U.S. patent number 4,628,026 [Application Number 06/552,090] was granted by the patent office on 1986-12-09 for method and apparatus for automated double fluorochromization analysis in lymphocytotoxicity testing.
Invention is credited to Dietlind Gardell, Gail Rock.
United States Patent |
4,628,026 |
Gardell , et al. |
December 9, 1986 |
Method and apparatus for automated double fluorochromization
analysis in lymphocytotoxicity testing
Abstract
An automated system for rapid sequential photometric analysis of
a collection of double fluorochrome stained lymphocyte specimens,
useful for antibody screening or lymphocytotoxicity analysis. The
specimens are sequentially alternately irradiated with light of two
distinguishable wavelengths, producing fluorescence at two
distinguishable wavelengths. The fluorescent emission light
intensity for each irradiation of each specimen is measured using a
photometer and computer. The computer controls the synchronization
of the irradiation through alternately selected condenser sets with
the sequential movement of specimens into the optical path of the
irradiating and detected light, and calculates the quotient of the
light intensities emitted from each specimen at the two selected
fluorescent light wavelengths. These quotients are compared against
a control ratio (for lymphocytotoxicity analysis) to classify the
specimen. Also described is a method of preparing specimens for
such analysis, which requires that a complement be added to the
first staining solution after the latter is applied to the
specimens, then this combination agitated, and then the second
staining solution added and the specimen incubated.
Inventors: |
Gardell; Dietlind (Oxford
Station, Ontario, K0G 1T0, CA), Rock; Gail
(Rockcliffe Park, Ottawa, Ontario, K1L 5A2, CA) |
Family
ID: |
24203895 |
Appl.
No.: |
06/552,090 |
Filed: |
November 15, 1983 |
Current U.S.
Class: |
435/7.24;
250/461.2; 435/286.2; 435/287.3; 435/288.7; 435/29; 435/963;
435/966; 435/973; 436/519; 436/800; 436/805; 436/807; 436/808;
436/809; 436/821 |
Current CPC
Class: |
G01N
21/6428 (20130101); G01N 21/6458 (20130101); G01N
21/6452 (20130101); Y10S 436/821 (20130101); G01N
2021/6419 (20130101); G01N 2021/6421 (20130101); G01N
2201/0446 (20130101); G01N 2201/0484 (20130101); Y10S
436/807 (20130101); Y10S 436/80 (20130101); Y10S
436/808 (20130101); Y10S 435/963 (20130101); Y10S
435/973 (20130101); Y10S 436/809 (20130101); Y10S
436/805 (20130101); Y10S 435/966 (20130101) |
Current International
Class: |
G01N
21/25 (20060101); G01N 033/53 (); G01N
033/554 () |
Field of
Search: |
;435/29,34,7,287
;436/519,800,805,807,809,821 ;250/461.2 ;422/63,65,172 ;424/11 |
References Cited
[Referenced By]
U.S. Patent Documents
Other References
Chemical Abstracts, 101:86887h (1984). .
Abstract by Pablo Rubinstein, M.D. et al., entitled "Automated
Reading of Cytotoxicity with the Contrast Fluorescence Test (CFT)",
American Association for Clinical Histocompatibility Testing, Mar.
1981. .
Rotman and Papermaster, (1966) Proc. N.A.S., pp. 134-141. .
Edidin and Church, Transplantation, vol. 6, No. 9, pp. 1010-1014
(1968). .
LePecq and Paoletti, J. Mol. Biol. (1967) 27, 87-106. .
White, in Standardization in Immunofluorescence, Holborow Ed
(1970). .
Fong and Kissmeyer-Nielsen, Tissue Antigens (1972) 2.57-63. .
Elves, Journal of Immunological Methods 2 (1972) 129-136. .
Krisshan, The Journal of Cell Biology-vol. 66, 1975, pp. 188-193.
.
Goding, Journal of Immunological Methods, 13 (1976) 215-226. .
Bruning, Kardol and Arentzen, Journal of Immunological Methods, 33
(1980) 33-44. .
Edidin, The Journal of Immunology, vol. 104, No. 5, May 1970. .
Tagasugi, Transplantation, vol. 12, No. 2, (1971). .
Martel, Jaramillo, Allen and Rubinstein, Vox Sang., 27: 13-20
(1974). .
van Rood, van Leeuwen and Ploem, Nature, vol. 262, pp. 795-797
(1976). .
Bruning, Claas, Kardol, Lansbergen, Naipal and Tanke, Human
Immunology, 5, 225-231 (1982). .
Johnson, Goddard and Holborow, Journal of Immunological Methods, 50
(1982) 277-280. .
van Lambalgen, Bruning and Bradley, Transplantation Proceedings,
vol. XV, No. 1, pp. 69-71 (1983). .
Zeiss brochure, "IM 35 ICM 405 Photo-Invertoscopes". .
Leitz brochure, "MPV Compact MT"..
|
Primary Examiner: Marantz; Sidney
Claims
We claim:
1. For use in fluorescence assays of specimens exhibiting
fluorescence at two distinguishable wavelength bands when
irradiated respectively by light of two pre-selected
distinguishable wavelength bands; apparatus comprising:
a movable specimen container having spaced compartments for a
plurality of individual specimens to be assayed;
alignment means for moving the container to provide predetermined
sequential centered alignment of individual compartments of the
container with a predetermined optical path;
irradiating means for sequentially irradiating said specimens, as
each is aligned in the optical path, alternately with light of the
two pre-selected distinguishable wavelength bands beamed along the
optical path;
light collection means for receiving and distinguishing light
produced by fluorescence in each said specimen along the optical
path;
detector means for detecting and recording the intensity of the
light produced by fluorescence of each said specimen at each of
said two distinguishable wavelength bands;
analyzing means for calculating the quotient of the intensities of
the light sequentially received from each said specimen at the two
distinguishable wavelength bands and for comparing such quotient
with at least one control quotient thereby to classify such
specimen according to such comparison; and
synchronization means for controlling, coordinating and
synchronizing the operation of the alignment means, irradiating
means, light collection means, detector means and analyzing
means.
2. Apparatus as defined in claim 1, wherein the synchronization
means comprises a computer programmed to control, coordinate and
synchronize the operation of the alignment means, irradiating
means, light collection means, detector means, and analyzing
means.
3. Apparatus as defined in claim 2, wherein the analyzing means is
part of the computer.
