ONCOLOGY

CD34 Stem Cell Analysis on a

Routine Hematology Analyzer
John Sandri1, Donald Wright2
2

Scripps Clinic, LaJolla, California, U.S. Abbott Diagnostics Division, Santa Clara, California, U.S.

he Hematology laboratory at Scripps Clinic, LaJolla, CA,supports the Scripps Cancer Center which specializes in the treatment of hematologic malignancies. Scripps physicians perform more than 100 autologous and allogenic stem cell transplantations each year. Flowcytometric stem cell counting is needed to support these programs but the provision of this service can be challenging. These challenges include the inability to offer a routine stem cell count outside of normal operating hours as well as bearing the high costs of operating a dedicated flow cytometer. The opportunity to perform CD34 counting on a routine hematology analyzer, the CELL-DYN Sapphire, would potentially overcome some of these challenges. This study reports the outcome of a feasibility study to assess the ability of a routine hematology analyzer to generate accurate stem cell counts.

Materials and Methods

CELL-DYN Sapphire The CELL-DYN Sapphire (Figure 1) is an automated
Figure 2: (a) Typical layout of a inmmuno-flow cytometer. (b) Optical bench of the CELL-DYN Sapphire including the Blue (488 nm) diode pumped laser.

Figure 1: The CELL-DYN Sapphire Hematology Analyzer.

hematology analyzer incorporating multiple analytical methods that include MAPSS (multi-angle polarized scatter separation), threecolor fluorescence detection, hemoglobinometry, and focused flow impedance analysis. In addition to providing the complete blood count, the CELL-DYN Sapphire employs immuno-fluorescence analysis technology, similar to that used on a dedicated fluorescence flow cytometer (Figure 2), for analysis of MAb applications. A diode pumped solid-state laser provides 20 mW of 488-

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Figure 3: Peak spectral fluorescence of the CELL-DYN Sapphire achieved with the blue (488nm) diode-pumped solid-state laser.

Figure 4: CD34 automated sample processing.

nm light which is suitable for immuno-fluorochrome excitation. The optical bench detects four light scatter signals and incorporates three single optical filters corresponding to 530nm, 580nm, and 630nm wavelengths (Figure 3) permitting the measurement of three-color fluorescent detection. In its current configuration, the CELL-DYN Sapphire has the ability to perform two fully automated immuno-fluorescent assays. The single color, FITC labeled CD61 immuno-platelet count and the twocolor, FITC/ PE labeled CD3+CD4+ T-helper and CD3+ CD8+ T-suppressor lymphocyte subset analysis can be processed within the routine hematology workload. With the CELLDYN Sapphire's current hardware and software design, the opportunity exists to explore the feasibility of performing other monoclonal antibody assays. Patient samples Residual patient samples were obtained including a mix of leukophoresis product, bone marrow aspirate and EDTA anticoagulated peripheral blood. Sample age was less than 12 hours from time of collection. Twelve samples were initially analyzed, as reported in the abstract, however, the study is ongoing and an additional 4 samples were included in this reported data set. Monoclonal antibodies Purified monoclonal mouse antibodies to CD34 (conjugated with Rphycoerythrin (RPE)) and CD45 (conjugated with fluorescein isothiocyanate (FITC)) were obtained from Dako Cytomation, (DakoCytomation, Inc., Carpinteria, CA) for use in this study. Sample preparation and processing The samples required an offline preparation prior to

