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Published on: July 16, 2011
[Effect of RBC lysing solution on CD34(+) cell counting]
1Center of heamatological Diseases, Beijing Chlidren's Hospital, Capital University of Medical Science, Beijng 100045, China. gwylb2319@sina.com.cn
Zhongguo Shi Yan Xue Ye Xue Za Zhi
|June 22, 2010
Summary
The choice of red blood cell lysing solution significantly impacts CD34(+) cell counts. Different lysing reagents can alter flow cytometry results, necessitating careful selection for accurate cell enumeration.
Area of Science:
- Hematology
- Immunology
- Cell Biology
Background:
- Accurate enumeration of CD34(+) cells is crucial for stem cell transplantation and monitoring hematopoietic recovery.
- Red blood cell (RBC) lysis is a common pre-analytical step in flow cytometry analysis of peripheral blood apheresis products.
- The choice of RBC lysing solution may influence the integrity and detectability of target cells.
Purpose of the Study:
- To evaluate the impact of different commercially available and self-prepared RBC lysing solutions on the relative enumeration of CD34(+) cells.
- To assess the effect of RBC lysis on cell characteristics, including forward and side scatter (FSC and SSC) and CD45 expression.
Main Methods:
- Peripheral blood apheresis products from 37 patients were analyzed using flow cytometry.
- Cells were stained with CD34-PE and CD45-FITC antibodies.
- RBCs were lysed using two commercial solutions (FACS Lysing Solution, IOTest 3 Lysing Solution) and one self-prepared solution via a lyse-and-then-wash method.
- Analysis was performed using the ISHAGE gating strategy.
Main Results:
- The FACS Lysing Solution resulted in significantly lower CD34(+) cell percentages compared to the IOTest 3 Lysing Solution (0.50% vs 0.92%, p=0.004).
- No significant difference in CD34(+) cell counts was observed between the IOTest-treated and self-prepared solution groups.
- IOTest-treated samples showed higher FSC and SSC intensities and a lower proportion of CD45(+) cells compared to FACS-treated samples.
- White blood cell (WBC) count did not correlate with CD34(+) cell numbers.
Conclusions:
- The selection of RBC lysing solution critically affects the accuracy of CD34(+) cell enumeration by flow cytometry.
- Different lysing reagents can alter cell scatter properties and CD45 expression, potentially impacting gating strategies.
- Careful consideration and validation of RBC lysing reagents are essential for reliable FCM performance in clinical settings.
