Light chain expression by flow cytometry

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Nic's picture
Light chain expression by flow cytometry

Hello all,
When we are looking at HIV positive (human) samples for B-cell monoclonality, we often have difficulties differentiating the positive and negative staining of our kappa and lambda.  Our staining and gating strategy essentially is:
1. CD45 vs SSc
2. Gate on lymphocyte region
3. histogram plots of CD19 vs kappa and CD19 vs lambda
4 Gated on the CD19 positive lymphocytes: histogram plot of kappa vs lambda.

On most of our B-cell malignancy samples we get very good discrimination between the positive and negative populations. However on occassions (and mainly on HIV positive patients) we often see that the diffentiation between pos and neg is often difficult (even on plot 4 above).

Our current cell prep is:  ammonium chloride lysis, 2x washes, incubate at room temp with Ab, resuspend and analyse.  This is a typical protocol commonly used in flow labs.

Has anybody also seen this and how do you discriminate or improve the separation between pos and neg.

Many thanks

samm's picture
One quick suggestion is that

One quick suggestion is that you might want to do the incubation in a refrigerator (~8dC) for ~30-40 mins, followed by 2x wash with cold HBSS+1%BSA/0.5% FBS. (Do not incubate on ice (0-4dC) for this app). You can use the wash buffer as the Ab incubation buffer too. What colors are you using on the panel? Try and keep your primary markers well separated if your flow machine has the color capability (instead of just going FL1,2,3...)