0 1995 Wiley-Liss, Inc.
Cytometry 20341-348 (1995)
Evaluation of Red Blood Cell Lysing Solutions for the
Detection of Intracellular Antigens by Flow Cytometry
Maarit I. Tiirikainen
Blood Transfusion Service, Finnish Red Cross, FIN-003 10 Helsinki, Finland
Received for publication October 19, 1994; acccpted March 28, 1995
When analyzing leukocyte cell surface antigens
by flow cytometry, leukocytes are usually &st labeled in whole blood and the red blood cells are
Wally lysed with lysing solutions. The erythrocytes
are lysed, but the leukocytes are expected to remain
intact. Six commercial red blood cell lysing methods were investigated for possible leukocyte permeabilization effect. The effectiveness of permeabilization was studied by propidium iodide staining, and
the detectability of intracellular antigens was studied by using monoclonal antibodies toward two
model antigens. Most of the lysing methods caused
permeabilization of at least part of the leukocytes,
but only one method, already found in our previous studies, was applicable for complete permeabilization of leukocytes and for detection of intracellular antigens alone or simultaneously with the cell
s h c e antigens. o 1995 WUey-Liss, I~C.
When analyzing leukocyte cell surface antigens by flow
cytometry, the cells in whole blood are usually first labeled, and then the red blood cells are lysed either with
ammonium chloride or commercial whole blood lysing
solutions. When lysing red blood cells, the leukocytes are
expected to remain intact.
The FACS Brand Lysing Solution (Becton Dickinson,
San Jose, CA), intended for lysing red blood cells, was
found to permeabilize leukocytes and to be applicable for
detection of intracellular antigens in our previous studies
(FACS Lyse method) (10-12). In the present study, five
other commercial red blood cell lysing methods were
investigated for their permeabilizing effect and compared
to the FACS Lyse method. These included Ortho-mune
Lysing Reagent (Ortho Diagnostic Systems, Raritan, NJ),
Serotec Erythrolyse Red Blood Cell Lysing Buffer (Serotec, Oxford, UK), Coulter Clone Immuno-Lyse (Coulter
Immunology, Hialeah, FL), Lyse and Fix (Immunotech,
Marseille, France) and Optilyse B Lysing Solution (Immunotech).
The methods were evaluated for the effectiveness of
permeabilization (propidium iodide (PI) staining of the
nucleus) and for the detection of antigenic sites on two
different types of intracellular proteins, vimentin and myeloperoxidase. Vimentin is a cytoskeletal protein present
in all human cells (7), whereas myeloperoxidase (MPO)
is a granule-associated protein located in the cytoplasm
of monocytes and neutrophils ( 5 ) . The light scatter properties and the relative amounts of different leukocyte subsets after each procedure were also studied.
The power of flow cytometry in the analysis of separate cell populations is enhanced by simultaneous label-
ing of several antigens. The FACS Lyse method is directly
applicable to the simultaneous detection of cell surface
and intracellular antigens when surface antigens are labeled before the lysis, but in the case that the intracellular antigens are labeled with unconjucated monoclonal
antibodies followed by a second step anti-mAb-fluorochrome reagent, labeling with a mAb to a cell surface
antigen is possible only after the intracellular labeling.
This requires that the cell surface antigens are still detectable after the permeabilization. Therefore, the effect
of the FACS Lyse on common cell surface CD antigens
was studied. The possibility to label cell surface antigens
after the lysis and intracellular labeling broadens the
choice of antibodies of interest.
Key terms: CD antigens, propidium iodide, vimentin, myeloperoxidase, formaldehyde, diethylene
glycol
MATERIALS AND METHODS
Samples
Acid citrate dextrose (ACD) anticoagulated peripheral
blood (PB) samples were collected from healthy blood
donors by using commercial tubes (Venoject Evacuated
Blood Collecting Tubes with ACD sol. B, Terumo Europe,
Leuven, Belgium) and the samples were processed within
6 h of drawing. In addition, PB and bone marrow (BM)
aspiration samples from 30 patients with childhood acute
leukemia (lymphatic or myeloid) at presentation were
collected after informed consent.
