CA1200477A - Method for detecting luminescence in living cell system - Google Patents
Method for detecting luminescence in living cell systemInfo
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- CA1200477A CA1200477A CA000431930A CA431930A CA1200477A CA 1200477 A CA1200477 A CA 1200477A CA 000431930 A CA000431930 A CA 000431930A CA 431930 A CA431930 A CA 431930A CA 1200477 A CA1200477 A CA 1200477A
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6445—Measuring fluorescence polarisation
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- Investigating, Analyzing Materials By Fluorescence Or Luminescence (AREA)
Abstract
ABSTRACT OF THE DISCLOSURE
A method for determining a time dependent change in the luminescent polarization of a living cell-dye system having a luminescent probe associated with the living cells, where the living cell-dye system is dispersed in a medium. The method comprises introducing a dye into the medium to associate a luminescent probe with the living cells of the living cell-dye system where luminescent components of the dye, which are unassociated with the living cells, increase in concentration over time in the medium to provide a background luminescence. Polarized light in a distinct orientation having a significant vertical orthogonal component is irradiated onto the living cell-dye system and medium. The polarized light emissions from the luminescent probe associated with the living cells and luminescent components in the medium are analyzed in two distinct orientations representing horizontal and vertical orthogonal components of the polarized light emissions to determine the time dependence of the components of the polarized emissions from the luminescent probe associated with the living cells of the system and the luminescent components of the medium.
The steps of generating polarized light and analyzing the polarized light emissions are conducted during a period commencing at times greater than the luminescence lifetime of the living cell-dye system and continuing until the background luminescence from the system medium significantly affects the polarized light emission intensities from the luminescent probe associated with the living cells of the system.
A method for determining a time dependent change in the luminescent polarization of a living cell-dye system having a luminescent probe associated with the living cells, where the living cell-dye system is dispersed in a medium. The method comprises introducing a dye into the medium to associate a luminescent probe with the living cells of the living cell-dye system where luminescent components of the dye, which are unassociated with the living cells, increase in concentration over time in the medium to provide a background luminescence. Polarized light in a distinct orientation having a significant vertical orthogonal component is irradiated onto the living cell-dye system and medium. The polarized light emissions from the luminescent probe associated with the living cells and luminescent components in the medium are analyzed in two distinct orientations representing horizontal and vertical orthogonal components of the polarized light emissions to determine the time dependence of the components of the polarized emissions from the luminescent probe associated with the living cells of the system and the luminescent components of the medium.
The steps of generating polarized light and analyzing the polarized light emissions are conducted during a period commencing at times greater than the luminescence lifetime of the living cell-dye system and continuing until the background luminescence from the system medium significantly affects the polarized light emission intensities from the luminescent probe associated with the living cells of the system.
Description
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METHOD FOR DETECTI~ LUMINESCENCE IN LIVING CELL SYSTEM
FIEI,D OF THE INVENTION
_ _ _ _ _ _ .
This invention relates to a method and apparatus for determlning the time dependent change in luminescent polarization of a luminescent dye associated with a living cell-dye system~
BACKGROUND OF T~E INVENTION
In the study of living cells, it is often necessary to analyze and evalua-te various biological changes associa-ted with the living cells. There are many approaches to probing such biological changes associated with living cells, such as the use in photometric techni~ues of which flow cytometry is an example. However, such equipment is very expensive and painstaking to use.
Another approach to evaluating biological changes associated with a living cell is to associate a dye probe with the cell and study the changes in luminescence of this probe. This technique has been particularly applied to inducing fluorescence with a type of cell known as a lymphocyte to establish whether or not a malignant tumour is present in the donor from which lymphocytes ha~e been removed. The intensity of Eluorescent emission is studied to determine if a characteristic oE the lymphocyte is present, which characteristic is a result of the donor having cancer.
The original work on this technique began in the early 1970's. Drs. Cercek and Cercek developed a technique which had the abi]ity to differentiate lymphocytes from healthy donors from those of donors with cancer. The test was based upon differential lymphocyte response or lack oE response to either a protein isolated from malignant tumours or the mitogen phytohaemagglutinin.
The work by Cercek and Cercek is well published, the original technique being reported in Cercek L., Cercek B., and Franklin C. (1974), Brit. J. Cancer 29, 345-352.
The test to diEfexentiate healthy donors from unhealthy donors was thought to rely on changes in the structuredness of the cytoplasmic matrix ISCM~ of a specific population of blood lymphocytes stimulated by t~
phytohaemagglutinin (PHA) or cancer basic protein (CaBP)O Assuminy that there are changes in the matrix, then it was believed that such changes were detected by using polarized light to determine qualitatively the rate of change of orientatlon of fluorescent molecules within the living cell. As reported in the sritish Journal oE Cancer, the Cercek technique involved mixing fluorescein diacetate (E'DA) dye with a medium containing lymphocyte cells isolaked from donor b~ood. Enzymes of the cell hydrolize the FDA to produce fluorescein molecules which were thought to be formed within the cells. Such molecules are excited with polarized light.
Measurements are made of the degree of polarization of the emitted fluorescence by use of a f]uorescence spectrometer fitted with polarization accessories.
Calculations were made to de~ermine po]arization values which were then used in accordance with their disclosed formula to determine if the lymphocytes came from a healthy donor or a donor who has cancer. The technique was carried out over a period of at least six minutes in measuring fluorescent emissions. During this interval, the fluorescein molecules would increase in concentration in the medium, due to some of the fluorescein molecules ormed by enzymatic action leaking out of the living cells. The increasing concentration of fluorescein molecules in the medium affects the reading on the intensities of emissions from fluorescein molecules thought to be within the cells. To compensate for this increasing concentration of fluorescein molecules in the medium at the end of the testing period, the medium with cells would be filtered so as to remove the cells and then a reading would be taken of the intensity of the remaining fluorescein molecules in the medium. These values were used to adjust the previously recorded values to give the absolute value for the change in intensity of the polarized fluorescence from molecules within the cells.
The Cercek and Cercek technique was improved upon by many including Dr. J.A.V. Pritchard. Dr.
. . .
Pritchard developed an improved technique for harvesting the cells from the blood sample to provide more consistent results. His work is widely published, where a review of his most up-to-date work is found in Europ.
J~ Cancer Clin. ~ncol., Vol. 18, No. 7, pages 651 through 659 (198~). Pritchard discovered that a particular method of using a Ficoll-Triosil gradient provided two bands or zones of cells within the centrifuged medium~ The cells from the two zones were tested. Some of the cells from each zone are tested unstimulated as control samples. A similar number of cells from each zone are stimulated with PHA and tested to yield superior results in determining an index indicative of the donor's health. After stimulation of some of the Zone 1 and Zone 2 cells with PHA the fluorescein diacetate was added which was thought to be hydrolyzed to fluorescein within the cells to produce fluorescence when excited by polarized light. A
fluorescence spectrophotometer was used to analyze polarized emissions to give values for intensi-ties over time. At the end of the testing which took approximately six minutes as with the Cercek technique, again filtering was required to establish the eEfect of free fluorescein in the medium on the values for emission intensity from fluorescein thought to be within the cells.
A considerable problem exists with the filtration required in the Cercek and Pritchard techniques to establish the effect that the free fluorescein molecules in the medium has on the intensity readings. The Eiltration disturbs the cells and can cause breakage of the cells which gives an erroneous reading. Due to the filtration technique, relatively large samples of blood are needed for the test. When considering the number of tests that are usually conducted in determining the existence of cancer, the additional quantities required by these tests can become quite significant. Another problem wlth filtering is the resultant lack of consistency in the results.
. , .
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Another prob:Lem encounter~d wlth these techniques is that they are very much subject to variation in results due to technician judgment, varying protocol and reagents. The bias of the operator conducting the tests can cause erroneous or misleading results.
The method and apparatus, according to this invention, overcomes the above problems in providing a technique which is reliable and relatively inexpensive to conduct.
SUMMARY OF T~IE IN~ENTION
To establish if living cells are from a healthy donor or an unhealthy donor, the time dependent change in the lurninescent polarization of a living cell-dye system having a luminescent probe associated with the living cells is determin~d. The living cell-dye system is dispersed in a medium, where a dye is introduced into the medium to associate a luminescent probe with the living cells of the living cell-dye system. Luminescent components of the dye, which are unassociated with the living cells, increase in concentration over time in the medium to provide background luminescence. Polarized light in a distinct orientation haviny a siynificant vertical orthogonal component is generated. The polarized light irradiates the living cell dye system plus medium. The polarized llght emissions from the luminescent probe associated with the living cells and luminescent components in the medium are analyzed in two distinct orientations representing horizontal and vertical orthogonal components of the polarized light emissions to determine the time dependence of the components of the polarized emissions from the luminescent probe associated with the living cells of the system and the luminescent components of the medium.
