Thread by @ssfarouk

1/ A continuation #tweetorial from my last one introducing human leukocyte antigen (#HLA) , the cell surface antigen...

Below, let's briefly review basics of #Luminex testing & "cross-matching."

What/who is Luminex?

2/ In this #tweetorial, we'll answer 3Qs that help us assess a #kidneytransplant recipient's immunological risk:

1. Does this recipient have #DSA* (against HLA-antigens) ?
2. Can the antibodies bind?
3. Can the antibodies activate complement?

*DSA: donor specific antibodies

3/ Q1: How do know if the recipient's serum contains DSA against HLA-antigens?

A1: #Luminex testing, a solid-phase (BEAD) based assay. To perform the test, take the recipient's serum and pipette it into multiple wells - each well has beads coated with #HLA antigens...

4/ If anti-HLA antibodies are present in a potential recipient's serum, they will bind to the beads. After washing, a secondary anti-IgG fluorescent antibody is added.

Then, we can quantify the fluorescence using the MFI (mean fluorescent intensity) ⚡️⚡️⚡️

#Luminex #HLA

5/ Below: an example of a #Luminex result (class II HLA antigens). Notice that as all MFI values are < 10K (depends on the transplant center's unacceptable antigen cutoff), callculated panel reactive antibodies (cPRA) is 0%

Here's a #cPRA calculator: optn.transplant.hrsa.gov/resources/allo…

6/ #Luminex testing is performed as part of the pre-transplant evaluation.

Once we have a donor, we review the donor's HLA antigens against recipient's HLA & Luminex resulting. Are there #DSA, or elevated MFI against an HLA-antigen that is expressed by the DONOR's lymphocytes?

7/ If the answer is YES, we move onto Q2: can these #DSA bind to the donor's lymphocytes?

A2: The flow crossmatch (XM), or a cell-based assay = donor lymphocytes + recipient serum (may contain antibodies) ➡️ wash + fluorescent IgG ➡️ flow cytometry

8/A positive flow XM is reported as a "channel shift" or change in the fluorescence measured by the flow cytometer.

T-cell XM: HLA class I DSA, expressed by all nucleated cells + platelets
B-cell XM: HLA class I & II DSA

9/ So say you have a positive flow XM...move onto Q3: can these antibodies activate complement and actually kill the cells?

Enter: complement dependent cytotoxicity (CDC) XM

Basically, repeat the flow XM above. But instead of adding a fluorescent IgG, add #complement!

10/ After the test is completed, assess the number of dead cells. If this number is > 20%, you might want to rethink moving forward with this #kidneytransplant.

Remember that the flow and CDC XM may be positive if there are non-HLA antibodies against the donor's lymphocytes...

11/ In summary:

Q1⃣: Are there donor specific antibodies (DSA) against HLA-antgens? ➡️ #Luminex
Q2⃣: Do they bind? ➡️ Flow XM
Q3⃣: Do they activate complement (kill)? ➡️ CDC XM

👇🏽 A nice graphic from @NatRevNeph that describes both cell-based and solid-phase (BEAD) assays

12/ For review, here's the #tweetorial describing #HLA basics:

https://twitter.com/ssfarouk/status/1188464951708606467?s=20