In some cases the B cell kappa and lambda run together and are difficult to separate. How can this be fixed?

There are several factors to be considered in resolving this situation, and 3 different scenarios. If the kappa versus lambda histogram is showing two distinct populations, but they are not nicely in upper left and lower right quadrants, this is not really a problem and is easily resolved. It is best to assess lights chains in a way that assures you are looking at B cells: the histograms that show CD19 versus kappa and CD19 versus lambda (or CD20 instead of CD19) will accomplish this. If these two histograms show distinct populations in upper right quadrant, this goal has been accomplished. In peripheral blood and tissue specimens, the numbers of events in these two populations should add up to that of the total B-cell population, because there should not be any B cells that are dual positive for kappa and lambda. In bone marrow specimens, hematogones also need to be taken into account.

If the kappa versus lambda histogram is showing overlapping populations in the upper right quadrant, there may be a technical problem, generally in one or more of the following areas:

 
1. Specimen processing: Adequate washing and adding back sufficient protein are the usual suspects in a sufficiently cellular specimen. Sometimes a 37°C wash is most effective, especially in cases of polyclonal hypergammaglobulinemia. A process for preparing specimens may work very well for 4 or 5 color assays but need to be changed when switching to 8 or 10 colors.
 
2. Antisera cocktails: Cocktails must be titered and validated. The more antibodies/colors per cocktail the greater the total volume in relation to the specimen so more protein may be required. If preparing cocktails ahead they must be validated for how long they can be used, especially for panels with light chain antisera. Some kappa and lambda antisera are very sensitive and may require a very small amount per cocktail. Titering light chain antisera may show that more events overlap into the kappa/lambda dual-positive quadrant when there is too much antisera and these populations may move back to the single-positive quadrants as lesser amounts are used in the cocktail. However, it is essential not to sacrifice sensitivity for aesthetics. Polyclonal antisera tend to look “messier” and monoclonal antisera tend to look “cleaner” but the sensitivity may be very different and must be a primary factor. If the problem persists, consider evaluating other reagents for kappa and lambda.
 
3. Instrument settings: Correct voltage and color compensation must be established for each panel.

 

If the kappa versus lambda histogram is showing a tight diagonal population through the upper right quadrant, these are cells that are either dead or otherwise non-specifically taking up both antisera equally. Here again, CD19 versus kappa and CD19 versus lambda histograms should show the real B cells. This can sometimes be addressed through cleaner specimen processing or through gating, but often it just needs to be recognized for what it is.