The steps of generating polarized light and analy2ing the polarized light emissions are conducted during the period commencing at times greater than the luminescence lifetime of the living cell-dye system after mixing and continuing unti] the background luminescence from the system medium significantly affects -the polariæed light emission intenslties from the luminescent probe associated with the living cells of the sys-tem. This avolds the need for physical separation of the living cells from the luminescent dye unassociated with the living cells.
Such dye acts as a luminescent probe which may be an organic or inorganic substance for absorbing light energy and produce fluorescent emissions which have a particular func-tional relationship with a cellular organism.
The method is particularly suitable for analyzing polarized fluorescent light emissions from the fluorescent probe associated with the liviny lymphocyte cell-dye system in the technique of detecting whether or not the cells are from a healthy donor or a donor who has cancer. The dye, which may be used is a weakly fluorescent dye which, when acted upon by the enzymes within the cells produces a highly fluorescent analog of the dye. According to a preferred aspect of the invention, the dye used is fluorescein diacetate which has fluorescein as its highly Eluorescent analog.
BRIEF DRESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention are described with reference to the drawings wherein:
Figure 1 is a graph demonstrating a plot of Intensity versus Time of measured intensities at various times for po~arized vertical and horizontal components of the emissions;
Figure 2 is a graph demonstrating a plot of calculated values oF the Polarization Index versus Time;
Figure 3 is a schematic showing the optics for the kinetic polarization fluorometer;
Figure 4 is a top plan view of the fluorometer schematically shown in Figure 3;
Figure 5 is a block diagram of the electronic control device for the fluorometer; and Figure 6 is a schematic representation of the gradient of the centrifuged treated blood sample for isolating the two zones of lymphocyte cells.
: ' DETAILED _ESCRI_TION OF_THE PREFERRED EMBODI~ENTS
The chemical or biological mechanism of the living cells depending on the health of the donor which affects the intensity of polarized luminescent emissions over time is not fully understood. However, the method of this invention provides consistent results in distinguishing living lymphocyte cells of healthy donors from donors which have cancer. An index is calculated based on the intensity of luminescence emitted from the living cells in two distinct horizontal and vertical orientations which represen-t orthogonal components of polarized light emission. The consistency in the results is at least in part attributed to the method obviating the need to filter the medium containing the cells in determining the effect of unassociated luminescent components ~background luminescence) in the medium on the intensity of emissions from a luminescent probe associated with the cells.
It is understood that the method and apparatus of this invention may be used to determine the time dependent change in the lumi~escence polarization of a luminescent dye associated with the living cells in any form of living cell system. To demonstrate the invention by way of a preferred embodiment, reference is made to the method as applied to determinin~ polarized fluorescent light emissions from a fluorescent probe associated with living lymphocyte cells. The time dependent change of the fluorescence can be analyzed to determine if the lymphocyte cells are from healthy donors or donors with cancer. In order to evaluate the lymphocyte cells, they are extracted from blood which is collected from the donor. The blood is processed in a particular way to provide the lymphocyte cells in a form which can be stimulated with a mitogenic or antigenic agent~ such as PHA (phytohaemagglutinin). Such cells, after being stimulated by PHA are brought into contact with a dye such as FDA (fluorescein diacetate~. Non-specific carboxyesterase enzymes of the cell hydrolize the weakly ~luorescent fluorescein diacetate by deacetylating the dye to produce highly fluorescent ..J'.~ ;J 7 fluorescein which becomes associated with living cells and when exposed to appropriate light energy, fluoresces to produce fluorescent emissions.
In order to isolate the lymphocyte cells from collected blood, the ~ollowing procedure is preferred.
The venous blood sample is collected in appropriate containers and allowed to cool to room temperature.
Iron carbonyl is added to the blood to remove phagocytes and platlets by use of magnets in accordance with standard techniques in a temperature controlled water bath. A Ficol]-Triosil gradient is prepared and temperature equilibriated in a water bath so that the density of the gradient will be exactly 1.081 m/ml.
Aliquots of the treated blood are layered carefully onto the gradient and are separated into two zones in a temperature controlled centrifuge. The lymphocyte cells are removed from the two Zones (1 and 2~ of the gradient and are suspended in a modified phosphate buffered saline (PBS) at 37C. at a cell concentration of 1.0 x 106 cells/ml.
In order to determine the time dependent change in the fluorescent emissions from the cells of Zones 1 and 2, a kinetic polarization fluorometer may be used.
The fluorometer generates polarized light in a distinct orientation having a significant vertical component. By significant, it is meant that the intensity of the vertical component is sufficient to generate the desired orientation of excitation in the living cell-dye system to provide emissions which can be analyzed. The polarized light is directed onto the living cell-dye system to generate fluorescence from the dye associated with the cells and in the medium. The light emissions from the dye associated with the living cells and in the medium is polarized into two distinct orientations which represent horizontal and vertical components of the polarized emissions. The intensities of the polarized emissions in the orthogonal directions of the vertical plane and in the horizontal plane are analyzed to determine the health of the donor.
As shown in Figure 1, there is a plot of measured intensities in the vertical (Iv) and horizontal (IH) orientations versus time~ In this representative Figure, the intensities vary in a generally linear manner. The details in which the intensities are measured will be discussed with respect to the apparatus. However for purposes of understanding how the data is analyzed to determine the health of a donor, Figure 1 is representative of intensities measured commencing shortly after the fluorescence lifetime. The fluorescence lifetime is the time required when excitation ceases for the intensity of the fluorescent light emission to decrease by e 1, where e is a constant of 2.71~28. This is a short time, so that measurements may commence within approximately one to twenty seconds after the cells are mixed with the fluorescein diacetate dye. At time tl the intensity of the vertical component of the emission is measured.
At time t2, the horizontal component is measured and similarly the vertical and horizontal components are measured for times t3 through to tlo. The measurements are continued only as long as needed to get a fair representation of the variation of intensity over time.
Although several precautionary steps may be taken to maintain stability in the FDA solution, the dye solution will on its own degenexate into fluorescein.
Additionally, the fluorescein produced by enzymatic action of the lymphocyte cells does not become totally associated wi-th the lymphocyte cells as a fluorescent probe. The fluorescein, which is not associated with the living cells and is free in the medium, is referred to as background fluorescence. As more and more fluorescein is produced during the testing period, the concentration of free fluorescein in the medium increases until i-t is at a level which distorts attempts to determine the fluorescent intensities of the fluorescent probes associated with the lymphocyte cells.
That is the measurements are of little if any value once ~2~ t,~7 the concentration of fluorescent components unassocia-ted with the living cells in the medium begins to si,gnificantly affect the measurement of the intensity of polarized light emission intensiti~s from fluorescein which is associated with the living cells. With the lymphocyte cell system, it has been found that measurements taken usually beyond 90 seconds are of no value in further computations.
It is not understood how the dye associates with the cell and how enzymes of the cell hydrolyze the FDA
to fluorescein. It is postulated that the FDA is either absorbecl into the living cells to form fluorescein in the cells, or produced fluorescein is located in the living cell membrane, or the produced fluorescein is attached to the surface of the living cell. From test results, however, regardless of how the produced fluorescein is associated with the living cells, the characteristic imparted to the lymphocyte cells by a donor having cancer affects the time dependent changes of the polarized fluorescent emissions from the fluorescent probe associated with the lymphocyte cells.
The polarized fluorescence can be evaluated by way of an index. The generated polaxization index for each sample tested is used in the following formula -to generate a Lymphocyte Fluorescent Polarization Index (LFPI)o This index is calculated in accordance wi~h the following formula:
LFPI'= LPA2-LPAl FORMULA 1 wherein LPA is Lymphocyte-PHA Activity Indicator and LPAl = P(System l)_- P(System 2) P(System l) FORMWLA 2 and LPA2 = P(System 3) ~ P(System 4) P(System 3) FORMULA 3 P(System l...) is the polarization index for each system analyzed wherein:
. ., System 1 consists of phosphate buf~ered saline (PBS), fluorescein diacetate (FDA) and lymphocytes from 20ne 1.
System 2 consists of PBS, FDA, lymphocytes from Zone 1 as stimulated by the pre~erred mytogenic agent phytohaemagglutinin (PHA).
System 3 consists of PBS, FDA and lymphocytes from Zone 2.
System 4 consists of PBS, FDA, lymphocytes from Zone 2 as stimulated by P~A.
Thus the cells from Zone 1 and 2 are divided up into four groups. Each group of cells is then tested to produce a series of values for measured ~luorescent emission intensity versus time as exemplified in Figure 1. In order to calculate the polarization index from this data, the following formulae are developed:
For the vertical and horizontal measured intensities of Figure 1, the plots are approximated by the following polynomial series IV aO.+ alt + a2t + ..... , FOR~IULA 4 I~l bo blt + b2t + ~ FORMULA 5 wherein I~ is measured vertically po:Larized fluorescent intensity IH is measured horizontally polarized fluorescent intensity : al, a2 .... are constants representing the change of IV vs. t, b1, b2 ~ are constants representing I~
vs. t t is the time which commences with the dye being i.ntroduced to the cells aO is the IV intercept at t=0 bo is the IH intercept at t=0 ; 35 The verti.cally and horiæontally oriented components of the polarized emissions are dete.rmined with respect to the plane defined by the excitation and emission axes which intersect at the sample of living cells being analyzed in the fluorometer.
