|Title of Invention||
AN APPARATUS FOR CANCER DETECTION BY OPTICAL ANALYSIS OF BODY FLUIDS
|Abstract||An apparatus for optical analysis of body fluids comprising;- - a light source for generating light rays, - an excitation wavelength determination means, - a grating for receiving optical rays from the body flyids, said optical rays being received at right angles to the optical rays incident on the said body fluids, - an optical conversion means for receiving optical rays of from the said grating and conmverting the said optical rays to electrical signals, a computer for receiving and processing said thje electrical signals.|
AiN AKrAKA I Lb tUK LANCtK Uh 1HCTION BY OPTICAL ANALYSIS OF
FIELD OF THE INVENTION
The present invention relates to an apparatus for optical anahsis of body fluids. More particularly the present invention relates to an apparatus for cancer detection for optically analyzing body fluids to diagnose the presence of carcinogenic cells found in any part of a human body.
BACKGROUND OF THE INVENTION
Cancer has always been a dreaded disease. In spite of the advances in science and medical care, cancer is curable only when detected early. There are some techniques already in practice for detecting and staging of cancer. Some of them are surgical biopsy, protein sequence analysis (PSA) tests, DRE tests, computerised axial tomography (CAT or CT) scan, magnetic resonance imaging (MRI) scan, ultra-sound scan, bone scan, positron emission tomography (PET) scan, bone marrow test, barium swallow , endoscopy, cytoscopy test, T/Tn antigen test, mammogram etc. Each one of the above mentioned tests has its own merits and demerits but none of them can be used effectively for mass screening.
The diagnostic procedures for cancer are done in three levels. The primary
level is when the patient meets the clinician, a report is taken from the patient and the doctor does a physical examination. If a tumor is suspected, at the primary level, the patient is referred to specific tests such as barium swallow, PSA etc. This is the secondarv level. For further confirmation before launching the treatment methods, a set of tertiary' lexel test such as biopsy involving histopatholog}" and cytopathology can be carried out.
The in\'ention provides an apparatus for optical analysis of body fluids in the primary and secondary- level of diagnostic testing of cancer. The tests are noninvasive and non-specific, hence cancer of any type, in any part of the body can be detected in a simple manner. More advantageously the apparatus according to the present invention is relatively simple and can be used for mass screening. Also it consumes very low power.
SUMMARY OF THE INVENTION
The present invention provides an apparatus for the optical analysis of body fluids. More particularly, the present invention relates to an apparatus for optically analyzing body fluids in order to diagnose the presence of carcinogenic cells present in any part of human body. This is based on fluorescence of biomolecules found in body fluids like blood, urine and their extracts. The analysis can be carried out based on fluorescence emission spectra, fluorescence excitation spectra and synchronous spectra of the body fluids. The apparatus consists of an optical source such as a lamp or laser at a predetermined wavelength and spectral
vvidth. The rays trom the optical source are directed to an excitation wavelength determining means (say a grating or a filter) and are focused on a sample of body fluids. The bod> fluid sample is kept in a transparent curvette. The fluorescence optical rays from the curvette, which are at right angles to the light incident on the body fluid sample in the curvette, are focused with the help of a focusing means (lens) to a grating. From here the optical rays are passed through a slit. The wavelength of the optical rays required is isolated with the help of the slit and the grating. The fluorescence optical rays of a required predetermined wavelength are given as input to an opflcal conversion means which converts the optical rays into analog electrical signals. The analog signals are then digitized and given as input to a computer for processing the results of the analysis carried out by the apparatus according to the present invention. When the grating is scanned, signals are collected at different wavelength and we get fluoroscence bands. These bands are fingerprints of the disease.
Accordingly the present invention provides an apparatus for optical
analysis of body fluids for cancer detection comprising: an optical source for
generating optical rays, an excitation wavelength determining means which
receives the optical rays fi*om said optical source and transmits the optical rays at
a predetermined wavelength, a curvette for holding body fluids and for
receiving the optical rays fi:"om the excitation wavelength determining means,a grating for receiving the optical rays from the curvette which are at right angles to the opflcal rays incident on the curvette and transmitting the optical rays with a
firedetermined wavelength, a slit being provided between the said grating and an optical conversion means for directing the optical rays receixed at a particular wavelength from the grating to the optical conversion means, said optical conversion means being provided for converting the optical rays reciex'ed from the said grating to electrical signals, a computer for receiving and processing the said electrical signals.
