The following paper appeared in Leukemia (2000) 14, 335-346 Correspondence: D Campana, Department of Hematology-Oncology, St. Jude Children's Research Hospital.

Simple methods for the rapid exchange of flow cytometric data between remote centers

TO THE EDITOR

The flow cytometric analysis of cellular immunophenotypes and DNA Content has become indispensable in the modern classification of leukemia and Lymphoma. The multiple applications of this technology include determination of the lineage association of malignant cells, assessment of their clonality, identification of prognostic indicators and monitoring of minimal residual disease.^1-3

Methods for cell separation, staining and analysis are becoming increasingly uniform,^4-6 but there is still Considerable debate on critical issues such as the optimal markers for disease classification, usefulness of isotype-matched control antibodies and setting of cut-off points to determine positivity (as an example, see the discussion that followed the paper by Bene et al). The achievement of broad Consensus may be, at least in part, hindered by the difficulty of precisely Communicating gating procedures, measurements of staining intensity and criteria for establishing percentage of positive cells. Thus, results obtained in different centers are often difficult to compare and to reproduce. This problem is particularly relevant to Collaborative multicenter studies, which require uniform classification criteria and could benefit from mutual or centralized review of data. Centers that have relatively new flow cytometric capabilities would also clearly benefit from timely monitoring of their results by more experienced investigators.

Traditionally, exchange of information between flow cytometrists has required travel to remote locations or the mailing or faxing of plots and histograms. However, travel iv expensive and time-consuming, and printed data presentations can be difficult to interpret accurately because they do not permit further analysis, for example, by changing of the marker settings and back-gating. To date, the most effective Communication between centers has been accomplished by mailing computer diskettes that contain data formatted to allow further analysis by the recipient (eg in list model. However, physical transport inevitably introduces delays, which Conflict with the requirements of modern clinical laboratory protocols.

We explored alternative methods of accomplishing flow cytometric Communication between remote centers.

We used the software Timbuktu Pro for Mac OS (Farallon Communications, Alameda, CA, USA) and direct dial mg to establish Communication between a FACScalibur flow cytometer equipped with the CellQuest software (Becton Dickinson, San Jose, CA, USA) located in Memphis, Tennessee (USA) and an identical instrument located in Guatemala City (Guatemala; Option 1 in Table 1). Timbuktu Pro allowed Control of the remote Computer by the operator in Memphis, with no need for data transfer. By using this software, we were able to analyze data placed in specific folders on the remote Computer. We were also able to change the settings of the remote flow cytometer while samples were being run, and to set the threshold levels and gains on the remote instrument. Because typed messages can be displayed on the Computer screen during this process, remote operators can confer while performing these operations. This method also allows the operator of the remote Computer to establish selective criteria and passwords to restrict access to selected individuals.

Direct analysis of previously acquired data wav also possible using the above software, but we found that file transfer wav relatively slow (more than 45 mm for 20 CellQuest files). Because the cost of a long-distance telephone call may be prohibitive for routine analysis of remote data or transmission of numerous files, we tested an alternative approach that uses the Internet (Option 2 in Table 1). The files containing flow cytometric data were Compressed by using the MacZip software (downloaded free of charge from the Website www.download.com) and were uploaded To the personal Web site of the operator in Guatemala City (created using tools available through the World-wide Web at www.geocities.com and www.guate.net. The operator in Memphis downloaded the files from the Web site, decompressed them by using MacZip, and analyzed them with the CellQuest software. This approach did not allow remote manipulation of instrument settings or real-time remote sample acquisition and analysis. However, it did allow virtually instantaneous transfer of data at a minimal cost (that of a local telephone call). We have used this method to exchange results of three-Color immunophenotyping and DNA content analysis performed in over 20 diagnostic bone marrow samples obtained from patients with suspected hematological malignancy, and found it to be fast and reliable. Unauthorized access To The data is unlikely because The files are labeled by code numbers and can be opened only by CellQuest software (available only To operators of Becton Dickinson flow cytometers). Further, full access To The Web site can be restricted To password holders. The use of Web publishing software allows the addition of supplemental information, such as images illustrating cell morphology, cytothemivtry, karyotype, etc, To the Web site To aid in The interpretation of The flow cytometric data.

In this study, we used Computer hardware which is commonly available as part of The Becton Dickinson flow cytometerv. Using methods identical To those described, we Could also Control The flow cytometer using a laptop Computer (Powerbook G3 with a 266 G3 MHz processor and a standard built-in 56 K modem. Thus, investigators can analyze flow cytometry data Collected in Their laboratories while they are away. In addition To The software that we used, there are other utilities that we did not test but are likely to allow effective links between remote flow cytometerv or between a flow cytometer and a laptop Computer. For example, SpeedShare Pro (Qdea, St Paul, MN, USA), Apple Remote Access (Apple Computer, Cupertino, CA, USA) and NetPresenz (Stairways Software, Booragoon, WA, Australia) should allow remote Control of The flow cytometer settings and/or data transfer. Other permutations, such as links with Timbuktu Pro via Internet could also be useful and need to be explored.

The simple and economical methods described here should greatly facilitate interactions between flow cytometry centers. Although our primary interest is the exchange of data used by the immunophenotyping of leukemia, these methods are clearly applicable to all uses of flow cytometry. We believe that these methods would particularly benefit multicenter clinical trials by allowing the transfer of flow cytometric data to a central site for uniform analysis. They would also allow experienced operators to train or closely monitor less experienced operators in conducting analyses, thus improving the accuracy of diagnosis while providing continuing education. Finally, the systematic comparison of the parameter used for disease classification in multiple centers should help the achievement of widely accepted consensus criteria, and the development of automated classification programs.⁸

Acknowledgements

We wish to thank Jose A. Galindo, Gustavo Lara, Andres Pappas and James Houston for their help in developing the connection between two medical centers. This work was supported by grants RO1-CA60419 and P30-CA21765 from the National Cancer Institute, and by the American Lebanese Syrian Associated Charities (ALSAC).

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