~y~
Assuming that a~... and b2... are both equal to zero, the fluorescence polarization at times approaching zero will be equal to (IV ~ aO) - G ~Ih bo) FORMUL~ 6 (IV ~ aO) + ~ (Ih ) wherein P is the polarization index, G is the constant for the machine Iv, I~, aO, bo are as defined in Formulas 4 and 5.
From Formulas 4, 5 and 6, it can be derived that:
~al/b2) - G
P = _ _ FORMULA 7 (al/b2) + G
G is the correction for the instrument bias in measuring the vertically and horizontally polarized emission intensities. G is determined experimentally by using exciting light which is horizontally polarized with respect to the laboratory plane and measuring IH~ and IHH, where first letter denotes the orientation of the excitation polarization and the second letter denotes the orientation of the emission polarization analyzer.
HV
G =
IHH
By determing the slopes al and bl from the plot of intensity versus time, a value for the polarization index P can be derived. Using Formula 1, the index representative of the health of the donor can be determined. From this a positive index indicates that the cells are from a donor having cancer which is indicative of a greater response in Zone 2. A negative index is indicative of a normally healthy donor where the greater response is in Zone 1.
Although Formula 7 is satisfactory in many instances for calculating P, particularly when the plot of intensity versus time approximates linearity, an alternate approach to calculating P has proven equally if not slightly superior for calculating va~ues of P.
The values of the intensity for the vertical and horizontal components are calculated by interpolation between the times measured for the vertical intensities to give an estimated value for the vertical in-tensity at the same time as the horizontal reading is obtainedO
Values for P are then calculated for each time, as shown in Figure 2 by the Formula:
(IVl - aO) ~ G~IHl - bo) FORMULA 8 (IVl - aO) G(IHl 0 As shown in Figure 2, the plot of P versus Time produces a curve and by the least square method a value for PO
may be extrapolated for which is the approximate polarization index at Time = 0. The values for PO for various systems is then used in Formulas 1, 2 and 3 to calculate the LFPI index.
~ eferring to Figur~ 3, the optical arrangement for the kinetic polarization fluorometer is shown. A
xenon light source 10 directs light onto a mirror 12 which is angled in a manner to reflect light onto an interference filter 14. This filter only transmits light that is at a particular wavelength. A beam splitter 16 provides two beams 18 and 20. Beam 13 is directed onto a photo-multiplier tube 22 which provides a reference for the excitation beam 20 in determining the intensity of emissions from the sample 24. A
rotating polari2er 26 is provided in front of the sample 24 to selectively transmit light from beam 2Q in a distinct orientation having a vertical component. In this embodiment, the orientation is vertical. The excitation beam directed onto the sample 24 causes the florescein associated with the living cells and in the medium to fluoresce. The fluorescent emission is analyzed along beam 28. A second polarizer 30 is provided, so that the emissions can be selectively anal~zed as the orthogonal components of vertical and horizontal polarized light. An iris 32 is provided to control the emissions and a lens 34 focuses the emission beam onto an interference filter 3~. A photo~multiplier tube 38 detects the intensity of the particular orthogonal component of the polarized fluorescent emission from the sample 24.
The structure of the kinetic polarization fluorometer is shown in E`igure 4~ The actual structure of the fluorometer includes a cabinet ~0 housing the components of the device. The sample of either stimulated or unstimulated lymphocyte cells is placed in the sample holder 42 where the sample is releasably held in the holder and may be released by linkage 44. There are light sources available such as an arc lamp light source 46 which produces a broad spectrum of wavelengths in the range of 300 nm. up to 1000 nm. The mirror 48 reflects the heam onto the filter 50 which passes the light onto a beam splitter 52. According to this embodiment, the interference filter 50 has a peak transmission at 470 nm and at a spectral band width at one-half peak intensity of 20 nm. This filter also incorporates appropriate blocking filters. A portion of the beam is reflected onto a photo-multiplier tube sensor 5~ for detecting the intensity of the excitation beam which passes through the beam splitter 52 onto a rotatable polarizer 55. The polarizer 55 is positioned to provide a distinct vertical orientation component of the excitation radiation. It is appreciated that there are alternatives to the rotatable polarizer 55, such as a light source which emits the desired orientation of polarized light at the desired wavelength.
The beam passing through the polarizer 55 impinges on the sample in the holder 42. The fluorescent emissions are detected at right ang]es to the incident beam. The emissions are passed through a rotatable polarizer 56 to selectively provide the horizontal or vertical polarized components of the emissions. The filter and lens are provided at 58 with a shutter 600 The fllter consists of an interference filter together with appropriate hlocking filters~ The pre~erred peak transmission of this filter is at 510 nm. and it has a spectral band width at one-half pea~ intensity of 10 nm.
The intensity of either the horizontal or vertical emissions is detected by a photo~multiplier tube ~PMT) 62. The power supplies to the vaxious units are shown where a water cooled power supply 6~ is provided for the axc lamp. The power supply for the photo-multiplier tube (P~T) 62 is provided at 66 and the power supply for the photo-multiplier tube ~PMT) 54 is provided at 68.
The photo-multiplier tube or other appropriate intensity measuring device 62 is capab:le of generating a value corresponding to the intensity of the emissions being measured over a period of time. It is appreciated that the measuring device can have digital or analog output. In the event that the output is analog, the signal is digitized before being analyzed by the computer. The photo-multiplie~ tube has a digital output which is passed through a signal shaping device and into a computer. The computer reads the information stored in the counter. As shown in Figure 1, the device may be adapted to measure the accumulated intensity value in an interval t definecl by times tl, t2, t3, etc. where the timing of the measurement is synchorized with the emission polariæer being in either the horizontal or vertical orientations. This may be accomplished manually. However to provide for automatic operation, a programmable controller may be used of the type shown in Figure 5. The unit has t:he capability of analyzing the data measured by the photo~multiplier tube 54 or 62. The intensity is measured over periods of time in accordance with the controller program and that accumulated intensity is stored for subsequent analysis in determining the slopes and thus the polarization index as per the output of the type of Figure 1. The controller may be programmed to plot the measured intens.ities versus time on an appropriate print-out or read-out on a cathode ray tube.
~ s described, four different system samples are to be tested in the fluorometer. The appropriate quantity of FDA and PBS is introduced into the spectrofluorometer cuvette and equilibriated to the controlled temperature. The sample from Zone 1 or 2 as stimulated with PHA or unstimulated is introduced into the cuvette. This is marked on the computer time base as t . The fluorescent intensity of vertical and horizontal components is measured by the device. The vertical component may be determined over a period of one to ten seconds and similarly the horizontal cornponent may be determined over the same time span.
lS The polarizer is switched from one attitude to the other under control of the programmable controller. Once the run is complete within approximately one and a half minutes, the values for the intensities are analyzed in accordance with Formulas 1, 2 and 3.
The programmable controller may be in the form oE a computer which includes a sub routine to analyze the intensities and provide va].ues for P~ The computer may also be programmed to generate values for PO to come up with the best fit to the generated data in determining the polarization index for the time dependent change of the fluorescent emissions from the fluorescent probe associated with the :Living cell-dye system.
The programmable computer of Flgure 5 is capable of analyzing the intensity readings and generating an index indicative of the health o -the donox. The intensity of the fluorescent emission at any time t is amplified by an amplifier 70. A signal-shaping device, discriminator 72 passes the information from amplifier 70 to a counter 7~ which counts the intensity of the emissions at any time T. to is established on the computer time base by pressing foot pedal switch 97 once the dye is mixed with the lymphocytes. The information from the counter is fed to a counter interface 76 which hlends the results of the intensities measured from the emissions wlth the intensity measured Erom the reference photo-multiplier tube 54. As with the information from the emission detector of the photo-multiplier tube 62, the reference photo-mul-tiplier tube 54 has its signal amplified by amplifier 78 which is passed to discriminator 80 and onto a counter ~2 which feeds its information into the counter interface 76. In accordance with the program in read-only memory 84, the central processing unit 86 analyzes the data transmitted from the counter interEace 76~ The program with which the central processing unit 86 analyæes the data i5 provided in the read-only memory 84. The data, as measured by the photo-multiplier tubes~ is accumulated over time in the random access memory 88. In order to compensate for variations in the intensity of the exciting beam, the channel counts from the photo-multiplier tube 62 is divided by the reference channel counts from photo-multiplier tube 54. This ratio normalizes the results to compensate for any variation in the intensity of the incident beam.
The central processing unit ~6 then calculates the data values for P. The calculated values for P are stored in the random access memory. By way of the input/output interface 90, information is fed to the video terminal 92, the plotter 94 ancl the printer 96.