The optical source may be a coherent light source (laser) or an incoherent light source (halogen lamp). The excitation wavelength determining means may be an interference filter, a notch filter or a gradng. The optical conversion means may be a photo detector (photo diode, photo multiplier or CCD array).
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows a schematic diagram of an apparatus for optical analysis of body fluids according to the invention.
Figure 2 shows a schematic diagram of a preferred and advanced embodiment of an apparatus for optical analysis of body fluids according to the invention.
Figure 3 shows the fluorescence emission spectra for tests performed on extracts of formed elements for a healthy sample.
Figure 4 shows the fluorescence emission spectra for tests perfonned on extracts of formed elements for a cancer diseased sample.
Figures Da, 5b and 5c shows the fluorescence emission spectra of blood plasma based on tests performed for showing different stages of cancer in healthy stage, early stage and advanced stage respecti\ely.
Figures 6a and 6b shows the fluorescence emission spectra of urine analysis done for a health} patient and a diseased patient respecti\'ely.
Figure 7 shows the fluorescence emission spectra based on urine analysis at a different wavelength.
Figures 8a and 8b shows the fluorescence emission spectra based on tests performed on urine extracts in a healthy patient and a diseased patient respectively.
Figure 9 shows the fluorescence excitation spectra based on tests performed on extracts of formed elements or urine.
Figures 10a and 10b show the fluorescence synchronous spectra based on blood plasma analysis of a healthy patient and a diseased patient respectively.
Figures 11 and 12 show the fluorescence synchronous spectra based on blood plasma analysis of a healthy patient and a diseased patient respectively, at different wavelengths.
Figures 13 and 15 show the fluorescence excitation spectra based on urine analysis for a healthy patient at different wavelengths.
Figures 14 and 16 show the fluorescence excitation spectra based on urine analysis for a diseased patient at different wavelengths
DETAILED DESCRIPTION OF THE INVENTION
The apparatus shown in figure 1 consists of an optical source (T). The optical source can be an incoherent light source like halogen lamp. mercur> lamp, xenon lamp or tungsten lamp. It can also be a coherent light source like a diode laser, helium- cadmium laser, frequency doubled Titanium (Ti) Sapphire laser, or a tunable dye laser at different levels of sophistication. The optical rays from the source (1) is directed to an excitation wavelength determining means (F). In this embodiment of the invention the excitation wavelength determining means (F) may be an interference filter or a notch filter. The optical rays from the excitation wavelength detennining means (F) correspond to a predetermined wavelength. These rays are focused on the sample of body fluids collected from a patient, through a first focusing means (LI). The body fluids are placed in a transparent quartz curvette(2).
The body fluids that are collected may be various substances based on the type of diagnosis required. The curvette(2) may provide about 10mm path length. Once the optical rays from the excitation wavelength determining means(F) falls on the body fluid sample kept in the curvette(2), fluorescence optical rays are given off fi"om the body fluid sample kept in the curvette(2) at different angles. The fluorescence rays that are received fi'om the curvette(2) at an angle of 90^ (right angles) from the rays incident on the curvette(2) alone are received. Once the fluorescence optical rays which are at an angle of 90^ are received, it is
focused by second focusing means (L2) to a grating (G), The grating (G) may be a ruled or a holographic grating of 1200 or 2400 hnes per mm. The optical rays are then dispersed out from the grating (G). The tluoroscence is often a band of wavelength of width 50 to lOOnm depending upon the sample. The combination of grating G and the rectangular slit (2mm x 1mm) selects fluorescence signal of 5nm band width. When the grating G is rotated, at a typical speed of 500nm per minute, the entire fluoroscence emission signal from the sample is scanned. The fluoroscence band thus obtained is the signature of healthy or abnomial sample. The optical rays from the slit(S) is given to an optical conversion means(3) such as a photo multiplier tube or a photodiode placed after the rectangular slit(S). The optical conversion means(3) converts the received optical rays of a specific wavelength to electrical signals. These signals are in the fomi of analog signals. The analog signals are then converted to digital signals with the help of a digitizer (4). The digital signals ft'om the digitizer (4) are fed to a computer (5). The computer (5) processes information collected based on the analysis performed by the apparatus and supplies the result of the optical analysis. Information processing in the computer is done with the help of a specially designed statistical analysis software customized to categorize and extract the results of the analysis performed.
