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.