The plot of the intensity versus time as accumulated is presented on the plotter 94 when called for through input via the keyboard 98. Any additional required information may be provided on the printer such as the readings for the vertical and horizontal components of the fluorescent emissions, calculated values for P and PO along with identification of the sample and the donor.
The rotation of the polarizers 55 and 56 is controlled by a controller 100. The controller has input to rotational polarizer motors 102 and 104. Motox 102 controls the rotatable polarizer 55 which determines the orientation of the excita-tion beam. Motor 104 controls the rotation of polarizer 56 which determines 3 ~'7 the orientation of the polarized fluorescent emission from the sample. The motors also have feed back to the controller lO0 to signal the position of the respective polarizers and thus by coordination through the central processing unit, in accordance with the program in read-only memory 8~, the values detected by the photo-multiplier tubes 54 and 62 are identified for subsequent analysis in accordance with the previously noted Formulas. In this way, the information as measured is coordinated and synchronized with the position of the polarizer 56, so that the values for the po]arization index ma~ be consistently and accurately calculated by the computèr program.
The system provides an index which is representative of the health of the donor~ Dependent upon the manner in which the samples are prepared, the index may be arrived at in one or more ways. To calculate the lymphocyte fluorescence polarization index ¦I.FPI) and other indicators of lymphocyte-PHA activity (LPA), there are several formulas which can be used.
The exact choice of parameter used depends on the preparation of the systems. The significance of the values of a particular choice of parameter indicators must be established by a controlled study of patients who have and patients who do not have cancer.
The following examples are provided to demonstrate preferred aspects of the invention and the results which can be obtained. Such examples are understood to be in no way limiting to the scope of the 3~ invention and the appended claims.
Example l -A blood specimen was collected from a normally healthy donor by way of two lO millilitre vacutainers, each containing 1~3 USP units of sodium heparin. The actual volume drawn in each sample was approximately 8 to 9 mls. The blood specimen was allowed to rest at room temperature for two hours to reach room temperature.
Phagocytes and platlets were removed from the specimen hy carbonyl ion sedimentation. 0.1 grams of carbonyl iron powder was transferred into a 25 ml.
screwcap container~ Five drops of normal saline was mixed with the iron powder. The blood in the heparin tubes was gently introduced into the screwcap container.
The sample was rotated on an inclined plane of a blood mixer inside a 37C. incubator. After twenty minutes the sample was removed from the incubator and the top was tightly sealed. The screwcap container was placed on a weak magnet in a temperature controlled water bath for 15 minutes. ~pproximately 17 mls. was removed from the screwcap container and transferred to another screwcap container which was placed on a strong magnet to remove any remaining phagocytes. From this sample, 5 ml. aliquots were slowly layered onto temperature equilibriated gradients. The Ficoll-Triosil gradients were prepared in accordance with standard procedure and temperature equilibriated to 1~.5C.
The gradient/blood tubes were centrifuged in a temperature controlled centrifuge for 20 minutes at 1,100 g. The temperature of the water bath and centrifuge was such that the density of the gradient was exactly 1.081 at 18.5C. temperature~
After centrifugation was complete, a lymphocyte cell interface was observed in the gradient tube, as shown in Figure 6. All of the cells in Zone 1, a~
identi~ied in Figure 5, were removed. Similarly all of the cells in Zone 2, as identified in Figure 6, were removed. The cells recovered from Zone 1 and Zone 2 were washed twice with normal saline and once with PBSo The isolated cells were counted for each Zone. The number of cells in each Zone was adjusted to a concentration of l.OxlO per ml. by adding an appropriate amount of modified PBS solution ~o each collected group of cells. The cells were allowed to remain undisturbed for a period of another one half hour ; at 35C. before testing.
Several cuvettes for the kinetic polarization fluorometer were washed and the fluorometer brought up to an operating temperature of 27C. A fresh solution o~ FDA was prepared and warmed to 27~C. in a water bath The groups of cells which were to be stimulated were removed from containers which have the cells collected from ~ones l and 2. The selected cell sample of .25 mls was drawn up in a syringe and was added to a 50 microlitre l/5 dilution of PHA in a small Falcon tube. This was incubated for 40 minutes at 37C. and then mixed with the prewarmed FDA substrate and tested.
The lymphocyte cells were handled in a manner to reduce physical shock to the cells in the form of temperature, osmolality, pH and ion concentrations of the medium.
These parameters were controlled so that the cells were not subjected to any temperature extremes or to solutions which cause changes in the cells by changes in osmosis. Throughout this procedure the pH of the solutions was maintained at 7.4 and osmolality of the PRS solutions was maintained at 330 mllliosmolar.
: Several individual samples of cells were tested in the apparatus of Figure 4. The intensity versus time of the vertical and horizontal components were measured as prescribed by the discussion of the apparatus. The data was analyzed to give the following P values based on the slopes of intensity versus time.
; The intensity readings were taken approximately ; every 5 seconds for the vertical and horizontal orientation of the emission fluorescent intensity from the sample. The readings began at approximately 20 seconds after the FDA was introduced into the sample and completed after approximately lO0 to llO seconds.
P Value P P Value P
o o System 1 .162 .160 System 3 .159 .159 .170 .173 .152 .153 .175 .176 Avge. .165 .166 10 System 2 .134 .131 System 4 .145 .146 .116 .115 .130 .132 Avge. .127 .126 In accordance with the computer program based on the calculation to be carried out uncler Formulas 7 and 8, the use of the P values produced an index of -14.
PO values produced an index of -16. In accordance with prior testing procedure, the significant negative index in each case indicates a normally healthy donor.
Examp]e ~
The procedure of Example 1 was followed with blood samples collected from a different donor to provide cells from Zones 1 and 2 of the collected and treated blood sample which were analyzed in accordance with the procedure of Example l with the apparatus of Figure 4. The following results were found from an analysis of the data in determining the variants of fluorescent i-ntensity over time in the sample. In each instance, measurement of the intensity commenced at approximately 20 seconds after the cell sample was contacted with the dye solution and from that point, approximately every 9 seconds the intensity of the other component was measured.
P Value PO P Value PO
System 1 ~ .163 System 3 .159 .168 .153 .165 .153 .163 Avge. .159 .164 ; System 2 .151 .157 System 4 .116 .125 .138 .134 .12~ .121 Avge~ .138 .140 In accordance wi-th the computer program based on the calculation to be carried out under Formulas 7 and g, the P values produced an index of +14. PO values gave an index of ~11. In accordance with the prior testing procedure, the significant posltive index in each case indicates a donor who has cancer.
It is appreciated that in situations where it is not desirable to rotate the emission polar.iæer 56 bac]c and forth to select the polarized verti..cal and horizontal components, two photo-rnultiplier tubes may be used where each is located to a side of the sample. A
polarizer may only pass a horizontal component of the emissions onto one photo-multiplier tube and similarly the other polarizer would only pass the vertical component of the emission through to the other photo-multiplier tube. The data then collected from these two separate photo-multiplier tubes can be interpreted in the normal way in providing a graph of the intensity versus t.ime for the vertical and horizontal components to provide the necessary polarization index values which are subsequently used to determine the health of the donor~
The method according to this invention in measuring the intensities of fluorescent emissions from the cells at very early times, that is in the range of 10 to 110 seconds after the living cells are contacted with a dye solution and before bac~ground fluorescence '7 can signicantly affect the polarized light emission intensities from a fluorescent probe associated with the li.ving cells, provides a consistent and accurate technique for determining the health of the donor of the lymphocyte cells.
Although various preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope oE the appended claims.
METHOD FOR DETECTI~ LUMINESCENCE IN LIVING CELL SYSTEM
FIEI,D OF THE INVENTION
_ _ _ _ _ _ .
This invention relates to a method and apparatus for determlning the time dependent change in luminescent polarization of a luminescent dye associated with a living cell-dye system~
BACKGROUND OF T~E INVENTION
In the study of living cells, it is often necessary to analyze and evalua-te various biological changes associa-ted with the living cells. There are many approaches to probing such biological changes associated with living cells, such as the use in photometric techni~ues of which flow cytometry is an example. However, such equipment is very expensive and painstaking to use.
Another approach to evaluating biological changes associated with a living cell is to associate a dye probe with the cell and study the changes in luminescence of this probe. This technique has been particularly applied to inducing fluorescence with a type of cell known as a lymphocyte to establish whether or not a malignant tumour is present in the donor from which lymphocytes ha~e been removed. The intensity of Eluorescent emission is studied to determine if a characteristic oE the lymphocyte is present, which characteristic is a result of the donor having cancer.
The original work on this technique began in the early 1970's. Drs. Cercek and Cercek developed a technique which had the abi]ity to differentiate lymphocytes from healthy donors from those of donors with cancer. The test was based upon differential lymphocyte response or lack oE response to either a protein isolated from malignant tumours or the mitogen phytohaemagglutinin.
The work by Cercek and Cercek is well published, the original technique being reported in Cercek L., Cercek B., and Franklin C. (1974), Brit. J. Cancer 29, 345-352.