Referring to the preferred embodiment shown in figure 2, the optical rays from the optical source (1) are allowed to fall on a grating (Gl) instead of the
invention, two gratings are present instead of one. The grating provided in front of the optical source(l) is the excitation grating(Gl) and the grating behind the body tluid sample kepi in the curvette(2) is the emission grating (G2). The light rays from the optical source(l) are incident on the excitation grating(Gl). The wavelength of the rays to be focused on the cur\'ette(2) are isolated with this grating(Gl). Once the predetermined emission wavelength is identified, the optical rays corresponding to that particular wavelength transmitted from the grating(Gl) are focused on a mirror(M). These rays are then reflected from the surface of the mirror(M) and are directed to the body fluid sample kept in the curvette(2) through a slit (SI) placed in front of the said curvette(2). The remaining features of this embodiment are the same as already described in figure I .
The results obtained from analysis done by the above mentioned apparatus give distinct signature and features of the bio-molecules specific to cancer. In order that the apparatus, according to the present invention, to fiinction in the manner as required by the user for the analysis of different body fluids, various procedures should be followed before placing the body fluid sample in the transparent curvette. Based on the types of samples tested and the wavelengths selected, various types of tests can be carried out. Typical tests are described below.
TESTS BASED ON FLUORESCENCE EMISSION SPECTRA OF BODY FLUIDS
EXTRACT OF FORMED ELEMENTS:
Step I: A disposable syringe is used to taice 5 ml venous blood from the subject and put it in a sterile vial containing ethylene diamide tetra acetic acid (EDTA) anticoagulant.
Step 2: The blood is centrifuged at 4000 rpm for 15 min and the supernatant plasma is separated out and collected in a sterile vial.
Step 3: The formed element containing mostly cells such as erythrocyte is treated with acetone in the ratio of 1:2 (i.e., to the 1 ml of formed elements 2 ml of acetone is added). The sample is vigorously shaken 100 times and then centrifuged at 4000 rpm for 15 min.
The supernatant thus obtained is a clear solution containing the bio-molecules that are tumor markers. It is subjected to the optical analysis as described before.
Step 4: The wavelength of excitation is fixed at 405nm by adjusting the interference filter and obtain fluorescence spectrum in the range of 425 to 720 nm.
With reference to a typical result shown in figure 3 for healthy sample and figure 4 for cancer diseased sample, the spectrum consists of 4 bands
1) Around 460 nm, due to Raman scattering of acetone.
2) Fluorescence band at around 505 nm most probably due to riboflavin or a bile component.
3) Fluorescence band at around 585 nm due to anionic species of porphyrin.
4) Fluorescence band at around 630 nm due to neutral species of porphyrin.
5) Fluorescence band at around 695 nm due to cationic species of porphyrin.
Step 5: The intensities of the bands are measured and denoted as I460.1505,1585, l630,
The ratio of intensities are denoted as.
Ratio (Ri) = (l630/l585)
r.5 2.25 Ri > 3 it implies that the patient is at the advanced stages.
We denote ( R2) =" 1595 /1585
( R3 ) "^ ^630 / I505 ( R4 ) ^ I585/I46O ( R5 ) "^ I5O5 / I46O
These fluorescence intensity ratio parameters are propotional to the ratio of concentration of above cited biomolicules. These are in different ratio for healthy and diseased samples. They are summariesed in table 1.
H - Healthy
HR - High Risk
E - Early stages of cancer
A - Advanced cases
C - Contrast Parameter
Ri, R2 and R3 are common for all types of cancer since it depends upon the concentration of porphyrin, a bio-molecule involved in heme metabolism. This is found at higher concentration in cancer patients than in healthy subjects because of the abnormal cell proliferation in the patients. This is in general the basis for laser based photodynamic therapy, which is in practice all over the world.
