Unit 2 Flashcards
What is antithetical
antigens that are products of pairs of genes like Fya and Fyb
^a and ^b indicate _____ expect for in the ____ system
antithetical, lewis system
What is a type and screen
type- test for ABO and Rh phenotypes
screen- test to find if there are any notable antibodies present in plasma
What is front type vs reverse type
front- finding what blood type you are
reverse-finding what blood type you are not
What is in the Front type test mixture
reagents anti A, B and D + your antibodies
if there is a reaction ex with anti A, you are type A blood
What is in the mixture for reverse type testing
reagents A and B antibodies + your plasma
if there is a reaction, this is what antibodies you’re body has, revealing what blood type you are not
if reagent B reacts, you must be blood type A
What is a crossmatch
test to see if blood it compatible with patient, mixing donor blood with patient plasma, we want this to be negative, so that the blood can be considered compatible
What is the difference between IS and Ext crossmatching
IS- directly add donor to patient cells after spinning
Ext- extended, add patient+donor plasma, incubate for 15 min, add AHG, centrifuge and read rxn, lastly use check cell if -
What type of antibodies are in AHG?
antihuman IgM antibodies
What is Anti-Sera
monoclonal or polyclonal manufactured antibodies
What are reagent cells
manufactured human cells with known antigens
Describe what a + rxn, - rxn and cell button look like
+ agglutination or hemolysis
- no agglutination or hemolysis
cell button- clump of red cells after centrifugation
What is FFP pRBCs PLTs CRYO LK IRR
FFP- fresh frozen plasma- has clotting factors
pRBCs - packed RBCs
PLTs- platelets suspended in a little plasma
CRYO- concentration of coag F VIII, fibrinogen, F XIII and VW factor
LK- leukoreduced
IRR- irradiated
Why would we need to LK blood?
leukoreduce- to prevent donor from mounting immune response against recipient. Can’t introduce donor WBCs into patient, or the donor WBCs will attack everything as foreign
Describe what a negative test in a tube looks like
Initially there is a pellet, but when spun slightly it dissipates back into a homogenous solution
Describe what a positive result looks like in a test tube
Starts with a pellet that does not fully dissipate, instead has clumps and never fully becomes a homogenous solution again
What are the 4 basic types of reagents
commercial RBCs with known antigens
Antisera with monoclonal or polyclonal antibodies
antiglobulin- with anti human Anti-IgG or anti-complement
antibody enhancers
What are polyclonal antibodies
antibodies that can recognize multiple epitopes
AHG- could be anti IgG and IgM
What are polyspecific reagents
AHG that has anti IgG and complement C3b
Anti-A, Anti-B and Anti-IgG are examples of what type of antibodies
AHG monoclonal antibodies
Monoclonal antibodies are secreted by:
a single clone of antibody producing B cells
Polyclonal antibodies are secreted by:
several different clones of antibody producing B cells
What reagents are used for ABO typing
anti-A and anti-B antisera reagents monoclonal
What colors does the ABO typing reagent have
anti A- Blue
Anti B- Yellow
anti AB- clear
What is in the polyclonal Anti D blend
human IgM anti-D
polyclonal IgG anti-D
Explain the D antigen typing procedure
anti-D mixed with patient and donor RBCs
if agglutination occurs, the D antigen is present
if it does not, there is no D antigen
a negative control is done to make sure it is not a false positive result
If a monoclonal control for anti-D testing control is _____ the test is invalid
positive,
it should give a negative result
What is in low protein anti D reagents
monoclonal ANTI D antibodies (IgM) or polyclonal
6% bovine albumin
replaced high proteins reagents
What blood type result requires a control run
AB+
What is low protein reagent used for
control, should show no agglutination,
protein is in mixture to detect the positive reaction from protein abnormalities rather than RBC antigens
If RBCs agglutinates with all ABO antisera, then a separate control must be done with what reagent and what test result for a forward blood type, assuming the patient is truly AB+
negative-low protein reagent
If positive- something else is causing the + rxn
Practice writing out the results to the following table
anti A Anti B Anti D Rh control
A+
A-
B+
O-
AB+
AB-answer Ch4 slide 26
What testing is done with A1 and B cells
reverse typing- find what you are not
Practice filing out the following table for reverse typing
reagent A1 B O+ A- AB+ B+ A+ AB- O- O+
answers Ch4 slide 30-31
What if forward typing results don’t match the reverse typing?
the issue is probably the reverse
Are A1 and B cell reagents also positive for Rh antigens?
no, they are negative for Rh to keep specificity
What concentration are screening cells usually in
2-5% concentration
What are screening cells for
antibody screening, for detection of unexpected abs, patient serum is added to see if agglutination occurs, result should be negative, comes with an antigram that shows the antigen profile
sets of 2-3 vials
What are panel cells
reagent cells in 10+ vials, used to ID abs in an antibody panel.
What do antiglobulin test reagents contain
anti IgG
and/ or anti-C3b complement
What is a direct antiglobulin test?
