How does one set up a validation study for lower limit of enumeration?

In 2015, CAP came out with a new checklist item (FLO.30800) that requires that the lower limit of enumeration must be validated for rare event flow cytometric assays such as Paroxysmal Nocturnal Hemoglobinuria (PNH) clone testing or minimal residual disease (MRD). This checklist item does not pertain to the CD34 stem cell enumeration. The lower limit of enumeration (LLOE) can be defined as the minimum number of events that constitutes a recognizable population of interest (POI) divided by the total number of events collected.

The preferred method for the LLOE validation is to serial dilute a patient sample containing the POI with the normal (or negative) diluent of the same specimen type (blood, bone marrow aspirate, etc). It sounds simple but there are a few things to consider in determining the flow cytometric LLOE. First, an immunophenotype of the POI is identified by several markers and is not always easily distinguished from the normal population. Therefore, the minimum number of events to identify the POI can vary greatly from case to case. The majority of flow cytometry laboratories are comfortable identifying the POI with about 50-100 events. From a statistical standpoint, it is advised to have a minimum of 50 events for good reproducibility and low coefficient of variation (CV).

The second issue is to determine the appropriate number of samples needed for the LLOE validation. It is unrealistic (and not expected) for a flow cytometry laboratory to test all different immunophenotypes. Each laboratory should make their own decision on what the appropriate number of samples is.

The third challenge is to condition the diluent. In determining the LLOE, the POI needs to be serially diluted while the total white blood count remains constant. Since the positive and diluent samples often have different white blood cell counts (WBC), calculating volumes to make the dilutions can be difficult. This problem can be fixed by diluting the positive and diluent samples separately so that they have the same WBC. The positive sample is then serially diluted 10-fold with the diluent. All dilutions are stained and run under the same conditions (antibody cocktail, total number of events collected, etc). The LLOE is the lowest dilution that still has a detectable POI.

Additional Resources:

1. Barnett D, Louzao R, Gambell P, De J, Oldakeer T, Hanson CA; on behalf of the ICSH/ICCS Working Group. Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS- Part IV- Postanalytic Considerations. Cytometry Part B 2013; 84B: 309-314.

2. Wood B, Jevremovic D, Bene MC, Yan M, Jacob P, Litwin V; on behalf of the ICSH/ICCS Working Group. Validation of Cell-based Fluorescence Assays: Practice Guidelines from the ICSH and ICCS- Part V- Assay performance criteria. Cytometry Part B 2013; 84B: 315-323.