorbed onto another RBC
milk
Autoantibodies
o Sda substance derived from human or
guinea pig urine - Autoantibodies can be cold or warm reacting.
- A positive autocontrol or DAT may indicate
Chemical Treatment
that an auto-antibody is present.
- Proteolytic Enzymes - Sometimes the autocontrol may be positive,
o Can be used to enhance or destroy but the antibody screening may be negative,
certain blood group antigens meaning antibody is already coating the RBC.
o Examples: DAT
 Ficin (figs)**
 - The direct antiglobulin test (DAT) tests for
the in vivo coating of RBCs with antibody.
- AHG is added to washed patient red cells
directly.
- Tests the attached IgG inside the body
- Naka coat na ang antibody sa cell pagka
extract

IAT

- In vitro attachment
- Patient serum + patient RBC  LISS  wash
3x  AHG
- If positive for IAT = antibody is present but do
not automatically attach, may be possibly
cold or warm which depends to patient
- Antibody is not automatically attach

o AIHA condition
 Diagnostic test for AIHA: IAT
or DAT

Cold Autoantibodies

- React at room temperature with most (if not

all) of the panel cells and give a positive
autocontrol
- The DAT is usually positive with anti-C3 AHG
(detects complement).
- Could be due to Mycoplasma pneumoniae,
infectious mononucleosis, or cold agglutinin
disease.

Warm Autoantibodies

- More common that cold autoantibodies

- Positive DAT due to IgG antibodies coating
the red cell
- Majority of panel or screening cells will be
positive
- The Rh system (e antigen) seems to be the
main target although others occur.
- Causes of warm autoantibody:
o Idiopathic
o Known disorder (SLE, RA, leukemias,
ulcerative colitis, pregnancy,
infectious diseases, etc.)
o Medications