Application Note No. 16

© 2017 ACEA Biosciences, Inc. All rights reserved. 6779 Mesa Ridge Road Ste 100 San Diego, CA 92121 | 866.308.2232 | www.aceabio.com

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NovoCyte® Flow Cytometer

Hematopoietic stem cell transplantation (HSCT) is an important clinical procedure used for treatment of blood malignancies and disorders. The majority of HSCTs performed use peripheral blood as the source of hematopoietic stem and progenitor cells (HSPC) and success of hematopoietic stem cell transplantation is associated with the quality of progenitor cells infused. Successful long-term hematopoietic transplantation highly correlates with the number of CD34+ HSPCs transferred. Therefore, CD34+ cell counts are used as a quality control for transplantation and CD34+ enumeration is essential for HSCT.

Flow cytometry is used to quantify HSPCs because the workflow is both rapid and simple. Previous advancements in CD34+ counting include the development of a single-platform protocol allowing all analysis to be performed using a flow cytometer. The International Society of Hematology and Graft Engineering (ISHAGE) has adopted an internationally accepted protocol to enumerate CD34+ cells in blood using multi-parameter flow cytometry. Reference fluorescent counting beads are used to accurately determine the absolute count of CD34+ cells, replacing the need of a hematology analyzer. The Single Platform ISHAGE protocol has become the standard gating strategy used for CD34+ cell analysis in clinical labs internationally. A common concern with the standardized protocol is the variability in reference counting bead concentration that can occur resulting from differences in sample buffer, handling, and instrumentation. Therefore, the possibility of direct cell counting by a flow cytometer may improve upon the current methodology for CD34+ enumeration. The NovoCyte® flow cytometer uses a high precision syringe pump that accurately measures the volume of the sample without the need of reference beads to achieve absolute cell counts. Here we demonstrate that the NovoCyte flow cytometer can be utilized for the ISHAGE multi-parametric staining protocol as well as direct enumeration of CD34+ cells.

Direct enumeration of CD34+ cells in blood using the NovoCyte® Flow Cytometer

Andy Filby, Ph.D.Director, Flow Cytometry Core Facility Newcastle University

ISHAGE Protocol for CD34+ enumeration on the NovoCyte

The NovoCyte flow cytometer has a multi-laser format capable at detecting up to 15 parameters allowing easy multi-parameter analysis necessary for the ISHAGE protocol. The ISHAGE protocol determines the frequency of viable HSPCs in blood through a Boolean gating strategy including FSC, SSC, a viability stain (7-AAD) and antibody staining of CD45 and CD34. To demonstrate detection of CD34+ cells in whole blood, the ISHAGE protocol was applied to cells acquired on the NovoCyte, analyzed using NovoExpress, and described below (Figure 1). Cells that do not express CD45 are excluded eliminating platelets and erythrocytes retaining only the leukocyte population (Plot 1). Next, CD34+ cells are identified within the CD45+ leukocytes (Plot 2).Validation of the previous gating is performed by ensuring that all identified CD34+ cells fall within a small cluster (Plot 3). CD34+ HSPCs have a similar FSC and SSC to the lymphocyte population, so both populations are compared (Plot 4 & 6). CD34+ cell viability is determined by 7-AAD staining and compared to live cell staining of all cells (Plot 7-8). Finally, to accurately quantify absolute cell count of CD34+ cells the frequency of fluorescent reference beads is determined (Plot 5). Traditional flow cytometers require these counting beads to obtain cell counts within samples, however, when using the NovoCyte, counting beads are not necessary (Figure 2). This complex gating strategy has been established as the most effective way to ensure the proper determination of the frequency of CD34+ cells in clinical laboratories.

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For life science research only. Not for use in diagnostic procedures.

© 2017 ACEA Biosciences, Inc. All rights reserved. 6779 Mesa Ridge Road Ste 100 San Diego, CA 92121 | 866.308.2232 | www.aceabio.comNovoCyte and NovoExpress are trademarks of ACEA Biosciences, Inc. in the US and other countries. All other product names and trademarks are the property of their respective owners.