4. Apparatus as defined in claim 1, wherein the light collection
means includes a pair of barrier filters each alternately
selectable to pass light of a respective one of said two
distinguishable wavelength bands to the detector means.
5. Automated apparatus for observing fluorescence stimulated by a
light source in a plurality of specimens in a transparent specimen
holder containing said specimens in a planar array, each of said
specimens exhibiting fluorescence at a first emission wavelength
when irradiated with light of a first exciting wavelength and
fluorescence at a second emission wavelength when irradiated with
light of a second exciting wavelength, comprising:
(i) an inversion microscope including a scanning stage operable to
retain said specimen holder and to be sequentially positioned to
place each of said specimens sequentially at a selected centered
position within the objective field of such microscope to collect
for observation fluorescent radiation from such specimen;
(ii) power means operable to drive said scanning stage in
orthogonal directions within a plane perpendicular to the focal
axis of the objective of said microscope;
(iii) first and second optical systems operable when said first and
second optical systems are respectively positioned in the path of
light from said source, to direct sequentially light of said first
incident wavelength and said second incident wavelength,
sequentially to the specimen located at said selected position, and
to transmit light of said first emission wavelength and said second
emission wavelength, respectively, from said specimen located at
said selected position to the ocular of said microscope for
observation;
(iv) motorized means for alternately positioning said first optical
system and said second optical system in the path of light from
said source; and
(v) control means operatively connected with each of said power
means and said motorized means for moving said scanning stage in
incremental steps to place each of said specimens sequentially at
said selected position within the objective field of said
microscope for a predetermined period of time and to actuate said
motorized slide means during said predetermined period of time so
that fluorescence light of said first emission wave length and of
said second emission wave length is observed by means of said
microscope for each of said specimens;
(vi) photodetector means coupled with said microscope and operable
to produce an amplified signal proportional to the intensity of
radiation emitted by a sample located at the selected position
within the optical field of said microscope; and
(vii) a computer programmed to collect and store signal data from
said photodetector measuring the fluorescence light intensity at
said first emission wavelength and at said second emission
wavelength from each said sample, and to transform said data into
quotients which are directly related to reference data for positive
and negative control standards of a given double-stained sample,
said computer being programmed to coordinate said control means so
that each said specimen is successively placed within the optical
field of said microscope where it is alternatively irradiated with
light of said first and second incident wavelengths.
6. Apparatus according to claim 5, wherein said first and second
optical systems are epi-fluorescence condenser sets and said
motorized slide means comprises
(i) a housing to hold said epi-fluorescence condenser sets in
parallel side-by-side alignment, said housing being axially movable
between two stop positions to alternately place said
epi-fluorescence condenser sets in the path of light from said
source, and
(ii) a motor operatively connected to said housing for moving said
housing between said two stop positions.
7. Apparatus according to claim 6, wherein said specimen holder is
a tray having identical U-shaped, flat bottom or round bottom wells
therein for holding specimens in a rectangular array of rows and
columns.
8. Apparatus according to claim 7, wherein said power means and
said control means are operable to place each of said identical
wells sequentially at said selected position within the objective
field of said microscope so that each of the wells is centered
within the optical field of said microscope when fluorescence from
said well is being observed.
9. Automated apparatus for the photometric analysis of a plurality
of specimens of cellular material, each such specimen having been
stained with a first fluorochrome exhibiting fluorescence at a
first emission wavelength when irradiated with light at a first
incident wavelength and a second fluorochrome exhibiting
fluorescence at a second emission wavelength contrasting with first
emission wavelength when irradiated with light at a second incident
wavelength, comprising:
(i) a microtest tray operable to hold said specimens and
appropriate fluorescence control standards within an array of
discrete wells;
(ii) an inverted epi-fluorescence microscope, comprising:
(a) a motorized scanning stage operable to retain said microtest
tray and to be sequentially positioned to place each of said wells
sequentially at a selected centered position within the optical
field of said microscope to collect fluorescent radiation from the
specimen in said well,
(b) first control means operatively connected with said motorized
scanning stage for moving said scanning stage in incremental steps
to place each of said wells sequentially at said selected
position,
(c) a light source operable to irradiate any of said specimens
located at said selected position within the optical field of the
microscope,
(d) first and second epi-fluorescence condenser sets operable to
admit light of said first incident wavelength and said second
incident wavelength, respectively, and to be interposed between
said light source and said specimen located at said selected
position within the optical field of the microscope,
(e) motorized filter-changing means operable to alternate the
interposition of said first condenser set and said second condenser
set between said light source and said specimen,
(f) second control means operatively connected with said motorized
filter-changing means for successively irradiating each sample
located at said selected position in the optical field of said
microscope first with light of said first incident wavelength and
then with light of said second incident wavelength for
predetermined intervals of time;
(iii) photodetector means coupled with said microscope and operable
to produce an amplified signal proportional to the intensity of
radiation emitted by a sample located at the selected position
within the optical field of said microscope;
(iv) a computer programmed to collect and store signal data from
said photodetector measuring the fluorescence light intensity at
said first emission wavelength and at said second emission
wavelength from each said sample, and to transform said data into
quotients which are directly related to reference data for positive
and negative control standards of a given double-stained sample,
said computer being programmed to coordinate said first control
means and said second control means so that each said specimen is
successively placed within the optical field of said microscope
where it is alternatively irradiated with light of said first and
second incident wavelengths.
10. Apparatus according to claim 9, wherein said specimens comprise
mixtures of live and dead cells in unknown proportions and a number
of positive and negative control standards having majority
proportions of live or dead cells, respectively, and wherein said
computer is programmed to transform original intensity fluorescence
data into quotients directly related to reference intensity
fluorescence data for said positive and negative control standards
and to compute and display data expressed as the percentage of live
and dead cells in each said sample.
11. For use in fluorescence assays of specimens of cellular
material exhibiting fluorescence at two distinguishable wavelength
bands when irradiated respectively by light of two pre-selected
distinguishable wavelength bands; an automated method
comprising:
selecting and ordering specimens containing substantially uniform
quantities of cellular material;
sequentially aligning individual ones of said specimens with an
optical path;
irradiating the specimens sequentially and alternately with light
of the two pre-selected distinguishable wavelength bands beamed
along the optical path;
receiving and distinguishing light produced by fluorescence caused
by the sequential and alternate irradiation of each said specimen
along the optical path; and
detecting the intensity of the light produced by fluorescence of
each said specimen at each of said two distinguishable wavelength
bands;
calculating the quotient of the intensities of the light
sequentially received from each said specimen at the two
distinguishable wavelength bands; and
comparing the said quotient for each specimen against a
predetermined control quotient thereby to classify the specimen
according to such comparison;
said method including automatically synchronizing and coordinating
all of the foregoing steps other than the selection and ordering of
specimens.