Whole Blood Lysis
Whole blood (WB) lysis samples were prepared from
100 p1 aliquots of normal PB with Ortho-mune Lysing
Reagent, FACS Brand Lysing Solution (FACS Lyse), Serotec Erythrolyse Red Blood Cell Lysing Buffer, Coulter
342
TIIRIKAINEN
Clone Immuno-Lyse, Lyse and Fix, and with Optilyse B
Lysing Solution following the recommended procedures.
All the lysing procedures mentioned, except the Orthomune method, include formaldehyde as fixative. Orthomune induces red blood cell lysis due to an accumulation
of ammonium chloride within the cells and contains no
fixative, FACS Lyse and Serotec Erythrolyse contain formaldehyde and an unspecified amount of diethylene glycol.
htracellular Labeling
Lysed whole blood cells were incubated in 10%human
AB serum at 37°C for 15 min, whereafter a pretitrated
amount of a monoclonal antibody (mAb) to vimentin or
mAb to MPO (DAKOPATTS A / S , Glostrup, Denmark) was
added. After 25 min incubation on ice, the cells were
washed once with PBS containing 1%BSA, and a secondary antibody (fluorescein isothiocyanate (FITC) conjugated F( ab' ), fragment of rabbit antimouse immunoglobulins or R-phycoerythrin (R-PE) conjugated affinity
isolated goat antimouse immunoglobulins, DAKOPATTS
A / S ) was added. After a second 25 min incubation and
one wash, the cells were analyzed ( 11,12). To stain the
nucleus after permeabilization, PI (Sigma Chemical Co.,
St. Louis, MO) at a concentration of 5 pg/150 pI cell
suspension was incubated for 15 min at room temperature, whereafter the tubes were immediately analyzed.
The unprocessed PB and BM samples containing one
million leukocytes obtained from patients with acute leukemia were lysed and permeabilized with FACS Lyse,
whereafter the cells were labeled for the nuclear terminal
deoxynucleotidyl transferase (TdT) with pretitrated
amounts of rabbit anti-TdT and FITC conjugated goat antirabbit immunoglobulins (Supertechs, Bethesda, MD)
(10).
Labeling of Cell Surface CD Antigens
Three sets of samples containing 180 p.1 aliquots of
normal PB were prepared. Two sets were prepared as
follows. The PB samples were incubated 15 min at room
temperature with pretitrated amounts of mAbs to CD antigens and lysed with FACS Lyse (prelysis labeling). The
first set of the tubes was analyzed immediately on the
flow cytometer and the second set was passed through
the intracellular labeling procedure, but no further antibodies were added (prelysis labeling with intracellular
labeling incubations). The third set of samples was first
lysed and permeabilized with the FACS Lyse as explained
above and labeled with mAb to vimentin or idiotypic
control mAb of class IgG 1 (Immunotech S.A.) using the
intracellular labeling procedure. After the last wash, the
cells were incubated with 15% human AB serum at 37°C
for 15 min, labeled with mAbs to CD antigens as explained above (postlysis labeling), and finally washed
once with PBS containing 1% BSA.
Two sets of samples obtained from patients with acute
leukemia were prepared. One set was labeled before lysis
for surface antigens as explained above and labeled further for the intracellular TdT antigen. The other set of
samples was first labeled for the TdT antigen, whereafter
the cells were incubated with mAbs to CD antigens in the
presence of AB serum as explained above (postlysis labeling).
FITC conjugated mAbs to CD3 (Leu-4) and CD7 (Leu9), phycoerythrin (PE) conjugated mAbs to CD3 (leu-4),
CD13 (Leu-M7), CD33 (Leu-MB), CD34 (a-HPCA-2), and
peridinin chlorophyll protein (PerCP) conjugated mAb
to CDl9 (Leu- 12) were all purchased from Becton Dickinson. FITC and PE conjugated mAbs to CDlO (CALLA,
SS2/36) were purchased from DAKOPATTS.
