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Journal of Appplied Hematology 2010

108
H
ematopoietic stem cells (HSCs) and hemo-
poietic progenitor cells (HPCs) obtained
from either the bone marrow (BM) or mo-
bilized peripheral blood (PB) are used for autologous
transplantation after high-dose chemotherapy in pa-
tients with hematological malignancies. These cells
are characterized by the expression of the adhesion
receptor CD34. The clinical value of determination
of the accurate absolute numbers of CD34+ cells
is the most important parameter for evaluating stem
and progenitor cell content in hematopoietic trans-
plantation. Flow cytometric enumeration of CD34+
cells has become widely accepted as the technique of
choice to quantify HSCs for the clinical management
Hemopoietic Progenitor Cell (HPC)
Enumeration: A Comparitive Study between
Flow Cytometry and Sysmex XE-2100
Hematology Analyzer
Salem H. Khalil, Barbara Geng
From the Hematology Section
Department of Pathology and Laboratory Medicine
King Faisal Specialist Hospital and Research Centre
Riyadh, Saudi Arabia
Corresponding author:
Salem H. Khalil, MD, FRCPA, FCAP
Consultant Hematopathologist
Assistant Professor, Al Faisal University
Department of Pathology and Laboratory Medicine
(MBC-10)
King Faisal Specialist Hospital and Research Centre
P. O. Box 3354, Riyadh 11211
Kingdom of Saudi Arabia
T: +966-1-4424296
F: +966-1-4424280
khalil@kfshrc.edu.sa
The decision to harvest peripheral blood stem cells (PBSCs) is frequently
based on the enumeration of CD34+ cells by flow cytometry by using either
single- or dual-platform methods. The aim of this study at the Department
of Pathology and Laboratory Medicine of King Faisal Specialist Hospital and
Research Centre (General Organization) was to evaluate an alternative meth-
od for circulating hemopoietic stem cell quantification, namely by means of
the hemopoietic progenitor cell (HPC) parameter generated by the Sysmex
XE-2100 automated hematology analyzer available in the Hematology Section.
Two hundred and ninety two (292) samples of peripheral and cord blood were
analyzed using single-platform flow cytometry and the immature myeloid in-
formation (IMI) parameter of the Sysmex XE-2100 hematology analyzer in
parallel runs.
The objective of this study was to minimize the number of CD34 determina-
tions routinely performed by flow cytometry. A strong correlation was found
between HPC and CD34+ in peripheral blood [r
2
(0.692), P value <0.0001]
and cord blood [r
2
(0.555), P value <0.0001]. Therefore, with HPC levels below
the detection limit of 00/mm
3
or count of >30/mm
3
, it is reasonable not to
await the CD34+ results before harvesting. These two situations account for
more than 66% of the CD34+ determinations carried out by flow cytometry
in our laboratory.
KEYWORDS: HPC, Stem cell, Sysmex XE-2100, Flow cytometry
of stem cell transplantation. Until recently, absolute
CD34+ cell counts were generally derived from the
so-called two-platform analysis in which the percent-
age of CD34+ cells is frst enumerated by fow cy-
tometry against the total CD45+ cells as the denomi-
nator. The white blood cell (WBC) derived from the
hematology analyzer to generate the absolute CD34+
cell count then multiplies this percentage of CD34+
cells. The single-platform method, which utilizes
TruCount absolute-count tubes, reduces variability by
eliminating the need for the cell count from an auto-
mated hematology analyzer.
1-4
The aim of this study
was to validate an alternative method for circulating
HSC quantifcation, namely by means of the HPC pa-
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Journal of Applied Hematology 2010
109
rameter generated by the Sysmex XE-2100 automated
hematology analyzer.
Materials and Methods
Patients and study design
Patients were included in the study provided the HPC
and CD34 enumeration were determined simultane-
ously at least on the presumed day of the harvest
and in some cases, on the days before. A total of 292
samples were analyzed using single-platform fow cy-
tometry and the immature myeloid information (IMI)
parameter of the Sysmex XE-2100 hematology ana-
lyzer. These included 77 peripheral blood samples of
patients with different types of hematological malig-
nancies and 215 samples of cord blood.
Counting Methods
The CD34+ cells were quantifed using a single-plat-
form fow cytometry procedure at the Hematology
Section, Department of Pathology and Laboratory
Medicine, King Faisal Specialist Hospital and Research
Centre (General Organization), Riyadh, Saudi Arabia.
The procedure uses TruCount [Beckton Dickinson
(BD)] kits on a four-color FACScalibur (BD) fow cy-
tometer by a single-platform method (Figures 1 and 2).
