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Heterogeneity of peripheral blood reticulocytes A flow cytometric analysis with monoclonal antibody HAE9 and thiazole orange.

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American Journal of Hematology 38:61-63 (1991)
Heterogeneity of Peripheral Blood Reticulocytes :
A Flow Cytometric Analysis With Monoclonal
Antibody HAES and Thiazole Orange
Eugene B. Mechetner, Daniel D. Sedmak, and Rolf F. Barth
Department of Genetics, The University of Illinois at Chicago, Chicago (E.B.M.); and Department of Pathology, Ohio State University,
Columbus, OH (D.D.S., R.F.B)
The expression of a human erythroid cell surface antigen recognized by monoclonal
antibody (mAB) HAES has been studied on peripheral blood reticulocytes by one- and
two-color flow cytometry. Total reticulocyte count was determined using Thiazole Orange
(TO) and flow cytometry. In normal individuals, 4.56% of reticulocytes were stained by
FITC-labeled mAB HAE9. The correlation between reticulocyte percentage by TO and
HAES staining was 0.828 (P < 0.0001) in patients with hematocrits less than 0.25. A
HAE9-positive reticulocyte percentage of 6 4 4 % was observed when analyzed by
two-color flow cytometry with TO and mAB HAE9. These findings, in conjunction with
previous studies, suggest that mAB HAES recognizes an early, less differentiated
population of peripheral blood reticulocytes. Enumerationof immature reticulocytes may
be of clinical utility.
Key words: reticulocytes, membrane antigens, flow cytometry
INTRODUCTION
ascitic fluid as described previously [3] and conjugated to
fluorescein isothiocyanate (FITC) according to [ 5 ] .The
In previous reports [1,2,3], an inter-species 70 kD
ratio of fluorescein to protein absorbance was 0.6.
membrane glycoprotein specific for mammalian erythSpecificity of the conjugate was confirmed by direct IF
roid cells, in particular, erythroblasts (Ag-Eb) was deflow cytometry of human adult bone marrow, umbilical
scribed. MAB HAE9 directed against a human epitope of
cord, blood, and erythroleukemic K562, HEL, and
Ag-Eb is reactive with 36% of late committed erythroid
SPI-801 cell lines. IF staining was verified by microprogenitors (CFU-E), almost 100% of proerythroblasts,
scopic examination and compared to indirect IF staining
erythroblasts, and normoblasts. Normal stem cells (CFUwith pure mAB HAE9 and secondary goat anti-mouse
GEMM), early erythroid progenitors (BFU-E) as well as
IgM (Sigma) at a dilution of 1: 100. 5 p1 samples of fresh
non-erythroid cells do not react with mAB HAE9 [3].
blood were washed 2 times by phosphate-buffered saline
Recently, we have used this mAB as an anti-erythroid
(PBS) and stained by HAE9-FITC conjugate ( 5 kg of
probe for immunophenotyping human leukemias [4].
antibody in 100 pl of PBS with 1% bovine serum
However, the expression of Ag-Eb on reticulocytes has
albumin, BSA) for 30 min at room temperature. After
not been studied in detail. The aim of this study was to
two additional washings, cells were resuspended in PBS
investigate reactivity of mAB HAE9 with peripheral
blood reticulocytes.
Received for publication November 29, 1990; accepted April 4, 1991.
MATERIALS AND METHODS
To reduce non-specific binding and provide greater
sample accuracy, direct immunofluorescent (IF) flow
cytometry was used for evaluation of reticulocyte staining. MAB HAE9 (mouse IgM, kappa) was purified from
0 1991 Wiley-Liss, Inc.
Address reprint requests to Eugene B. Mechetner, Ph.D., Department
of Genetics (MIC 669), University of Illinois at Chicago, 808 South
Wood Street, Chicago, IL 60612.
The work was performed at the Department of Pathology at the Ohio
State University.
62
Case Report: Mechetner et al.