In the present invention, we are concerned with the concentration of porphyrin carried in the blood stream and excreted through urine. Higher the concentration of this fluorophore, higher the tumor activity or tumor volume.
There are some special cases to this also.
rfR4 0.5 Assuming Ratio (R5) - (I505/1460)
If, R5 0.5 0.75 This factor is distinct in pancreatic cancer with obstruction into the liver.
A few types of cancer detection tests w^ere carried out based on the present invention. They are as follows.
1. Animal Models
150 albino mice were studied in which squamous cell carcinoma had been induced using chemical carcinogen DMBA. These mice were sacrificed at different stages of cancer and blood samples taken were subjected to the sample analysis outlined above. The healthy blood and the (cancer) diseased blood showed distinct features .It was seen that Ri = I^so / h^s increases as the disease
2. Field study on Human patients
424 human patients were tested as a field stud}'. The details of disease and diagnosis score are given below. The score was done with reference to the conventional histopathology.
Test II A (Fresh Urine Sample)
2ml of urine is dropped in a quaitz curvette. The excitation wavelength is set at 325 nm and the fluorescence spectrum is obtained from 350 to 600 nm. There is a smooth fluorescence band with a peak around 420 to 440 nm with a high intensity for healthy urine. There is a weak shoulder around 550nm. The intensity' ration may be given as follows:
For cancer diseased patient there are two bands one around 440 nm and another around 550nm with an intensity' ratio (R^) = (I550 / I440) varies from 0.4 to 1. This is shown in figure 6(a) for healthy sample and figure 6(b) for diseased sample.
But these two bands are at least 50 times weaker than the fluorescence of healthy urine.
The fluorescence band around 550nm is most probably due to bilirubin. This is at least two times higher in concentration in cancer patients as compared to the healthy subjects.
Next, the excitation wavelength is set at 405nm and obtain fluorescence spectrum from 425 to 725nm. This is shown in figure 7. There are five bands. 470nm. 500nm, 550nm, 580nm and 620nm .The notation may be given as follows:
(R7) = (l6:o /1550) and (R^) - (I,2o /1580)
If R7 0.5 R7> 0.8 and Rg >1.2 it implies advanced stage of
Test II B
Extract of Urine
A reagent of ethyl acetate and acetic acid is prepared in the ratio of 4:1 (40 ml ethyl acetate to 10 ml of acid). In a test tube 2ml of reagent and 1 ml of urine are added. After shaking well, it is allowed to settle for 10 minutes. Take the upper layer (about 1ml) which has extracted porphyrin. It is then subjected to optical analysis.
The wavelength is set at 405 and obtain spectra from 425 to 720 nm. Four bands
are obtained as tbllovvs.This is shown in figure 8(a) for health}- patient and figure 8(b) for diseased patient.
1) 460 nm due to Raman Scatterins: of reasents
2) 550 nm due to bilirubin
3) 575 nm and 620 nm due to porphyrin.
The intensity of all bands are measured. The notation mav be sixen as follows:
(R9) = (I620/I550)
If 0.75 R9> 1.5 it implies that is advanced.
Thus nine parameters are obtained for mass screening of cancer from body fluids. The porphyrin, and also the bilirubin, found in higher concentration in the body fluids of cancer patients than the healthy subjects, are the tumour markers. These biomolecules, porphyrin and billirubin are involved in heme metabolism, which appear to be considerably altered by cancer. These biomolecules are the cancer specific fingerprints in laser or light induced fluorescence.
With the two above mentioned body fluids and the above mentioned
parameters cancer mass screening can be done with a reliability factor of 80%.
TESTS BASED ON FLUORESCENCE EXCITATION SPECTRA AND SYNCHRONOUS SPECTRA OF BODY FLUIDS
Analysis based of the excitation and synchronous spectra can also be carried out to improve the specificity and reliability. Typical tests are described below.
TEST III A
This is the inverse of fluorescence spectrum of the sample and under optimized conditions gives the absorption spectrum.
