(M) Antibody detection testing Flashcards
Antibodies that are not expected for
the patient to have.
Unexpected/
irregular
antibodies
Type of antibodyes
In ABO, a patient who is type a is
expect to have anti-B
Expected
antibodies
Also known as clinically significant
antibodies. These antibodies are
produced in response to RBC
stimulation and when an individual is
exposed to the foreign RBC antigens.
Immune
alloantibodies
- Antibodies that are present without the
RBC stimulation but probably due to
exposure to environmental sources like
pollen or bacteria (structure similar to
some RBC antigens)
naturally occurring antibodies
- Antibodies that are pre-formed
already in another individual and are
passed onto the other individual. - E.g., Rhogam, which are Rh antibodies
or immunoglobulins that are injected to
a Rh negative mother who just gave
birth to a Rh positive baby. To be able
to neutralize the Rhd antigens present
on the mother’s circulation, these
passively acquired antibodies will now
coat the Rh positive fetal RBCs in the
maternal circulation, thereby
preventing the sensitization of the
mother.
passively acquired antibodies
- Antibodies that are directed against
antigens expressed on their own
RBCs.
autoantibodies
Significance of antibody detection
A critical in pretransfusion compatibility testing to test for _________________–
clinically significant unexpected antibodies
antibody detection testing is a tool for investigating which (3) diseases?
- hemolytic transfusion reaction
- immune hemolytic anemias
- HDFN
at what temperature and phase does clinically significant unexpected antibodies react at?
37C at the AHG testing phase
What is the red cell concentration needed for:
- tube method
- gel method
- 2-5%
- 0.8%
The reagents may come in 2 or 3 vial / cell suspension, ideally the antigen expression should be (homozygous / heterozygous) to enhance the detection of the antibodies
homozygous
Why are homozygous antigens preferred over heterozygous in antibody screening?
There are antibodies that have a dosage effect that heterozygous antigens may not be able to detect
What is the sample used for:
- tube method
- gel method
- solid phase adherence
- serum / plasma
- plasma
- serum / plasma (antibodies)
RBC (antigens)
whaat is the procedure used in the tube method of antibody screening?
IAT
what are the three phases of testing in the tube method?
- immediate spin phase
- 37C phase / thermal phase / protein phase
- AHG phase
What antibody does each phase detect?
a. IgG
b. IgM
c. both
d. neither
- immediate spin phase
- 37C phase
- AHG phase
- b
- a
- d
this phase utilizes potentiator
a. immediate spin phase
b. protein phase
c. both
d. neither
b. protein phase / thermal phase / 37C phase
What is done after getting a negative AHG test result?
check cells / coombs control cells are added
This comes with the purchased known cell reagent, determining which antigen is present in the vial
antigram / antigen sheet
T or F
the Rh phenotype is included in the antigram or antigen sheet
T
T or F
all antigrams may share LAT numbers, making it easier to remember
F (LAT-specific)
What indicates a positive result in antibody screening: tube method
agglutination in any of the tubes
I. When interpreting the result of an antibody screening, all tubes are interpreted separately.
II. Therefore if only one of the tubes, in all phases, has hemolysis or agglutination, it is interpreted as negative
a.only the first statement is true
b. only the second statement is true
c. both statements are true
d. both statements are false
d.
I. When interpreting the result of an antibody screening, all
tubes are interpreted as one.
II. Therefore, if any one of the
tubes, in any phases, has HEMOLYSIS or
AGGLUTINATION, it is interpreted as POSITIVE for
antibody screening.
Tube method
1. What is considered as a STRONG positive reaction in antibody screening?
2. What is the disadvantage of using the tube method in antibody screening?
- hemolysis
- It is subjective as reading may differ from one MT to another
this method of antibody screening is based on the principle of controlled centrifugation of RBCs
through a DEXTRAN-ACRYLAMIDE GEL which will
determine whether positive, with agglutination, or negative,
without agglutination.
gel method
Gel method of antibody screening
- ________ acts as a sieve which prevents agglutinated cells from passing through the gel.