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Resolution of CD34 staining on the NovoCyte Flow Cytometer

To ensure consistent CD34 staining on different flow cytometers, CD34+ cell staining in blood was acquired on both the NovoCyte and competitor instruments. The staining index was measured on all instruments to quantify the resolution of CD34 detection. Stain index (SI) is a measure of the brightness of a fluorescent stain calculated by the separation of the positive and negative populations. The frequency of CD34+ cells (Figure 2A) and CD34 stain index (Figure 2B) are comparable on all instruments. Thus, the NovoCyte flow cytometer is an effective way to evaluate CD34 staining.

Enumeration of CD34+ cells on the NovoCyte Flow Cytometer

1. Philip T, Guglielmi C, Hagenbeek A, Somers R, Vanderlelie H, Bron D, et al. Autologous bone-marrow transplantation as compared with salvage chemotherapy in relapses of chemotherapy-sensitive non-Hodgkins-Lymphoma. N Engl J Med. 1995;333:1540–5. 2. Attal M, Harousseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JF, et al. A prospective, randomized trial of autologous bone marrow transplantation and chemotherapy in multiple myeloma. N Engl J Med. 1996;335:91–7. 3. To LB, Levesque JP, Herbert KE, Winkler IG, Bendall LJ, Hiwase DK, et al. Mobilisation strategies for normal and malignant cells. Pathology. 2011;43:547–65. 4. Brando B, et al. The “Vanishing Counting Bead” Phenomenon: Effect on Absolute CD34+ Cell Counting in Phosphate-Buffered Saline-Diluted Leukapheresis Samples. Cytometry. 2001;43:154–160. 5. Sutherland, D.R., Anderson, L., Keeney, M., Nayar, R., Chin-Yee, I. The ISHAGE guidelines for CD34+ cell determination by flow cytometry. J Hematother. 1996;3:213–226.

References

Competitor Instrument NovoCyte Stain IndexA B

Figure 2: Analysis of CD34+ cells in peripheral blood Human blood was analysed using the ISHAGE protocol on the NovoCyte as well as competitor’s instruments. Acquisition was done on ACEA NovoCyte 3000 or a competitors instrument (A). Stain Index was calculated for CD34 staining on each instrument (B). All data was analysed using the NovoExpress software.

Direct volumetric counting offers an alternative to cell counting with fluorescent counting beads, simplifying assay procedures. CD34+ enumeration was assessed with a NovoCyte flow cytometer and competitor instruments by reference counting beads and direct volumetric counting (Figure 3). CD34+ cells were quantified using a commercially available standard sample with a known concentration of CD34+ cells. The concentration of CD34+ cells was determined using fluorescent reference beads; all instruments determined the concentration within the threshold range established from the manufacturer. Next, direct volumetric counting of the standard was performed. Cytometers capable of volumetric counting (Cytometer 1 and NovoCyte) accurately determine the standard concentration. Cytometer 1 does determine cell concentration correctly by both methods, although on the lower end of the acceptable threshold. The absolute cell counts quantified by the NovoCyte are consistent with cell counts obtained by reference beads. Therefore, accurate CD34+ cell counts can be achieved using direct volumetric counting on the NovoCyte Flow Cytometer eliminating the need for expensive reference counting beads.

Figure 3: Enumeration of CD34+ cells using reference beads or direct volumetric cell counts Absolute numbers of CD34+ cells were quantified in standard of know concentration using fluorescent reference beads (left panel) or direct volumetric cell counting (right panel). Cytometer 2 did not have volumetric counting capabilities. Red lines represent a determined concentration within the acceptable range. Cell count is number of CD34+ cells/µL.

ConclusionCD34+ enumeration is a critical step for successful transplantation and it is essential to correctly calculate the number of CD34+ cells to be transferred to the recipient. Accurate enumeration of CD34+ cells in blood without the need for fluorescent reference beads can be done on the NovoCyte Flow Cytometer through the use of a high precision syringe pump for sampling to determine particle concentration. The NovoCyte provides an accurate, reproducible, and economical option for the enumeration of CD34+ cells.

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