Hi Guys
I`m in need of your opinions on interpretation of HI resuts
I`ve attached a couple of annotated plates where I seem to have four different outcomes. Could you read them and say if you agree with my interpretations
What titre you would give samples 354-361
I`m using type-O human RBCs and V-bottom 96-well micro-titre plates.
It seems many but not all studies use U-bottom plates for human RBCs and V-bottom for mostly avain RBCs, have you any experience on differences this makes?
From the literature for V-bottom plates and avian RBCs, antibody positive is recorded as complete tearing/streaming of the RBCs which settle at the bottom of the well rim when the plate it tilted 60o, which is the guide I`ve been using
For U-bottom plates and human RBCs, antibody positive is said to be recorded as formation of an RBC "halo". There is no mention of tilting the plate and observing streaming.
I`ve not seen what I would call a halo, but I do see clear steaming when I do tilt the plate
Any comments would be gratefully received
The use of V or U bottom plate depends on how consistent results one gets. For us, we use U bottom plate for mammalian red cells and V bottom plates for avian red cells. So the plate selection varies though most of them use what you mentioned.
Using U bottom plate we can see ring of RBC (creating a halo) so no need for tilting the plate. Since you are using V bottom plate, you cannot see halo.
I don't mind whether the stream/ tear formation is slight or more pronounced. So, for me the blue circles are positive for antibody. Moreover, sample 358 seems to have antibody titre of 40. I suggest it would be easier to interpret the result if you can run the assay in quadruplet (which we generally do).
Hoe this helps.
Hello again Prem
I've switched to U-bottom and the results are comparable to v-bottom but clearer.
Could you look at the attached HI plate and let me know which dilutions you would call positive
I'm particularly interested in your opinion on the faint hallo's in the following samples and dilutions:
372, 1:40
371, 1:80
369, 1:320
373, 1:40
Looking forward to your answers
Hello Mark
I can't open the file. Can you upload it again, better in ppt.
Hi Prem
Ppt attached, let me know what you think, lookingforward to your verdict
Hi Mark
For me its 80, 40, 40, and 40 for wells 375-378 respectively.
These interpretations are subjective, so what I suggest is set your own criteria, and it would be better if you include red cell control and compare your result with it.
Good luck
I am concerned that you don't get the "button" look of rbcs when you don't get agglutination. What is your protocol? Do you wash the cells in PBS. Do you use BSA (I recommend against it)? What percent of blood cells do you use? I tend to use 1% of the total volume then let it rest in the cold room for 1-2 hours... see the attached picture.
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