The test to diEfexentiate healthy donors from unhealthy donors was thought to rely on changes in the structuredness of the cytoplasmic matrix ISCM~ of a specific population of blood lymphocytes stimulated by t~
phytohaemagglutinin (PHA) or cancer basic protein (CaBP)O Assuminy that there are changes in the matrix, then it was believed that such changes were detected by using polarized light to determine qualitatively the rate of change of orientatlon of fluorescent molecules within the living cell. As reported in the sritish Journal oE Cancer, the Cercek technique involved mixing fluorescein diacetate (E'DA) dye with a medium containing lymphocyte cells isolaked from donor b~ood. Enzymes of the cell hydrolize the FDA to produce fluorescein molecules which were thought to be formed within the cells. Such molecules are excited with polarized light.
Measurements are made of the degree of polarization of the emitted fluorescence by use of a f]uorescence spectrometer fitted with polarization accessories.
Calculations were made to de~ermine po]arization values which were then used in accordance with their disclosed formula to determine if the lymphocytes came from a healthy donor or a donor who has cancer. The technique was carried out over a period of at least six minutes in measuring fluorescent emissions. During this interval, the fluorescein molecules would increase in concentration in the medium, due to some of the fluorescein molecules ormed by enzymatic action leaking out of the living cells. The increasing concentration of fluorescein molecules in the medium affects the reading on the intensities of emissions from fluorescein molecules thought to be within the cells. To compensate for this increasing concentration of fluorescein molecules in the medium at the end of the testing period, the medium with cells would be filtered so as to remove the cells and then a reading would be taken of the intensity of the remaining fluorescein molecules in the medium. These values were used to adjust the previously recorded values to give the absolute value for the change in intensity of the polarized fluorescence from molecules within the cells.
The Cercek and Cercek technique was improved upon by many including Dr. J.A.V. Pritchard. Dr.
. . .
Pritchard developed an improved technique for harvesting the cells from the blood sample to provide more consistent results. His work is widely published, where a review of his most up-to-date work is found in Europ.
J~ Cancer Clin. ~ncol., Vol. 18, No. 7, pages 651 through 659 (198~). Pritchard discovered that a particular method of using a Ficoll-Triosil gradient provided two bands or zones of cells within the centrifuged medium~ The cells from the two zones were tested. Some of the cells from each zone are tested unstimulated as control samples. A similar number of cells from each zone are stimulated with PHA and tested to yield superior results in determining an index indicative of the donor's health. After stimulation of some of the Zone 1 and Zone 2 cells with PHA the fluorescein diacetate was added which was thought to be hydrolyzed to fluorescein within the cells to produce fluorescence when excited by polarized light. A
fluorescence spectrophotometer was used to analyze polarized emissions to give values for intensi-ties over time. At the end of the testing which took approximately six minutes as with the Cercek technique, again filtering was required to establish the eEfect of free fluorescein in the medium on the values for emission intensity from fluorescein thought to be within the cells.
A considerable problem exists with the filtration required in the Cercek and Pritchard techniques to establish the effect that the free fluorescein molecules in the medium has on the intensity readings. The Eiltration disturbs the cells and can cause breakage of the cells which gives an erroneous reading. Due to the filtration technique, relatively large samples of blood are needed for the test. When considering the number of tests that are usually conducted in determining the existence of cancer, the additional quantities required by these tests can become quite significant. Another problem wlth filtering is the resultant lack of consistency in the results.
. , .
~ .
~ 4~
Another prob:Lem encounter~d wlth these techniques is that they are very much subject to variation in results due to technician judgment, varying protocol and reagents. The bias of the operator conducting the tests can cause erroneous or misleading results.
The method and apparatus, according to this invention, overcomes the above problems in providing a technique which is reliable and relatively inexpensive to conduct.
SUMMARY OF T~IE IN~ENTION
To establish if living cells are from a healthy donor or an unhealthy donor, the time dependent change in the lurninescent polarization of a living cell-dye system having a luminescent probe associated with the living cells is determin~d. The living cell-dye system is dispersed in a medium, where a dye is introduced into the medium to associate a luminescent probe with the living cells of the living cell-dye system. Luminescent components of the dye, which are unassociated with the living cells, increase in concentration over time in the medium to provide background luminescence. Polarized light in a distinct orientation haviny a siynificant vertical orthogonal component is generated. The polarized light irradiates the living cell dye system plus medium. The polarized llght emissions from the luminescent probe associated with the living cells and luminescent components in the medium are analyzed in two distinct orientations representing horizontal and vertical orthogonal components of the polarized light emissions to determine the time dependence of the components of the polarized emissions from the luminescent probe associated with the living cells of the system and the luminescent components of the medium.
The steps of generating polarized light and analy2ing the polarized light emissions are conducted during the period commencing at times greater than the luminescence lifetime of the living cell-dye system after mixing and continuing unti] the background luminescence from the system medium significantly affects -the polariæed light emission intenslties from the luminescent probe associated with the living cells of the sys-tem. This avolds the need for physical separation of the living cells from the luminescent dye unassociated with the living cells.
Such dye acts as a luminescent probe which may be an organic or inorganic substance for absorbing light energy and produce fluorescent emissions which have a particular func-tional relationship with a cellular organism.
The method is particularly suitable for analyzing polarized fluorescent light emissions from the fluorescent probe associated with the liviny lymphocyte cell-dye system in the technique of detecting whether or not the cells are from a healthy donor or a donor who has cancer. The dye, which may be used is a weakly fluorescent dye which, when acted upon by the enzymes within the cells produces a highly fluorescent analog of the dye. According to a preferred aspect of the invention, the dye used is fluorescein diacetate which has fluorescein as its highly Eluorescent analog.
BRIEF DRESCRIPTION OF THE DRAWINGS
Preferred embodiments of the invention are described with reference to the drawings wherein:
Figure 1 is a graph demonstrating a plot of Intensity versus Time of measured intensities at various times for po~arized vertical and horizontal components of the emissions;
Figure 2 is a graph demonstrating a plot of calculated values oF the Polarization Index versus Time;
Figure 3 is a schematic showing the optics for the kinetic polarization fluorometer;
Figure 4 is a top plan view of the fluorometer schematically shown in Figure 3;
Figure 5 is a block diagram of the electronic control device for the fluorometer; and Figure 6 is a schematic representation of the gradient of the centrifuged treated blood sample for isolating the two zones of lymphocyte cells.
: ' DETAILED _ESCRI_TION OF_THE PREFERRED EMBODI~ENTS
The chemical or biological mechanism of the living cells depending on the health of the donor which affects the intensity of polarized luminescent emissions over time is not fully understood. However, the method of this invention provides consistent results in distinguishing living lymphocyte cells of healthy donors from donors which have cancer. An index is calculated based on the intensity of luminescence emitted from the living cells in two distinct horizontal and vertical orientations which represen-t orthogonal components of polarized light emission. The consistency in the results is at least in part attributed to the method obviating the need to filter the medium containing the cells in determining the effect of unassociated luminescent components ~background luminescence) in the medium on the intensity of emissions from a luminescent probe associated with the cells.
It is understood that the method and apparatus of this invention may be used to determine the time dependent change in the lumi~escence polarization of a luminescent dye associated with the living cells in any form of living cell system. To demonstrate the invention by way of a preferred embodiment, reference is made to the method as applied to determinin~ polarized fluorescent light emissions from a fluorescent probe associated with living lymphocyte cells. The time dependent change of the fluorescence can be analyzed to determine if the lymphocyte cells are from healthy donors or donors with cancer. In order to evaluate the lymphocyte cells, they are extracted from blood which is collected from the donor. The blood is processed in a particular way to provide the lymphocyte cells in a form which can be stimulated with a mitogenic or antigenic agent~ such as PHA (phytohaemagglutinin). Such cells, after being stimulated by PHA are brought into contact with a dye such as FDA (fluorescein diacetate~. Non-specific carboxyesterase enzymes of the cell hydrolize the weakly ~luorescent fluorescein diacetate by deacetylating the dye to produce highly fluorescent ..J'.~ ;J 7 fluorescein which becomes associated with living cells and when exposed to appropriate light energy, fluoresces to produce fluorescent emissions.
In order to isolate the lymphocyte cells from collected blood, the ~ollowing procedure is preferred.
The venous blood sample is collected in appropriate containers and allowed to cool to room temperature.
Iron carbonyl is added to the blood to remove phagocytes and platlets by use of magnets in accordance with standard techniques in a temperature controlled water bath. A Ficol]-Triosil gradient is prepared and temperature equilibriated in a water bath so that the density of the gradient will be exactly 1.081 m/ml.
Aliquots of the treated blood are layered carefully onto the gradient and are separated into two zones in a temperature controlled centrifuge. The lymphocyte cells are removed from the two Zones (1 and 2~ of the gradient and are suspended in a modified phosphate buffered saline (PBS) at 37C. at a cell concentration of 1.0 x 106 cells/ml.
In order to determine the time dependent change in the fluorescent emissions from the cells of Zones 1 and 2, a kinetic polarization fluorometer may be used.