1.0 and analyzed by flow cytometry. Staining of reticuloN=35
cytes by irrelevant anti-Leu-7 (IgM) FITC-labeled antii
r=0.828
c
body (Becton Dickinson, Mountain View, CA), at the
p~0.0001
same concentration, was shown in preliminary experiments to be an adequate non-specific control for HAE9FITC. Nonspecific control values were subtracted from
values obtained for HAE9-FITC. Negative controls,
which consisted of incubation with PBS-BSA (autofluorescent background) and/or polyvalent FITC-labeled antisera, were also used in some experiments. Total reticulocyte counts were determined by flow cytometric
0
1
2
3
4
5
6
analyses [6] with TO (Retic-COUNT, Becton Dickinson,
Reticulocytes, X
Mountain View, CA) at room temperature for 30 min.
For two-color flow cytometry, 5 p1 peripheral blood Fig. 1. Correlation between the percentage of HAES-posisamples were stained by pure mAB HAE9 (5 pg/lOO pl tive reticulocytes and the total reticulocyte percentages
of PBS-BSA), then treated by PE-labeled rat anti-mouse determined by TO.
kappa antibody (Becton Dickinson, Mountain View, CA)
20 pl in 100 pl of PBS-BSA, and, finally, incubated with
TO (4 samples studied). All incubations were performed
at room temperature for 30 min. Before each step, the
samples were washed twice with PBS. Similar incuba- DISCUSSION
tions with 1) irrelevant anti-Leu-7 FITC-labeled antiMAB HAE9 is highly reactive with human nucleated
body, 2) TO, 3) mAB HAE9 PE-labeled secondary red cells [3]. The results of this study indicate that this
antibody, and 4) PBS-BSA PE-labeled secondary an- antibody binds to a small proportion (5-10%) of periphtibody were used as controls for auto- and non-specific eral blood reticulocytes. This finding, in conjunction
green and red IF. An Epics C flow cytometer (Coulter with previous studies, suggests that mAB HAE9 recogCorp., Hialeah, FL) with Reticulocyte Enumeration and nizes immature reticulocytes which are immediately
STAT software which excluded nucleated blood cells post-normoblasts in erythroid differentiation. Recently,
from analysis, was used. IF was determined on a similar pattern of reactivity has been observed with
minimum of 50,000 cells using a 256 channel log scale.
another mAB directed against differentiating red cells (81.
Samples were obtained from the Hematology Laboratory
The enumeration of early reticulocytes may be of
at the Ohio State University Hospital. Normal samples
clinical significance. Davis et al. [9] has shown that a
were defined as those with normal age, sex values for
reticulocyte maturity index proportional to the amount of
hematocrit, hemoglobin, red cell count, and white blood
RNA in reticulocytes, but not the reticulocyte percentage
cell count [7]. For correlation studies, samples with low
or absolute counts, was the earliest predictor of bone
hematocrit value (<O. 25) were randomly chosen from
marrow engrafment in bone marrow transplant (BMT)
non-leukemic patients.
recipients. Similarly, mAB HAE9 could be used as a
convenient and reliable phenotypic marker of erythropoiRESULTS
etic activity in BMT patients. Additional studies on
In 16 samples of normal peripheral blood, 4.56 & patients with different hematopoietic disorders and on
2.56% (S.E.M.) of reticulocyte, as defined by TO
BMT patients may more accurately define the clinical
staining, were HAE9-positive. In order to assess the
applicability of this mAB and clarify the relationship
relationship between the number of reticulocytes enumerbetween the health status of patients and HAE9 antigen
ated by TO and by mAB HAE9, 35 samples with expression.
hematocrit less than 0.25 were studied. The correlation
(Fig. 1 ) between reticulocyte percentage by TO and by
HAE9-positivity was 0.828 (P < 0.0001, y = 0.156 X
ACKNOWLEDGMENTS
0.105). The average percentage of HAE9-positive cells
The authors thank Jo Ellen Bohman, M.S., Debra
in this group of patients was 10.2 5.74%. Using
dual-immunofluorescent cytometry (4 samples studied), Walsh, Karen Halas, and Tom Miller for excellent
the mean percent of TO-stained (green IF) reticulocytes technical assistance. This study was supported by
positive for HAE9-staining (red IF) was 24.3 16.9% Yamagiwa-Yoshida International Cancer Study Grant
(6.0%, 15.5%, 31.7%, and44%).
from the International Union Against Cancer (Geneva).
0)
+
+
*
*
Case Report: Heterogeneity of Reticulocytes
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flow, periphery, hae9, reticulocyte, heterogeneity, monoclonal, analysis, cytometric, antibody, thiazol, orange, blood
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