ABO detection and ID Flashcards
The test used to detect antibodies is called an
antibody screen
Antibody screens are used for
Patients needing a transfusion
– Pregnant women
– Patients who have had transfusion reactions
– Blood(red cells) and plasma donors
Antibody Screen
Indirect Antiglobulin Test:
Uses the patient’s plasma or serum against reagent
RBCs to detect unexpected antibodies
Indirect Antiglobulin Test:
Unexpected antibodies
found in addition to the expected anti-A or anti-B antibodies
result of RBC stimulation (transfusion, pregnancy)
maybe
– Clinically significant (IgG)
– Not clinically significant (IgM)
Other Antibodies
Naturally occurring antibodies
may form as a result of exposure to
environmental sources (e.g., pollen, fungus, and bacteria), which have
structures similar to some RBC antigens.
Other Antibodies
Passively acquired antibodies
are produced in one individual and then
transmitted to another via plasma-containing blood components or derivatives
Other Antibodies
Autoantibodies,
when present, may complicate the detection of clinically
significant antibodies
In blood banking, we test knowns with
“unknowns”
KNOWN: UNKNOWN:
Reagent RBC + patient serum
Reagent antisera + patient RBCs
When detecting and/or identifying antibodies, we test the patient
When detecting and/or identifying antibodies, we test patient serum (unknown)
with reagent red cells (known)
Indirect Antiglobulin Test
Incorporates multiple PHASES of testing
IS = immediate spin (not required)
37oC = after incubation at 37oC with or without potentiators
AHG = anti-human globulin added (anti-IgG) after washing antibody/antigen reaction
Coomb’s Control Cells (aka Check Cells) =
used with negative reactions to ensure
proper washing technique = must be positive
“IAT”
Antigen Source
= commercial red cells
IAT
Antibody Source =
patient’s serum/plasma
Autocontrol
Tests a patient’s serum with their
own RBCs
(+) mean autoantibody
Autocontrol
Autocontrol is incubated with the
antibody screen (or antibody panel)
Autocontrol
The AC and DAT can help in determine if the antibodies are directed
are directed against
the patient’s cells or transfused cells (allo- or autoantibody)
Autocontrol
If a laboratory technician uses an autocontrol with a screen and it is positive,
the technician may run a DAT (patient cells plus AHG) to detect in vivo coating
If autocontrols (-)
then you have a alloantibody
DAT could be negative=
look up why
Direct Antiglobulin Test
antigen and antibody sources
Only incorporates 1 phase
AHG
Antigen Source = patient’s red cells
Antibody Source = Immunoglobulin attached to patient’s red cells
IgG or C3 coating patient’s red cells in vivo
Clinically
Significant
Antibodies
- Usually IgG
-React best at 37° C and
during the antihuman
globulin (AHG) phase
(indirect antiglobulin test
[IAT])
- Clinically significant
antibodies are associated
with hemolytic transfusion
reactions (HTRs) and HDFN
Performing
an Antibody
Screen
- Patient’s plasma or
serum is incubated with
screening cells
-After incubation, an IAT
is performed using AHG
reagent
- Want to detect any
IgG antibodies
Screening cells are
are single or pooled donor group O cells;
however, single-donor vials offer increased sensitivity
Screening Cells
group O cells are used so that
Group O cells are used so that anti-A and anti-B antibodies will not
react
Screening Cells
Screening cells come in sets of 2 or 3 vials each
● Donor Testing vs Patient Testing
● Each vial (donor) has been phenotyped for each antigen
Screening Cells
how many antigens are required on at least one of the vials
18 antigens are required on at least one of the vials:
D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k, Jka, Jkb, Fya, Fyb
The screen only uses
2-3 cells
An antibody panel usually includes at least.
10 panel cells
Panel
Each of the panel cells has been antigen-typed (shown on
antigram)
+ refers to the presence of the antigen
0 refers to the absence of the antigen
An autocontrol should also be run with
ALL panels
The same phases used in an antibody screen are
used in a panel
A tube is labeled for each of the panel cells plus
one tube for AC
one drop of panel cells
+
two drops of serum
IS Phase
Perform immediate spin (IS) and grade agglutination; inspect for
hemolysis
Potentiators
Used in antibody detection and identification to enhance an antigen–antibody
reaction
Saline
(may only enhance
if incubated for a
long time)
Low-ionic-strength
solution (LISS):
common
Bovine serum
albumin (BSA)
Polyethylene glycol
(PEG)
Proteolytic
enzymes
(can destroy some
antigens)
(LISS) 37°C Phase
2 drops of LISS are added, mixed and incubated at 37oC for 10-15 minutes
Centrifuge and check for agglutination
Record results
IAT Phase (or AHG)
were testing whether or not _________
to do this we use the
Indirect Antiglobulin Test (IAT) – we’re testing whether or
not possible antibodies in patient’s serum will react with
RBCs in vitro
To do this we use the Anti-Human Globulin reagent
(AHG)
Polyspecific (anti-IgG, C3)
Anti-IgG
Anti-complement (anti-C3)
AHG Phase steps
Wash cells at least 3 times with saline (manual or
automated)
Add 2 drops of AHG and gently mix
Centrifuge
Read
Record reactions
All cells are
negative at
AHG, so
add
“Check”
Cells
- 29 y.o. female admitted to Family Birthing Center for labor
progression. All laboring patients will have an active blood bank
specimen during their admission. The patient has been determined to
be a high risk for hemorrhage. The doctor wants to order 2 units of
red cells to be kept at bedside.