The fluorometer generates polarized light in a distinct orientation having a significant vertical component. By significant, it is meant that the intensity of the vertical component is sufficient to generate the desired orientation of excitation in the living cell-dye system to provide emissions which can be analyzed. The polarized light is directed onto the living cell-dye system to generate fluorescence from the dye associated with the cells and in the medium. The light emissions from the dye associated with the living cells and in the medium is polarized into two distinct orientations which represent horizontal and vertical components of the polarized emissions. The intensities of the polarized emissions in the orthogonal directions of the vertical plane and in the horizontal plane are analyzed to determine the health of the donor.
As shown in Figure 1, there is a plot of measured intensities in the vertical (Iv) and horizontal (IH) orientations versus time~ In this representative Figure, the intensities vary in a generally linear manner. The details in which the intensities are measured will be discussed with respect to the apparatus. However for purposes of understanding how the data is analyzed to determine the health of a donor, Figure 1 is representative of intensities measured commencing shortly after the fluorescence lifetime. The fluorescence lifetime is the time required when excitation ceases for the intensity of the fluorescent light emission to decrease by e 1, where e is a constant of 2.71~28. This is a short time, so that measurements may commence within approximately one to twenty seconds after the cells are mixed with the fluorescein diacetate dye. At time tl the intensity of the vertical component of the emission is measured.
At time t2, the horizontal component is measured and similarly the vertical and horizontal components are measured for times t3 through to tlo. The measurements are continued only as long as needed to get a fair representation of the variation of intensity over time.
Although several precautionary steps may be taken to maintain stability in the FDA solution, the dye solution will on its own degenexate into fluorescein.
Additionally, the fluorescein produced by enzymatic action of the lymphocyte cells does not become totally associated wi-th the lymphocyte cells as a fluorescent probe. The fluorescein, which is not associated with the living cells and is free in the medium, is referred to as background fluorescence. As more and more fluorescein is produced during the testing period, the concentration of free fluorescein in the medium increases until i-t is at a level which distorts attempts to determine the fluorescent intensities of the fluorescent probes associated with the lymphocyte cells.
That is the measurements are of little if any value once ~2~ t,~7 the concentration of fluorescent components unassocia-ted with the living cells in the medium begins to si,gnificantly affect the measurement of the intensity of polarized light emission intensiti~s from fluorescein which is associated with the living cells. With the lymphocyte cell system, it has been found that measurements taken usually beyond 90 seconds are of no value in further computations.
It is not understood how the dye associates with the cell and how enzymes of the cell hydrolyze the FDA
to fluorescein. It is postulated that the FDA is either absorbecl into the living cells to form fluorescein in the cells, or produced fluorescein is located in the living cell membrane, or the produced fluorescein is attached to the surface of the living cell. From test results, however, regardless of how the produced fluorescein is associated with the living cells, the characteristic imparted to the lymphocyte cells by a donor having cancer affects the time dependent changes of the polarized fluorescent emissions from the fluorescent probe associated with the lymphocyte cells.
The polarized fluorescence can be evaluated by way of an index. The generated polaxization index for each sample tested is used in the following formula -to generate a Lymphocyte Fluorescent Polarization Index (LFPI)o This index is calculated in accordance wi~h the following formula:
LFPI'= LPA2-LPAl FORMULA 1 wherein LPA is Lymphocyte-PHA Activity Indicator and LPAl = P(System l)_- P(System 2) P(System l) FORMWLA 2 and LPA2 = P(System 3) ~ P(System 4) P(System 3) FORMULA 3 P(System l...) is the polarization index for each system analyzed wherein:
. ., System 1 consists of phosphate buf~ered saline (PBS), fluorescein diacetate (FDA) and lymphocytes from 20ne 1.
System 2 consists of PBS, FDA, lymphocytes from Zone 1 as stimulated by the pre~erred mytogenic agent phytohaemagglutinin (PHA).
System 3 consists of PBS, FDA and lymphocytes from Zone 2.
System 4 consists of PBS, FDA, lymphocytes from Zone 2 as stimulated by P~A.
Thus the cells from Zone 1 and 2 are divided up into four groups. Each group of cells is then tested to produce a series of values for measured ~luorescent emission intensity versus time as exemplified in Figure 1. In order to calculate the polarization index from this data, the following formulae are developed:
For the vertical and horizontal measured intensities of Figure 1, the plots are approximated by the following polynomial series IV aO.+ alt + a2t + ..... , FOR~IULA 4 I~l bo blt + b2t + ~ FORMULA 5 wherein I~ is measured vertically po:Larized fluorescent intensity IH is measured horizontally polarized fluorescent intensity : al, a2 .... are constants representing the change of IV vs. t, b1, b2 ~ are constants representing I~
vs. t t is the time which commences with the dye being i.ntroduced to the cells aO is the IV intercept at t=0 bo is the IH intercept at t=0 ; 35 The verti.cally and horiæontally oriented components of the polarized emissions are dete.rmined with respect to the plane defined by the excitation and emission axes which intersect at the sample of living cells being analyzed in the fluorometer.
~y~
Assuming that a~... and b2... are both equal to zero, the fluorescence polarization at times approaching zero will be equal to (IV ~ aO) - G ~Ih bo) FORMUL~ 6 (IV ~ aO) + ~ (Ih ) wherein P is the polarization index, G is the constant for the machine Iv, I~, aO, bo are as defined in Formulas 4 and 5.
From Formulas 4, 5 and 6, it can be derived that:
~al/b2) - G
P = _ _ FORMULA 7 (al/b2) + G
G is the correction for the instrument bias in measuring the vertically and horizontally polarized emission intensities. G is determined experimentally by using exciting light which is horizontally polarized with respect to the laboratory plane and measuring IH~ and IHH, where first letter denotes the orientation of the excitation polarization and the second letter denotes the orientation of the emission polarization analyzer.
HV
G =
IHH
By determing the slopes al and bl from the plot of intensity versus time, a value for the polarization index P can be derived. Using Formula 1, the index representative of the health of the donor can be determined. From this a positive index indicates that the cells are from a donor having cancer which is indicative of a greater response in Zone 2. A negative index is indicative of a normally healthy donor where the greater response is in Zone 1.
Although Formula 7 is satisfactory in many instances for calculating P, particularly when the plot of intensity versus time approximates linearity, an alternate approach to calculating P has proven equally if not slightly superior for calculating va~ues of P.
The values of the intensity for the vertical and horizontal components are calculated by interpolation between the times measured for the vertical intensities to give an estimated value for the vertical in-tensity at the same time as the horizontal reading is obtainedO
Values for P are then calculated for each time, as shown in Figure 2 by the Formula:
(IVl - aO) ~ G~IHl - bo) FORMULA 8 (IVl - aO) G(IHl 0 As shown in Figure 2, the plot of P versus Time produces a curve and by the least square method a value for PO
may be extrapolated for which is the approximate polarization index at Time = 0. The values for PO for various systems is then used in Formulas 1, 2 and 3 to calculate the LFPI index.
~ eferring to Figur~ 3, the optical arrangement for the kinetic polarization fluorometer is shown. A
xenon light source 10 directs light onto a mirror 12 which is angled in a manner to reflect light onto an interference filter 14. This filter only transmits light that is at a particular wavelength. A beam splitter 16 provides two beams 18 and 20. Beam 13 is directed onto a photo-multiplier tube 22 which provides a reference for the excitation beam 20 in determining the intensity of emissions from the sample 24. A
rotating polari2er 26 is provided in front of the sample 24 to selectively transmit light from beam 2Q in a distinct orientation having a vertical component. In this embodiment, the orientation is vertical. The excitation beam directed onto the sample 24 causes the florescein associated with the living cells and in the medium to fluoresce. The fluorescent emission is analyzed along beam 28. A second polarizer 30 is provided, so that the emissions can be selectively anal~zed as the orthogonal components of vertical and horizontal polarized light. An iris 32 is provided to control the emissions and a lens 34 focuses the emission beam onto an interference filter 3~. A photo~multiplier tube 38 detects the intensity of the particular orthogonal component of the polarized fluorescent emission from the sample 24.
The structure of the kinetic polarization fluorometer is shown in E`igure 4~ The actual structure of the fluorometer includes a cabinet ~0 housing the components of the device. The sample of either stimulated or unstimulated lymphocyte cells is placed in the sample holder 42 where the sample is releasably held in the holder and may be released by linkage 44. There are light sources available such as an arc lamp light source 46 which produces a broad spectrum of wavelengths in the range of 300 nm. up to 1000 nm. The mirror 48 reflects the heam onto the filter 50 which passes the light onto a beam splitter 52. According to this embodiment, the interference filter 50 has a peak transmission at 470 nm and at a spectral band width at one-half peak intensity of 20 nm. This filter also incorporates appropriate blocking filters. A portion of the beam is reflected onto a photo-multiplier tube sensor 5~ for detecting the intensity of the excitation beam which passes through the beam splitter 52 onto a rotatable polarizer 55. The polarizer 55 is positioned to provide a distinct vertical orientation component of the excitation radiation. It is appreciated that there are alternatives to the rotatable polarizer 55, such as a light source which emits the desired orientation of polarized light at the desired wavelength.