Analysis by Flow Cytometry
Labeled cell samples were analyzed on a FACScan flow
cytometer (Becton Dickinson) equipped with a 15 mW
air-cooled 488 nm argon-ion laser. FLl (FITC) signals
were detected through a 530130 nm band pass (BP) filter, FL2 (PE and PI) signals were detected through a
585/42 nm BP filter, and FL3 signals (PerCP and PI)
through the >650 nm longpass filter. Electronic compensation was used to remove spectral overlap. Ten-to
fifteen-thousand events were recorded and analyzed using the Lysys I1 software (Becton Dickinson). Data were
analyzed after gating the lymphocyte, monocyte, or granulocyte area (or the blast population) in a dot plot displaying the linear forward (FSC) and side scatter (SSC)
properties of the cells. The expression of fluorescence of
the cells was analyzed in a fluorescence or SSC/fluorescence dot plot using a logarithmic scale.
Quality control of the flow cytometer was carried out
twice a week using CaliBRITE'" Flow Cytometer Beads
(Becton Dickinson).
Statistical M e t h o d s
The repeatability (variability between repeated analyses made by using the same PB sample from one donor)
and the reproducibility (variability between analyses
made by using PB samples from several donors) of intracellular labeling after FACS Lyse was demonstrated by
the Quality Control Graph of the Gricket Graph program
(Gricket Graph, version 1.2.1, Cricket Software, Malvern,
PA).
RESULTS
Light Scatter Properties of W B Lysed PB Cells
All the red blood cell lysing methods resulted in three
separate leukocyte populations in the FACScan FSC/SSC
dot plot (Fig. 1) (Table 1). Because unlysed whole blood,
when analyzed in the FACScan, results in a dot plot comprising mostly of red blood cells that overlap the lymphocyte-monocyte area of the leukocyte population, unlysed
blood could not be used as a control. Instead, whole
blood cells lysed with the Ortho-rnune Lysing Reagent
served as an example of unfixed leukocytes (Fig. 1, row
1). When lysing WB samples with Serotec Erythrolyse,
5-11% of the leukocytes (mainly lymphocytes and
monocytes) formed repeatedly two small populations of
apparently bigger cells in the FSC/SSC dot plot (Fig. I ,
row 3).
DETECTION O F INTRACELLULAR ANTIGENS BY FACS
343
Permeabilization and Intracellular Staining Results
m '. . . .
8
.... .. .. .... .... 1
288 488 668 868 ld8@
-.
~
"-
The Ortho-mune Lysing Reagent did not permeabilize
leukocytes (no PI staining of the nucleus), and thus the
intracellular molecules remained inaccessible (Fig. 1,
row 1). All the other five lysing reagents permeabilized
30-100% of the leukocytes, as determined by high intensity PI staining (Fig. 1) (Table 2). The efficiency of
intracellular labeling with antibodies did not, however,
directly correlate with the effectiveness of permeabi lization (Table 2).
Complete permeabilization was achieved with two
methods, FACS Lyse and Optilyse B, but the PI staining
pattern was more diffuse after Optilyse than after FACS
Lyse. Intracellular antigens were detectable with both antibodies as expected after FACS Lyse, but only a very
weak signal was achieved after the Optilyse procedure. A
proportion of the leukocytes remained unpermeabilized
after Serotec Erythrolyse, even when the volume of the
lysing solution was raised. The rest, a major part of the
cells, stained well with PI and the intracellular antigens
were detectable (Table 2). When using Immuno-Lyseand
Lyse and Fix methods, the staining results of successive
analyses were inconsistent.
Cell Loss and the Change of FSC/SSC Properties
During the Intracellular Labeling Steps after FACS
Lyse W B Permeabilization
FL2-HV'ropidiun Iodide
FIG. 1. Light scatter properties (left) and PI staining results (right) of
PB cells after WB lysis with the following reagents: Ortho-mune ( l ) ,
FACS Lyse (2), Serotec Erythrolysr ( 3 ) . I.yse and Fix ( 4 ) , Coulter Immuno-Lyse ( 5 ) , and Optilysc B (6).
