We investigated about flow cytometry (FCM) currently performed as a reference for standardizing immunophenotyping of hematologic neoplasia with 3 Commercial Clinical Laboratories in Japan, 3 cancer research institutes in the U.S.A., and 1 cancer research institute in Europe. Investigation followed quality control (QC) of the instrument, the cluster of differentiation (CD) panel, the gating method, and the data analyzing method. About QC, the method of managing mean fluorescence intensity (MFI) using a fluorescence bead in all institutions was taken. The fluorescence bead to be used had the institution which uses only CaliBRITE Beads, and the institution which is using together Rainbow Calibration Particle which can check five kinds of fluorescence peaks. About the CD panel, the feature was accepted by each purpose, diagnosis of the neoplasm cell before treatment, minimal residual disease (MRD) detection after treatment, monitoring of the mature process of the cell in bone marrow after treatment, and the analysis of a special case. About the gating method, FSC-SSC dot-plot gating and CD45 blast gating were used together with most of institutions. With a European institution, CD45 blast gating was not made indispensable. With one institution in the U.S.A., CD45 blast gating which used CD45 Log-SSC dot-plot was used. Although 2-dimensional analysis was performed with most of institutions about the data analyzing method, multi-dimension analysis was performed with two facilities in the U.S.A.. Establish the standard method of immunophenotyping of hematologic neoplasia is an important subject in order to raise the clinical meaning of FCM.

Assessment of immunophenotype is essential for diagnosis and risk-directed therapy in pediatric leukemia. Although flow cytometry has been widely used for diagnosis of hematological malignancies, the complexity of multiparameter analysis techniques and the multitude of sample preparation methods and available monoclonal antibodies demand a standardization of protocols for the use of flow cytometry in clinical laboratories in order to achieve interlaboratory reproducibility. Therefore, the Working Group for Standardization of Immunophenotypic Diagnosis in Pediatric Hematological Malignancies has been started in order to establish a consensus protocol for flow cytometric diagnosis of pediatric leukemia in Japan, as a basis for quality assurance and support for upcoming technologies such as immunological detection of minimal residual disease. Fact-finding inquiry was made to reveal actual situation of immunophenotypic diagnosis of pediatric leukemia in Japan. The survey results indicated majority of institutes tend to use central flow cytometric centers by clinical study groups as a principal laboratory if they have available central flow cytometric laboratories. In contrast, institutes without available central laboratory look commercial laboratory as a principal diagnostic laboratory. In most of institutes, immunophenotypic analysis was submitted to some laboratories, and pediatric hematology-oncologist with experience of flow cytometric analysis was thought to make a final immunophenotypic diagnosis. Inquiry was also made against the central laboratories of leukemia study group and commercial laboratories. It was showed diverse protocols for sample preparation and analysis as well as insufficient systems for a guarantee of examination quality. Based on these results, we indicated the proposal panels for diagnosis of pediatric leukemia, and also form a plan of external quality control survey for major pediatric immnophenotyping laboratories in Japan. To establish precise and reproducible immunophenotypic diagnostic protocol will helps to choose adequate treatment and improve outcomes of pediatric leukemia throughout the activities of working group.

Flow cytometric analysis has become essential in the diagnosis and management of hematologic neoplasms. The cellular markers commonly used for immunophenotyping of leukemia are CD13, CD33, CD117,cytoplasmic myeloperoxidase for myeloid lineage, CD10, CD19, CD20, CD22, CD23, cytoplasmic CD79a for B-cell, CD2, CD3, CD7, CD4, CD8, cytoplasmic CD3, TCRα/β, TCRγ/δfor T-cell, CD16, CD56 for NK-cell lineage, CD41, CD42, CD61 for megakaryocytic lineage and CD235a for erythroid lineage. CD34, HLA-DR and TdT are also used. Immunophenotyping is especially necessary for the diagnosis of biphenotypic acute leukemia and FAB M0 of AML. Blasts of AML M0 are histochemically negative for myeloperoxidase, but flow cytometry (FCM) demonstrates myeloid markers such as CD13, CD33 and myeloperoxidase. Biphenotypic acute leukemia is characterized by the presence of blasts coexpressing lymphoid and myeloid antigens. In addition, FCM is useful in diagnosing the extramedullary infiltration of leukemia by analysing specimens such as ascites, pleural effusion, cerebrospinal fluid and solid tissue specimens. The evaluation of mininal residual disease (MRD) by FCM is useful for leukemic blasts with antigenic abnormalities, such as AML with B- or T-cell associated markers or CD56. Sensitivity of 10^-2～10^-4 can be achieved by identifying blasts on SSC/CD45 dot plots and data acquisition from a large number of events.

Flow-cytometry (FCM) of cell surface markers is routinely used for the clinical diagnosis of hematopoietic malignancies in our institution. This test is important for the diagnosis and for the planning of treatment schedule but, presently, we face with the problem of loss (absence) of standardization of the laboratory procedures. Similar to internal quality control, the external quality control is essential to guarantee the compatibility of data at an inter-institutional level. However, at present, there is no basic manual, and therefore, no standardization. “Guidelines for performing surface antigen analysis on hematopoietic malignant cells.” (JCCLS H2-A V1.0) was extracted from FCM-WG (Chairperson: Shiro Miwa) of the Japanese Committee for Clinical Laboratory Standards (JCCLS) for the guarantee of precision, accuracy and standardization in 2003. The present report aims to introduce the “JCCLS H2-P V1.0 guideline” as an example of standardization for the tests for the analysis of hematopoietic tumor cell surface markers in adult leukemia.

CD10 expression assessed by flow cytometry (FCM) is useful in diagnosis of acute B-cell lymphoid leukemia, angioimmunoblastic T-cell lymphoma (AITL) and follicular lymphoma (FL). However, in a considerable number of patients with AITL and FL, expression level of CD10 is so low that it cannot be detected with antibodies. We compared the sensitivity of seven commercially available CD10 antibodies, and investigated association of translocation of bcl-2 and CD10 expression in FL. There are significant difference in the several antibodies in fluorescent level. Fluorescence intensity of all five phycoerythrin (PE)-labeled antibodies are higher than those of two fluorescein isothiocyanate (FITC) labeled antibodies. In FL, detection rate of CD10 was 32/56 (57%) with FITC-labeled antibodies, and 36/42 (86%) with PE-labeled antibodies, respectively. In 41 patients with FL, with PE-labeled antibodies, CD10 expression rate was 35/41 (85%), and 24 patients (58%) harbored translocation of bcl-2. None showed translocation of bcl-2 without expression of CD10. In regard to detection of CD10 expression in FL by FCM, sensitivity depends on the sort of antibody.

Because RNA is easily degraded by RNase that is present in tissues handled by routine methods of surgical pathology, it is sometimes difficult to obtain satisfactory results in in situ hybridization for mRNA on archival formalin-fixed, paraffin-embedded tissue sections. In situ reverse transcription-polymerase chain reaction (in situ RT-PCR) is a technique that allows in situ amplification of reverse-transcribed complementary DNA (cDNA) originating from mRNA. Because in situ RT-PCR amplifies cDNA on a tissue section, it is considered theoretically more suitable for in situ detection of target mRNA than conventional in situ hybridization. In the present article, we reviewed and discussed in situ RT-PCR with special reference to the technical aspects.