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Correlation of hematopoietic progenitor cell count determined by the SE-9000тДв automated hematology analyzer with CD34+ cell count by flow cytometry in leukapheresis products.

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American Journal of Hematology 67:42–47 (2001)
Correlation of Hematopoietic Progenitor Cell Count
Determined by the SE-9000™ Automated Hematology
Analyzer With CD34+ Cell Count by Flow Cytometry in
Leukapheresis Products
Keon Uk Park,1* Sang Hee Kim,2 Cheolwon Suh,2 Shin Kim,2 Sun Jong Lee,2 Jung Sun Park,2
Hwa Jung Cho,2 Kang Wook Kim,2 Keehyun Lee,2 Hyo Jung Kim,2 Jinny Park,2
Young Joo Min,2 Jeong Gyoon Kim,2 Taewon Kim,2 Je Hwan Lee,2 Sung Bae Kim,2
Sang We Kim,2 Kyoo Hyung Lee,2 Jung Shin Lee,2 Woo Kun Kim,2 Chan Jeong Park,3 and
Hyun Sook Chi3
1
Department of Medicine, Dongguk University College of Medicine, Kyongju, Korea
Department of Medicine, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea
3
Department of Clinical Pathology, University of Ulsan College of Medicine, Asan Medical Center, Seoul, Korea
2
The yield of stem cell collection after mobilization is crucial for autologous peripheral
blood stem cell (PBSC) transplantation. Quantitative determinations of CD34+ cells using
flow cytometry or stem cell culture have been used, but these methods require much
time, technical experience, and expensive reagents. The automated hematology analyzer
(Sysmex SE-9000™, TOA, Japan) equipped with the Immature Information (IMI) channel
for immature myeloid cells can detect IMI+ cells within 90 sec. Detection is made possible
by the combination of a special reagent system and direct current/radiofrequency biosensors. We studied the relation of IMI+ cells and variable cell counts with CD34+ cell yield
in autologous stem cell harvest. In a series of 32 patients (median age, 44 years; M:F =
11:21), 184 leukaphereses were performed after mobilization regimens with chemotherapy and G-CSF or G-CSF alone. Full blood cell counts were enumerated on peripheral
blood (PB) samples taken prior to each leukapheresis. Mononuclear cell (MNC) and IMI+
cell counts by automated hematology analyzer and flow cytometry based CD34+ cell yield
were measured on the harvested product. The relationship among PB white blood cells
(WBC), PB monocytes, IMI+ cells, MNC, and CD34+ cell yield in a single leukapheresis was
estimated by Pearson correlation analysis. PB WBC count showed no correlation with
CD34+ cell yield in a single leukapheresis (r = 0.02, P = 0.81). PB monocyte count showed
a weak correlation (r = 0.21, P = 0.01) and MNC in harvest also showed a weak correlation
(r = 0.36, P = 0.0001) with CD34+ cell yield. In contrast, CD34+ cell yield correlated well with
IMI+ cell count (r = 0.68, P = 0.0001), and data could be fitted by a linear regression
equation, y = 0.330 + 0.974x. IMI+ cell assay by the automated hematology analyzer
correlated well with the CD34+ cell yield in a mobilized autologous stem cell harvest. The
IMI+ cell count might be used as a simple and efficient indicator of blood stem cell
mobilization and collection. Am. J. Hematol. 67:42–47, 2001.
© 2001 Wiley-Liss, Inc.
Key words: peripheral blood stem cell; flow cytometry; CD34+ cells; automated hematology analyzer
INTRODUCTION
Peripheral blood stem cell (PBSC) transplantation is
now used extensively to provide rapid and durable hematopoietic reconstitution after high-dose chemotherapy
for malignant disease [1,2]. Mobilization techniques include the administration of hematopoietic growth factor
after chemotherapy [3]. It is widely accepted that the
CD34+ cell count is generally a good predictor of the rate
of engraftment. A number of studies have demonstrated
© 2001 Wiley-Liss, Inc.
that reinfusion of a stem cell dose of ⱖ5 × 106 CD34+
cells/kg increases the probability of rapid engraftment,
leading to reduced use of supportive measures, short hos*Correspondence to: Keon Uk Park, M.D., Department of Medicine,
Dongguk University College of Medicine, 1090-1 Sukjang-Dong,
Kyongju, Kyongbuk 780-350, South Korea.