12. The method of claim 11, wherein the specimens are selected for
IF measurement of the cytotoxic reaction between prepared
lymphocytes and selected identical quantities of antisera, and
wherein the specimens are prepared according to the following
sequential steps:
(a) adding the washed lymphocytes to a first staining solution to
prepare a suspension having a known concentration of lymphocytes,
said first staining solution comprising a first fluorochrome
selectively staining living cells,
(b) adding a selected quantity of said suspension to each of said
antisera and immediately thereafter adding to each of the
antiserum-lymphocyte specimens a selected quantity of
complement,
(c) agitating the specimens at room temperature for a selected
period of time,
(d) adding to each of the specimens a selected quantity of a second
staining solution comprising a second fluorochrome selectively
staining dead cells and exhibiting fluorescence over a range of
wavelengths contrasting with the range of fluorescence exhibited by
said first fluorochrome, and
(e) incubating the specimens for a selected period of time.
13. A method as defined in claim 12, comprising the additional
step:
(f) adding to each of the incubated specimens a stabilizer to
inhibit further cytotoxic reaction between the antibodies of said
antisera and the antigens of said lymphocytes.
14. A method as defined in claim 13, wherein said first
fluorochrome is selected from the group consisting of fluorescein
diacetate, fluorescein isothiocyanate and carboxyfluorescein
diacetate.
15. A method as defined in claim 13 or claim 14, wherein said
second stain is selected from the group consisting of ethidium
bromide, propidium iodide, rhodamine and rhodamine
isothiocyanate.
16. A method as defined in claim 12 or 13, wherein the lymphocytes
are washed in a non-autofluorescing wash solution before they are
added to the first staining solution.
17. A method as defined in claim 13, wherein said first
fluorochrome is fluorescein diacetate and said second fluorochrome
is ethidium bromide.
18. A method as defined in claim 17, wherein said first staining
solution comprises acetone and fluorescein diacetate having a stock
concentration lying in the range 3-6 mg fluorescein diacetate per
ml of acetone and said second staining solution contains 1 .mu.g of
ethidium bromide per 1 ml of Veronal buffer having a pH of 7.0.
19. A method as defined in claim 18, wherein the final
concentration of the first staining solution is of the order of 50
ng per .mu.l acetone/HBSS/BSA mixture per 2.times.10.sup.6
cells.
20. A method as defined in claim 19, wherein said selected quantity
of said suspension added to said antisera contains a known quantity
of lymphocytes lying in the range of 1.5.times.10.sup.3 to
3.0.times.10.sup.3 per .mu.l of antiserum for each of said
antisera.
21. A method as defined in claim 20, wherein said complement is
rabbit C' and said selected quantity of complement lies in the
range of 1-2 .mu.l per .mu.l of antiserum.
22. A method as defined in claim 21, wherein said agitation of
antiserum-lymphocyte-complement specimens at room temperature is
effected by agitation of the specimens at a rate of 10-50 cycles
per minute for a period of 30-80 minutes.
23. A method as defined in claim 22, wherein subsequent to the
addition of said second staining solution to said specimens the
specimens are incubated for a period of time lying in the range of
7-15 minutes.
24. A method as defined in claim 21, claim 22, or claim 23, wherein
said stabilizer is EDTA-NaCl solution having a concentration lying
in the range of 0.2 to 1.2%.
Description
FIELD OF THE INVENTION
This invention relates to an automated system for the rapid
sequential photometric analysis of double fluorochrome stained
cellular and non-cellular structures from the same selected object
area of a specimen. With a plurality of specimens, fluorescence
light intensities of different wavelengths emitted from a selected
site in each specimen are quantitated in fast, sequential measuring
steps.
More particularly, the invention relates to an automated method and
apparatus for antibody screening, or for the testing of
lymphocytotoxicity by double fluorochromization analysis which
facilitates tissue typing procedures by providing objective
standardized data, and to a method of preparing specimens for such
testing.
The invention also relates to automated apparatus for observing
fluorescence from a plurality of specimens each of which exhibits
fluorescence at a first emission wavelength when irradiated with
light of a first excitation wavelength and fluorescence at a second
emission wavelength when irradiated with light of a second
excitation wavelength, and to a method of preparing an automated
lymphocytotoxicity assay simultaneously employing two contrasting
fluorochromes.
DESCRIPTION OF THE PRIOR ART
The basic principles of immunofluorescence and their application in
immunoenzyme analytical techniques are well known. Suitable
fluorochromes, such as fluorescein (F) demonstrate the existence of
biological structures, which are extra--or intracellular parts of
cells or tissues, by creating fluorochromasia. Fluorescence
reactions are observed with light of longer wavelength within the
visible spectrum (green, yellow, orange or red) than that of the
exciting light (green, blue or UV).
In 1966 Rotman and Papermaster (Biochemistry 55, 134-141, 1966)
demonstrated that certain fluorescein esters of fatty acids, such
as the diacetate FDA, used to stain cellular specimens, resulted in
intracellular fluorescein retention due to hydrolysis of FDA to F,
and highly intensive fluorochromasia.
As the degree of staining with FDA and like compounds depends
directly, inter alia, on the integrity of the cell surface
membrane, the intensity of fluorescence may be used in a visual
differentiation of live and dead cells in a stained specimen.
Methods of visualizing FDA stained live cells or of quantitating by
measuring different fluorescence light intensities while simply
assuming cell death and lysis in respect of the residual cellular
material--described as monofluorochromatic assays--are
unsatisfactory in that they only yield an estimate of the numbers
of living and dead cells in a specimen, thereby failing to
differentiate accurately between 100% killed (lysed) cells and
merely absent cells.
In the NIH technique for testing lymphocytotoxicity the visual
differentiation of live and dead cells is effected through the use
the non-fluorescent stain eosin Y, which stains dead lymphocytes
and not viable cells. Lymphocytes are added to the various typing
sera in a microtyping tray, and the cell-serum mixtures incubated
with rabbit complement. To each specimen the stain and buffered
formaldehyde are sequentially added. Reactions of the
cell-serum-complement-dye mixture are then read visually using a
microscope, living lymphocytes appearing as small refractile bodies
and dead reactive lymphocytes appearing as larger stained bodies or
lysed cells. The reactions are evaluated by calculating the
percentage of live cells in control negative and positive specimens
and establishing whether or not a distinct increased staining (cell
death) of lymphocytes occurs in the test specimens.