The cell distribution after red blood cell lysis with
FACS Lyse was comparable to the reference method, Ortho-mune (Table 1). However, cell losses occurred during the following steps of the intracellular staining procedure, apparently due to repeated cell washes and
centrifugations (1). The cells lost were mostly lymphocytes as 2 5 4 0 % of the cells in the FSCBSC lymphocyte
gate were lost. The loss of lymphocytes was also confirmed by CD antigen labeling of ungated samples. However, the cell loss among the lymphocytes was not selective since the relative amounts of T and B cells in the
lymphocyte gate was unchanged after the intracellular
staining (Table 3) (Fig. 2).
When the leukocytes were labeled after permeabilization for intracellular antigens without surface antigen label ing, the lymphocyte, monocyte and granulocyte populations could be analyzed separately by utilizing the
FSC/SSC properties of the cells. The monocyte and granulocyte populations became less distinguishable, however, when leukocytes were labeled for both intracellular
and cell surface antigens (either pre- or postlysis), as the
monocytes gained more SSC and the granulocytes partly
lost their granularity (Fig. 2). The similarity in the side
scatter properties resulted in a gain of CDlO positive cells
in the FSC/SSC monocyte gate (R2 in Fig. 2) after the
intracellular labeling procedure (Table 3). Of the cells in
the monocyte gate, 8%were positive for CDlO when the
samples were labeled, lysed, and analyzed immediately
(surface labeling). When the samples were further passed
through the intracellular antigen staining procedure
(without antibodies), 26% of CDlO positive cells were
TIIRIKAINEN
344
Table 1
Proportions of Major Leukocyte Populations after Lysing Red Blood Cells with Commercial Lysing Solutions"
Lysing solution
Cell gate
Lymphocyte
Monocyte
Granulocyte
Ortho-mune
FACS Lyse
Erythrolyse
Lyse and Fix
Immuno-Lyse
Optilyse B
24.8 f 4.1
24.9 5.8
25.7 f 6.0
36.3 f 15.2
33.2 f 9.3
34.2 t 8.1
6.6 f 2.8
5.3 f 2.0
4.3 f 1.4
5.4 f 2.2
5.5 f 1.3
5.8 f 1.8
68.8 f 4.6
64.5 f 5.9
41.7 2 12.9
45.6 f 13.6
*
51.0 2 11.6
46.9
2
9.4
Total cells
95.3 f 1.8
94.7 2 1.1
71.7 12.5
87.3 5.6
89.8 6.2
86.9 t 8.0
*
*
*
n
10
10
10
9
9
10
aValues represent the mean-% f 1s (s = standard deviation) of cells in a gate when 9 or 10 (n) peripheral blood
samples from healthy blood donors were lysed with each method.
Table 2
Proportions of Propidium Iodide (PI), Anti-Vimentin PE and Anti-Myeloperoxidase (MPO) PE Labeled Cells in
Cell Gates after Lysing Red Blood Cells with Commercial Lysing Solutions'
Target
Lysing solution
Nucleus
(PI)
Ortho-mune
FACS Lyse
Er ythrolyse
Lyse and Fix
Immuno-Lyse
Optilyse B
Ortho-mune
FACS Lyse
Er ythrolyse
Lyse and Fix
Immuno-Lyse
Optilyse B
Ortho-mune
FACS Lyse
Erythrolyse
Lyse and Fix
Immuno-Lyse
Optilyse B
Vimentin
MPO
Cell gates
LympKocyte
*
0.1 0.1
98.2 f 1.2
95.4 f 2.6
65.4 k 27.6
45.7 f 28.4
92.6 ? 3.1
0.2 0.2
95.7 f 1.5
87.2 k 5.2
41.0 f 24.9
*
28.9 f 16.1
15.5 k 4.1
0.2 k 0.1
12.9 f 6.0
45.7 k 12.5
1.5 2 1.8
1.4 ? 1.0
1.7 k 3.3
Monocyte
0.2 0.3
98.0 1.3
89.8 f 6.5
42.2 2 31.9
30.6 f 25.5
95.5 2.5
0.7 0.6
92.3 f 2.2
74.9 f 8.4
26.5 f 19.0
14.0 7.3
33.8 10.6
0.9 -t- 0.5
73.8 16.6
63.4 19.0
9.0 f 7.1
8.1 2 5.1
20.0 15.2
*
*
*
*
*
*
*
*
Granulocyte
0.4 2 0.1
99.6 f 0.2
98.4 f 1.3
60.1 % 29.5
73.7 f 20.8
99.7 f 0.2
0.3 f 0.2
94.8 2.3
92.0 5.0
31.9 2 17.0
34.1 f 23.0
9.9 5.6
0.4 t 0.2
93.3 2 5.8
95.6 f 1.9
33.6 f 23
39.7 17.2
16.7 f 8.9
n
5
8
9
8
8
8
*
*
5
7
9
*
8
8
*
7
5
5
8
5
5
6
aValues represent the mean-% f 1s of labeled cells in a gate when 5-9 (n) peripheral blood samples from blood
donors were analyzed. The results of the Erythrolyse method comprise the analyses made by excluding the unpermeabilized cells (see Fig. 1).