The sample (extract of formed elements, urine etc) is prepared and taken in the quartz cuvette. The emission grating is fixed at 630nm and the excitation grating is scanned fi"om 350 to 600nm and the spectrum is recorded. The excitation spectrum has a primary peak around 398nm, with a few secondary peaks. The intensity of I398 is measured. Then the emission grating is tlxed at 585nm and scanned usin2 sratino Gl from 300 to 550nm. This sives another excitation band.
very similar to the previous one, but at 410nm. These two are the excitation spectra of t\vo species of porphyrin. This is shown in figure 9. The peak intensit}^ of these two band (I410) are measured. The ratio of these intensities are denoted as
If Rio 0.8 Rio > 1.5 Advanced stages of cancer
Test III B
With suitable modifications in the svstem as mentioned before , one more t\^pe of spectra is obtained ie. Synchronous spectra for the same sample. This becomes an additional window of analysis. This is a compounded spectrum of fluorescence emission of many molecules but each molecule being excited at the absorption peak. It gives a better resolution and identification of weakly fluorescing, submerged fluorophore.
SYNCHRONOUS SPECTRA OF BLOOD PLASMA
The plasma sample is prepared and placed in the cuvette as before. The
excitation Grating is set at 200nm and emission Grating is set at 210rkn with a wavelength difference lOnm. Then synchronously both gratings are scanned. The fluorescence obtained with the excitation of 200nm is collected from 210nm onwards. Then the excitation grating moves to 210 and s\nchronously the emission grating moves to 210 and collects tluorescence
Such synchronous spectra obtained for any sample show distinct and marked differences between healthy and diseased fluid.
There are well defined bands around 311nm. 365nm. 450nm, 505nm, 550nm, and 620nm. As plasma contains a host of free and enzyme bound flurophores (biomolecules) we can only tentatively assign the bands to the fluorophores: Out of these, 311nm is the sharp Raman Band of back ground plasma medium. 365 nm is most likely due to tryptophane; 450nm due to NaD(P)H; 505nm due to riboflavin and 555nm due to bilirubin, 585nm and 625nm due to porphyrins. Comparing the healthy and diseased spectra one can see that these bio molecules are out of proportion in diseased blood. (See fligures 10a for healthy and 10b for the deseased)
For example, the ratio of band at 311nm (due to Raman spectra of water) and at 365nm (due to tryptophane) is 0.7 for healthy and 1.8 for the advanced stage of cancer. (So the contrast parameter is 2.6); this ratio is 1.05 for early cancer and 0.83 for high risk or hyperplasia. Another important ratio is the concentration between tryptophane and porphyrin. This ratio (1365 1-^85) is 2.3 for
healthy, 3.5 for high risk cases; 4.5 for early cancer and is 8.7 for advanced cases. Other similar fluorescence ratios are given in Table III shown below
H - Healthy
Hr - High Risk
E - Early stages of cancer
A - Advanced cases
C - Contrast Parameter
Next set instrument for synchronous spectra with the phase lag between two grating as 30nm. Run the spectra as we did above. Here we get two bands of flouroscence, one at 355nm and another at 440nm and third one at 500nm. Intensity ratio (Rp) = (hoofhss )• This is about 0.4 for healthy, 0.6 for early cancer and greater than 1 for advanced cancer. Similarly (Rig) = (I500 / I440 ) ■ This is shown in figure 11
If this is
1-1.5 - Early Cancer
> 1.5 - Advanced Cancer
Now set the phase las between two eratins as 70nm. We set three bands one at 340nm another 450nm and third 500nm. This is shown in figure 12.
(R19) - (I440/1340) > 0.6 for diseased.
(R20) = (I500 /1450) > 0.7 for diseased
SYNCHRONOUS SPECTRA OF URINE:
2ml of Urine is dropped in the Cuvette and run synchronous Spectrum from 200nm to 800nm, with a phase lag of lOnm.
The Intensities at 390nm and 490nm ( corresponding to trv^ptophane and riboflavin) are picked out. This is shown in figure 13 for healthy patient and figure
14 for diseased patient.
We denote the ratios
I 490 / I 390 ~ R21
of R:; > 2 > 4 - Advanced Cancer
Synchonous spectrum is scanned for urine from 200 - 700nm and again with a phase lag of 70nm in order to obtain the spectra .This is shown in figure 15 for healthy patient and figure 16 for diseased patient.