Patient T/S Results:
Blood Type: A positive
ABSC: Positive
What needs to happen next?
Interpreting Antibody Panels
There are a few basic steps to follow when interpreting
panels
- “Ruling out” means crossing out antigens that did not react
- Circle the antigens that are not crossed out
- Consider antibody’s usual reactivity
- Look for a matching pattern
An antibody will only react with
cells that have the
corresponding
antigen; antibodies will not react
with cells that do not have the
antigen
Ruling out
Can only rule out on
completely negative
cells
cannot
use for
rule
outs
Ruling out
cross off on what
Cross off on homozygous only only except: K and Lu^a
Ruling out
E could be + on
Heterozygous
Ruling out
Everytime a Rxn happens
Lea is present
Ruling out
Le^a is
IgM
Ruling out
Le^a is normally a
Cold-Reacting antibody (IgM), so it makes
sense that we see the reaction in the IS phase of testing;
The E antigen will usually react at warmer temperatures
Guidelines:
Autocontrol
Negative - alloantibody
Positive – autoantibody or DTR (i.e.,alloantibodies)
Guidelines
Phases
IS – cold (IgM)
37° - cold (some have higher thermal range) or warm reacting
AHG – warm (IgG)…significant!!
Guidelines
Reaction strength
1 consistent strength – one antibody
Different strengths – multiple antibodies or dosage
About reaction strengths……
The strength of reaction may be due to
Dosage
About reaction strengths……
If panel cells are homozygous,
a strong reaction may
be seen
About reaction strengths……
If panel cells are heterozygous,
reaction may be weak
or even non-reactive
About reaction strengths…..
Panel cells that are heterozygous should not be
crossed
out because antibody may be too weak to react
Matching the pattern
Single antibodies
usually shows a pattern that matches one of the
antigens (see previous panel example)
Multiple antibodies
are more difficult to match because they often
show mixed reaction strengths
The rule of three must be met to confirm the presence of
the antibody
Rule of three
P value and confidence interval
A p-value ≤ 0.05 must be observed
This gives a 95% confidence interval
Rule of three
Patient serum must be
Positive with 3 cells with the antigen
Negative with 3 cells without the antigen
What if the “rule of three” is not fulfilled?
If there are not enough cells in the panel to fulfill the
rule, then additional cells from another panel could be
used
Most labs carry different lot numbers of panel cells
Patient History
GET THE HISTORY!
- Mixed RBC populations from a previous transfusion can remain for up to
3 months
– Patient may have come from another hospital
– Some diseases are associated with antibodies
– Some antibodies occur at a higher frequency in some races
– Learn about the diagnosis, age, and race of the patient
Autocontrol is postive or DAT indicates
Positive autocontrol or DAT indicates that autoantibody or
alloantibody is present to recently transfused cells
Negative autocontrol indicates
alloantibody
Interpretation Guidelines
Phases
- IgM antibodies react at room temperature or during immediate-spin
(IS) crossmatching; anti-Lea, anti-Leb, anti-M, anti-N, anti-I, anti-P1
antibodies - IgG antibodies usually react at 37° C or with AHG
- Reactions at different phases may indicate IgM and IgG
Interpretation Guidelines
Reaction strength
– Reactions with varying strengths indicate multiple antibodies
– Strength is also affected by dosage
Interpretation Guidelines
Ruling out antibodies
– Panel cells that are negative in all phases can be used to rule out
antibodies
– Begin with the first panel cell that is negative in all phases and
cross out any antigens present
– Panel cells that are heterozygous should not be crossed out,
because the antibody may be too weak to react
Interpretation Guidelines
Matching the pattern
– The pattern of reactions matches one of the antigen columns when a
single antibody is present
– Multiple antibodies show varying patterns
Interpretation Guidelines
Rule of three
– To ensure valid results, 3 antigen-positive cells must react and 3
antigen-negative cells must not react with the patient’s serum or
plasma
– If this does not occur, additional panel cells (selected cells) are used
Interpretation Guidelines
Phenotyping the patient
Another way to confirm the presence of antibody is to
determine the patient’s phenotype for antigen
Interpretation Guidelines
Individuals do not make alloantibodies toward
antigens on their
own RBCs
Interpretation Guidelines
If the patient has recently been transfused, what techniques should be done
RBC separation
techniques should be used before phenotyping is done