The HPCs are identifed in the IMI-HPC area of
the IMI channel of the Sysmex XE-2100 on the basis
of their resistance to the lysing reagent, their volume
(direct current), and their internal structure (radio-
frequency). HPCs, like all immature cells, are resistant
to the lytic reagent used and are located within a spe-
cifc gated area of the scattergram.
Results
All data were entered properly coded into prepared
Microsoft Excel 2007 worksheets. Data were exam-
ined for competencies and accuracy prior to export-
ing to Statistical Package for Social Sciences, Volume
16 (SPSS, Chicago, Illinois, USA) for statistical analy-
sis. Using Spearmans rank test, a strong correlation
was found between HPC and WBC [r
2
(0.395), P val-
ue=0.0004] Figure 3 and between HPC and CD34+
in peripheral blood [r2 (0.692), P value <0.0001] and
cord blood [r
2
(0.555), P value <0.0001] Figures 4
and 5. Up to 45.45% of samples showed HPC levels
of more than 30/mm
3
and 20.78% with HPC levels
below detection limits (00/mm
3
), representing more
than 66% of the samples studied. However, the levels
of HPC ranging between 0/mm
3
and 30/mm
3
have
been detected in up to 33.77% of samples.
Figure 1. Single-platform CD34 enumeration in peripheral
blood.
Figure 2a. Single-platform CD34 enumeration in cord blood
pre-gradient volume reduction protocol (Pre-GVRP).
Figure 2b. Single-platform CD34 enumeration in cord blood
post-gradient volume reduction protocol (Post-GVRP).
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110
Figure 3. Correlation between white blood cell (WBC) and hemopoietic progenitor
cell (HPC) in peripheral blood. r
2
(0.395), P value=0.0004.
Figure 4. Correlation between CD34+ and hemopoietic progenitor cell (HPC) in
peripheral blood. r
2
(0.692), P value <0.0001.
Figure 5. Correlation between CD34+ and hemopoietic progenitor cell (HPC) in
cord blood. r2 (0.555), P value <0.0001.
Discussion
HSCs can be mobilized from the bone marrow to the
peripheral blood by chemotherapy, by a combination
of chemotherapy and hematopoietic growth factors,
as well as by hematopoietic growth factors alone.
Today, mobilization is performed with chemotherapy
in combination with growth factors, since it provides
for the most effcient stem cell mobilization, in addi-
tion to the eradication of malignant cells.
The stem cell yield may be increased up to 100
times with the application of a growth factor [e.g.,
granulocyte colony-stimulating factor (G-CSF), gran-
ulocyte macrophage colony-stimulating factor (GM-
CSF)]. The administration of a growth factor alone,
without pre-chemotherapy, e.g., with healthy alloge-
neic donors, has the advantage of exactly planning
the time of apheresis.
An accurate determination of the available amount
of stem cells before transplantation will be required.
This will be done routinely by the number of CD34+
cells measured by different methods of fow cytom-
etry.
5-6
A special parameter of HPC cell quantifcation per
microliter of blood was developed in the IMI chan-
nel of the Sysmex XE-2100 hematology analyzer.
The objective was to provide an alternative method
of the daily CD34+ monitoring by fow cytometry,
which was not only costly but also entailed great ex-
penditures in time and personnel. The IMI channel
of the Sysmex system lyses mature and erythropoietic
cells by means of a special lyse reagent. The remain-
ing immature cells will be two-dimensionally analyzed
by volume and intracellular structure. Cells in the area
of low volume and a reduced plasma/nucleus relation
are detected as HPC cells and analyzed using a special
HPC software program.
7

Since the introduction of the HPC software in
1997, many studies of HPC determination versus
CD34+ cell enumeration have been performed and
reported with different levels of correlation.8-14
From this study, HPC determination has been shown
to be helpful in two situations. First, during the fol-
low-up of patients after mobilization, the CD34+
count does not need to be assessed before HPCs are
detectable. Second, if the HPC count is >30/mm
3
, it
is reasonable not to await the CD34+ results before
harvesting.
These two situations account for more than 66%
of the CD34+ determinations carried out by fow cy-
tometry in our daily practice.
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References
Acknowledgments
The authors would like to thank the Flow cytometry
staff (Mr. Faisal Rawas and Ms. Ameena Saifaldeen) at
King Faisal Specialist Hospital and Research Centre,
Riyadh for their technical support and also Dr.
Mohamed Shoukri, PhD for statistical analysis.
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