Flow cytometric enumeration of CD34+ hematopoietic stem cells: A comparison between single- versus dual-platform methodology using the International Society of Hematotherapy and Graft Engineering protocol

Abstract

BACKGROUND: Flow cytometric enumeration of CD34+ hematopoietic stem cells (HSC) is the reference point for undertaking apheresis and evaluation of adequacy for peripheral blood stem cell (PBSC) engraftment. AIMS: To determine whether single platform correlates with dual platform methods in CD34+ enumeration using ISHAGE protocol. METHODS: Retrospective analysis of CD34 Enumeration assays on both peripheral blood and PBSC product samples using Beckman Coulter FC500 Flow Cytometer. The t test and correlation study was used to study the difference between single and dual platform methods in CD34+ enumeration. RESULTS: We present our data on 152 samples comprising 41 peripheral blood samples collected before apheresis procedure and 111 samples collected from PBSC product. We observed strong positive correlation between single and dual platform methods for CD34+ counts in peripheral blood sample ( r = 0.92; P < 0.001) and PBSC product sample ( r = 0.85; P < 0.001). CONCLUSION: In our study, both single versus dual platform had similar results in CD34+ cell counts. The single platform provides rapid results with ease of procedure. Errors with dual platforms are relatively common with respect to denominator. We recommend to use mean of total leukocyte count from two different hematology analyzer to minimize variation in dual platform.

Determination of mononuclear cell count using peripheral smear and flow cytometry in peripheral blood stem cell products

“…As single-platform assays have the potential to be more effective in reducing interlaboratory variation than dual-platform techniques in CD34 enumeration, determining MNC count through CD34 enumeration plot by flow cytometry shall provide instant value with ease of procedure. [ 3 6 ] As absolute MNC count in harvest was an easily accessible and alternative variable to assess the adequacy of hematopoietic stem cell collection, it gains importance in centers where CD34+ cell enumeration was outsourced. The study observed a significant difference between MNC doses estimated between these two methods ( P = 0.047) [ Table 2 ].…”

“…[ 1 ] Hematopoietic stem cells are identified by dim CD45 expression/low side scatter and CD34+ population using sequential Boolean gating strategy in flow cytometry. [ 2 3 ]…”

Standard operating Protocol (SOP) of Haematopoietic Stem Cells Enumeration

“…This cell surface markers are mostly glycoproteins referred to as clusters of differentiation (CD) markers which aid in differentiating cell subpopulations [2], Example CD44 + , CD38 -, CD38 -CD90 + (thy-1), CD117 + (c-kit), CD34 + and CD45 + CD49f + and lineage markers (CD14 -, CD8 -) This report describes the SOP for enumeration of HSCs in PB in other to evaluate the patient need for transplantation, which is dependent on donor and recipients matching, graft-versushost response or leukaemia effect [3]. Accurately, enumerating HSCs will assist in the subsequent plans for apheresis collection [4].…”

Non-Classical Effects of the cAMP Accumulation Activators In Vivo

“…Blood samples were taken from the tail vein on days 1, 10, 30 and 60. The proliferative activity of CD34 + stem cells was studied using a CD34 Countkit 24,25 kit for cytofluorimetry via a FACS Calibur flow cytofluorometer. The digital material was processed using the analysis of variance, Student's/ Bonferroni modification (Figure 1).…”