- If the result is positive, the agglutinated cells are found on the _____ of the gel or column
- ______ or ______ of several microtubules allows performance of several tests simultaneously
- Dextran-acrylamide gel
- top
- strip or card
Gel method of antibody screening
- For how many days is storage allowed for this method of antibody screening?
- _____ are small tubules with a reaction chamber on top
- It is incubated for _____ to _____ minutes before centrifugation
- 3 days
- microtubes
- 10 to 15 minutes
Gel method reaction
a. positive
b. negative
- agglutination of the RBCs in the chamber, remaining at the top
- settlement of the red cells at the bottom of the tube
- a
- b
Please read! Gel method - AB screening
- Identify the patient sample.
- Dispense 50 uL of screening cells to microtubes labelled
as I, II, and III. - Dispense 25 uL of patient’s plasma or serum.
- Incubate at 37 ‘C for 15 minutes.
- Centrifuge for 9 minutes.
- Read the results. `
What are the advantages of antibody screening: tube method
- inexpensive
- easy
- readily available
What are the advantages of antibody screening: gel method
- no washing or tube shaking needed
- 98% sensitivity
- cards can be saved for up to three days
- simplifies cross training
- improves productivity and workflow efficiency
I. No washing is required since the reaction occurs in the reaction chamber and the AHG will not be neutralized since it is within the gell
II. even if unbound antibodies are present, it will not affect the AHG since it stays at the bottom of the tube
a.only the first statement is true
b. only the second statement is true
c. both statements are true
d. both statements are false
a
II. Even if unbound antibodies are present, it won’t affect
the AHG since it stays at the TOP of the tube.
Grading results in the gel method
- Non-agglutinated cells pass freely through gel and pellet at the (top/ bottom) of the microtubule
- Agglutinated cells are too large to enter the gel matrix and remain at the (top / bottom) of the column
- bottom
- top
Grading results in the gel method
All are correct except:
a. 4+ = solid band of red cells on top of the gel
b. agglutinated cells in the lower half of the gel column
c. 2+ red cell agglutinates through the entire length of the column
d. 1+ RBC agglutinates mainly in the lower half or lower 1/3 of the gel coloumn
e. all cells pellet at the bottom
b. agglutinated cellls in the UPPER half of the gel column
Grifols gel method / system
- ABO typing gel confirm card is used in this method and comes with ___ cards
- This gel card is good for ___ donors
- 8
- 2
In 1978, __________ and coworkers13 were the first to apply
the principle of solid-phase immunoassay to RBC typing &
Ab screening
Rosenfield
in this antibody screening method, RBC antigens or antibodies are embedded or immoblized in microplate wells to detect antibody-antigen reaction
- the only thing that is needed to do is to add the patient sample
solid phase adherence
Solid phase adherence sample
- If you’re detecting antibodies
- If you’re detecting antigens
- serum / plasma
- RBC
Please read on the procedure of the solid phase method
thanks
Solid Phase Adherence Result Interpretation
- well is coated fully with antibodies, and the indicator cell will now attach to the antibody
- there’s pellets found at the center of the tube
- Tube 1 is coated with antibodies while there’s pellet formation in tubes 2 and 3
- strong positive
- negative
- positive
Solid Phase Adherence
- what are we trying to detect?
- What is done after the identification of a positive result?
- clinically insigmnificant / irregular / unexpected antibodies in the px sample
- identification of the antibody detected
Antibody identification
- (clinically significant / insignificant / all) antibodies should be identified
- If antibodies are clinically significant and can cause HTR and HDFN, antigen (positive/negative) red cell components are selected
- patients with clinically significant antibodies should receive red cells that have been tested and found to (have / lack) the corresponding antigen
- all antibodies should be identified
- antigen negative red cell components are selected
- to lack the corresponding antigen
Antibody identification, antibody screening, and cross matching
follows the same principle of indirect antiglobulin testing, the only
difference lies in the __________
antigen source
What is the source of antigen for antibody screening and antibody identification?
comercially prepared reagent cells
- what is the procedure used in antibody identification
- what is the sample used?
- IAT
- serum or plasma
What are the information needed in the patient’s medical history for antibody identification?
