Week 5 - Antibody detection Flashcards
(34 cards)
CONDITIONS THAT AFFECT ABO/RH TESTING
Antigen Problems:
1) Genetic Mutations resulting in abnormal antigens (Weak D)
2) Disease states resulting in changes to antigens (abnormal or lack the antigen)
3) Antisera that cross-react with other antigens
-affects lab testing (CAD, warm AUAB)
-can affect pt - if they dont have a blood group or AB it can delay getting blood ready them
-in emergency situation you can give uncross matched blood
CONDITIONS THAT AFFECT ABO/RH TESTING
Polyagglutination:
-red cells are agglutinated by all human sera.
-due to exposure to a normal “hidden antigen” or a red cell antigen mutation.
-If you test these cells with human antisera, they will show agglutination regardless of the antibody specificity
-Patients will be grouped as A and B Rh-positive even if they do NOT have those antigens
-Acquired T activation is caused by a bacterial or viral infection (seen in children diagnosed with Streptococcus Pneumoniae)
What are ALLO ANTIBODIES (NON SELF ANTIGENS)
- Produced as a normal immune response
- Patient’s history of previous transfusions and pregnancies is essential in antibody investigations.
- Patient diagnosis is vital information (Sickle cell patients are more likely to produce Antibodies).
What are AUTO ANTIBODIES
-formed when body doesnt recognize self
–These antibodies react with almost all human red cells; they are usually non-specific or specific to a very high incidence antigen (Anti-e).
-Cold Autoantibodies –IgM and react at RT or 4˚C
-Warm Autoantibodies- IgG and react at body temperature
-Autoantibody can be detected in the patient’s plasma and attaches to the patient’s own red cells
-Antibodies attached to the red cells in vivo can be detected with the Direct Antiglobulin Test (DAT)
-difference between DAT and IAT is the incubation period, IAT needs the incubation to join AB to AG
ANTIBODY DETECTION
-detects antibodies in the patient’s plasma- Reverse grouping , they are naturally occurring (RA/RB reagents)
-if positive then AB are present
-Rh antibodies are Red Cell Immune
-OBG antibodies are Red Cell Immune and depend on the production and stimulation of IgG or IgM forms
-Rh and OBG antibodies are not expected to be present in the patient’s plasma if they are then its from exposure to RBC from transfusion or pregnancy
STIMULATION PROCESS
-first exposure, you may or may not develop an antibody
1-Antigens on Red Cells are introduced to the Recipient.
2-Patient Lacks the Antigen. The immune System recognizes
antigens as Foreign.
3-Antibody Production
IgM or IgG
4-Memory Cell, Secondary Response, and Antibody levels react faster.
5- Significant Antibodies destroy red cells.
Antibody production depends on:
- Immunogenicity of the antigen: The ability of the antigen to elicit an
immune response or to stimulate the production of antibodies. - Rh and Kell are most immunogenic
- Immune system of the individual
- Protocol is to test all patients who have the potential to be transfused and pregnant
ABO AND RH TESTING
All pts are tested with
1) Anti-A
2) Anti-B
3) Anti-A,B (Michener)
4) Anti-D1/D2
5) Reverse ABO performed on all patients > 4 months of age
6) Weak D performed on Rh-negative babies with Rh-negative moms, and donor testing (at CBS)
7) Rh Control not routinely done (Discrepancy on D1/D2, AB Rh positive patients, and Weak D testing)
8) In the Hospital, no reverse testing and only confirmation testing is performed on donor units
- Rh Control added to all patients who initially type as AB Rh positive
- Need to rule out spontaneously agglutinating cells
TESTS TO DETECT ANTIBODIES
***
all testing is based on AB-AG reactions
-reaction is either agglutination or sensitization
-one is known and other is unknown
SCREENING CELLS
-commercially prepped
-2 or 3 cell suspensions
-each row is a different donor
-Group O cells are chosen to prevent a reaction with the patient’s Anti-A,
Anti-B, and Anti-A,B (ABO antibodies).
-each cell is phenotyped with results on the antigram
-+/0 is the presence or absence of AG
-needs pos RBC for all blood type
-should be able to detect all ABs
-homo is prefered because it is the strongest AG expression to detect weak ABs for dosage
- (Duffy/MNSs) Destroyed by enzymes
-known part of reaction is AG in panels
-lot number and exp date are specific to the set of cells used
-antigrams give you an AG profile
SCREENING CELLS
phenotype for cell 1 and 2
11.Rh Phenotype Cell 1 is always R1R1 and cell 2 is always R2R2.
12. This provides a homozygous cell for each Rh antigen (C, E, c, and e).
13.Antibodies to C, E and c are common.