The beam passing through the polarizer 55 impinges on the sample in the holder 42. The fluorescent emissions are detected at right ang]es to the incident beam. The emissions are passed through a rotatable polarizer 56 to selectively provide the horizontal or vertical polarized components of the emissions. The filter and lens are provided at 58 with a shutter 600 The fllter consists of an interference filter together with appropriate hlocking filters~ The pre~erred peak transmission of this filter is at 510 nm. and it has a spectral band width at one-half pea~ intensity of 10 nm.
The intensity of either the horizontal or vertical emissions is detected by a photo~multiplier tube ~PMT) 62. The power supplies to the vaxious units are shown where a water cooled power supply 6~ is provided for the axc lamp. The power supply for the photo-multiplier tube (P~T) 62 is provided at 66 and the power supply for the photo-multiplier tube ~PMT) 54 is provided at 68.
The photo-multiplier tube or other appropriate intensity measuring device 62 is capab:le of generating a value corresponding to the intensity of the emissions being measured over a period of time. It is appreciated that the measuring device can have digital or analog output. In the event that the output is analog, the signal is digitized before being analyzed by the computer. The photo-multiplie~ tube has a digital output which is passed through a signal shaping device and into a computer. The computer reads the information stored in the counter. As shown in Figure 1, the device may be adapted to measure the accumulated intensity value in an interval t definecl by times tl, t2, t3, etc. where the timing of the measurement is synchorized with the emission polariæer being in either the horizontal or vertical orientations. This may be accomplished manually. However to provide for automatic operation, a programmable controller may be used of the type shown in Figure 5. The unit has t:he capability of analyzing the data measured by the photo~multiplier tube 54 or 62. The intensity is measured over periods of time in accordance with the controller program and that accumulated intensity is stored for subsequent analysis in determining the slopes and thus the polarization index as per the output of the type of Figure 1. The controller may be programmed to plot the measured intens.ities versus time on an appropriate print-out or read-out on a cathode ray tube.
~ s described, four different system samples are to be tested in the fluorometer. The appropriate quantity of FDA and PBS is introduced into the spectrofluorometer cuvette and equilibriated to the controlled temperature. The sample from Zone 1 or 2 as stimulated with PHA or unstimulated is introduced into the cuvette. This is marked on the computer time base as t . The fluorescent intensity of vertical and horizontal components is measured by the device. The vertical component may be determined over a period of one to ten seconds and similarly the horizontal cornponent may be determined over the same time span.
lS The polarizer is switched from one attitude to the other under control of the programmable controller. Once the run is complete within approximately one and a half minutes, the values for the intensities are analyzed in accordance with Formulas 1, 2 and 3.
The programmable controller may be in the form oE a computer which includes a sub routine to analyze the intensities and provide va].ues for P~ The computer may also be programmed to generate values for PO to come up with the best fit to the generated data in determining the polarization index for the time dependent change of the fluorescent emissions from the fluorescent probe associated with the :Living cell-dye system.
The programmable computer of Flgure 5 is capable of analyzing the intensity readings and generating an index indicative of the health o -the donox. The intensity of the fluorescent emission at any time t is amplified by an amplifier 70. A signal-shaping device, discriminator 72 passes the information from amplifier 70 to a counter 7~ which counts the intensity of the emissions at any time T. to is established on the computer time base by pressing foot pedal switch 97 once the dye is mixed with the lymphocytes. The information from the counter is fed to a counter interface 76 which hlends the results of the intensities measured from the emissions wlth the intensity measured Erom the reference photo-multiplier tube 54. As with the information from the emission detector of the photo-multiplier tube 62, the reference photo-mul-tiplier tube 54 has its signal amplified by amplifier 78 which is passed to discriminator 80 and onto a counter ~2 which feeds its information into the counter interface 76. In accordance with the program in read-only memory 84, the central processing unit 86 analyzes the data transmitted from the counter interEace 76~ The program with which the central processing unit 86 analyæes the data i5 provided in the read-only memory 84. The data, as measured by the photo-multiplier tubes~ is accumulated over time in the random access memory 88. In order to compensate for variations in the intensity of the exciting beam, the channel counts from the photo-multiplier tube 62 is divided by the reference channel counts from photo-multiplier tube 54. This ratio normalizes the results to compensate for any variation in the intensity of the incident beam.
The central processing unit ~6 then calculates the data values for P. The calculated values for P are stored in the random access memory. By way of the input/output interface 90, information is fed to the video terminal 92, the plotter 94 ancl the printer 96.
The plot of the intensity versus time as accumulated is presented on the plotter 94 when called for through input via the keyboard 98. Any additional required information may be provided on the printer such as the readings for the vertical and horizontal components of the fluorescent emissions, calculated values for P and PO along with identification of the sample and the donor.
The rotation of the polarizers 55 and 56 is controlled by a controller 100. The controller has input to rotational polarizer motors 102 and 104. Motox 102 controls the rotatable polarizer 55 which determines the orientation of the excita-tion beam. Motor 104 controls the rotation of polarizer 56 which determines 3 ~'7 the orientation of the polarized fluorescent emission from the sample. The motors also have feed back to the controller lO0 to signal the position of the respective polarizers and thus by coordination through the central processing unit, in accordance with the program in read-only memory 8~, the values detected by the photo-multiplier tubes 54 and 62 are identified for subsequent analysis in accordance with the previously noted Formulas. In this way, the information as measured is coordinated and synchronized with the position of the polarizer 56, so that the values for the po]arization index ma~ be consistently and accurately calculated by the computèr program.
The system provides an index which is representative of the health of the donor~ Dependent upon the manner in which the samples are prepared, the index may be arrived at in one or more ways. To calculate the lymphocyte fluorescence polarization index ¦I.FPI) and other indicators of lymphocyte-PHA activity (LPA), there are several formulas which can be used.
The exact choice of parameter used depends on the preparation of the systems. The significance of the values of a particular choice of parameter indicators must be established by a controlled study of patients who have and patients who do not have cancer.
The following examples are provided to demonstrate preferred aspects of the invention and the results which can be obtained. Such examples are understood to be in no way limiting to the scope of the 3~ invention and the appended claims.
Example l -A blood specimen was collected from a normally healthy donor by way of two lO millilitre vacutainers, each containing 1~3 USP units of sodium heparin. The actual volume drawn in each sample was approximately 8 to 9 mls. The blood specimen was allowed to rest at room temperature for two hours to reach room temperature.
Phagocytes and platlets were removed from the specimen hy carbonyl ion sedimentation. 0.1 grams of carbonyl iron powder was transferred into a 25 ml.
screwcap container~ Five drops of normal saline was mixed with the iron powder. The blood in the heparin tubes was gently introduced into the screwcap container.
The sample was rotated on an inclined plane of a blood mixer inside a 37C. incubator. After twenty minutes the sample was removed from the incubator and the top was tightly sealed. The screwcap container was placed on a weak magnet in a temperature controlled water bath for 15 minutes. ~pproximately 17 mls. was removed from the screwcap container and transferred to another screwcap container which was placed on a strong magnet to remove any remaining phagocytes. From this sample, 5 ml. aliquots were slowly layered onto temperature equilibriated gradients. The Ficoll-Triosil gradients were prepared in accordance with standard procedure and temperature equilibriated to 1~.5C.
The gradient/blood tubes were centrifuged in a temperature controlled centrifuge for 20 minutes at 1,100 g. The temperature of the water bath and centrifuge was such that the density of the gradient was exactly 1.081 at 18.5C. temperature~
After centrifugation was complete, a lymphocyte cell interface was observed in the gradient tube, as shown in Figure 6. All of the cells in Zone 1, a~
identi~ied in Figure 5, were removed. Similarly all of the cells in Zone 2, as identified in Figure 6, were removed. The cells recovered from Zone 1 and Zone 2 were washed twice with normal saline and once with PBSo The isolated cells were counted for each Zone. The number of cells in each Zone was adjusted to a concentration of l.OxlO per ml. by adding an appropriate amount of modified PBS solution ~o each collected group of cells. The cells were allowed to remain undisturbed for a period of another one half hour ; at 35C. before testing.
Several cuvettes for the kinetic polarization fluorometer were washed and the fluorometer brought up to an operating temperature of 27C. A fresh solution o~ FDA was prepared and warmed to 27~C. in a water bath The groups of cells which were to be stimulated were removed from containers which have the cells collected from ~ones l and 2. The selected cell sample of .25 mls was drawn up in a syringe and was added to a 50 microlitre l/5 dilution of PHA in a small Falcon tube. This was incubated for 40 minutes at 37C. and then mixed with the prewarmed FDA substrate and tested.
The lymphocyte cells were handled in a manner to reduce physical shock to the cells in the form of temperature, osmolality, pH and ion concentrations of the medium.