E-mail: kupark@dumc.or.kr
Received for publication 10 March 2000; Accepted 4 October 2000
Technique: Correlation of Hematopoietic Progenitor Cell Count
pital stays, and decreased costs [4]. Although the yields
of PBSC after mobilization are crucial for engraftment,
no conventional method to identify the presence of PBSC
in the recovered mononuclear cell fraction has been
available. Quantitative CD34+ analysis performed by
flow cytometry is used as a guide to determine a target
number of progenitor cells needed for timely hematopoietic recovery. The best established method to identify the
quantity of PBSC is CFU-GM assay [5]. However, these
methods require time (flow cytometry, 2 hr; CFU-GM
assay, 14 days), technical expertise, and expensive reagents.
The automated hematology analyzer (Sysmex SE9000, TOA Medical Electronica Co. Ltd, Japan) is
equipped with a special detection unit for immature
white blood cells (WBC), called the Immature Information (IMI) channel. Detection of immature WBC is made
possible by the combination of a special reagent system
and direct current/radiofrequency biosensors. The reagent system contains detergents, which are capable of
lysing more mature white blood cells because of their
higher membrane lipid content while immature cells remain relatively intact. Because various types of immature
WBC react differently to the reagent, they also occupy
distinct areas on the bivariate matrix of the IMI scattergram. Thus it is possible to distinguish blast cells from
less immature WBC and, in turn, immature granulocytes
such as myelocytes and metamyelocytes from left-shifted
neutrophils [6,7]. This technique, which does not require
any pretreatment of the blood sample or specific operator
skills, can be carried out with a 90-sec turnaround time to
completion. We study here the correlation of IMI+ cell
count determined by the SE-9000™ automated hematology analyzer with CD34+ cell by flow cytometry in
PBSC harvesting products. Also, the relationships of
WBC and monocyte counts in peripheral blood with
CD34+ cells by flow cytometry were evaluated.
MATERIALS AND METHODS
Patients
Between December 1998 and October 1999, 32 patients [breast cancer, 14; non-Hodgkin’s lymphoma
(NHL), 11; multiple myeloma (MM), 5; ovarian cancer,
1; and medulloblastoma, 1] following at our institution
underwent PBSCs harvesting after chemotherapy: either
cyclophosphamide alone (2–4 g/m2) on Day 1 or other
specific tumor-oriented chemotherapies. After chemotherapy, the first dose of G-CSF (5–10 ␮g/kg/day) was
given on Day 7 or 8. All patients received the dose of
G-CSF administered subcutaneously until the last harvesting day of PBSC. PBSC collection was started when
white cell (WBC) recovery reached 10 × 109/l or the
monocyte count reached 1 × 109/l. Leukocyte subsets
43
were monitored frequently (every 1–3 days) from the
start of mobilization until the end of the leukapheresis
procedure.
Methods
Leukapheresis was performed with continuous flow
blood cell separator (Fenwal CS-3000 plus, Baxter,
USA). Venous access was established by central venous
catheter. Anticoagulant, consisting of heparin at 10 U per
ml of ACD-A, was infused at a ratio of 1 ml of anticoagulant to 30 ml of whole blood. Inlet flow rate was
maintained at 50–80 ml per minute in the small-volume
leukapheresis procedure and 80–100 ml per minute in the
large-volume leukapheresis procedure. The collection
rate was maintained at 1–2 ml per minute. A set volume
of 14 l per leukapheresis was used. The criteria for adequate PBSC collection was a target number 3 × 106
CD34+ cells/kg. Leukapheresis was continued daily in an
attempt to achieve that goal.
The leukocyte count in the sample was determined
with an automated hematology analyzer (Sysmex SE9000, TOA Medical Electronica Co. Ltd, Japan). Differential counts were done microscopically on Giemsastained cell smear. The mononuclear cell count was
obtained by multiplying the number of leukocyte with
the sum of the percent of lymphocytes and monocytes
from the differential count. The CD34+ cells were enumerated by flow cytometry (FACScan威 Becton Dickinson, Fullerton, CA). Leukapheresis products were used
without Ficoll-Hypaque centrifugation. The cell suspensions were stained with the following antibody combination5: CD34 FITC + CD14 PE and clgG1 FITC (negative isotope control) + CD14 PE. In addition, a sample
was stained with CD45 FITC as a marker for leukocytes.
