BB - Day 1 Flashcards
Washing cells is important before using this particular reagent. What does the washing step remove?
Antihuman globulin (AHG) reagent
Unbound IgG and other free globulin molecules/proteins that create false reactions
-AHG binds to bound IgG, causes agglutination
What does the DAT test demonstrate? What reagent is used to check for this?
In-vivo coating of RBCs with globulins (IgG and/or C3)
AHG - binds to IgG or C3, which are bound to RBCs, causing agglutination
-detects in vivo hemolysis
Why is AHG needed to agglutinate RBCs?
IgG and C3 are so small, they cannot agglutinate RBCs by themselves
-AHG binds to IgG or C3, which are bound to RBCs
When check cells are added to a negative tube, assuming the tube really is negative, what kind of reaction grade should we expect? What happens if this does not happen?
2+ agglutination = true negative (1+ for DAT)
Repeat test
True/False - both negative and positive reactions should be tested with check cells
False
- only negative reactions should be tested
- positive reaction means washing step and AHG binding worked, so no need to waste reagent
DAT positive and DAT negative test results - what is the agglutination result and check cell result?
DAT positive = agglutination in test tube, no check cell needed
DAT negative = no agglutination in test tube, check cells agglutination
Incompatible ABO donor blood type mixing with patient blood causes what kind of hemolysis?
Intravascular hemolysis
- ABO are IgM antibodies
- triggers complement cascade
An Rh-positive cell has this antigen on its surface…
D antigen
Anti-D made in an Rh negative mother can cause HDFN in an Rh ___ child
Positive
Difference between forward type and reverse type. Which test is the confirmatory test?
Forward type looks for A, B, or AB antigens on patient’s RBCs
Reverse type looks for anti-A, anti-B, or anti-A,B antibodies in patient’s serum
-aka “backtype”
Reverse type is the confirmation of the forward type
Which antibodies are “naturally-occurring” antibodies? What immunoglobulin class(es) are they predominantly?
anti-A and anti-B = predominantly IgM
- called saline reactive antibodies because they do not need a high protein medium to react
- react at room temperature or lower
anti-A,B = predominantly IgG
Which of the following antibodies are saline reactive and which are protein reactive: anti-D, anti-A, anti-B, anti-A,B. Which are IgM and which are IgG
anti-A, anti-B, anti-A,B = saline-reactive
anti-D = protein-reactive
anti-A, anti-B = IgM
anti-A,B, anti-D = IgG
The weak D test is performed on tubes with these results…
Tubes that are negative for anti-D and Rh control
-some cells express D antigen so weakly they do not agglutinate with most anti-D reagents
What is done in a weak D test to enhance agglutination?
Incubation (15 mins at 37C)
Followed by addition of AHG
Weak D is an example of a direct or indirect agglutination test?
Indirect - cells sensitized in vitro at 37C with antiserum before antiglobulin testing
Which class of antibodies can across the placenta? Which region of the antibody is required to aid in the transport?
IgG
Fc
Most clinically significant class of antibodies
IgG
-some IgM also clinically significant (ABO), but mostly it’s IgG
Pentameric IgM are joined together by the…
J chain
- covalent bonds can be cleaved to detect IgG
- IgM removed with beta-mercaptoethanol or DTT
This blood group system makes antigens in tissue that eventually find their way onto RBCs. Is the blood group system clinically significant?
Lewis blood group system
No
Binding of RBC antibodies to complement causes…
Intravascular hemolysis
- caused by IgG antibodies
- exception: paroxysmal cold hemoglobinuria (PCH) caused by IgM
Extravascular hemolysis happens in the… Due to…
Spleen, liver
RBCs coated with antibodies are removed by macrophages
Which red blood cell antigen is the most immunogenic?
D antigen
-70% of D-negative people will be sensitized if exposed to D antigen
Kell blood group system - is it clinically significant? Which antigen is the most immunogenic after the D antigen?
Yes
-IgG
K
If you naturally make an RBC antigen, do you also make the antibody against it?
No
-autoimmunity if this happens
This blood group system is notorious for falling below detectable limits. Is it clinically significant?
Kidd
-IgG
Yes
The most important step before starting any new test is to know…
Patient’s history
DAT vs IAT - which is in vitro and which is in vivo? Both tests use this reagent to bind to antibodies to help RBCs agglutinate…
DAT = in vivo
-detects antibodies bound to RBCs
IAT = in vitro
- HDFN, weak D
- take patient’s serum and add test RBCs
AHG = Coombs reagents
Factors influencing agglutination reactions (6)
22% albumin
LISS
PeG
Proteolytic enzymes – ficin (fig), papain (papaya), trypsin (pig stomach), bromelin (pineapple)
Chemical reduction – DTT, B-2-ME – thiol reducing agents
ZZAP – thiol reducing + proteolytic enzyme – causes dissociation of IgG molecule from the RBC surface and alters the surface antigen
Monospecific AHG detects… Polyspecific AHG detects…
IgG
IgG and complement (C3)
Monospecific AHG targets this particular region of the antibody…
Fc heavy chain region
A DAT is performed when we suspect a patient is having ___. Two cases when we would perform a DAT…
Hemolysis
-due to antibodies binding to RBCs, causing intra/extravascular hemolysis
Transfusion reaction, newborns (HDFN)
What are check cells
Sensitized cells that will agglutinate unbound AHG remaining in a negative test tube
- AHG did not/could not bind to Fc of antibody on test RBC, so free in solution to bind to check cells
- only use for negative tubes
Rouleaux formation in the blood usually indicates too much… How do we get rid of it?
Protein (ie multiple myeloma)
More washes
If DAT is positive, what is the auto-control result?
Positive
-remember that DAT checks for hemolysis. Auto-control uses patient’s RBCs plus serum containing the antibodies that target antigens on these RBCs
When grading RBC agglutination, the point at which the background looks turbid with tiny agglutinates is graded as…
1+
At 2+, the background is clear
