What is the best approach to triage low cellularity specimens?

Flow cytometric immunophenotyping of paucicellular specimens poses a diagnostic challenge, as it often limits the number of markers that can be utilized in the analysis. Common paucicellular specimens include, but are not limited to fine needle aspirates, small biopsies, and some body fluids such as cerebrospinal or ocular fluids.

Paucicellular specimens should be handled with great care to minimize cell loss. Upon collection, these samples should be placed immediately in a buffering medium such as RPMI-1640, Earle's balanced salt solution, or TransfixTM. These buffering media have been shown to prevent rapid cell loss, but laboratories should perform their own validation to determine their effect on cell viability, as well any interferences with antigen expression. Even with the help of a buffering medium, paucicellular specimens should be processed and analyzed as soon as possible, preferably less than 24 hours after collection.

Another way to reduce cell loss is to limit washing and centrifuging steps. Cerebrospinal fluids generally lack free immunoglobulins and therefore the washing step prior to surface antibody staining is unnecessary. After centrifugation, the supernatant should be aspirated instead of decanted.

If possible, only one or two panels of multiple surface monoclonal antibodies are used to screen. Intracellular staining causes significant cell loss and should only be used when necessary.

A stacked panel in which paired antigens expressed by non-overlapping cell subsets on a single fluorochrome are combined can be used. For example, CD4 and kappa can be stacked with the same FITC fluorochrome; and CD8 and lambda with PE. The stacked panel allows the use of more antibodies beyond the fixed number of colors in a cytometer.

If there is a known disease history (e.g., non-Hodgkin's lymphoma, B lymphoblastic leukemia, or acute myeloid leukemia), a focused panel including antibodies to detect the particular disease should be used instead of a broad screening panel.

References:

1. Stacchini, A., Demurtas, A,. and Aliberti, S. 2014. Immunophenotyping of Paucicellular Samples. Current Protocols in Cytometry. 68:9.46:9.46.1-9.46.14.
2. de Graaf MT, de Jongste AHC, Kraan J, Boonstra JG, Sillevis Smitt PAE, Gratama JW. Flow cytometric characterization of cerebrospinal fluid cells. Cytometry Part B 2011; 80B: 271-281.
3. Greig B, Stetler-Stevenson M, Lea J. Stabilization media increases recovery in paucicellular cerebrospinal fluid specimens submitted for flow cytometry testing. Cytometry Part B 2014; 86B: 135-138.