- patient’s clinical diagnosis
- history of transfusions, pregnancies
- recent drug therapy
What are the reagents used in antbody identification?
- reagent red cells (comercially prepared)
- antiglobulin reagent
- enhancement reagent
Reagent red cells
1. panel cells are group __
2. panel cells expressed antigens are associated with clinically (significant / insignificant) antibodies
3. purchased and come with a __ to __ % red cell suspension in a preservative medium for tube method
- group O
- significant antibodies
- 2 to 5%
Reagent red cells
- Concentration of red cells for gel method?
- stored in the _________ when not in use
- should not be used beyond ______ weeks
- 0.8%
- refrigerator
- beyond 2 to 4 weeks
Antigram of reagent red cell panel
- There are ___ vials
- The antigens present on the red cells can be observed as a _____ sign in the antigram
- 10 vials
- plus sign
Interpretation of results
name the method wherein nonreactive / negative antibodies in all phases of testing are excleded
exclusion / cross out / rule-out method
Crucial in antibody identification
a. negative results
b. positive results
c. both
d. neither
c
Interpretation of results
- Examine ________ of reactivity (grade of the reaction), there might be presence of mixed antibodies and not only one
- Examine _______ of reactivity, as it can guide us to tell whether we are dealing with IgG or IgM
- Examine ________ of reactivity, there should be uniformity of reaction
- strength
- phase
- pattern
I. Antibodies exhibiting dosage effect are also excluded in the exclusion method
II. Common blood group systems with antibodies that exhibit dosage include Rh, Kidd, Duffy, MNSs, and Lutheran
a. only the first statement is true
b. only the second statement is true
c. both statements are true
d. neither statements are true
b
Checking of antibodies that exhibit dosage effect is also
included in exclusion method.
o You cannot outright exclude them; you have to verify if
the antigens present are expressed in a homozygous
or heterozygous state.
Identify the antibody:
- reacts at room temp and the immediate spin phase
- clinically significant
- Reacts at 37C and the AHG phases of testing
- IgM
- IgG
- IgG
Homozygous
a. M+N+
b. Fy (a+b+)
c. both
d. neither
d. neither (they are the onlyl heterozygous phenotypes on the given chart)
Reacts at 37C
a. Fy
b. Le
c. both
d. neither
b. le
Study the interpretation of the red cell panel chart
di siya effective i-cards beh go na aralin mo na
may have Ig G and IgM complement
a. Lewis
b. M
c. both
d. neither
c
Familiarize yourself with the serologic clues
- Reactivity in tests at room temperature suggests:
o anti-H, -I, -P1, -P, -PP1Pk (-Tja) - Rh, Duffy, MNS, and Kidd systems most commonly
demonstrate dosage
o Make sure to not cross them outright if the result is
negative - Lysis of reagent red cells when testing with fresh serum
is characteristic of anti-P, -PP1Pk, and -Jk3.
o It is also seen with some examples of anti-H and -I
o When checked and there is presence of hemolysis then
most likely these are the present antibodies.
4 . Weak nebulous reactions in the antiglobulin phase are
often associated with anti-Kna, -McCa, -Yka, and -Csa. - Antibodies such as anti-U, -McCa, -Sla, -Jsb, -Hy, -Joa, -Tca, -Cra, and -Ata should be considered if the serum
is from a black individual because the antigen-negative
phenotypes occur almost exclusively in blacks.
2 main functions of crossmatching
- final check of ___ compatibility between donor and recipient
- Detect the presence of an _______ present in the recipitent directed against an _________ on the donor RBC
- ABO
- antibody, antigen
Familiarize the 4 types of crossmatching
- serological crossmatch
- immediate spin crossmatch
- antiglobulin crossmatch
- computer crossmatch
two types of serological crossmatching
a. major
b. minor
- Patient Cell (PC) + Donor Serum (DS)
- Patient Serum (PS) + Donor cell (DC)
- obsolete, not performed anymore
- b. minor
- a. major
- b. minor
Important in computer crossmatch:
to encode the _____ and ______ of both donor and patient
antigen and phenotype
When is the only time that computer crossmatch is performed?
if antibody screening is negative
What is the limitation of crossmatching
there is no current testing procedure
T or F
a compatible crossmatch and a negative antibody screening can guarantee that the transfused RBCs will survive normally in the patient
F ( cannot guarantee)
Identify the following:
- means that there are no detectable antibodies in the patient’s sample directed against antigens present on these particular screen cells
- means that there are no
detectable antibodies in the patient’s sample directed
against antigens present on this particular donor cells.
