NAITP Tutorial Flashcards
What is Flow Cytometry?
A technique by which rapid and complex analysis of large numbers of cells can be performed as they flow, single file, in a fluid stream suspended in sheath fluid and analysed using laser light and fluorochromes.
What sort of measurements can be taken with flow cytometry? Via what method?
Cell size through forward scatter and granularity via side scatter of a laser light as well as cell type through fluorochrome labelling.
How are flow cytometry measurements achieved?
Argon lasers excite fluorochromes to emit a wave length above that of the wavelength of light of the laser.
These signals are received through detectors such as photodiodes or photomultipliers.
Forward scatter and side scatter aids in size/ contents
What are the major drawbacks of flow cytometry?
Cost and availability of an instrument.You need highly skills operators, daily maintenance programmes and comprehensive service contracts.
What is the role of Human Platelet Antigen (HPA) system in NAITP?
Antibodies specific for platelet antigens inherited from the father which are absent in the mother
The foetal platelets are able to cross the placenta and this results in the recognition of these paternal antigens by the mothers immune system as non-self subsequently also-reactive antibodies of IgG class are generated which cross the placenta.
About 80% of NAITP cases implicate antibodies against platelet antigen HPA-1a. Commonly the father is HPA-1a/1a or 1a/1b and the mother is HPA-1b/1b and developing anti-HPA-1a antibodies. They are IgG so they can cross the placenta and enter the foetus.
What is the principle of the screening assay for NAITP?
A PIFT (platelet immunofluorescence test)
To look for an immunological reaction between anti-platelet antibodies in the maternal serum against paternal platelets.
The serum from the mother has all of the antibodies. The father has the platelets that possess the antigens not present in the mother. We will also use Anti Human IgG FITC because it is the brightest flurochorme which gives us the highest sensitivity and we pick IgG because it is the only one that can cross the placenta and enter the foetus’ bloodstream.
Why is AB serum used and a negative control?
It will not possess ABO/HLA/HPA antigens which are present in the platelets as AB donors don’t have these naturally present.
Why do we use Anti HLA as a positive control?
HLA-1 is present in all cells, and high volumes within Platelets
How do we identify platelets by flow cytometry?
Flow cytometry can use forward scatter (to identify size) and side scatter ( to identify components and fragments).
Platelets have low intercellular contents as they are very small, do not have a nuclei and have few granules.
Why use the median fluorescence?
It will not be affected by outliers or skewness as this gets removed when looking at the standard distribution
Why do we go to 3DP when calculating the Negative Cut Off?
Flow cytometry can only detect fluorescent to 3DP so a better accuracy cannot be achieved as you can’t claim an accuracy greater than you cannot measure.
Why use +/- 2SD as a Negative Cut Off?
This will enable us to cover 97.5% of the entire population.It ensures we can detect both weak positives and false positives and are able to rule them out through confirmatory testing
How can you interpret your PIFT results?
Any value that is equal or greater than your negative cut off value is a positive result.
What should be done next and why?
Confirm which Antibody caused the positive test as platelets have ABO/HLA/HPA
One needs to confirm with MAIPA which is the golden standard procedure. If both PIFT and MAIPA come out as positive the foetus will have NAITP.
If only PIFT is positive it could be a non-immune cause due to trauma, drugs like aspiration or a bacterial infection.
What if PIFT & MAIPA come back positive.
If both PIFT and MAIPA come out as positive the foetus will have NAITP. The next steps would be to council mum to have a C-section at future birth, no forceps or ventures delivery and monitor Anti-HPA titre through pregnancy.