Both tests, the above-described cytotoxic test employing the uptake
of a vital stain such as FDA and the NIH test, are slow (owing to
the requirement for painstaking visual analysis of the specimens)
and are sometimes subject to the inaccuracies inherent in assuming
cell lysis in the absence of visible cells and to those arising
from human error, e.g. the misreading of cells out of microscopic
focus.
Reports that fluorochromes such as ethidium bromide (EB) (LePecq
and Paoletti in Journal of Molecular Biology 27, 67-106, 1967),
propidium iodide (PI) (Bruning et al, Human Immunology 5, 225-231,
1982), and rhodamine (R) (van Rood et al, Nature 262, 795-797,
1976) would form fluorescent complexes with nucleic acids resulting
in luminescence within the red wavelength spectrum, suggested the
possibility of "double-staining immunofluorescence (IF)" to
differentiate between live and dead cells in a double stained
specimen using well contrasting fluorochromization in an immune
reaction. Dead cells can be selectively labelled with PI or EB, the
stained dead cells fluorescing red in the presence of green and
blue exciting light, respectively. The stain penetrates the
membrane of damaged cells rapidly, where it gives a bright
fluorescence with nuclear DNA.
The superiority of double staining IF with well contrasting images
to the monofluorochromatic assays referred to above was
demonstrated first by Edidin (J. Immunology 104-105, 1313-1315,
1970) and later by Tagasugi (Transplantation 12, 148-151, 1971).
Edidin used the sequential application of contrasting stains, first
differentiating live and dead cells by FDA fluorochromasia
(Transplantation 6, 1010-1014, 1968) and later adding EB for
spectrophotometrically assaying the dead cell IF in the
lymphocytotoxcity assay.
Some efforts towards automation were reported by Martel, J. C. et
al (Vox Sang 22, 13-20, 1974), using an FDA-EB double-staining
technique, although automated data were not shown in their report.
They too stressed the importance of actually measuring the numbers
of living and dead cells rather than only estimating them as in the
older FDA-monofluorochromatic assays.
In 1976, Van Rood et al employed a differential, double-staining
technique to examine specimens of lymphocyte populations, including
T and B cells. The cells were double-stained with FITC as a cell
surface membrane marker for B cells and EB or R as a nuclear
contrasting fluorochrome. The stained specimens were sequentially
excited to obtain green IF from FITC-conjugated antiimmunoglobulins
on B cells, contrasting with the red EB or R derived nuclear
fluorochromasia. While future options for automation were
discussed, none was shown in the reported results of Van Rood.
An attempt at achieving the highly desirable end of automation of
standardized lymphocytotoxicity testing by microphotometry of
double stained specimens was reported by Bruning et al (Human
Immunology 5, 225-231, 1982). The cell specimens to be analyzed
were held in Terasaki trays and studied with incident fluorescence
light excitation using a Leitz MPV compact MT fitted with
photomultiplier, motorized scanning stage, and a computer with
printer unit. However, two separate staining methods had to be
applied. A carboxyfluorescein fluorochromasia resulting in green
fluorescence of live cells was used. In a separate staining
procedure, the application of PI replaced the formaldehyde step of
the NIH lymphocytotoxicity test after the complement-mediated
lysis. The reported technique is not fully automated in that the
microscopic equipment used did not allow for the fast sequential
differentiation of one simultaneously dual-stained object, a
procedure which is necessary for any fully automated typing
technique.
SUMMARY OF THE INVENTION
Apparatus is provided according to the invention for use in
fluorescence assays of specimens exhibiting fluorescence at two
distinguishable wavelength bands when irradiated respectively by
light of two pre-selected distinguishable wavelength bands. The
apparatus comprises:
movable specimen containing means having spaced compartments for a
plurality of individual specimens to be assayed;
alignment means for moving the container to provide sequential
alignment of individual compartments of the container with an
optical path;
irradiating means for irradiating said specimens sequentially and
alternately with light of the two pre-selected distinguishable
wavelength bands beamed along the optical path;
light collection means for receiving and distinguishing light
produced by fluorescence in each said specimen along the optical
path; and
detector means for detecting and recording the intensity of the
light produced by fluorescence of each said specimen at each of
said two distinguishable wavelength bands.
The apparatus also preferably comprises a computer programmed to
control and synchronize the operation of the alignment means,
irradiating means, light collection means, and detector means. The
computer preferably also includes
quotient calculation means for calculating the quotients of the
intensities of the light sequentially received from each said
specimen at the two distinguishable wavelength bands; and
comparison means for comparing the said quotient for each specimen
against a predetermined control quotient thereby to classify the
specimen according to such comparison.
The light collection means may include a pair of barrier filters
each alternately selectable to pass light of a respective one of
said two distinguishable wavelength bands to the detector
means.
The movable specimen containing means may conveniently be a
transparent specimen holder such as a microtiter tray containing
the specimens in a planar array. The specimen holder may be movably
retained in a conventional microscope including a scanning stage
operable to retain said specimen holder and to be positioned to
place any one of the specimens at a selected position within the
objective field of said microscope, so as to collect for
observation fluorescent radiation from said specimen. A
conventional two-step motor can be provided which is operable to
drive the scanning stage in orthogonal directions within a plane
perpendicular to the focal axis of the objective of said
microscope.
Two separate optical arrangements are selectably and alternately
provided for each specimen. These arrangements are positioned to
direct irradiating light of the two selected wavelength bands
alternately in turn to each specimen (each specimen in turn being
located at the selected position within the objective field of the
microscope). These optical arrangements are also operable to
transmit light of the selected emission wavelength bands,
respectively, from each specimen in turn located at the selected
position to the ocular of said microscope for observation or to a
detector (e.g. a photomultiplier) which provides an output signal
which can be utilized as an input to the computer.
A motor is provided for alternately positioning the first optical
arrangement and the second optical arrangement in the path of light
from the source of light. The computer or other suitable control
means operatively connected with each of the motors causes the
scanning stage to move in incremental steps, preferably in a
meander path, to place each of the specimens sequentially at the
selected position within the objective field of said microscope for
a predetermined period of time. This actuates the optical
arrangement selection motor during said predetermined period of
time to position first one of the optical arrangements and then the
other in the optical path from the specimen so that fluorescence
light of the first emission wavelength and then of the second
emission wavelength can be observed by means of the microscope for
each of the specimens or can actuate the photomultiplier.