processing. In duplicate tubes (13mm x 75mm no additive tubes), 200_L of patient sample was pipetted into each tube followed by 10_L of anti-CD34 (PE) and 10L of anti-CD45 (FITC) monoclonal antibody. Tubes were then incubated in the dark at room temperature for 20 minutes. Processing the CD34 samples on the CELL-DYN Sapphire requires placing a blank tube in position (1-7) of a 10tube position sample rack, immediately followed by the two prepared stained sample tubes (Figure 4). The automated monoclonal antibody mode was used for processing assay. This mode is selected from the main menu and the sample processor is activated. Onboard analysis time is approximately 8 minutes. FCS file retrieval and offline analysis The CELL-DYN Sapphire stores the analyzed samples in list mode using a file structure that is compatible with fcs (flow cytometry standard) files. Once the sample processing is completed, retrieval of the .fcs file is made possible through a menu driven utility via the CELL-DYN Sapphire software and downloaded to a DVD. Each file contains the results of both analyzed tubes. Commercially available software, FCS Express version 3.00.0316, (DeNovo Software, Thornhill, ON, CAN) was used for the offline file processing. Analyzing the data from the fcs files required a two step process. Step one consisted of a straightforward merging of the events from tube one with the events from tube two creating a single file. This approach effectively doubles the number of cellular events used in the analysis. Step two involved making an initial layout for population gating. Once the template was made, processing of the files required little or no additional user manipulation. The

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population analysis strategy took into consideration the morphological and immuno-fluorescent file data (0, 7, FL1, FL2, and FL3) and the potential of spectral overlap of FL1, FL2 and FL3. As a result, a primary CD45+(FL1/FITC) regional gate was identified with a subsequent CD34+(FL2/RPE) nested gate. A compensation routine was utilized to minimize effects of spectral overlap (Figure 5). Statistical Analysis Data was analyzed by Passing-Bablok analysis performed with Analyse-It (Analyse-It Software Ltd, Leeds, England, U.K.).
Figure 5: CD34 Stem Cell population analysis routine.

Results
The 16 individual sample results were directly compared with the results obtained from the reference flow cytometer (Table 1). The Passing-Bablok regression plots for two data groups are shown in (Figure 6).
Table 1: CD34+CD45+ results from the CELL-DYN Sapphire hematology analyzer compared to reference flow cytometer. Study ID 144 147 156 187 232 233 238 249 312 279 343 362 363 394 395 398 N = 16 0.31 0.57 0.44 0.33 0.8 0.52 6.8 2.86 8.36 3.46 1.88 0.88 0.46 1.4 0.6 0.4 0.28 0.33 0.2 0.17 27.4 29.4 0.01 0.18 0.44 0.27 0.23 1.1 Peripheral Blood Leukophoresis Bone Marrow Sapphire (%)Flow (%) Sapphire (%)Flow (%) Sapphire (%)Flow (%) 6.06 8.6 0.63 1.05 Figure 6: Passing-Bablok regression analysis, (a) all samples, (b) sample values <10%.

Discussion
Although a relatively limited number of samples have been tested, the results from the initial 16 samples showed generally comparable results with the current laboratory flow cytometry method. The authors recognize the potential for an individual high count to make the correlation statistics look favorable. One sample in

particular had an extremely high CD34 count and the regression analysis was performed with (Figure 6 chart (a)) and without (Figure 6 chart (b)) this sample present. The absence of this sample did not cause a marked deterioration in the correlation statistics. Interestingly, the blood sample in question (# 0156 PB) had additional reference laboratory immuno-phenotyping markers performed which showed two CD34+ populations, of which, one coexpressed CD34+ and CD33+ (myeloid progenitor cell), often found in patients with M1 AML (Figure 7, plot (b)). Another notable sample was number # 0187 BM (Figure 9, plot (a)). The discrepancy between the reference flow result and the CELL-DYN Sapphire was >1%. Review of the reference flow scatterplot revealed a non-specific population that streamed through the two specific populations and was considered to be a potential artifact (such as non-lysed RBCs). This population was not apparent in the CELL-DYN Sapphire analysis. All sample scatterplots exhibited well defined populations (Figures 7, 8, 9 ). The additional data continues to show good agreement to the reference flow cytometer and warrants further sample testing.

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Figure 7: CD34+CD45+ scatterplots - peripheral blood samples.

Figure 8: CD34+CD45+ scatterplots - leukophoresis samples.

Figure 9: CD34+CD45+ scatterplots - bone marrow samples.

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