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Rh Typing reagents for Anti -D
- saline (low protein)
- high protein
- low protein
- monoclonal antibodies
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saline anti-D reagent
- IgM
- low protein based
- used to test for cells already coated with IgG antibody
- disadvantage: cost, long incubation time, cannot test for weak D
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high protein anti-D reagent
- faster
- can weak D test
- disadvantages: requires control tube, increased false positives
- if control is positive, test is invalid
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monoclonal anti-D reagent
- consists of a combination of various monoclonal anti-D reagents from different clones in order to ensure reactivity with a broad spectrum of Rh positive RBCs
- can be used for slide, tube, microwell and automated testing
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Gel technology
- pipette tip should not go past the gel
- 0.8% cell suspension for forward typing
- gel particles are composed of beads of dextran acrylamide
- advantage: more sensitive and more standardized than tubing
- disadvantage: cannot test hemolyzed samples. lipemic samples will clog the gel. Rouleux caused by high protein conct in serum with patients with multiple myeloma or waldenstrom's macroglobinemia
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why is there a mixed field in gel technology
- recently transfused
- bone marrow transplant
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Anti A reagent
- monoclonal antibody
- IgM
- blue
- Anti -A1 lectin: dolichos bilforus
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anti B reagent
- monoclonal antibody
- IgM
- yellow
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RBC reagents for AB screen
- group O
- cells are suspended in 2 - 5% diluent
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Enhancement/Potentiators Reagents
- 22% albumin
- LISS (low ionic strength solution)
- PEG (polyethylene Glycol
- AHG
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22% albumin
- reduces zeta potential allowing RBCs to approach each other and increase chances of agglutination.
- zeta potential: difference in electrical potential between RBC surface and outer layer of ionic cloud
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LISS (low ionic strength solution)
lowers zeta potential and increases uptake of antibody onto RBC, thus reducing incubation time
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PEG (polyethylene glycol)
- removes water from the test system, concentrating the antibodies present
- thus increasing the degree of RBC sensitization.
- more sensitive than LISS, albumin, saline.
- do not use PEG with pt specimen with increased protein concentration such as multiple myeloma
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AHG reagents
- allows for agglutination of incomplete antibodies
- contains antibodies to both IgG and complement
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negative result for AHG
must be controlled by adding Coombs control cells (check cells)
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check cells/ coombs control cells
- They are Rh + coated with anti D
- will react with anti IgG in the AHG reagent resulting in visible agglutination.
- Addition of coombs control cells proves that AHG is working properly
- must show a positive result to validate. if negative (no agglutination) the test must be repeated
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limitations from AB screening
- cannot detect low titer antibodies
- cannot detect low frequency antigens that are not present on any of the RBCs in the screen cell set
- false negative reactions occur due to postzone when antigen is in excess
- temperature and phase of reactivity
- length of incubation
- pH: antibodies react best at a neutral pH between 6.8 and 7.2
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what might give a positive DAT result
- transfused within past 3 months
- medications
- autoimmune disorders
- infections
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advantageous of molecular teting
- ability to screen for multiple antigens at one time
- no interference from recent transfusion or positives DAT on RBCs
- not limited due to lack of rare anti sera
- able to screen large numbers of donors in a short time
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what are the techniques used when ABID panel does not have clear cut specificity
- selected panel cells
- enzymes
- neutralization
- adsorption
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selected panel cells
- done after ABID
- testing additional panels with minimal overlap in antigens they possess
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enzymes
- treating panel cells with enzymes destroying certain antigens and enhancing expression of others
- sulfyhydryl - DTT, 2-ME. destroys kell
- ZZAP - denatures kell, MNS, Duffy
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autoadsorption
- when autoantibodies are suspected, its best to use the patients own RBCs to adsorb the autoantibody.
- autologous cells are first washed to remove unbound antibody
- cells are incubated with patients serum. temperature is dependent on adsorbing either cold or warm antibody.
- sample is inspected for signs of agglutination throughout incubation period. agglutination means that RBC binding sites are saturated with autoantibody.
- serum is then harvested and incubated several times with new aliquots
- if no agglutination is apparent during incubation the harvested serum is tested against patient's own RBCs
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