The two optical arrangements preferably comprise epi-fluorescence
condenser sets. The motorized slide arrangement may comprise
(i) a housing to hold the epi-fluorescence condenser sets in
parallel side-by-side alignment (the housing being axially movable
between two stop positions to alternately place said
epi-fluorescence condenser sets in the path of light from said
source), and
(ii) a motor operatively connected to the housing for moving said
housing between the two stop positions.
A method of preparing specimens for immunofluorescence measurement
of the cytotoxic reaction between suitably prepared lymphocytes and
selected identical quantities of antisera according to the
invention comprises the sequential steps of:
(a) adding the lymphocytes (which have preferably been washed in a
non-autofluorescing wash solution) to a first staining solution to
prepare a suspension having a known concentration of lymphocytes,
said first staining solution comprising a first fluorochrome
selectively staining living cells,
(b) adding a selected quantity of said suspension to each of said
antisera and immediately thereafter adding to each of the
antiserum-lymphocyte specimens a selected quantity of complement
(C'),
(c) agitating the specimens at room temperature for a selected
period of time,
(d) adding to each of the specimens a selected quantity of a second
staining solution comprising a second fluorochrome selectively
staining dead cells and exhibiting fluorescence over a range of
wavelengths contrasting with the range of fluorescence exhibited by
said first fluorochrome, and
(e) incubating the specimens for a selected period of time.
A stabilizer may be added to the incubated specimens to inhibit
further cytotoxic reaction between the antibodies of said antisera
and the antigens of said lymphocytes, thereby decreasing background
IF.
The first fluorochrome is preferably selected from the group
consisting of fluorescein diacetate, fluorescein isothiocyanate and
carboxyfluorescein diacetate, and the second stain is preferably
selected from the group consisting of ethidium bromide, propidium
iodide, rhodamine and rhodamine isothiocyanate (RITC).
The method according to the invention for fluorescence assays of
specimens exhibiting fluorescence at two distinguishable wavelength
bands when irradiated respectively by light of two pre-selected
distinguishable wavelength bands comprises the following steps:
sequentially aligning individual such specimens with an optical
path;
irradiating the specimens sequentially and alternately with light
of the two pre-selected distinguishable wavelength bands beamed
along the optical path;
receiving and distinguishing light produced by fluorescence in each
such specimen along the optical path; and
detecting and recording the intensity of the light produced by
fluorescence of each such specimen at each of said two
distinguishable wavelength bands.
The automated method additionally comprises
calculating the quotient of the intensities of the light
sequentially received from each such specimen at the two
distinguishable wavelength bands; and
comparing the quotient for each specimen against a predetermined
control quotient thereby to classify the specimen according to such
comparison.
According to the fully automated system of double
fluorochromization analysis in antibody screening or
lymphocytotoxicity testing of the present invention, only one
combined procedure is needed for the evaluation of live and dead
cells stained with two contrasting fluorochromes. The controls are
directly derived from the fluorescence light intensity (LI) raw
data and expressed on a scale analogous to the percentage scale for
cytotoxicity with percentages of live and dead cells. In a single
evaluation step, both the negative and positive control quotients
are simultaneously obtained from the same specimen object area. All
results reflect directly transformed raw data and represent an
interpretation of the double staining immunofluorescence
measurement expressed as light intensities. The data analysis
associated with the system includes a statistical validity check,
any data exceeding three standard deviations off the mean being
rejected and marked as such.
As a consequence of these features of the system of the invention,
a considerable saving of time and manpower over prior techniques of
analysis may be achieved while producing a quantitative assay using
standardized procedures that are easily checked.
BRIEF DESCRIPTION OF THE DRAWINGS
In the drawings which illustrate embodiments of the invention:
FIG. 1 is a perspective view of the modified epi-fluorescence
invertoscope and related components employed in a preferred
embodiment of apparatus according to the invention.
FIG. 2 is a schematic view of a preferred embodiment of apparatus
according to the invention.
FIGS. 3 and 4 are respectively a top plan view and a perspective
view of a prepared microtest well tray showing by means of an arrow
a preferred scanning sequence for the test wells when used in the
automated scanning system of the invention.
FIG. 5 is a portion of a specimen computer print-out of the type
obtained when the preferred automated embodiment and process of the
invention are utilized.
FIG. 6 comprises two graphic representations of immunofluorescence
intensity measurements obtained using the apparatus of the
invention in conjunction with the tray preparation method of the
invention in connection with a specific example discussed below, in
comparison with results obtained by utilizing a conventional
technique.
FIG. 7 is a schematic block diagram showing the optical path
transversed by incident exciting light and by fluorescent radiation
emanating from a sample being analyzed.
DESCRIPTION OF PARTICULAR EMBODIMENT
As mentioned, the present invention is concerned with a rapid
sequential photometric analysis of double fluorochrome stained
cellular and non-cellular structures. If of cellular origin, the
stained structures may be situated in the core, as in nuclear
stains, or on the cell surface, as is the case when membrane
markers are used.
In the apparatus of the invention, any of a variety of standard
trays and slides may be used to hold the stained cell culture
specimens, e.g. microscope slides. However, microtiter trays of the
kind illustrated as tray 1 in FIG. 1 are particularly well suited
for use with the apparatus of the invention where the automated
double-staining IF analysis of white blood cells is studied with
incident fluorescent exciting light.
Tray 1 of FIGS. 2 and 3 contains sixty identical wells 2 for
holding the specimens and standards to be analyzed. Automated
antibody screening or lymphocytotoxicity testing with the system of
the present invention is preferably performed in sixty or
seventy-two well microtest trays.
In lymphocytotoxicity testing, target lymphocytes having an unknown
antigenic pattern are reacted with selected quantities of antisera
that have been placed in identified wells of the microtest tray.
These antisera contain known antibodies and the IF data obtained by
the method of the invention provide a measurement of cytotoxicity.
Evaluation of cytotoxicity is employed in matching potential donors
to a recipient in clinical immunology, e.g. tissue transplantation
and blood transfusions where the known antisera may be those of the
recipient and the target lymphocytes those of the potential
donor.
In antibody screening, it is desired to identify the antibody
specificity of unknown antisera. For example, it may be important
to ascertain the antibodies present in the blood serum of a patient
who has received a blood transfusion. Known quantities of the
antisera are placed in the test wells of the microtest tray and are
reacted with selected quantities of lymphocytes having a known
antigenic pattern.
