Tissue typing Flashcards
What is the platelet associated antibody (PAA) test)?
- Screen to ensure that there is no antibody bound to platelets
- Generally only performed in cases of NAIT as antibody bound to platelets will compromise cross match results.
- In NAIT: both the maternal and paternal platelets are screened.
What is the PIFT test?
◊ Patient plasma is incubated with a panel of blood group O apheresis platelet donors. If antibodies are present, these will bind to the platelets .
◊ After washing, anti-human IgG conjugated to FITC is added which will bind to any antibodies present in the sample.
◊ A flow cytometer is used to measure the amount of FITC bound to the platelet. A “channel shift” to the right is indicative of bound antibody.
◊ Tells you if antibody is bound, not what antibody is bound.
What is the MAIPA test?
- Platelets from HLA typed group O apheresis donors are used and incubated with the test serum. If antibody is present to an antigen on the donor platelets, then the antibody will bind.
- Mouse anti-human IgG is added. The IgG is specific to a particular platelet glycoprotein or antigen (i.e. GPIIb/IIIa, GP1b, HLA)
- The antigen is then solubilised, along with any bound antibody- anti-human IgG that is bound.
- The solubilised lysate is then added to a plate that is coated with goat anti-mouse IgG. The anti-human IgG will bind to the plate.
- Horse radish conjugated antihuman IgG is then added and binds to the patients antibody. If binding occurs, colour develops denoting a positive result.
How is Luminex antibody testing performed?
- Different platelet antigens are bound to different (but distinguishable) beads.
- The beads containing antigen are incubated with the test plasma. If antibodies are present, these will bind. The sample is then washed and PE or FITC labelled anti-IgG is added which will bind to any human IgG that is bound to the beads. The sample is then washed again.
- The beads then pass through the luminex flow cell one by one. The red laser reads the bead and the green laser detects how much antibody is bound to that bead.
What antibodies are implicated in NAIT?
• In 80-90% of Caucasian cases: the antibody is directed against HPA-1a and in 5-15% of cases, directed against HPA-5b
○ CD36, HPA-3 and HPA-4 important in Asian populations.
What tests are performed to diagnose NAIT?
○ PAA, PIFT, MAIPA, cross match and phenotyping/ genotyping
Aim is to identify antibody in maternal plasma, demonstrate lack of that antigen on maternal platelets and presence of that antigen on paternal platelets, demonstrate reactivity between maternal serum and paternal platelets.
Need to genotype farther to determine risk in future pregnancies: if father homozygous at that allele then 100% of future pregnancies will be affected vs 50% if heterozygous at that allele.
What is the definition of platelet refractoriness?
- Platelet transfusion without the expected improvement in platelet count on at east two occasions:
- Corrected count increment (CCI)= (plt increment x BSA x10)/ (no. platelets transfused x 10^11). CCI <5000/uL= refractory
- Practically: failure to sustain an increment >10 x10^9
How is platelet refractoriness investigated?
○ An HLA class I type by molecular (PCR-SSP) and serology techniques must be performed if the patients HLA type is unknown
○ HLA class I antibody screen by LUMINEX: if HLA antibodies are detected then an urgent LUMSAGI test is performed to determine the specificity of the HLA antibodies demonstrated
○ PIFT and MAIPA screening can also be performed to assess for HPA antibodies.
What HLA antigens are most important to match in allogenic stem cell transplantation?
- A, B, C, DRB1: high protein expression- mismatch strongly associated with HCT outcome and GVHD risk.
- DQB1, DPB1: included in NGS high resolution typing (to get a 12/12 match). Lower protein expression. Single mismatch has a lower association with transplant outcome.
What HLA antigen is most important to match in solid organ transplant?
○ HLA-DR: most immunogenic, most important antigen for rejection followed by HLA-B, and -A
○These three loci are the most important for matching donor and recipient (with most emphasis on DR)
At what MFI are HLA antibodies considered to be positive?
○ >1500 MFI positive
○> 10000 MFI strongly positive.
What four methods of HLA typing are used at NZBS?
1) Sequence specific primers (SSP)
- Fast method, results available within 3-4 hours (deceased donors, platelet refractory patients)
- Primers are designed so the SNPs which differentiate the HLA alleles, are at the 3’ end of the primer.
- PCR product is read on gel electrophoresis.
- Depending upon the selection of primers, PCR-SSP can be used for low or high resolution typing.
- Not suitable for high throughput typing of samples.
2) Sequence specific oligonucleotides (SSO)
- SNP/ mutations lie within the probe rather than at the 3’ terminus.
- Chemiluminescent or colorimetric reaction can be used to reveal the presence of bound oligonucleotide
- Luminex LABType: Biotinylated primers, complementary SSO probes conjugated to beads, Streptavidin-phycoerythrin conjugate (SAPE) binds to biotin, phycoerythrin serves as the reporter molecule. Luminex analyser identifies any SAPE that is bound to the bead.
3) PCR and high resolution melt curve analysis
- Two pairs of primer (one for the HLA SNP and the other for a control sequence).
- SYBR green added, incorporated into double stranded DNA and fluoresces.
- When DNA is heated, it denatures into single stranded DNA, SYBR green dissociates and stops fluorescing.
- Different melting temps for control vs HLA SNP
- Used for deceased donor typing in solid organ transplantation (HLA-A, -B, -C, -DRB1/3/4/5)
- Low resolution
4) NGS
- High resolution
- High throughput
What is the CDC B-cell Panel Reactive Antibody (BPRA) test?
○ Serum from patient tested against a random selection of B lymphocytes from apheresis donors to obtain a %BPRA value
○ DTT added during incubation to denature IgM, ensure only IgG antibodies are identified.
○ Performed in the presence of complement
○ When antibodies bind, the complement dependent lysis of the B cells can be visualised by differential staining of lysed and unlysed cells.
- Dead cells fluoresce orange/red (ethidium bromide), live cells fluoresce green (acridine orange).
What is the Luminex LABScreen method
○ Microbeads coated with HLA antigen to detect HLA antibody and determine antibody specificity
- Patient sera is incubated with microbeads
- Bound IgG antibody detected by a secondary IgG specific antibody conjugated with phycoerythrin.
- Luminex laser identifies the bead and associated HLA antigen and detects fluorescence when an antibody is bound to that antigen.
- The screening assay uses beads that contain a mixture of different HLA antigens.
- The confirmation/ identification assay uses beads with a single HLA antigen.
How is HLA crossmatching performed?
1) Flow cytometry
○ The T and B lymphocytes are detected by their staining characteristics.
○ An increase in fluorescence is seen (shown by a right shift in the curve) when bound antibody is present.
2) Complement dependent lymphocytotoxicity
○ Lymphocytes are mixed with the patients serum
○ DTT added to denature IgM. Complement, acridine orange and ethidium bromide also added
○ Live and dead cells are differentiated by their staining characteristics under the microscope (orange/red= dead, green= alive)
