MULTIPARAMETRIC INVESTIGATION OF
THE IMMUNE SYSTEM DURING MAJOR
SURGERY
BY
LASER
SCANNING
CYTOMETRY
A. GERSTNER1, W. LAFFERS1, F. BOOTZ1, A. TÁRNOK2
1
Department of Otorhinolaryngology; 2Department of Pediatric Cardiology, Cardiac
Centre; University of Leipzig, Leipzig, Germany;
Address requests to: Dr. Andreas Gerstner, gersta@medizin.uni-leipzig.de
SUMMARY
Investigations of the immune system in the fast majority are based on flow
cytometry (FCM). The drawback of most of the FCM assays is that cells
cannot be analysed a second time and that the morphology of the cells cannot
be studied. This however is possible by using laser scanning cytometry (LSC).
We developed an assay for immunophenotyping of peripheral blood
leukocytes (PBLs) by LSC. The percentage of cells in different subsets
determined by LSC correlates very well with those determined by FCM but
only 25% of material is needed. We expect LSC to play a major role in further
investigations on the role of specific cell subsets in systemic immune
responses according to its unique facility to analyse the same cells repeatedly
by different assay.
INTRODUCTION
Many clinical situations demand repeated analyses of blood parameters but
permit only minimal amounts of peripheral blood to be taken. This includes
neonates with low birth weight, during extensive operations on young
children, or patients with restricted bone marrow function. In order to
determine the function of the cellular immune system, LSC is the ideal tool to
determine the distribution of different subsets of the PBLs in these cases.
A major advantage of LSC as compared to FCM is the fact that the cells are
fixed on the slide. The instrument is build around a standard research
fluorescence microscope. The cells on the glass slide are scanned by two
lasers (Ar + HeNe) and the emitted fluorescence is measured on a pixel-topixel basis; the analysis itself is performed on the resulting bit-map similar to
image analysis (1, 2). This process leaves the cells unchanged. The
immobilisation of the cells on the slide allows the direct morphological
control of the measured cells by relocating them on the slide. Additionally, it
reduces the amount of both, peripheral blood and reagents needed.
Furthermore, slides can be stored and re-analyzed later.
Existing assays (3) for immunophenotyping with the LSC are based on
triggering the analysis on the forward light scatter (FSC) of the cells. We
experienced substantial difficulties in achieving data of sufficient quality and
reliability by this strategy: on the one hand any particle or air bubble on the
slide could produce a FSC-signal indistinguishable of a cell. On the other
hand some cells might have virtually invisible scattering features and
consequently could be lost from analysis. We were therefore looking for an
alternative assay for immunophenotyping of PBLs by LSC and developed a
strategy where analysis is triggered on nuclear fluorescence.
MATERIALS AND METHODS
Blood was taken from patients undergoing resection of oro- and
hypopharyngeal cancer in advanced metastatic stages with reconstruction by
free microvascular reanastomosed flaps (forearm flap, latissimus dorsi flap,
lateral arm flap, scapular flap) 60-90min after onset of anaesthesia. For
analysis, full peripheral EDTA blood is aliquoted to 10µl and incubated with
different arrays of CD-antibodies (2,5µl each). After lysis of the erythrocytes
cells are fixed with paraformaldehyd and dispersed on a conventional
microscopic glass slide to pre-defined areas; this makes the analysis easier
and more standardised. The slides are allowed to air-dry and are incubated
with acetone. Cells are finally stained with the DNA-stain
7-aminoactinomycin-D (7-AAD; 5µg/ml) and are coverslipped. For analysis,
pre-set scan areas on the microscope slide are used. The fluorescence of the
nuclei is taken as the contouring triggering signal. This ensures that all
nucleated cells are included in the analysis. Furthermore, staining with
7-AAD allows the differentiation between PMNs and mononuclear cells (not
shown). The FSC is used to define the eosinophils (and basophils):
eosinophils are characterised by strongly light-scattering granula and high
autofluorescence and therefore would otherwise interfere with the specific
immunofluorescence. Further discrimination of monocytes and lymphocytes
is performed on the basis of their CD45-fluorescence.
In order to compare the analysis by LSC with FCM data the same staining is
run but starting with 40µl of peripheral full EDTA blood instead of 10µl. The
sample is split after fixation with paraformaldehyd: at that point three quarters
of the material are taken for FCM analysis in a Becton-Dickinson
FACSCalibur.