Whether the method of the invention used for lymphocytotoxicity
testing, is employed for determining antigens or antibodies, the
method of tray preparation is preferably as described below.
However, in the former case commercially prepared standarized
microtest trays containing the known antisera are available and may
be used. In the latter case it is necessary first to add known
quantities of the unknown antiserum or antisera to be tested to the
microtest tray wells. In either case, the use of known quantities
of antisera in reaction with known quantities of lymphocytes in the
same quantitative relationship for each specimen testing is
critical to obtaining reproducible quantitative measurements of
cytotoxicity.
In the preparation of a specimen tray for lymphocytotoxicity
according to the present invention, HLA antisera, including known
negative and positive control sera are placed in the respective
wells of non-autofluorescing microtiter trays. Viable target
lymphocytes are washed in a medium which must contain no colourant
capable of producing disturbing autofluorescence.
The target lymphocytes are then treated with a fluorochrome for
later differentation between live cells and contrast-stained dead
cells. The fluorochromization of dead cells occurs later following
a specific antigen-antibody reaction. The live-stained cells are
thoroughly washed to remove excess extracellular fluorochrome.
Identical minute quantities of the singly-stained incubated cells
are placed into selected wells of the tray wells. A selected
portion of the tray wells contains equal quantities of positive and
negative control standard antisera. These are known to be either
reactive or non-reactive, respectively, with the lymphocyte cell
surface membrane markers and serve as reference data in the
computations conducted in the system of the present invention.
To the singly stained incubated cells, antisera and control
standards, microquantities of complement are then added and the
mixtures agitated in the wells of the microtest tray for a period
of time which is sufficient for the antigen-antibody-complement
interaction. Thereupon, to each of the cell samples and control
standards a small quantity of a second staining agent is added. The
reaction may be terminated by adding a stabilizer and centrifuging
the trays.
The method of the invention is also applicable to antibody
screening of unknown sera using lymphocytes having a known
antigenic pattern. In that case, selected identical quantities of
the sera to be tested are first placed in identified wells of the
microtest tray. When choosing the two staining agents for the
cells, it is preferable to use those fluorochromes which emit
fluorescence light at different wavelengths, such as the well
contrasting fluochromization in the green and yellow-orange-red
ranges. Examples of dyes which fall within this definition are
fluorescein-derived green fluorochromes, such as FITC, FDA, CFDA,
on the one hand, or red fluorescent dyes such as EB, PI, R or RITC,
on the other hand.
According to a preferred embodiment of the present invention, the
automated evaluation of the prepared trays is performed using an
inverted epi-fluorescence microscope which may be a conventional
commercially available Zeiss* model IM 35 photo-invertoscope,
modified as discussed in greater detail below.
As seen in FIG. 1, the microscope indicated as 3 is provided with a
scanning stage 4 adapted to hold microtest trays 1 and to be
positioned to place any one of wells 2 at a fixed position
pre-focused within the viewing field of the microscope objective.
In the automated use of the system, as described below, scanning
stage 4 is motorized to move in (say) 50 .mu.m steps by X-Y drive
motor 5. These components are individually known and readily
commercially available.
The arrangement of optical elements in microscope 3 is illustrated
simply in FIGS. 2 and 7.
The source 7 of exciting radiation, is preferably an HBO 100 W high
pressure mercury lamp which radiates over the visible wavelength
spectrum. Light from the source may be selectively directed through
a selected one of two epi-fluorescence condenser sets integrated
into a single slideable dual condenser housing unit 8. In FIG. 7,
elements of housing unit are shown with one of the condenser sets 9
in operating position. On the assumption that FDA is utilized
(which will result in green immunofluorescence) and EB is selected
as the contrasting stain (which will result in red
immunofluorescence), then Zeiss* Models Nos. 48 77 10 and 48 77 15
may be used as the alternately selectable condenser sets.
Assume for the moment that the first of these condensers is
positioned in the optical path illustrated in FIG. 7, Zeiss*
condenser 48 77 10 comprises a blue exciter filter 10 (BP 450-490
nm), a chromatic beam splitter 11 for green fluorescence (FT 510
nm) and a barrier filter 12 (BP 520-560 nm) for green
fluorescence.
Light from the exciting source is filtered through exciter filter
10 and impinges at a 45.degree. angle on beam splitter 11. The
purpose of exciter filter 10 is to narrow the wavelength range of
the light impinging on the specimens to approximately the blue
range 450-490 nm suitable for excitation of specimens treated with
FDA. The reflected portion of the blue light, indicated by arrow
13, is directed at right angles to the incident beam, indicated by
line 14, and passes through fixed microscope objective 15, which
focuses the blue exciting radiation onto the stained prepared
specimen in well 2A of microtiter tray 1.
Back-scattered green fluorescence light, indicated by arrow 16, in
turn passes through objective 15 and impinges on beam splitter 11.
The transmitted portion of the green fluorescence is filtered
through barrier filter 12, and passes through a pin hole with a
diameter in the millimeter range onto the light-receiving aperture
of photometer 18 (preferably an S'F photometer with Hamamatsu HTV R
928 photomultiplier tube) optically aligned with well 2A and
objective 15 along an axis perpendicular to the beam of exciting
light from light source 7. The transmitted fluorescence passing
through barrier filter 12 may alternatively be observed visually
through the ocular (not shown) of the microscope. The provision for
direct visual microscopic examination of a specimen in a selected
well within the viewing field of microscope objective 6 is not only
to permit conventional visual specimen analysis when desired, but
also to allow initial visual control of the centering of the test
wells within the viewing field. The purpose of barrier filter 12 is
to pass only the green light approximately in the band 520-560 nm
which has been produced by fluorescein fluorochromasia.
As may be seen from FIG. 2, the movement of housing 8 at right
angles to the plane defined by the optical pathways brings either
selected one of the condenser sets into the operating position
shown in the figure, only one condenser set at a time being placed
in the optical path. The second condenser set is similarly
constructed to the first, comprising an exciter filter, chromatic
beam splitter and barrier filter, but operates to direct green
light on the sample in the tray well and to pass to the photometer
fluorescence in the red wavelength range. For this purpose, the
second condenser set can be a Zeiss Model No. 48 77 15 with an
exciter filter of BP 546-10, chromatic beam splitter FT 580, and
barrier filter LP 590.