Define R22 as the intensity ratios at I465 / 13(35. (again due to riboflavin and tryptophane)
R22 ~ I465 / I365
if R22 > 3 > 4 - Advanced Cancer
age 30-55 and 128 from diseased patients (mostly cancer of cer\'ix or breast). Our optical anahsis after diagnosis is more than 80% as shown below in Table IV.
Subjects Number Correct Optical Diagnosis False -ve False -
Healthy 50 45 5
Premalignant 15 12 ^ 3
Cancer 113 102
TOTAL 178 159 5 14
The Advantage of the apparatus for optical analysis of body fluids for cancer detection according to the present invention is that the apparatus is useful for mass screening of cancer similar to diabetics mellitus test. It is non-invasive and non-painful method to diagnose cancer. It allows ageneric optical test to diagnose any type of cancer.The diagnosis and result would take less than an hour. A statistical analysis model for turnkey analysis and report is incorporated. The apparatus
requires low capital investment and low maintenance with high throughput. The
Operation of the instrument by technicians is simple. It is highh potential for
marketing it in ever\' clinic and hospital. The apparatus developed is compact and
occupies approximately 3ft x 4ft. In case of emergency, or use in remote village.
12V car batter}'is enough as powder source. It is extremely simple to setup,
alien and train technicians.
1. An apparatus for cancer detection by optical analysis of body fluids
an optical source(l) for generating optical rays,
an excitation wavelength determining means (F,G1) which receives the optical rays from said optical source(l) and transmits the optical rays at a predetermined wavelength,
a curvette(2) for holding body fluids and for receiving the optical rays from the excitation wavelength determining means(F,Gl),
a grating(G,G2) for receiving the optical rays from the curvette (2) which are at right angles to the optical rays incident on the curvette (2) and transmitting the optical rays with a predetermined wavelength,
a slit(S,S2) being provided between the said grating(G,G2) and an optical conversion means (3) for directing the optical rays received at a particular wavelength from the grating (G,G2) to the optical conversion means(3), said optical conversion means(3) being provided for converting the optical rays recieved from the said grating(G,G2) to electrical signals,
a computer (5) for receiving and processing the said electrical signals.
2. The apparatus as claimed in claim 1, wherein a digitizer (4) is provided
between the said optical conversion means(3) and the said computer (5) for
means(3) to digital signals and for feeding the said digital signals to'the said computer (5).
3. The apparatus as claimed in claim L wherein the said optical conversion means(3) is a photo detector.
4. The apparatus as claimed in claim L wherein the said optical conversion means(3) is a photo multiplier.
5. The apparatus according to claim 1, wherein the said excitation wavelength determining means (F) is an interference filter.
6. The apparatus according to claim 1, w^herein the said excitation wavelength determining means(F) is a notch filter.
7. The apparatus according to claim 1, wherein the said excitation wavelength determining means is a grating (Gl).
8. The apparatus according to claim 1, wherein a first focusing means(Ll) is provided between the said excitation wavelength determining means(F) and the said curvette(2) for focusing the opfical rays recieved from the said exitation wavelength determining means(F) to the said curvette(2) and a second focusing
means (L2) is provided between the said curvette(2) and the said grating(G) for focusing the optical rays transmitted at right angles to the optical rays incident on the said curvette(2).
9. The apparatus according to claim 1 wherein a first focusing means (LI) is
provided between the said optical source (1) and the said grating (Gl) for focusing
the optical rays fi'om the said optical source(l) to the grating(Gl), and a second
focusing means (L2) is provided between the said curvette(2) and the said
grating(G2) for focusing the optical rays transmitted at right angles to the optical
rays incident on the said curvette(2).
10. The apparatus according to claims 9 wherein a slit (SI) is provided between
a mirror(M) and said curvette(2) for directing the optical rays to the curvette(2).
11. The apparatus for optical analysis of body fluids, substansially as herein
|Indian Patent Application Number||587/CHE/2003|
|PG Journal Number||38/2007|
|Date of Filing||22-Jul-2003|
|Name of Patentee||DR. VADIVEL MASILAMANI|
|Applicant Address||100 THENDRAL, ANNAI THERESA NAGAR, MADIPAKKAM, CHENNAI 91,|
|PCT International Classification Number||A61 B 19/00|
|PCT International Application Number||N/A|
|PCT International Filing date|