2006 Bethesda International Consensus recommendations on the immunophenotypic analysis of hematolymphoid neoplasia by flow cytometry: recommendations for training and education to perform clinical flow cytometry.

As clinical flow cytometry practices continue to expand and immunophenotyping for leukemia and lymphoma becomes more widespread, the need for defined guidelines for training of medical professionals is imperative. Standards of expected knowledge and skills are necessary to ensure reliable test results as well as provide direction to those who are considering adding flow cytometry to their clinical laboratory practice. Before now, no clear guidelines have been established for defining the areas of responsibility, education and training standards, and credentials that would be required to perform clinical flow cytometry for leukemia and lymphoma. As part of the 2006 Bethesda Consensus conference, a committee was formed to address this need and provide recommendations for training and education. The committee included laboratory professionals from private, public, and university hospitals as well as large reference laboratories that routinely operate clinical flow cytometry laboratories with an emphasis on lymphoma and leukemia immunophenotyping. This document represents the work of the committee. Categories of work responsibility are defined and the requisite education, training, and credentials, as well as measurement methods for assessing competency for each area of responsibility are provided. Additional recommendations are included that promote creating a specialty certification in flow cytometry, establishing benchmarks for training technologists and interpreters, and offer suggestions for minimum levels of experience to direct a clinical flow cytometry laboratory.

Flow cytometric diagnosis of X-linked hyper-IgM syndrome: application of an accurate and convenient procedure.

Hyper-IgM (HIM) syndrome encompasses a family of congenital immunodeficiency states characterized by frequent infections and markedly low serum levels of IgG, IgA, and IgE but normal or elevated levels of IgM. Many patients have neutropenia. The major defect shared by all forms of HIM syndrome is a failure of immunoglobulin isotype-switching. Recently, a flow cytometric assay was described in the immunology literature for diagnosis of patients with inherited X-linked (X-HIM) syndrome. Using this assay, activated CD4 peripheral blood T lymphocytes from two patients suspected of having HIM syndrome, and from their mothers, were subjected to immunofluorescent flow cytometric analysis for the expression of CD40 ligand (CD154 antigen). Test results established the diagnosis of X-HIM syndrome that was inherited in one patient and spontaneous in the other. The authors' experience illustrates that the flow cytometric assay used and described in detail here can facilitate an accurate and timely diagnosis of X-HIM syndrome. Because the assay can be carried out in most clinical flow cytometry facilities, it lends itself to use by pediatric hematologists in the standard evaluation of patients whose differential diagnosis includes that disorder. The authors hope this report will raise awareness of the value of this procedure.

Multicenter clinical experience with flow cytometric method for fetomaternal hemorrhage detection.

BACKGROUND: Enumeration of fetal red blood cells (RBCs) is important in the management of fetomaternal hemorrhage (FMH), particularly in situations of Rh incompatibility. METHODS: We evaluated results from three institutions using the flow cytometric method (FCM) to detect fetal RBCs based on the anti-hemoglobin F (HbF) monoclonal antibody method. RESULTS: During 1997-2001, 69 of 1248 patients (5.5%) had measurable fetal erythrocytes (RBCs) in maternal blood. Only 21 patients (1.7%) had more than 30 mL of fetal blood detected in maternal blood. Of the 11 patients with large FMH and clinical follow-up, 7 had fetal demise (64%). In positive samples, significant differences were found in the fluorescence intensity (FI) of anti-HbF antibody staining between HbF-negative erythrocytes (HbF-) and adult HbF containing erythrocytes (F cells; 4 +/- 0 versus 57 +/- 9 linear mean channels [LMC]; P < 0.001) and between HbF-cells and fetal RBCs (4 +/- 0 versus 433 +/- 136 LMC; P < 0.001). In addition, significant differences were observed in forward light scatter intensity between HbF-cells and fetal RBCs (298 +/- 15 versus 355 +/- 68 LMC, P = 0.03). The transportability of the test is also addressed by comparing results from two other laboratories. The experience of our three laboratories, as well as the results from the recently reinitiated College of American Pathologists survey, which compares FCM and manual methods, clearly documents the superiority of the FCM test over the manual Kleihauer-Betke (KB) test. CONCLUSIONS: The FCM is a simpler, more objective, and more precise alternative to the KB method in clinical testing. The high mortality rate associated with large FMH and therapeutic implications of these results should give laboratories motivation to abandon the KB method with more robust FCM to detect FMH.

Flow cytometric immunophenotyping test for staging/monitoring neuroblastoma patients.

Ten years ago, we made an incidental flow cytometric observation while immunophenotyping biopsy and marrow samples from children suspected to have leukemia/non-Hodgkin's lymphoma, but were subsequently diagnosed with neuroblastoma. The samples contained neoplastic CD45(-) cells that had an extremely bright CD56(+) (beyond the fourth decade on a four-decade scale) population distinguishable from CD45(+)CD56(usual density+) natural killer lymphocytes as well as other CD45(-)CD56(usual density+) nonhematopoietic tumors such as small cell carcinoma or melanoma. Following the "rare event" philosophy of selecting one negative and two positive antigens, we initially tried a "cocktail" of CD45(-)CD56(very bright+) neuron-specific enolase (NSE)(cytoplasmic+). We later modified the procedure to a more clinically applicable "lysed whole blood" CD45(-)CD56(very bright+) ganglioside GD2(+) cocktail to improve turnaround time (eliminating the cell permeabilization step for cytoplasmic NSE analysis), specificity, and sensitivity of the assay. A total of 123 marrow/tissue/fluid samples were analyzed by the various forms of the assay. Clearly interpretable samples had an 83% specificity and a 100% sensitivity. The three-color GD2 assay has successfully detected cells in marrow samples to a level of 0.002% (1 per 10(5) cells) using patient samples (not artificially "spiked" material). We added CD81 expression of the neuroblastoma cells as a fourth color and now use this rare event clinical test to help stage and monitor all patients with neuroblastoma.