found in the monocyte gate. The relative amount of
monocytes remained unchanged, however, as determined by the percentage of cells having a high intensity
reaction with mAb to CD33 (R4 in Fig. 2). Only few cells
reacting with the mAbs to CD13, CD33, and CDlO
(monocytes and granulocytes) were found in the lymphocyte FSUSSC gate after intracellular staining (Table
4).
Effect of FACS Lyse Permeabilization and
Intracellular Labeling on Detection of Cell SurEace
CD Antigens
Most of the CD antigens studied could be detected as
reliably after FACS Lyse (postlysis) and intracellular staining as before lysis (prelysis). The amount of CD33 labeled cells in the granulocyte gate was markedly decreased, but this was due to the analysis method, not to
the destruction of the antigen. No new epitopes for any
mAbs to the CD antigens studied appeared (Table 4).
To demonstrate that the same subset of leukocytes was
stained whether samples were labeled for the cell surface
antigens before or after lysis, double labeling of leukemic
cells was performed by using mAbs to CD antigens and a
polyclonal antibody to TdT antigen followed by antirabbit FITC conjugate, which enables the labeling of CD
antigens with mAbs either before or after intracellular
labeling. Practically the same amount of cells with simultaneous expression of, e.g., CDlO and TdT antigens was
detected when the cells were labeled with mAb to CDlO
either before or after FACS Lyse (Table 5 ) .
Repeatability and Reproducibility of Intracellular
Antigen Detection with the FACS Lyse
Permeabilization Method
For the evaluation of the repeatability of intracellular
labeling, 15 parallel analyses of intracellular vimentin and
MPO of PB leukocytes from the same blood donor were
performed. Vimentin was analyzed using the FSC/SSC dot
plot lymphocyte gate and MPO using the granulocyte
gate, respectively. The reproducibility of the method was
evaluated by analyzing separate PB samples from 15 dif-
DETECTION O F INTRACELLULAR ANTIGENS BY FACS
345
Table 3
Cell Loss During Intracellular Labeling: Proportions of T Cells ( W 3 , B Cells (CD19),
Myeloid Cells (CD13/33), and CDlO Positive Cells Directly after Lysis with FACS Lyse us. after
Additional Intracellular Labeling
CD Ag
CD3 FITC
CD3 FITC
CDl9 PerCP
CDl9 PerCP
CD13 PE
CD13 PE
CD13 PE
CD33 PE
CD33 PE
CD33 PE
CDlO FITC
CDlO FITC
CDlO FITC
Analysis gate
Ungated
LYmphoCyte
Ungated
Lymphocyte
Ungated
Monocyte
Granulocyte
Ungated
Monocyte
Granulocyte
Ungated
Monocyte
Granulocyte
"Values represent mean-%
blood donors.