Interpretation Guidelines
Reagent antisera (of the suspected antibody) is added to
to
the patient’s RBCs, a negative reaction should result
Multiple Antibodies
Matching the pattern is more difficult when
-multiple antibodies are
present
Varying reactions may also be observed
Multiple Antibodies
Special techniques can be used to help identify
multiple antibodies
Multiple Antibodies
Special techniques can be used to help identify multiple antibodies
– Selected cells
– Neutralization P1, I
– Chemical treatment
– Proteolytic enzymes
– Sulfhydryl reagents
– ZZAP
Selected Cells
Selected cells are chosen from
other panel or
screening cells to confirm or eliminate the antibody
Selected Cells
The cells are “selected” from other panels
because
of their characteristics
Selected Cells
The number of selected cells needed depends
on how may antibodies are identified
Selected Cells
Every cell should be positive for each of the
antibodies and negative for the remaining
antibodies
Selected Cells
For example:
Let’s say you ran a panel and identified 3 different
antibodies: anti-S, anti-Jka, and anti-P1
Selected cells could help…
Neutralization
Some antibodies may be neutralized as a way of
confirmation
Neutralization
Commercial “substances” bind to the
antibodies in the patient’s serum, causing
them to show no reaction when tested
with the corresponding antigen (in the panel)
Neutralization
The control contains
saline and serum (no substance)
and should remain positive
Neutralization
A control shows that a loss of reactivity is due to the
neutralization and not to the dilution of the antibody
strength when the substance is added
Neutralization
Manufacturer’s directions should be followed
and a _________ control should always be used
Manufacturer’s directions should be followed
and a dilutional control should always be used
Neutralization
Common substances
P1 substances
P1 substance (sometimes derived from hydatid cyst fluid)
Neutralization
Common substances
Lea and Leb substance
(soluble antigen found in plasma and saliva)
Neutralization
Common substances
I substance
can be found in breast milk
Neutralization
Common substances
Sda substance
derived from human or guinea pig urine
**You should be aware that many of these substances neutralize
COLD antibodies; Cold antibodies can sometimes mask more
clinically significant antibodies (IgG), an important reason to use
neutralization techniques
Proteolytic Enzymes
Enzymes are used to eliminate or enhance antibody activity
- Destroyed Antigens: Duffy and MNS
– Enhanced Antigens: Rh, Kidd, and Lewis, I, and P
Proteolytic Enzymes
Several enzymes exist:
Ficin (figs) Bromelin (pineapple) Papain (papaya)
Proteolytic Enzymes
Procedure Steps
One-stage test
Enzymes, RBCs, and serum are simultaneously incubated
Proteolytic Enzymes
Two-stage test
- Panel cells are pretreated with enzymes, incubated and washed
– Patient serum is added to panel cells and tested
Sulfhydryl Reagents cleaves
Cleave the disulfide bonds of IgM molecules and help
differentiate between IgM and IgG antibodies
Sulfhydryl Reagents
Good to use when you have both IgG and IgM
antibodies (warm/cold)
Dithiothreitol (DTT) is a thiol and will denature Kell antigens
2-mercaptoethanol (2-ME)
ZZAP
A combination of
proteolytic enzymes and DTT
ZZAP
Denatures
Kell, M, N, S, Duffy and other less frequent blood group antigens
ZZAP
Does not denature the
Kx antigen
ZZAP
Good for adsorption techniques
“frees” autoantibody off patient’s cell, so that autoantibody
can then be adsorbed onto another RBC
Antibodies to
High-Frequency
Antigens
High-frequency antigens occur
in the population at a
frequency of
98%
Antibodies to
High-Frequency
Antigens
Suspect an alloantibody to a
high-frequency antigen if most
panel cells are positive
Antibodies to
High-Frequency
Antigens
Additional testing may be
needed to confirm the
presence of the
antibody
enzymes to enhance or destroy the _________
DDT to destroy _________
*Enzymes to enhance or destroy
antigens
*Dithiothreitol (DTT) to destroy Kell
antigens
High-Titer, Low-Avidity Antibodies
react at
not implicated in _______
antibodies to ________
Antibodies to high-frequency
antigens that react weakly
React at AHG phase
Not implicated in transfusion
reactions or HDFN
Antibodies to
Low-Frequency Antigens
May occur alone or are suspected
when the screen is negative and
crossmatching is positive
Antibodies to
Low-Frequency Antigens
Only one reactive cell suggests
this type of antibody
Antibodies to
Low-Frequency Antigens
Common antibodies include
– Anti-Cw, anti-Wra, anti-V, anti-Cob, anti-Bga, anti-Kpa, and anti-Lua antibodies