- negative antibody screen
- compatible crossmatch
Majority of the results in antibody screening and crossmatching
negative screen, compatible crossmatch
antibody screen and cross match result that may have been caused by:
○ Donor RBCs are incompatible with patient’s blood type,
retyping the donor is done.
○ Subgroups: Anti-A, in serum of A2 or A2B patients.
○ Other alloantibodies reactive at RT (IgMs)
○ Cold autoantibodies
○ Rouleaux formation
* Most common cause is DONOR UNIT HAS POSITIVE DAT
ON DONOR UNIT.
Negative screen, incompatible crossmatch
If donor unit has a positive DAT, crossmatching result will always be positive. If the blood is DAT positive, it may be transfused to all patients.
a. first statement is true
b. second statement is true
c. all statements are true
d. neither statements is true
a.
if blood is DAT positive, it must be returned to the blood provider or discarded
All of the following are true regarding rouleaux formation except
a. property of serum that causes all cells tested to appear agglutinated at RT and 37 C
b. stacked coin appearance of RBCs
c.saline replacement is done to confirm for the presence of roleaux
d. none of the above
d
If agglutination remains after saline replacement is it due to Rouleaux formation or is it a true agglutination?
TRUE agglutination
What is the cause of a positive screen and incompatible crossmatch
presence of alloantibody
What is the cause of negative autocontrol and negative DAT?
presence of alloantibody
the following are true for the resolution of negative autocontrol and negative EXCEPT
a. proceed with antigen identification
b. perform panel study
c. look for antigen-negative units to be served to the patient
d. identify all possible antibodies present in the sample
a. proceed with ANTIBODY identification
fill in the blanks
- If autocontrol is positive, obtain_________
- If the presence
of alloantibodies and the patient’s antibodies are
coating the red cells, there will be _____________
reaction since the present red cells would be the
patient’s cells and donor’s cells coated with
antibodies. - If both allantibodies and autoantibodies are present: perform ________ to remove bound antibodies from cells and identify specificity
- paitent history
- mixed field reaction
- elution procedure
causes of Positive autocontrol (AC) and Negative DAT
- antibody to ingredient ofr component in enhancement media
- roleaux formation
the following are true in the resolution of of Positive autocontrol (AC) and Negative DAT except
a. get another batch / lot number of that particular reagent
b. due to drugs or additives in reagents
c. repeat the test with the same lot number of enhancement media
d. repeat the test but use a different method such as an enzyme technique or saline test
c. repeat the test with a different lot number of enhancement media
Causes of of Positive autocontrol (AC) and Positive DAT
✓ Alloantibody causing delayed HTR
✓ Passively acquired autoantibody (e.g.,
intravenous immune globulin).
✓ Cold- or warm-reactive autoantibody.
✓ Rouleaux formation.
Roleaux formation
a. Positive autocontrol and Positive DAT
b. Positive autocontrol and Negative DAT
c. both
d. neither
c
the following are true in the resolution of of Positive autocontrol (AC) and Positive DAT except
a. remove autoantibodies useing adsorption and elution
b. incubate all of the materials
c. warm auto-absorption is necessary
d. all of the above
c. cold auto-absorption is necessary
Antibody screen and crossmatch result indicated by:
- Presence of an alloantibody directed against antigen in
screen 1 BUT is not present in donor - Antibody dependent on reagent red cell diluent
- Antibody reacting with homozygous screen cells, donor
1 is heterozygous or negative.
positive screen, compatible crossmatch
What should be done in cases of positive screen, compatible crossmatch
- perform an antibody identification panel and test the donors for antigen
- crossmatch units negative for antigen
familiarize the causes of positive screen and compatible crossmatch
- Autoanti-H (autoanti-H) or anti-Leb and nongroup O units are
selected. - Antibodies dependent on reagent red cell diluent
- Antibodies demonstrating dosage and donor red cells are
from heterozygotes (i.e., expressing a single dose of antigen) - Donor unit is lacking corresponding antigen that is why the
screening test is negative
Miscellaneous tests
- Removing antibody free from the serum or plasma by _________ to red cells carrying the corresponding antigen
- You introduce a red cell with a ________ antigen attached to the red cell or carrying that corresponding antigen.