For staining, 30 ␮l of cell suspension was incubated with
each 20 ␮l of the monoclonal antibody combinations.
After being washed, the remaining red blood cells were
lysed by adding 1 ml of ammonium chloride lysis buffer
for 6 min at room temperature in the dark. Then, the cells
were washed, resuspended and examined with a FACScan flow cytometer (Becton Dickinson). They were then
analyzed by the FACScan research software (Becton
Dickinson).
The detection of immature granulocyte is possible
through the IMI channel with the automated hematology
analyzer (Sysmex SE-9000, TOA Medical Electronica
Co. Ltd, Japan). The lysis reagent used for the IMI channel (Stromatolyser-IM) contains a polyoxyethyleneseries nonionic surfactant and sulfur-containing amino
acid, both for fixation of blood cell cytoplasm and membrane, and an anionic surfactant for reduction of erythrocyte ghosts by damaging the red cell membrane. In the
IMI channel, the polyoxyethylene nonionic surfactant
first damages blood cells, with different degrees of dam-
44
Technique: Park et al.
age to different blood cell types. The normal mature
granulocyte contains lipids with an amount about 2 times
greater than that in the lymphocyte. This is because the
lipid content is nearly proportional to the cell size. For
both the normal mature granulocyte and lymphocyte, the
phospholipid content is about 35% and the cholesterol
count is about 10%. In contrast, the immature granulocyte has a lower cholesterol content than the mature
granulocyte, and its phospholipid composition has a relatively higher ratio of phosphatidylcholine and a lower
ratio of sphingomyelin. The difference in the degree of
damage among the blood cell types is attributable to the
difference in lipid contents and compositions. When exposed to polyoxyethylene nonionic surfactant, the mature
granulocytes cell membrane becomes damaged, resulting
in an exposed nucleus due to elution of its intracellular
components. The juvenile granulocytes cell membrane
also becomes damaged; however, before its intracellular
components are eluted, the polyoxyethylene nonionic
surfactant and sulfur-containing amino acid enters it
through its damaged cell membrane site and will eventually fix its cell membrane and intracellular components. In this process, the sulfur-containing amino acid
acts as a protector for the cell against the action of the
surfactant. The juvenile granulocyte is thus fixed while
retaining the intact cell membrane and cytoplasm. The
cationic surfactant then reduces erythrocyte ghosts and
mature leukocyte size, facilitating discrimination of juvenile granulocyte from erythrocyte ghosts and mature
leukocytes with exposed nucleus, which are then differently measured.
Statistical Analysis
The clinical and laboratory datas were retrieved from
the transplant data and analyzed using the SAS system
(SAS Institute, Cary, NC). The relationship between the
number of CD34+ cells and IMI+ cells was estimated by
linear regression and correlation analysis. Also, the relationships with WBC and monocyte count in peripheral
blood and harvest products were evaluated. The Pearson
rank correlation was used to evaluate the relationships. A
significant level of P < 0.05 was chosen. The receiver
operating characteristic (ROC) curve was used to set the
cutoff point of IMI+ cells when more than 1 × 106 CD34+
cells/kg were collected in leukapheresis product.
RESULTS
Patients’ Characteristics
Table I presents patients characteristics. A total of 184
PBSC components were collected from 32 patients
(male:female ⳱ 11:21). The median age was 44 years.
The median number of aphereses was 5 (range, 3 to 9).
TABLE I. Characteristics of Patients
Median age (range)
Sex (male/female)
Diagnosis
Breast cancer
NHL
MM
Medulloblastoma
Ovarian cancer
Mobilization method
Chemotherapy + G-CSF/GM-CSF
G-CSF/GM-CSF only
Numbers of leukaphereses (total)
Breast cancer
NHL
MM
Medulloblastoma
Ovarian cancer
44 (18–65)
11/21
14
11
5
1
1
30
2
184
111
49
21
3
10
The median count of collected CD34+ cells was 4.04 ×
106 cells/kg.
The 11 non-Hodgkin’s lymphoma patients were classified in accordance to the International Lymphoma
Study Group classification. Three patients had diffuse
large B-cell lymphoma, 2 had follicular center lymphoma, 3 had lymphoblastic lymphoma, 1 had angioimmunoblastic lymphoma, 1 had peripheral T cell lymphoma, and 1 had Burkitt’s lymphoma. The median
number of leukaphereses was 3.5 (range, 3 to 7). The
median count of collected CD34+ cells was 3.88 × 106
CD34+ cells/kg.
