A simple flow-cytometry method to evaluate peripheral blood contamination of bone marrow aspirates
Delgado JA (1), Guillén-Grima F (2), Moreno C (1), Panizo C (3), Pérez-Robles C (1), Mata JJ (1), Moreno L (1), Arana P (1), Chocarro S (1), Merino J (4).
(1) Department of Immunology, Clínica Universidad de Navarra, University of Navarra, Spain.
(2) Department of Preventive Medicine, Clínica Universidad de Navarra, University of Navarra, Spain.
(3) Department of Hematology, Clínica Universidad de Navarra, University of Navarra, Spain.
(4) Department of Immunology, Clínica Universidad de Navarra, University of Navarra, Spain.
Bone marrow (BM) aspirates used for flow-cytometry (FCM) studies are usually obtained from a second aspiration, as the primary aspirate is used for morphological assessment.
For this reason, the FCM samples unavoidably contain some blood; although, good-quality samples contain only a small amount. It is of utmost importance to assess the quality of samples prior to FCM analysis; yet, contamination with peripheral blood (PB) is not evaluated in most laboratories, possibly because the methods available are either qualitative or too complex for daily practice.
Here, we propose a simple FCM method to quantitatively evaluate PB contamination in BM aspirates, by analyzing the percentage of plasma cells and CD34+ cells - two cell populations nearly absent from PB - and CD10+ granulocytes, which comprise the majority of the PB granulocyte population.
We analyzed these three populations in 122 BM aspirates from subjects without hematological disease, and identified samples with PB contamination by performing a hierarchical cluster analysis. A discriminant analysis yielded a function, which we named the PB contamination index (PBCI).
This index value gives a quantitative indication about the degree of hemodilution of a given sample. A threshold was identified that discriminates low-quality samples.
The method and the threshold proved to be useful in BM aspirates infiltrated with malignant cells, with the exception of cases where hematological disease altered two of the three parameters included in the index. We have easily implemented the PBCI calculation in our daily routine, and find it very helpful for an accurate interpretation of FCM results in a large proportion of BM specimens. Limitations of the technique are discussed.
CITATION J Immunol Methods. 2017 Mar;442:54-58. doi: 10.1016/j.jim.2016.12.006