DAT- detects IgG or complement bound to RBCs in vivo
this is post transfusion to see if a bad transfusion occurred
What test is this?
RBCs washed, patient sample+ anti-IgG= agglutination
DAT
What does a positive DAT mean?
that there is sensitization occurring in a live person
What reasons are there for a DAT to be positive
transfusion rxn
hemolytic disease of the fetus and newborn
autoimmune hemolytic anemia
drug related rxn
If a DAT test is positive due to the donor cells being coated with IgG
the patient had a incompatible transfusion
If a DAT is positive because fetal RBCs are coated with IgG the explanation is
maternal antibodies have cross the placenta
What is an Indirect antiglobulin test
IAT- detects IgG or complement bound to RBCs in vitro
What type of test is this
IgG patient sample
antibodies incubated at 37C with RBC reagents
RBC washed then combined with AHG
IAT
What is IAT testing used for
to find the presence of and identify antibodies
antibody screening
crossmatch
antigen typing
What are the sources of false positives in IAT testing
red cells agglutinated before washing,
dirty glassware
over centrifugation
If during an IAT test you do not wash the cells correctly before adding AHG reagent
you could have a false negative because unbound human serum globulins have neutralized the AHG reagent
During an IAT test if you forget to add the reagent,
you would get a false negative
When do you need to use check cells or indicator cells
if AHG results are negative
control added to a negative test should cause agglutination
What are check cells made of
O+ RBCs coated with IgG antibodies
What might cause a false negative in check cell control testing
no AHG reagent added
reagent not reactive
did not wash RBCs correctly
What does LISS do
it is an antibody potentiator, increases rate of antibody uptake, enhances agglutination
What does PEG do
polyethlene glycol
concentrates the antibody in the test environment in LISS
What do proteolytic enzymes do
speed up agglutination by removing negative charges from RBC membrane, reducing zeta potential
What does bovine serum albumun do?
reduces repulsion between cells, does not shorten incubation time
What concentration does bovine serum albumin remain in
22-30%
What are lectins
seed extracts that are specific towards RBC antigens
What is CAT
column agglutination technology, a test where a dextran acrylamide gel has predispensed antigens.
What temperature should CAT testing be stored at
stored in fridge, has to be room remp before testing
What concentration do CAT tests have?
0.8-1% cell suspension- diluted
What blood type is on slide 50 ch 4
O-
What CAT test result is the following description
red cell agglutinates are dispensed throughout the length of the gel
2+
What CAT test results are described
majority of the red cell agglutinates are trapped in the upper half of the gel
3+
What are the disadvantages to CAT
need special equipment, cant use hemolyzed or lipemic samples. higher risk of rouleaux
what is rouleaux?
stacked coin appearance on RBCs could be due to a disease present
What are the advantages of CAT
no wash step
more specific and sensitive
standardization
What is solid phase Red cell adherence
a test that indicates if a reaction occurs, it there is a cell button- negative
if no button- +
Explain the procedure for solid phase red cell adherence
well has antigen on sides already embedded add patient plasma and LISS incubate at 37 wash well with saline add antiG coated RBCs centrifuge
If a solid phase test shows cells adhered to the sides and bottom of well and there is no button this is a
positive
If a solid phase test shows blood settling to the bottom of well and create a cell button, this is a
negative result
Practice labelling slide 55 Ch4
center image
What are the advantages of solid phase testing
well defined endpoints, longer shelf life
What are the disadvantages of solid phase testing
too much could be added making it unreadable
need special equipment
How are monoclonal reagents made
spleen lymphocytes from immunized mice that are fused with myeloma cells
A reagent that makes a lot of one type of antibody that reacts with one specific epitope is a
monoclonal reagent
How are polyclonal reagents made
injecting animals with human globulin components then collecting the resulting antihuman antibodies
A reagent that is directed against multiple epitopes is
polyclonal
List the test procedure for DAT
EDTA tube collected
3-5% RBC suspension mixed with 0.9% saline
1 drop of suspension washed 3 times with saline
add 2 drops of AHG readent
centrifuge and read
if negative rxn, add indicator cells
What temperature and what specific antibody is immediate spin IAT testing looking for? and extended ?
cold-IgM
warm-IgG
What is the procedure for IAT testing
looks for IgM 2 drops of serum or plasma added to 1 RBC reagent drop read at initial spin ------------------- Looks for IgG 2 drops of LISS incubate at 37C for 15 minutes centrifuge and read wash 3 times with buffered saline add 2 drops of AHG reagent centrifuge and read if negative, add check cells-
What is the purpose of washing the RBCs?
washing removes any unbound abs and the AHG reagent will make agglutination visible
What are the possible reasons a DAT could show a false negative
inadequate washing
undercentrifugation
loss of reagent reactivity
In a coombs control, what should the reaction result be if it is a valid test when the check cells are added
there should be a reaction
check cells are O+ with Anti-D
all blood types will react with this
if no reaction, something else is causing agglutination
Which IAT test does not require check cells
gel testing