- adsorption
- positive
Familiarize the uses of adsorption
- Separating multiple antibodies present in a single serum.
- Removing autoantibody activity to permit detection of coexisting alloantibodies.
- Removing unwanted antibody (often anti-A and/or anti-B) from serum that contains an antibody suitable for reagent use.
- Confirming the presence of specific antigens
- Confirming the specificity of an antibody
Miscellaneous tests that ees antibody molecules already ATTACHED to
sensitized red cells. You do not want the autoantibodies
present so you have to do elution techniques.
elution
______ or ______ solvent methods are used for elution of warm-reactive auto- and alloantibodies
acid or organic solvent methods
elution is done by
- changing the ______ of Ag-ab reactions
- _______ or _______ forces of attraction that hold Ag-Ab complexes togerher
- ________-__ the structure of the Ag-Ab binding site
- thermodynamics
- neutralizing or reversing
- Disturbing
Factors affecting elution technique
- Incorrect techniques
- Incomplete washing
- Binding of proteins to glass surfaces
- Dissociation of antibody before elution
Uses of elution
- Investigation of positive DAT
- Concentration and purification of antibodies, detection of
weakly expressed antigens and identification of multiple
antibody specificities - Preparation of antibody free red cells for phenotyping
studies
familiarize the elultion methods (3)
- temperature dependent elution mehtods
- pH mehtod
- organic solvent method
Elution method
- The easiest procedure among the elution techniques
- Change the temperature of the antigen-antibody
environment by incubating the sample at 56°C for heat
method.
o By that, the attached antibody will dissociate or will be
free from our supernatant. Do the heat method if
you’re dealing with cold antibodies.
o If you’re dealing with warm antibodies, decrease to
18°C or colder (Lui freeze-thaw method).
o If you do this, you CANNOT use your red cells
anymore because the antigens present on the red
cells will be destroyed. All that’s left will be the
antibodies present on the system.
Temperature-dependent methods
Elution method
- If you want to elute (remove the bounded antibodies), you
have to wash the antibody-coated cells and then
mix/subject it with a glycine acid solution at a pH of 3.
o In this way, you change the thermodynamic as well and
then you free the antibodies from its attachment to the
antigens on the red cells.
pH method
What should be the pH of your test system for pH mehtod of elution
neutral: 7.2 to 7.4
Elution method
- With the use of dichloromethane, xylene, and ether.
- Just add these reagents and the antibodies are released now
from its attachment to the antigen. - This is not commonly used due to its toxicity
organic solvent methods
BGS that are enhanced by enzyme treatment
Rh, Kidd, Lewis, P1, I
BGS that are inactivated by enzyme treatment
Duffy, MNS, Xga
This quantifies the antibody that is present or determines Ab concentration levels
antibody titer
methods to get the antibody titer
- flow cytometry
- RIA
- EIA
the highest dilution with reaction or with greater than or equal to 1+ agglutination
titer
Provide the scores of each agglutination grade
○ 4+ =
○ 3+ =
○ 2+ =
○ 1+ =
○ 4+ = 12
○ 3+ = 10
○ 2+ = 8
○ 1+ = 5
To interpret if the titer is significant, get the previous titer
and compare it with the current titer. It is a SIGNIFICANT
VALUE if there is:
- Fourfold or greater increase in titer from the previous
titer - Even if it did not increase 2 tubes more, but the score
has increased to 10 or more, it is also SIGNIFICANT
- Once antibodies are identified and the patient has multiple
antibodies, we need to figure out how many units of blood
need to be cross matched to find a compatible match. - Obtained by multiplying the frequencies of antigen
negative
percentage of compatible blood
