MDIPA whole blood staining automation

[shwetank]

Hello,
Has anyone tried implementing automation for PROT1 fixed whole blood?
The PROT1 protocol suggests, using 25ml lysis buffer wash post staining but it's too difficult to implement that high a volume with automation platforms.
Please let me know if you have tried using 1-2ml multiple wash instead of one 25ml lysis buffer wash to lyse RBCs post PROT1 fixation of whole blood?
Best
Shwetank

[rhannan]

I've made the mistake of trying to process PROT1 blood in 15mL conicals before, and it took at least three ~8mL lysis buffer washes before all the blood was lysed. I would guess you could achieve lysis using smaller lysis volumes, with a concomitant increase in number of washes required. I don't *believe* that the lysis is particularly harsh on non-erythrocyte populations or their antigen . I will keep your question in mind and try out a small-volume lysis next time I am thawing PROT1 blood for ab validation.

[mleipold]

HI Shwetank,

For the IMPACC COVID study, we had 270uL WB+ 420uL PROT1 = 690uL of sample.

We used 5mL 48-well deepwell blocks: Axygen® 48-well Clear V-Bottom 5 mL Polypropylene Rectangular Well Deep Well Plate, 5 per Pack, Nonsterile (P-5ML-48-C) - Fisher Scientific, cat# 14-222-938

4. While samples are thawing, load 48-well deep-well blocks with 3mL 1X Thaw-Lyse buffer per well.
5. Once samples are thawed, transfer each sample to a well on the deep-well block and mix thoroughly.
6. Incubate at RT for 10 min.
7. Centrifuge at 400 g for 5 min at RT and aspirate supernatant.
8. Resuspend in an additional 4 mL 1X Thaw-Lyse buffer.
9. Incubate at RT for 10 min.
10. Centrifuge at 400 g for 5 min at RT and aspirate supernatant.
11. Resuspend cells in 4mL CSB, spin at 250g x 7 minutes ("platelet depletion" wash), aspirate supernatant.
*** If thawing multiple sets of 10 samples, pause after first resuspension in CSB until all sets have reached this point, and then begin first centrifugation.
12. Repeat “platelet depletion” wash 2x more, aspirate supernatant.

So, 1x3mL, 1x4mL Thaw-Lyse, then 3x4mL CSB "platelet depletion" washes. Generally, the two ThawLyse were sufficient to lyse what would be lysable (ie, not clumped, not over-fixed with PROT1), and then the platelet depletion washes were surprisingly effective at cleaning up a lot more of the reddishness that was removable.

Really, anything remaining pink (let alone red) at that point wasn't going to clean up, and would be tossed at a later step.


Mike