Housing 8 may be moved manually in the sense described above to
alternately position the two condenser sets in the required optical
path and to permit visual reading of the specimens through an
eyepiece (e.g. the ocular of microscope 3) instead of taking
photometric measurements of fluorescence intensity. In the
automated system, housing 8 is actuated by a drive motor 20 in two
stop movements for each sample measurement of green and red
immunofluorescence, such drive motors being conventional and
commercially available. In the automated process, the actual object
measuring time of less than one second for each excitation
wavelength does not result in any significant fading of
fluorescence intensity. Accordingly, a single specimen can be read
many times without loss of accuracy.
Earlier techniques of fluorochromization analysis employed
transmitted light microscopy. The arrangement of an invertoscope
with incident light fluorescence excitation, including a turn-back
illumination carrier 21, has the advantages of providing a large
working distance for use with all microtiter trays, and of a
pre-focused selected position for a specimen in the objective
field.
In automated operation, tray 1 is scanned by the motorized scanning
stage so that each specimen or control well is successively brought
into the focal position along the "meander" path indicated by the
line 17 of FIGS. 3 and 4. For reproducibility of results, it is
critical that each U-shaped well of the tray be successively very
accurately centered at the pre-focused position within the optical
field of the microscope objective. The meander path referred to
above is best adapted to the requisite control of well positioning
using an X-Y drive motor for the scanning stage holding the
tray.
The automated system further includes computer assisted control
means 19 (by way of example ZONAX* Intelligent Systems) for
directing stepwise movement of X-Y drive motor 5 to center the
specimens in the microtiter tray successively in a selected
prefocused position of the microscope objective. The alternating
motion of the movable filter housing is also synchronized under the
control of the computer with the motion of the tray. The filter
housing automatically successively interposes between the
irradiated sample and the photometer the first barrier filter
transparent to the green immunofluorescence light emanating from
the FDA fluorochromasia and the second barrier filter transparent
to the red immunofluorescence light emanating from the EB
fluorochromasia, as previously described. The interface between
control means 19 and drive motors 5 and 20 is conventional, and may
comprise a buss connector and an analogue to digital converter.
Fluorescence light from each sample or control standard is measured
as the output from photometer 18. The output is analyzed by the
computer which also controls tray movement and switching of the
filters. The computer-directed automatic measuring process uses the
photomultiplied fluorescence light intensities as raw data and
effects a computation resulting in quotients of arbitrary LI units
derived from the two original IF measurements. The final human
leukocyte antigen (HLA) score number represents a direct quotient
transformation of the original dual staining IF data from the same
object measuring field.
SPECIFIC EXAMPLE OF TRAY PREPARATION AND ANALYSIS RESULTS
In a preferred method of tray preparation for lymphocytotoxicity
testing according to the invention, each well of a
non-autofluorescing non-static microtiter tray is filled with 4
.mu.l petrolatum (liquid) and 1 .mu.l of HLA antiserum. Known
negative and positive control sera are included. Lymphocytes are
obtained from heparin blood by gradient centrifugation and three
subsequent washes in phenol-red free Hank's balanced salt solution
(HBSS), pH 7.2 to which 5% bovine serum albumin or fetal calf serum
are added. Other washing solutions such as cell culture media may
be used so long as their colour additives do not result in
autofluorescence.
1 ml HBSS is added to a 10 .mu.l aliquot of a FDA/acetone
-20.degree. C. stock solution. The concentration of this stock
solution may be in the range of 3-6 mg FDA per ml of acetone, but
preferably 5 mg FDA/ml acetone. 100 .mu.l of the FDA/acetone/HBSS
mixture per 1.5-3.times.10.sup.6, preferably 2.times.10.sup.6
washed cells are incubated for up to 5 minutes, preferably for 4
minutes, then washed twice, 100 times diluted, in HBSS/BSA for 5
minutes at 2000 g, and adjusted to 2.times.10.sup.3 cells/.mu.l. 1
.mu.l of the suspension is dropped into each well. 1-2 .mu.l of
complement (C') are added after a minimum of 30 minutes incubation
time. The use of freeze-dried rabbit C', immediately prepared
before use is recommended, as are equal amounts of fresh C'. More
preferably, 1 .mu.l of C' is combined immediately with the nuclear
stained cells and continuously agitated at room temperature. The
speed of the agitation may range from 10-50 cycles per minute and
the corresponding range of time of agitation from 30-80 min.
Preferably, the mixtures of antiserum, singly stained cells and C'
are rotatively agitated at 30 rpm for 45 minutes. A 0.2-0.8 .mu.l
drop of ethidium bromide from an EB stock solution of 50 .mu.g EB
per 50 ml Veronal buffer, pH 7.0 is then added to each well and
incubated for 7-15 minutes, preferably about 12 minutes. 1 .mu.l of
an disodium ethylene diamene tetraacetate/sodium chloride
(EDTA-NaCl) solution of concentration in the range 0.2-1.2%,
preferably 0.4%, may be added and the tray is rotated in a
centrifugal movement at 200 g for 2-8 minutes, preferably about 3
minutes. The prepared tray is closed with its lid. No slide and/or
oil is used to cover the test wells. The tray is then to be kept at
4.degree. C. and may be read up to 24 hours after an initial period
of "settling down" of the cells. The reading may be performed at
room temperature with or without tray lid. However, trays should be
stored at 4.degree. immediately after reading.
FIG. 6 presents graphical representations of the green and red IF
intensities, respectively, measured using the above-described
apparatus and method of the invention for lymphocytotoxicity
testing of target lymphocytes against standard antisera containing
known antibodies. In each of the figures, the Y-coordinate
represents the fluorescence intensity in arbitrary LI units. Each
data point represents the average of ten to twelve calculated mean
intensities, each of which is in turn the calculated average of
fifteen original intensity measurements employing a damping factor
of 15.
In FIG. 6 (both graphs), the X-coordinate is a measure in .mu.l of
the varying amounts of complement C' used in the test system. The
circular data points were obtained by adding C' to the test wells
immediately after addition of the FDA-stained cells to the antisera
and slight agitation of the mixture. The square data points
represent results obtained by the conventional procedure of
incubating FDA-stained cells with the antisera for about 30 minutes
prior to adding C'. It may be seen from the figures that the
preferred method of tray preparation according to the invention
gives rise to higher fluorescence light intensities than are
conventionally obtained, with attendant decrease in percentage
error of measurements.
