Special Techniques Flashcards
What are the sulfhydryl reagents?
DTT - dirhiothreitol
2-ME - 2-Mercapto-Ethanol
AET - 2 aminoethylisothiouronium
How do sulfhydryl reagents work?
Reduce the double bond of the cysteine residue which breaks the secondary structure of the polypeptide
What is 0.01 M DTT used for?
Disperse autoagglutinins from the cell
Differentiate IgM and IgG
What is 0.2 M DTT used for?
Denatures antigens (KEL, LU, DO, ect.) Used in ZZAP or WARM reagent
What is the procedure to disperse autoagglutinins?
RBC’s coated with IgM cold auto antibodies can spontaneously agglutinate causing false positive rxns
DTT breaks the disulfide bond in the IgM
- Equal part 0.01 M DTT + 50% RBC suspension (PBS)
- Incubate 37C for 15 min
- Wash cells w/ saline and make 2-5% cell suspension
- Include a 6% albumin or mono control in testing
What is the procedure to differentiate IgM and IgG?
DTT breaks the S-S bonds of monomeric bonds of IgM
-Equal parts of 0.01 M DTT + serum/plasma
-Dilution control = Equal PBS and Serum/Plasma
-Incubate 37C for 30-60 min
-Test DTT treated serum using standard methods.
If DTT treated plasma is neg and ctr is positive = IgM
If DTT is positive and ctr is positive = IgG
If both ctr and DTT are negative = Invalid (diluted)
What is the procedure for using DTT to denature antigens?
Useful to destroy k, Kpb, Jsb, Lub, and other high’s
- 4 volume of 0.2 M DTT + 1 volume of washed Packed RBC’s
- Incubate 37C for 30-60 min
- Wash with saline
- Resuspend cells in 2-5% cell suspension
- Ctr = Treat K+ w/ DTT and test with anti-K
What is in ZZAP?
0.5 mL of 1% ficin or papain
2.5 mL of 0.2 M DTT
2.0 mL of PBS
AKA WARM made by imunocor
Store at 4C for 5 days
What is ZZAP used for?
Used for warm auto adsorption
- DTT removes bound IgG; free up binding sites
- Ficin/Papain removes part of they glycoprotein structure enhancing antibody binding and adsorption
- Can be used in allo adsorption to enhance binding
What antigens are destroyed by ZZAP?
DTT destroys YT, DO, CR, IN, LU, JMH, KN, LW, KEL
Enzyme destroys Fya/Fyb, MNS(s), EnaTS, EnaFS, Ch/Rg, XG, JMH, YT, Ge2, Ge3, IN, KN (KN resistant to papain)
Chido and Rodgers eXcluded John MH Yet GE2 and Ge INcluded Knops
What is the procedure for using ZZAP in adsorptions?
2 vol of ZZAP + 1 volume of washed packed RBC’s
Incubate 30 min at 37C
Wash cells x3 and pack RBC’s
Add patient plasma to packed RBC’s and mix
Incubate for 30-45 min at 37C
Centrifuge and test plasma or repeat adsorptions if needed
What are examples of how ZZAP can be used?
Can be used in place of enzymes or DTT
- Make K0 cells to ID high incidence KEL antibody
- Identify Kel allo antibodies in AIHA
- Characterize Kell auto antibodies
- Characterize Kell antigens
What is AET used for?
Used like DTT
- Destroy Kel antigens
- Characterize New Kell antigens (if not destroyed = not Kell)
- Determine if auto antibody has Kell specificity
- Anti-Gerbich reacts strongly with AET treated cells
- Kx is not destroyed, anti-Kx reacts strongly with DTT/AET treated cells
How does AET work?
No one knows!
AET treated cells look like PNH cell
PNH cells have increased sensitivity to C’ lysis but have normal K antigens
What is the procedure to use AET?
AET is a dry powder = 6% AET + distilled water and adjust pH to 8.0 with NaOH
One volume of washed packed RBC’s + 4 vol of AET
Incubate at 37C for 20 min
Wash cells 5-7x and test cells with anti-K as ctr
What is EGA?
EDTA glycine-Acid 10% EDTA 0.1 M glycine HCL 1 M of TRIS (buffer) EDTA is used instead of saline because saline would cause hemolysis Buffer is used to bring pH back to 7-7.5
What is EGA used for?
Treating cells with low pH dissociates bound IgG for phenotyping with IAT
Inactivates antigens in the Kell, Bg, and Era
Does not affect Duffy or MNS antigens
What is the procedure for using EGA?
Wash cells 6x with saline
Mix Glycine and EDTA in a 20:5 ratio
Add 20 volumes of EGA to 10 volumes of RBC’s
Mix and incubate at RT for 2 min then add TRIS
Centrifuge and remove supernant
Wash RBC’s 4x with saline
DAT is done to verify IgG is removed
What are the limitations of EGA?
May not work (works about 85%)
May over treat cells and causing tanning or clumping of cells
Does not remove C so use anti-IgG
What is CDP used for?
Chloroquine Diphosphate
Dissociate cell bound IgG w/o damaging antigens
Allows for cell phenotyping
Also removes Bg antigens
What is the procedure for using CDP?
0.2 mL of wash packed RBC’s + .8 mL of CDP
Incubate for 30 min at RT
Do DAT on cells to ensure IgG is removed
IF DAT is positive incubate for another 30 min
What is the disadvantage of CDP?
May take 2 hours and may not completely remove antibody from DAT positive cells
What chemicals are commonly used to differentiate IgG and IgM?
DTT and 2-ME
*2-ME has strong odor so DTT is more common
What chemicals are used to disperse cold autoagglutinins from the RBC’s?
DTT and 2-ME
What chemicals are used to destroy red cell antigens?
DTT and AET destorys KEL,LU, LW, DO, CR, YT, KN, IN, JMH?
Compare EGA and CDP?
EGA is more effective but destorys Kell and Era antigens
EGA is faster
Why must autologous cells be treated before adsorption procedures?
Autologous cells antigen binding sites may be blocked with IgG so antibody can’t bind. DTT, enzymes, ZZAP, AET, CDP, and heat elution can be used to remove bound IgG
How can you enhance antibody uptake in adsorption procedures?
Using enzymes or ZZAP will enhance antibody binding by removing glycoprotein from the RBC membrane making antigens more accessible
Can increase proportion of RBC to plasma
Can increase the number of adsorptions but risk diluting allo antibodies
PEG or LISS can be used to decrease incubation time
How do you determine if antibody has be adsorbed out?
Test the adsorbed plasma with group O cells or DAT neg auto cells to determine if antibody has been removed
What cells should be selected for warm auto adsorption?
Patients own cell if not recently transfused or pregnant
If recently transfused do a phenotypically similar cell if possible.
If phenotype not available use R1R1, R2R2, and rr cells that have at least one K-, Jka-, Jkb- if using enzymes to Destroy Duffy and MNS antigens.
Which auto antibodies will not be removed enzyme treated cells are used?
auto antibody specific for Duffy, MNS, Ena, Ge, JMH, Ch/Rg, Inb will not be removed with enzyme treated cells because the antigen is destroyed.
How does the acid elution remove bound antibody
It disrupts the antibody-antigen binding forces by lowering the pH
Glycine acid dissociates the bound antibody
When should an eluate be preformed?
When patient has a positive DAT and has been transfused in the last 3-4 weeks if may detect a newly formed antibody or DHTR
Can also be done on patients suspected of have immune mediated hemolysis
Can you use potentiators in the eluate testing?
LISS and bovine albumin is not necessary because eluate is prepared in a low ionic substrate
PEG or GEL can be used if desired.