ProDortion of labeled cells. Mean-% & lsa
Surface
Surface and intracellular
labeling
labeling procedure
17.3 2 2.4
68.9 2 6.7
3.3 k 1.4
14.5 2 7.0
72.2 2 4.3
89.4 2 14.5
97.9 & 1.0
74.5 & 3.6
93.3 2 4.4
88.40 2 9.7
62.9 f 7.8
7.9 k 6.0
72.9 2 16.7
12.3 2 1.4
68.7 f 6.0
2.4 2 1.1
14.3 f 6.9
79.3 2 3.9
90.7 ? 12.8
98.5 & 1 . 1
79.0 2 4.7
88.8 f 4.8
91.3 f 5.6
67.0 f 8.1
25.7 f 18.4
72.2 C 23.42
* 1s of labeled cells in the peripheral blood samples of six different
ferent blood donors for MPO and vimentin. The results
are shown in Figure 3.
DISCUSSION
Three major populations of normal leukocytes-lymphocytes, monocytes, and granulocytes-can be analyzed separately by flow cytometry. The size and granularity of the cells result in different kind of forward (FSC)
and side (SSC) scatter properties and thus give separate
populations in the flow cytometric FSC/SSC dot plot. In
order to take advantage of that powerful property also
when analyzing intracellular antigens, the light scatter
properties of the cells should remain unchanged despite
permeabilization.
During the last 10years, various methods for leukocyte
permeabilization have been published, first, for the analysis of DNA and also for the detection of intracellular
antigens. The permeabilization methods published so far
are intended for the permeabilization of separated leukocytes, obtained either by the lysis of red blood cells with
ammonium chloride or by Ficoll density gradient separation (2-4,6,8,9).
In this study, six commercial red blood cell lysing
methods were evaluated for the detection of intracellular
leukocyte antigens. All the lysing methods studied except
the Ortho-mune Lysing Reagent contained formaldehyde
as fixative. The methods with a fixative caused permeabilization of 30-100% of the leukocytes as determined
by the number of PI stained cells with high intensity.
Leukocytes were completely permeabilized, however,
only with FACS Lyse and Optilyse B.
Vimentin and MPO were well stained after lysis with
FACS Lyse. A weak reaction of lymphocytes with antiMPO was encountered after lysis with FACS Lyse, but the
staining intensity of monocytes and granulocytes was
still markedly stronger. Despite the good permeabilization with the Optilyse B reagent, only very weak staining
with the monoclonals was achieved probably due to still
inadequate permeabilization and/or loss of the antigenic
epitopes.
In conclusion, among the commercial red blood cell
lysing methods, only the FACS Lyse method was found
completely to permeabilize leukocytes without altering
the intracellular antigenic sites, and thus to be the only
method applicable for the labeling of intracellular antigens. When compared to conventional permeabilization
methods, simultaneous red blood cell lysis and leukocyte
permeabilization makes the method simple and rapid and
enables analysis of unseparated whole blood and bone
marrow samples.
The FACS Lyse method is also applicable to simultaneous detection of intracellular and cell surface antigens,
which enables specific identification of the permeabilized cells with mAbs to CD antigens. Most of the CD
antigens studied could be detected as reliably after FACS
Lyse as before the lysis; the only antigen that was difficult
to detect after FACS Lyse was the CD33 antigen on the
surface of granulocytes. According to the results presented in Table 4 , CD33 detectability was decreased on
granulocytes and to some extent also on monocytes
when the leukocytes were labeled after FACS Lyse. This
was not, however, due to the destruction of the antigen
as might be presumed, but to the increased binding of the
PE conjugated control Mab to granulocytes in some but
not all samples after FACS Lyse. Even when labeling intact
granulocytes, the fluorescence intensity was low due to
low CD33 antigen expression on granulocytes, and thus
the detection of positive fluorescence after FACS Lyse
was not possible when the fluorescence of control was
increased. Monocytes, however, have a high expression
of CD33 and positive fluorescence was markedly higher
than control fluorescence. A decrease in the amount of
positive cells was also observed in the rnonocyte gate
when labeling CD33 antigen after FACS Lyse, but this was
346
TIIRIKAINEN
n
I
I
.@
d
.-I
Q,
I
I
0
m
(D
I
u
co
m
0
200 400 688 888 10018
eo
eo
h
I
I
.@
%
I
.-I
Q,
I
u
m
co
::
8
v)
FsC-H\Fsc-H~ight --->
FLe-H\FLe-Height
--->
FLl-H\FLl-Height
--->
FIG.2. FACS analysis of FACS Lysed WB PB leukocytes. A1-A3: Cells
analyzed directly after CD antigen labeling and FACS Lyse (prelysis labeling). Bl-B3: Cells analyzed after C D antigen labeling, FACS Lyse and
intracellular staining incubations, successively (prelysis labeling with
incubations). C143: Cells analyzed after FACS Lyse, intracellular labeling, and CD antigen labeling (postlysis labeling). A l - C l : Distribution of
the cells into the lymphocyte, monocyte and granulocyte gates. Note the
partial intermixing of monocytes and granulocytes (A and C) when intracellular labeling was combined with surface antigen labeling. AZ-
C2: High intensity C D 3 3 labeling of monocytes. The relative amount of
monocytes (R4/ALL and R4/MGG) was similar after each procedurc. R4
= absolute number of high intensity labeled monocytes, ALL = absolute
number of ungated leukocytes, MCG = absolute number of cells in the
monocyte and granulocyte gates. A3-C3: CD3 FITC labeling of T cells.