These parameters were controlled so that the cells were not subjected to any temperature extremes or to solutions which cause changes in the cells by changes in osmosis. Throughout this procedure the pH of the solutions was maintained at 7.4 and osmolality of the PRS solutions was maintained at 330 mllliosmolar.
: Several individual samples of cells were tested in the apparatus of Figure 4. The intensity versus time of the vertical and horizontal components were measured as prescribed by the discussion of the apparatus. The data was analyzed to give the following P values based on the slopes of intensity versus time.
; The intensity readings were taken approximately ; every 5 seconds for the vertical and horizontal orientation of the emission fluorescent intensity from the sample. The readings began at approximately 20 seconds after the FDA was introduced into the sample and completed after approximately lO0 to llO seconds.
P Value P P Value P
o o System 1 .162 .160 System 3 .159 .159 .170 .173 .152 .153 .175 .176 Avge. .165 .166 10 System 2 .134 .131 System 4 .145 .146 .116 .115 .130 .132 Avge. .127 .126 In accordance with the computer program based on the calculation to be carried out uncler Formulas 7 and 8, the use of the P values produced an index of -14.
PO values produced an index of -16. In accordance with prior testing procedure, the significant negative index in each case indicates a normally healthy donor.
Examp]e ~
The procedure of Example 1 was followed with blood samples collected from a different donor to provide cells from Zones 1 and 2 of the collected and treated blood sample which were analyzed in accordance with the procedure of Example l with the apparatus of Figure 4. The following results were found from an analysis of the data in determining the variants of fluorescent i-ntensity over time in the sample. In each instance, measurement of the intensity commenced at approximately 20 seconds after the cell sample was contacted with the dye solution and from that point, approximately every 9 seconds the intensity of the other component was measured.
P Value PO P Value PO
System 1 ~ .163 System 3 .159 .168 .153 .165 .153 .163 Avge. .159 .164 ; System 2 .151 .157 System 4 .116 .125 .138 .134 .12~ .121 Avge~ .138 .140 In accordance wi-th the computer program based on the calculation to be carried out under Formulas 7 and g, the P values produced an index of +14. PO values gave an index of ~11. In accordance with the prior testing procedure, the significant posltive index in each case indicates a donor who has cancer.
It is appreciated that in situations where it is not desirable to rotate the emission polar.iæer 56 bac]c and forth to select the polarized verti..cal and horizontal components, two photo-rnultiplier tubes may be used where each is located to a side of the sample. A
polarizer may only pass a horizontal component of the emissions onto one photo-multiplier tube and similarly the other polarizer would only pass the vertical component of the emission through to the other photo-multiplier tube. The data then collected from these two separate photo-multiplier tubes can be interpreted in the normal way in providing a graph of the intensity versus t.ime for the vertical and horizontal components to provide the necessary polarization index values which are subsequently used to determine the health of the donor~
The method according to this invention in measuring the intensities of fluorescent emissions from the cells at very early times, that is in the range of 10 to 110 seconds after the living cells are contacted with a dye solution and before bac~ground fluorescence '7 can signicantly affect the polarized light emission intensities from a fluorescent probe associated with the li.ving cells, provides a consistent and accurate technique for determining the health of the donor of the lymphocyte cells.
Although various preferred embodiments of the invention have been described herein in detail, it will be understood by those skilled in the art that variations may be made thereto without departing from the spirit of the invention or the scope oE the appended claims.
Claims (18)
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A method for determining a time dependent change in the luminescent polarization of a living cell-dye system having a luminescent probe associated with the living cells, said living cell-dye system being dispersed in a medium comprising introducing a dye into said medium to associate a luminescent probe with the living cells of said living cell-dye system where luminescent components of the dye which are unassociated with said living cells increase in concentration over time in the medium to provide background luminescence, generating polarized light in a distinct orientation having a significant vertical orthogonal component, irradiating said living cell-dye system plus medium with such light and analyzing the polarized light emissions from said luminescent probe associated with said living cells and luminescent components in said medium in two distinct orientations representing horizontal and vertical orthogonal components of the polarized light emissions to determine the time dependence of said components of the polarized emissions from said luminescent probe associated with said living cells of the system and the medium, said steps of generating polarized light and analyzing the polarized light emissions are conducted during the period commencing at times greater than the luminescence lifetime of the living cell dye system after mixing and continuing until said background luminescence from the system medium significantly affects the polarized light emission intensities from the luminescent probe associated with the living cells of the system, thereby avoiding physical separation of said living cells from the luminescent dye unassociated with said living cells.
2. A method of claim 1 adapted to analyze the polarized fluorescent light emission from a fluorescent probe associated with living lymphocyte cells comprising introducing a weakly fluorescent dye into said medium, enzymatic action by the cells producing a component of said dye which is capable of fluorescence, said step of analyzing the polarized fluorescent light emissions continues until fluorescent dye components unassociated with the cells significantly affects the value for polarized fluorescent light emission intensities from the fluorescent probe associated with the living cell-dye system.
3. A method of claim 2, wherein said step of analyzing polarized fluorescent emissions from said system and medium comprises measuring the intensities of the polarized fluorescent emissions at relatively frequent intervals.
4. A method of claim 2, wherein the non-fluorescent dye mixed with the system is fluorescein diacetate, non-specific carboxyesterases of the living cells deacetylating the dye to produce fluorescein which becomes associated with the living cells.
5. A method of claim 3, wherein the step of measuring the intensities of the polarized fluorescent emissions continues for approximately one and half minutes after the dye is mixed with the living cell-dye system.
6. A method of claim 3, wherein the step of analyzing the polarized light emissions, the intensity of the emissions in the two distinct orientations is measured, the measurements are alternated frequently between the two distinct orientations for the light emissions during said period.
7. A method of claim 6, wherein the frequency of alternating between the two distinct orientations is approximately every one to ten seconds.
8. A method of claim 7, wherein said period extends for approximately one and a half minutes.
9. A method of claim 6, wherein an index representative of the time dependent change of fluorescent polarization is a function of a ratio of the measured accumulated intensities for the two distinct orientations over a period of time during said period of measurement before background fluorescence significantly affects the polarized light emissions from the living cells and determining the intensities at any predetermined time by interpolation of measured intensity values at times before and after said predetermined time.
10. A method of claim 9, wherein said index is a function of the ratio of the slopes of the plots of intensities versus time for the orthogonal components of measured intensities of the polarized emissions.
11. A method of claim 9, wherein polarization indexes at several predetermined times are evaluated and extrapolated to provide an index value at approximately the time when the dye commences to fluoresce within the living cells.
12. A method of claim 1, wherein said dye is in solution, adjusting the temperatures, osmolality, pH and ion concentrations of the living cell system medium and the dye solution to be compatible and mixing said system medium and said dye solution whereby shock to the living cells is prevented.
13. A method of claim 6, wherein the intensities of the orthogonal components of the polarized light emissions are measured by a single photomultiplier device which produces a digital signal representative of the measured intensity at any predetermined time for the particular orthogonal component being measured.
14. A method of claim 13, wherein an index representative of the time dependent change of fluorescent polarization is a function of a ratio of the measured accumulated intensities for the two distinct orientations over a period of time during said period of measurement before background fluorescence significantly affects the polarized light emissions from the living cells, transmitting each such digital signal to a computational means for determining the respective intensity at any predetermined time for an orthogonal component by computational interpolation of measured intensity values at times before and after said predetermined time.
15. A method of claim 14, wherein means for selecting two distinct orientations representing orthogonal components of the polarized light emissions by movement of said selector means from a first position to a second position, said computational means controlling the movement of said means for providing the distinct orientations to coordinate thereby the computation of the respective intensities at any predetermined time.
16. A method of claim 1, wherein each distinct orientation of polarized light emissions is measured by a light sensitive device, generating an analog signal from said light sensitive device representative of the measured intensity for the particular orthoganol component being measured and converting said analog signal into a digital signal for computational analysis.
17. A method of claim 2, wherein the two distinct orientations of polarized light emissions are continuously generated.
18. A method of claim 17, wherein the two distinct orientations of polarized light emissions are continuously measured by two photomultiplier devices spatially located to measure said two distinct orientations of emissions, producing two digital signals representative of the measured intensity for the respective orthoganol component being measured.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000431930A CA1200477A (en) | 1983-07-06 | 1983-07-06 | Method for detecting luminescence in living cell system |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CA000431930A CA1200477A (en) | 1983-07-06 | 1983-07-06 | Method for detecting luminescence in living cell system |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA1200477A true CA1200477A (en) | 1986-02-11 |
Family
ID=4125625
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA000431930A Expired CA1200477A (en) | 1983-07-06 | 1983-07-06 | Method for detecting luminescence in living cell system |
Country Status (1)
| Country | Link |
|---|---|
| CA (1) | CA1200477A (en) |
-
1983
- 1983-07-06 CA CA000431930A patent/CA1200477A/en not_active Expired
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