Following the analysis in the LSC the coverslips are removed, the slides are
placed in PBS, and a conventional H&E staining is performed. Slides are
coverslipped again, placed in the microscope stage, and single cells of interest
are localised to confirm and document the morphology of the cells (see
figures 1&2). Slides can be stored afterwards without any special
requirements.
RESULTS
The protocol described above yields reliable and consistent data. Processing
of the material must start within 60min after taking the specimen, the total
time of the protocol is 4h 15min. The specimens can be left overnight after
fixation in paraformaldehyd. The measurement in the LSC should be carried
out within 48h because of significant loss of fluorescence with storage. With
this method we can determine the percentage of eosinophils (and basophils),
neutrophils, monocytes, and lymphocytes, and can divide the lymphocytes
into CD3+CD4+-, CD3+CD4–-, CD3–CD19+-, and CD3–CD19–-cells in a single
scan using 10µl whole blood (see fig. 2). The percentage of the subsets
correlates very well with those determined by FCM on the same material (see
tab. 1). The ratio of the integrated fluorescence values of distinct cell subsets
(CD3+CD4+ vs. CD3+CD4–, e.g.) on the LSC is optimised to the range yielded
on FCM. This shows that the resolution of the fluorescence intensities by LSC
is equivalent to that by FCM. Relocating single cells of interest after H&E
staining worked very reliably.
CONCLUSIONS
We expect the LSC to prove its capacity for multi-parameter analysis of PBLs
in minimal amounts of peripheral blood. Its unique feature of relocating cells
allows to perform additional investigations on the same cells. This will be
especially helpful in investigating the role of specific cell subsets of the
immune system in systemic responses post-operatively e.g. after cardiopulmonary-bypass: we are working on an assay that allows consecutive
immunophenotyping, intra-cellular chemokine detection, and TUNEL-assay.
REFERENCES
1
2
3
KAMENTSKY et al.: Slide-based laser scanning cytometer. Acta Cytol
41:123-143, 1997
REEVE et al.: New technology in the analytical cell sciences: the laser
scanning cytometer. Eur J Surg Oncology 23:445-450, 1997
CLATCH et al.: Simplified immunophenotypic analysis by laser
scanning cytometry. Cytometry (Clin. Comm.) 34:3-16, 1998
ACKNOWLEDGEMENT
Reprint permission kindly provided by Monduzzi Editore, Bologna, Italy,
2000.
Eosinophils
(100x objective)
1
2
Fig. 1: PBLs stained with CD45-APC, CD3-FITC, CD4-PE, CD14-PE, CD19-PE, and
7-AAD; dot plot in the inset shows area versus FSC Max Pixel for all measured
events. Single cells are gated; region 1 is gated on eosinophils, region 2 is set on
the remaining single cells. Cells in region 1 were relocalised after H&E staining
and correspond to eosinophils (photographs taken with 100x objective).
Monocytes
(40x objective)
3
Neutrophils
4
Fig. 2: Same material as in fig. 1 but additionally gated on region 2. No compensation
is set. Dot plot of PE (CD4-CD14-CD19) vs. APC (CD45). Upper line: cells
within the region 3 correspond to monocytes (see photographs). Lower line: cells
within the region 4 correspond to neutrophils (see photographs).
Patient 1
Patient 2
LSC
FCM
LSC
FCM
57,8%
59,6%
79,4%
79,2%
Eosinophils + Basophils
2,5%
2,1%
1,4%
1,7%
Monocytes
7,0%
5,8%
3,0%
3,9%
32,3%
29,3%
Neutrophils
Lymphocytes all
+
CD3
-
CD3
70,2%
29,7%
16,1%
15,8%
+
13,8%
15,7%
-
86,2%
84,3%
68,8% CD19
31,1%
CD19
Tab. 1: Comparison of the percentages of cells in different cell subsets as determined
by LSC and by FCM (left and right column, resp.) in blood of two different
patients (Pat.1 and Pat.2).
CD3+CD4+
Lymphocytes
5
6
CD3+CD4–
(40x objective)
Fig. 3: Same material as in fig. 1, but additionally gated on cells with bright CD45fluorescence (not shown). Compensation is set. Dot plot showing CD3 vs.
CD4-CD19. Cells within region 5 correspond to CD3 +CD4+ lymphocytes (see
photographs in upper line), cells within region 6 correspond to CD3+CD4–
lymphocytes (see photographs in lower line).