A proportion of the T cells was lost during the intracellular staining steps
(RS/ALL), but the relative amount of T cells in the lymphocyte gate
(RYR1) was similar after each procedure. The percentages represent
the mean values of six separate analyses of different blood samples.
due to the increased presence of low intensity labeled
granulocytes in the monocyte gate.
The CD13/33 prelabeling and postlabeling results
were similar when labeling the samples from patients
with acute leukemia (Table 5). This suggests that the
positive cells were expressing the CD33 antigen with
high intensity similar to normal monocytes or that the
positive fluorescence was mostly due to the expression
of the CD13 antigen, the detection of which was not
altered when performing postlysis labeling. N o new
epitopes for any mAbs to CD antigens studied appeared
after FACS Lyse (Table 4). When using postlysis labeling
the possible cytoplasmic expression of, e.g., the CD3 an-
tigen in some forms, the antigen in some forms of leukemia should be considered. Whether the cytoplasmic antigen epitopes is detectable after the permeabilization
with FACS Lyse was not investigated in this study.
As mentioned in Results, the lymphocyte, monocyte,
and granulocyte populations could be analyzed separately after FACS Lyse and intracellular labeling by utilizing the FSC/SSC properties of the cells. The monocyte
and granulocyte populations became less distinguishable,
however, when leukocytes were labeled for both cell surface and intracellular antigens, as the monocytes gained
more SSC and the granulocytes partly lost their granularity due to the additional processing (Fig. 2). Therefore,
Table 4
Effect of FACS Lyse and Intracellular Lubeling on the Detectability of Cell Membrane CD Antigen?
Monocvte
Post %
Pre %
2 . 0 * 1.0
2.2 % 1.4
2.0 1.3
4.1 t 1.6
4.5 t 2.2
1.5 2 0.4
0.7 t 0.5
0.7 f 0.4
Lymphocyte
Pre %
Post %
68.7 t 6.0
69.9 t 6.1
68.6 t 6.2
68.3 t 7.0
73.4 t 9.5
71.2 t 11.1
CD Ag
CD3 FlTC
CD3 PE
CD7 FITC
CD19 PerCP
CD13 PE
CD33 PE
CDlO FlTC
CD74 PE
14.3 k 6.9
3.0 2 1.6
4.5 t 1.9
0.8 t 0.7
0.5 0.1
*
Granulocvte
Pre %
0.8 t 0.4
0.6 2 0.4
0.5 2 0.2
0.1 2 0.2
98.5 t 1.1
91.3 t 5.6
*
11.7k 4.5
3.8 k 2.7
2.6 t 1.7
1.5 2 1.2
1.2 1.1
90.7
88.8
* 12.8
85.4 f 9.3
78.5 % 7.0
26.0 2 17.8
1.3 1.4
* 4.8
25.7 2 18.4
2.7 2 1.4
*
72.2
1.3
*
Post %
n
-
0.9 k 0.3
1.4 t 0.7
1.1 t 0.41
0.4 t 0.4
96.9 ? 1.2
27.4 t 20.4
68.1 t 28.9
0.9 t 0.8
* 23.4
* 1.6
6
6
6
5
6
6
5
6
"Peripheral blood leukocytes of healthy blood donors were labeled for CD antigens either before lysing the red blood cells with FACS
Lyse, which was followed by intracellular labeling without antibodies (prelysis labeling with intracellular labeling incubations), or after
FACS Lyse and intracellular labeling (postlysis labeling). The values represent mean-% 2 1s of labeled cells in 5-6 ( n ) different blood
samples. See Discussion for explanation of altered CD33 detectability on granulocytes when performing postlabeling.
