Some cases of acute leukemia are not easy to diagnose, doubts arise due to the complexity of the disease, the variability of the immunophenotypic pattern, the variability in marker expression, and overlapping immunophenotypes. To address the complexity and subjectivity of multiparametric data analysis, EuroFlow™ has proposed the development of reference databases containing representative sets of flow cytometry data from both controls and patients for the most representative reagent panels like the ALOT.
Overview of the general automated analysis process for ALOT samples including AG&I, Compass orientation and Automatic Report as described below:
- Compatible FCS file
Acquired using the EuroFlow™ SOPs for instrument set-up, sample preparation and antibody panel.
All events are automatically identified by clustering algorithms.
- Identification with EuroFlow Databases (AG&I)
Infinicyt™ automatically compares the clusters with the EuroFlow™ Database:
– Clusters identical to cell populations included in the database are directly renamed with the name of those populations.
– Clusters that are not identical to the ones included in the database are assigned to Check Populations. The user has to review them and decide whether they are normal or abnormal.
- Comparison of the abnormal population(s) identified with a reference database (Compass Classification Tool)
Once the abnormal blast population is identified the software automatically performs a comparison (using the Compass tool) with a reference database containing abnormal (B, T and Myeloid) leukemic cells.
- Automatic Report includes (click here to see an example):
– Frequency of each population with reference ranges.
– Relevant information for clinical routine automatically updated for each sample.
– Available in 13 different languages.
Automated Gating and Identification
Infinicyt™ Automated Gating and Identification tool provides a complete analysis of a given sample. For Acute Leukemia Orientation, two separate databases are used: one ALOT Database for peripheral blood samples and another ALOT Database for bone marrow samples. All cell populations present in the sample are compared with the normal reference database and classified either unequivocally as normal, or to be reviewed by the expert. This, together with warnings and alarms related to the frequency of all sample populations, is meant to help the user when identifying the abnormal blast population.
How was the AG&I ALOT reference database built?
EuroFlow™ collected normal peripheral blood and bone marrow samples in different centers to reflect biological and technical variability regarding different laboratories, instruments and operators.
EuroFlow™ experts analyzed those normal samples and created a library of reference populations. When comparing a new sample with this database, all populations of the sample will be automatically identified.
ALOT Classification Database
Once the abnormal blasts population is identified, the software automatically performs a comparison using the Infinicyt™ Compass tool, with a reference Classification Database containing abnormal (B, T and Myeloid) leukemic cells.
The ALOT classification database is developed for the lineage assessment of the abnormal immature hematopoietic cells and proper characterization panel selection in samples with suspicion of acute leukemia. This database uses the Compass tool and compares the abnormal precursors with the database groups containing B-ALL, T-ALL and AML abnormal blast populations, independent of sample type (bone marrow or peripheral blood).
How was the ALOT Compass Classification database built?
This database contains pathological cases proceeding from different EuroFlow™ centers and analyzed by flow cytometry experts. The building process started with 747 pathological cases of typical B, T and Myeloid acute Leukemia diseases. After a process of evaluation of EuroFlow™ standards each category was reduced in number of cases. Then, all this cases were merged and reviewed again to discard outliers to finally obtain the total cases that constitute the ALOT database.
Schematic overview of the construction of the EuroFlow database. Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia. Van der Velden. Leukemia 2018.
How to interpret the Compass results?
The following images represent different possible outcomes:
- Almost all events are similar to the cases from one group in the database.
- The selected events belong to two distinct lineages; part of the events are similar to the cases from one group in the database and part are similar to the cases from a second group in the database.
- Most of the events have a mixed phenotype between the groups indicated.
- Some of the events are similar to the cases from one group in the database and the rest have mixed phenotype/transitional potential.
- Lhermitte L, et al. Automated database-guided expert-supervised orientation for immunophenotypic diagnosis and classification of acute leukemia. Leukemia. 2017 Apr; 2(4): 874-81. Go to publication.
- van Dongen JJ, et al. EuroFlow antibody panels for standardized n-dimensional flow cytometric immunophenotyping of normal, reactive and malignant leukocytes. Leukemia. 2012 Sep; 26(9):1908-75. Go to publication.