Five multiple myeloma patients received four cycles of
the vincristine–doxorubicine–dexamethasone (VAD) regimen before mobilization. The median number of leukaphereses was 3 (range, 2 to 6). The median count of collected
CD34+ cells was 6.18 × 106 cells/kg. One medulloblastoma and one ovarian cancer patient were included.
Correlation Between Peripheral Blood WBC,
Monocytes, MNC in Apheresis Products, and
CD34+ Cell Yield
Most patients in our study underwent serial procedures, and the median WBC count in the blood for patients treated with a chemotherapy-containing regimen
was 16.0 × 109/l (range, [1.0–55.5] × 109/l). The median
number of CD34+ cells in a leukapheresis product was
0.37 × 106 CD34+ cells/kg. The WBC counts in peripheral blood (PB) showed poor correlation with CD34+ cell
yield (r ⳱ 0.02, P ⳱ 0.81) (Fig. 1). The median monocyte counts in the peripheral blood was 0.63 × 109/l
(range, [0–4.2] × 109/l). The PB monocyte counts
showed a weak correlation with CD34+ cell yield (r ⳱
0.21, P ⳱ 0.01) (Fig. 1). The median MNC in a leukapheresis product was 1.37 × 108 cells/kg (range, [0.14–
3.63] × 108 cells/kg). There was also a weak correlation
between MNC in leukapheresis product and CD34+ cell
yield (r ⳱ 0.36, P ⳱ 0.0001) (Fig. 2).
Technique: Correlation of Hematopoietic Progenitor Cell Count
45
Fig. 1. (A) Relationship between the peripheral blood WBC (X axis) and CD34+ cells × 106 per kg of patient in apheresis
products (Y axis). (B) Relationship between the peripheral blood monocyte (X axis) and CD34+ cells × 106 per kg of patient
in apheresis products (Y axis).
Fig. 2. (A) Relationship between the mononuclear cell (MNC) × 108 per kg of patient (X axis) and CD34+ cells × 106 per kg
of patient in apheresis products (Y axis). (B) Relationship between the IMI+ cell counts (X axis) and CD34+ cells × 106 per
kg of patient in apheresis products (Y axis). The relationship is described by the regression equation y = 0.330 + 0.974x.
Correlation Between IMI+ Cells and CD34+
Cell Yield
The median IMI+ cell count was 0.32 × 109/l (range,
[0–14.72] × 109/l). A significant correlation between the
IMI+ cells and CD34+ cells in a leukapheresis product (r
⳱ 0.68, P ⳱ 0.0001) was found, and the data could be
fitted into a linear regression equation, y ⳱ 0.330 +
0.974x (Fig. 2). To set the cutoff point, we used the
receiver operating characteristic (ROC) curve. The ROC
curve graphically portrays the trade-offs involved between either a test’s sensitivity or its specificity. An ideal
test is one that reaches the upper left corner of the graph
(100% sensitivity and 100% specificity). We graphed
sensitivity as a function of [1 − specificity]; this latter
quantity is sometimes called the false-positive rate. The
ROC curve showed that the best cutoff point for high
sensitivity and specificity was 0.66 × 109/l (Fig. 3).
When we selected the cutoff value as 0.7 × 109/l, the
sensitivity and specificity to obtain more than 1 × 106
CD34+ cells/kg were 83.3% and 83.1% (Table II), respectively.
DISCUSSION
To make PBSC transplantation a cost effective procedure, it is necessary to optimize the conditions for priming, collection, storage, and engraftment of the leukapheresis products. Cytotoxic chemotherapy with or
without hematopoietic growth factors has been used to
mobilize hematopoietic progenitors into the peripheral
blood, and it is known that chemotherapy and growth
factor act synergistically to increase the number of hematopoietic progenitors [11]. The more advanced the
mobilization schemes become, the more important are
the precise determination of the optimal timing and frequency for harvesting. Through optimal harvesting time
prediction, it is possible to spare the patient from poten-
46
Technique: Park et al.
Fig. 3. Receiver operating characteristic (ROC) curve for
IMI+ cell counts to obtain more CD34+ cells in apheresis
products.
TABLE II. Possibility of Obtaining More Than 1 × 106 CD34+
Cells/kg of Patient in Apheresis Products When Selected
Cutoff Point Is 0.7 × 109/l.