SCREENING CELLS
what do we prefer for OBG AG
For OBG Antigens we prefer to have Homozygous expression, but this is
not always possible.
What happens if person for AB screen has weak Anti-Jkb
-may not detect
-example of dosage in which the heterozygous Jk(a+b+) cell has too few Jkb antigens to demonstrate agglutination.
SCREENING TESTS
Three methods for Antibody Screen:
1)Tube Method - (iat) - detects cold AB
2)Micro Typing System or MTS (Gel column method) -no washing or CCC
3)Solid Phase Red Cell Adherence (Capture R)
donor cell is tested with the patient’s plasma - unknown Abs
All 3 use the same concept of screening cells, but the red cell suspensions differ.
Antigrams are used in all 3 methods.
The purpose of ALL 3 methods is to pick up any significant antibodies in the patient’s plasma
SCREEN RESULTS
-No guarantee that the antibody is NOT present
-AB can be weak and can be undetected
-Weak Antibodies mean fewer IgG Once exposed a second time, the immune system is stimulated and produces more antibodies.
-This is the one LIMITATION in pretransfusion testing.
-May not be able to detect antibodies unless restimulated after being transfused.
-different methods detect different AB
-Tube is IS = IgM is detected
-MTS and Capture R have AHG phase, warm and IgG AB (can miss cold AB like M and N clinically insignificant)
-screening wont pick up rare AB
What antibody can fall below detectable levels?
-Kidd in delayed transfusion reactions
-they fall below detectable levels and are restimulated with transfusion of packed cells where the AB will be present in high titers
POSITIVE SCREEN RESULTS
-Identify all AB detected, whether they are significant or not because one could mask another
-Test plasma with Panel cells to identify the antibody
-Screening cells only have 2-3 cells, while the panel cells can have 10-11 cells.
-Positive cells (agglutination) can help identify a pattern of the antibody(
MTS method is known as
Gel technology.
-uses Dextran acrylamide gel with LISS to trap agglutinated red cells; the gel acts as a medium to separate the agglutinated red cells from those that did not agglutinate
-uses 0.8 suspension of RBC for the AB-AG ratio
-used for pt plasma with kown red donor cells in a panel with 2 cells on a screen and 11cells on a panel
-less variation than tube
-gel particles get combined with diluent/reagents
-NO washing , adding CCC/Rh
-good for identifying IgG immunoglobulins, but it may miss cold-reacting antibodies.
Used for ABO/Rh, Screening, Antibody detections, and even phenotyping.
SCREENING TESTS
MTS Method
-6 microtubes.
- in upper chamber where you add the cells/plasma/antisera that is where sensitization occurs during incubation
-red cells added first to wide part, be super to pipette on side of wall
-the cells that fall through the gel get coated with AB and react with AHG in the column
-unagglutinated red cells settle to the bottom (negative) and agglutinated red cells are trapped in the upper portion (pos)
add cells, plasma, incubate and spin
gel cards can be reused, so remove enough foil for what you need
What causes mixed field
-strong cold antibody that starts to agglutinate immediately. Cells agglutinate at the top of the column where the temperature is coldest, while the rest of the red cells fall to the bottom (E.g., Anti -I).
-cells added started falling before plasma was added.
-Could be fibrin in plasma binding to
RBC at the top (older samples).
SCREENING METHOD
Capture R Testing
-still AB-AG reaction
-only for IgG AB
-Red cells (target antigen) are bound to the bottom of the microplate wells.
- Protein is bound irreversibly to polystyrene in microplate wells.
-Red cells are lysed, antigens are bound
-Plates come preloaded with RBC antigens
-2 or 3 cells, different LOT# with different antigrams.
-Add liss
-add pt serum with bound RBC AG
-incubate at 37
-plate is washed to removed excess immunoglobulin
-add indicator cell - Anti-IgG bound by FC portion (helps to visualize)
-centrifuge
What is a positive and negative reaction of Capture R
Positive Reaction: The patient’s IgG reacts with the antigen on the wells. Indicator cells stick to platebound IgG, and the wells are ‘coated’ with indicator cells.
-Negative Reaction: No antibody, No IgG on antigens. Indicator cells fail to adhere to well. Cells form a
button after centrifugation.
Used for antibody screening, antibody ID, ABO testing, compatibility testing, and even phenotyping.
What would the results of a typical panel in capture R look like
First is the screen (3-cell screen), which always runs a positive control.
The panel includes 14 cells and a positive and negative control.
Controls are important because they indicate that the plates are coated properly with the antigens
When are enzyme panels used
for exclusion purposes if the ag is not destroyed by the enzyme
DONT use enzyme panels to rule out any of the DUFFY or MNSs ab
