Leukocyte morphology on an anti-CD antibody microarray for acute leukemia diagnosis: morphology rejuvenated

Abstract

Acute leukemia diagnosis steadily shifts towards automated analysis and precise molecular methods banishing the methods resistant to complete automatization (such as cytochemistry and bone marrow morphology). The reasons for this lie above all in the desire to eliminate the human factor as well as to find a way to cope with increasing work load. The better understanding of the biology of the disease achieved with the advanced molecular technologies led to the inclusion of new recurrent cytogenetic abnormalities, mutations and gene fusions into the revised WHO classification of tumors of the hematopoietic and lymphoid tissues. Wherever possible the acute leukemias are classified by mutations rather than by underlying subtypes according to French-American-British (FAB) classification. 1 FAB classification is retained only in the "not otherwise specified" category. These changes give a probably involuntary message that as mutations prevail over morphologic and immunophenotypic characteristics of leukemic cells the latter at a certain point will not be needed for the diagnosis.On the other hand, as the amount of diagnostic methods involved in acute leukemia diagnosis increases, there is a rise in demand for cost-optimizing strategies, and FAB classification 2 is used to narrow the range of molecular tests to be performed on any particular bone marrow sample.According to the established guidelines 3,4 the diagnosis of acute leukemia starts with the analysis of morphology, cytochemistry and immunophenotype of the bone marrow aspirate. If more than 20% of the total nucleated cells in bone marrow aspirate are immature leukocyte precursors (blasts), the patient is diagnosed with acute leukemia. Leukemia subtype is than determined by blast attribution to different leukocyte lineage.The blast enumeration and identification are performed separately by morphology and cytochemistry in a bone marrow aspirate film and by surface and intracellular CD determination by flow cytometry after gating by side-scattering properties and the levels of CD45 expression. The results from both methods are then compared and integrated to establish the presence of acute leukemia and to determine its subtype (ALL BI-BIV, ALL TI-TIV or AML M0-M7). As morphology and flow cytometry use different methods to define the blast population, blasts detected by these two methods are not necessarily the same. 5,6 This fact can lead to differences in the size of blast population determined by morphology and flow cytometry, as well as pose problems in attributing the leukemic cells to different lineages and determining the leukemia subtype.To solve this problem and to integrate the morphology and immunophenotyping in the acute leukemia diagnosis we would need a method permitting the morphology study and the surface clusterof-differentiation antigen determination on the same cells. Several approaches have been tried to this end. Imaging flow cytometry is a beautiful technique that has a great potential, however, it is not yet able to enumerate cells with blast...