ⱕ0.7 × 109 IMI+ cell/l
>0.7 × 109 IMI+ cell/l
ⱕ1 × 106
CD34+ cells/kg
>1 × 106
CD34+ cells/kg
103 (83.06%)
21 (16.94%)
9 (16.67%)
45 (83.33%)
tial complications of leukapheresis and reduce treatment
costs. The optimal timing and frequency for leukapheresis have not been established on a well-designed study
base. It is difficult to make a decision on when leukapheresis should be started and how many times it will
be done. Many collection centers use WBC or monocyte
count in peripheral blood as a predictor for the timing of
leukapheresis, delaying collection until the WBC count
exceeds 3 × 109 or 5 × 109 or even 10.0 × 109/l [13–15].
As shown in this study, our center started leukapheresis
when WBC recovery reached 10 × 109/l or the monocyte
count reached 1 × 109/l. Based on our analysis, WBC or
monocyte counts could not predict the number of CD34+
cells in the leukapheresis products. The minimum threshold of CD34+ cells is achieved in most patients, but an
optimal collection usually requires at least three harvests.
Prior chemotherapy might adversely affect mobilization.
Optimal timing of collections is important, because as
few as two leukapheresis procedures are enough to obtain an adequate progenitor cell dose.
We retrospectively analyzed the datas from 184 PBSC
collections to study the predictive value of IMI+ cell
count in the peripheral blood.
The pluripotent stem cell has yet to be identified and
isolated positively. Indirect laboratory assays provides
limited information about the hematopoietic potential of
collected hematopoietic progenitor cells. Mononuclear
cell numbers, although easily determined, are not reliable, and colony culture assays, although reliable, remain
poorly standardized, time-consuming, and are difficult to
interpret for clinical purposes. Because culture methods
cannot be evaluated for 2 weeks or longer, these assays
are generally most helpful for retrospective analysis or
for quality control of peripheral blood stem cell collection. Quantitative CD34 analysis may be quickly performed by flow cytometry. A number of CD34+ cell
counts and CFU-GM are closely correlated, and the number of CD34+ cells in the leukapheresis components is
associated with engraftment kinetics [8–10]. Quantitative
CD34+ cells analysis showed a significant positive correlation with CFU-GM, and the number of infused
CD34+ cells correlates with hematopoietic recovery [12].
CD34+ cell counts by flow cytometry are not standardized and usually are not available for the next 24 hrs. In
addition, flow cytometry measurements have required at
least 2 hr and expensive reagents. It has caused prolonged apheresis and placed economic burdens on patients.
It is possible that hematopoietic progenitor cells were
included in IMI+ cells that were simply detected by an
automated hematology analyzer (Sysmex SE-9000, TOA
Medical Electronica Co. Ltd, Japan) with a usual detection technique, and the total infused IMI+ cells, like the
CD34+ cell count, also provides a useful indication of
recovery of hematopoietic function in PBSC transplantation. The pluripotent stem cell, capable of differentiation as well as self-renewal and ultimately responsible
for all hematopoietic function, is currently presumed to
be present in small numbers primarily in the bone marrow. We studied the possibility of a simple and easy
identification of the stem cells in leukapheresis products
using a conventional automated blood cell counter with
the function of white cell differentiation. The SE-9000
(TOA Medical Electronics Co. Ltd, Japan) analyzes the
five normal WBC populations using radiofrequency (RF)
and direct current (DC) detection methods, as well as
separating of eosinophil and basophil channels. The IMI
channel method reduces the waiting time for analysis
results of stem cells: from 2 hr with flow cytometry down
to 90 sec. The analysis can be performed on a routine
hematology analyzer, no special operator skills are required, and the results are available anytime of the day.
No special pretreatment of the sample is required except
when the total leukocytes concentration exceeds 5 × 109/
l, and then a simple dilution step is required. The test may
be used to determine the appropriate timing for leu-
Technique: Correlation of Hematopoietic Progenitor Cell Count
kapheresis and may also be used to monitor the effectiveness of hematopoietic progenitor cell harvest procedures while in progress. To improve the timing of
leukapheresis, we are studying the correlation between
the IMI+ cell count in peripheral blood and CD34+ cell
yield in leukapheresis products. The information provided by the IMI channel appears to be useful in determining the optimal timing of PBSC harvest because it
includes semiquantitative datas.
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