Table 5
CD Antigen Labeling befme and after FACS Lyse PmeabiCization and Intracellular TdT Labeling:
Double LabeI Criteria fm the Identification of Labeled Cellf
CD Ag positive cells
CD3 PE
CDlO PE
CD19 PerCP
CD13/33 PE
CD34 PE
CD Ag/TdT double positive cells
Pre %
Post %
Pre %
Post %
n
24.2 -1- 29.6
62.8 t 35.5
57.0 t 34.1
27.7 t 29.1
6 3 . 6 t 31.0
23.7 2 27.5
62.5 35.7
50.6 2 31.5
27.7 2 28.1
64.2 2 34.9
0.9 2 0.7
53.5 2 35.6
38.7 t 33.9
15.0 t 17.6
41.0 t 34.1
1.0 % 0.9
51.5 t 33.8
10
15
10
10
6
*
35.8 2 32.4
16.6 f. 21.9
37.3 t 33.8
aBone marrow cells of patients with childhood acute leukemia were labeled for CD antigens before (pre)
or after (post) leukocyte permeabilization and nuclear TdT antigen labeling. The values represent the
mean-% t 1s of labeled cells in the lymphocyte/blast gates of separate samples when 5-1 5 (n) samples were
analyzed.
Repeatability of MPO Detection
Repeatability of Vimentin Detection
105
3
V
2
s
100
P
EP
-
105
c)
c)
Mean94.4 %
s 0.5
RSD 0.5
95
8
6
100
Mean
'2s
Mean 95.8 %
s 0.7
RSD 0.8
95
F
3
90
e;l
5
0
10
loo
0
Mean + 2s
-
s 3.4
Mean 91.5 9%
s 3.1
RSD 3.4
Mean - 2s
I
I
1
Mean 94.5 %
18 90 85
15
Reproducibility of MPO Detection
'05
Mean + 2s
#
10
Number of repeated analyses, same sample
Reproducibility of Vimentin Detection
3
5
0
15
Number of repeated analyses, same sample
Y
I
I
I
5
10
15
Number of samples analysed
FIG. 3. The repeatability and the reproducibility of the intraccllular
vimentin labeling in lymphocytes and of the intracellular MPO labeling
in granulocytes was determined by labeling 15 peripheral blood samples
from the same blood donor (repeatability) or from 15 different blood
RSD 3.5
Mean - 2s
85
!
0
Y
I
I
1
5
10
15
Number of samples analysed
donors (reproducibility) after permeabilization with FACS Lyse. The
mean-%, standard deviation s, and the relative standard deviation (RSD
= 100 x s h e a n value) are given.
348
TIIRIKAINEN
when double labeling of both the cell surface and the
intracellular antigens of monocytes or granulocytes is
wished, an (additional) labeling with specific mAbs to
monocyte/granulocyte related CD antigens is recommended (triple labeling). Only a few CD13, CD33, and
CDlO positive cells (monocytes or granulocytes) were
found in the lymphocyte gate after intracellular and surface staining procedure (Table 4), which enables analysis
of lymphocytes by utilizing solely the FSC/SSC properties
of the cells even when both surface and intracellular antigens are studied.
ACKNOWLEDGMENTS
The financial support of the Finnish Cancer Foundation, the Foundation of Nona and Kullervo V&e, and the
Instrumentarium Scientific Fund, Instrurnentarium Corporation Finland, is gratefully acknowledged.
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