The raw IF data and HLA scores for lymphocytotoxicity testing as
presented by the ZONAX computer programmed according to the system
of the present invention is illustrated below in a reproduction of
a typical printer display (see also FIG. 5):
______________________________________ PLATE NUMBER: 101 PLATE
NAME: R. Richards A B C D E F
______________________________________ 1: 184.6 143.5 214.3 145.2
272.1 129.4 : 65.5 74.9 64.3 48.2 63.6 62.7 2: 204.5 255.0 216.9
192.6 177.7 223.6 : 50.0 67.5 44.5 44.9 45.1 59.6 3: 161.7 208.7
218.3 161.7 157.3 157.4 : 80.8 59.9 112.9 92.7 77.7 68.1 4: 175.2
141.5 140.3 150.1 158.5 182.3 : 45.1 42.9 43.3 44.1 43.3 65.2 5:
150.5 161.7 177.8 159.1 174.6 226.5 : 76.3 59.1 59.9 62.0 62.9 97.9
6: 124.5 125.5 133.1 143.9 126.6 149.2 : 75.2 82.5 62.0 63.4 60.4
61.2 7: 147.4 207.1 188.5 176.1 175.9 166.8 : 46.9 64.8 53.7 48.6
45.1 44.0 8: 135.8 127.4 143.9 142.5 158.1 143.3 : 77.3 62.9 80.7
80.3 81.4 96.2 9: 193.3 194.0 207.7 236.9 177.1 190.8 : 61.8 67.1
63.6 65.9 51.3 50.9 10: 154.3 256.2 158.8 157.4 247.7 157.9 : 83.1
89.7 77.0 81.7 61.5 87.3 1: 2.818 1.916 3.333 3.012 4.278 2.064 2:
4.090 3.778 4.874 4.290 3.940 3.752 3: 2.001 3.484 1.934 1.744
2.024 2.311 4: 3.885 3.298 3.240 3.404 3.661 2.796 5: 1.972 2.736
2.968 2.566 2.776 2.314 6: 1.656 1.521 2.147 2.270 2.096 2.438 7:
3.143 3.196 3.510 3.623 3.900 3.791 8: 1.757 2.025 1.783 1.775
1.942 1.490 9: 3.128 2.891 3.266 3.595 3.452 3.749 10: 1.857 2.856
2.062 1.927 4.028 1.809 1: 1 8 1 1 1 8 2: 1 1 1 1 1 1 3: 8 1 8 8 8
4 4: 1 1 1 1 1 1 5: 8 1 1 2 1 4 6: 8 8 6 6 8 4 7: 1 1 1 1 1 1 8: 8
8 8 8 8 8 9: 1 1 1 1 1 1 10: 8 1 8 8 1 8
______________________________________
In obtaining the above data, a single Falcon sixty-well plate was
processed containing specimens identified by numerals 1 to 10 in
the leftmost column of each printed table of data and the six
letters A-F across the top of each table.
In the first table of data, the raw IF intensity data are given in
arbitrary LI units, red IF above and green IF below. For example,
specimen 8C produced red IF of 143.9 units and green IF of 80.7.
Well 1A of the tray shows the negative control readings (viable
cells), well 1B the positive control readings (dead cells) obtained
by the same automated photometric action.
The second table of data presents red IF/green IF quotients and the
third table HLA scores expressing the percentages of live and dead
cells as integers from 1 to 8, each integer representing a selected
range of cytotoxicity comparable to the percentage scale 0-100%
derived from the standard NIH technique. Thus the HLA score of 1
for negative control 1A refers to cytotoxicity lying in the range
0-20% and the HLA score of 8 for positive control 1B refers to
cytotoxicity in the range 81-100%. However, the automated LI
measurements require an open scale due to the possibility of
further increased green and red fluorescence intensities beyond
those evaluated in the negative and positive control wells.
The program governing the system computer through the entire
analysis procedure enables for the first time a completely
automated tray analysis. Once the first well is centered in the
object field and the system is set running, no further manual
interference is needed for the evaluation of a prepared microtiter
tray.
Functionally, the program includes a number of sub-programs,
namely:
1. A plate measuring sub-program to determine the requisite
accurate measuring steps for correct centering of tray wells to
avoid deviations during tray evaluations. This sub-program permits
the use in the system of different tray sizes and allows for
dimensional variations between the multiple-well trays of different
manufacturers and different lots of the same manufacturer. Further,
the program permits the measurement of pre-selected areas of a tray
in a single scan, instead of a complete scan through all tray
wells.
2. A data acquisition sub-program. This includes parameters for
identifying particular test trays, for controlling light source
parameters such as high voltage and gain, damping factor, for
controlling the motorized filter housing, including the sequence of
fluorescence measurements, for controlling the motorized microscope
stage and centering a well system within the focus of the
microscope objective, routines for the measurement of
photomultiplied IF and for the direct presentation of numerical LI
data on the computer terminal screen.
3. A data analysis program which includes and prints pairs of raw
green and red IF data, performs statistical evaluations and
rejection of unacceptable data, transforms data into quotients,
establishes a control range from highest and lowest absolute
quotients, checks the actual negative and positive control well
data with respect to their validity (rejecting them in the case of
an unacceptable control range and replacing them with the highest
and lowest measuring data), rearranges the control range according
to the percentage range for lymphocytotoxicity, and provides for
the arbitrary choice of a nomenclature for expressing the light
intensity ranges of live and dead cells.
4. An antibody name program which provides for the co-ordination
between serum and cell identification (name) in each tray well and
IF measurement results, expressed as percentage of live over dead
cells.
By means of control of the system through this program, each tray,
regardless of size, number of sera, serum frequency within a tray
or manufacturer of the tray may be correctly assayed. All results
reflect directly transformed raw data and represent an
interpretation of the double staining IF measurement expressed as
light intensities. It is apparent that a number of alternative sets
of program instructions might be employed in conjunction with the
apparatus of this invention to effect the controls and measurements
described above. Included as Appendix A to this disclosure is a
listing of the plate measuring and data acquisition programs used
by the applicants in conjunction with the ZONAX unit. Included as
Appendix B is a listing of the data analysis program.
While a particular embodiment of applicants' invention has been
described and shown with reference specifically to fully automated
double fluorochromization analysis in lymphocytotoxicity testing,
it is to be understood that this embodiment is illustrative only,
and that the present invention is not limited thereto, but includes
all embodiments falling within the scope of the appended claims.
##SPC1##
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