US 3856930 A
A process is provided for expediting the diagnosis of an array of tissue specimens suspected of being diseased by eliminating from the array a large portion of the clearly disease-free specimens so that only a relatively small number of specimens need be examined closely for the presence or absence of disease. In the process, a specimen of cells is homogenized after extraneous cellular material such as protein is removed. A predetermined quantity of cells is counted and separated from the specimen and are treated with a radioactive dye which corresponds to the nature of the disease to be diagnosed. The cells uptake the dye in relation to their condition of health, i.e., diseased cells take up more dye and hence radioactive material than normal cells. The excess radioactive material is rinsed from the quantity of cells, and the quantity is solubilized. The level of radioactivity of the specimen cells is sensed and is compared with the level of radioactivity of a normal cell sample which had been identically prepared to obtain a ratio. If the level of activity is below a prescribed ratio, as determined empirically, the specimen need not receive further examination.
Claims available in
Description (OCR text may contain errors)
United States Patent Nodine et al.
[451 Dec. 24, 1974 METHOD OF SCREENING TISSUE SPECIMENS FOR DIAGNOSTIC EXAMINATION Inventors: John H. Nodine, Wayne, Pa.; John Herbert Waite; Herbert Waite, both of Haddonfield; Martin J. Fletcher, Belle Mead, all of NJ.
 US. Cl..... 424/1, 23/230 B, 250/303  Int. Cl. A6lk 27/04, GOln 23/00  Field of Search 424/1; 23/230 B;
252/30l.l R; 250/106 T, 71.5 R, 303, 304
 References Cited UNITED STATES PATENTS 3,673,410 6/1972 Waite et al. 424/1 X 3,678,148 7/1972 Caiola 424/1 Primary Examiner-Benjamin R. Padgett Attorney, Agent, or Firm-Dorfman, Herrell and Skillman  ABSTRACT A process is provided for expediting the diagnosis of an array of tissue specimens suspected of being diseased by eliminating from the array a large portion of the clearly disease-free specimens so that only a relatively small number of specimens need be examined closely for the presence or absence of disease. In the process, a specimen of cells is homogenized after extraneous cellular material such as protein is removed. A predetermined quantity of cells is counted and separated from the specimen and are treated with a radioactive dye which corresponds to the nature of the disease to be diagnosed. The cells uptake the dye in relation to their condition of health, i.e., diseased cells take up more dye and hence radioactive material than normal cells. The excess radioactive material is rinsed from the quantity of cells, and the quantity is solubilized. The level of radioactivity of the specimen cells is sensed and is compared with the level of radioactivity of a normal cell sample which had been identically prepared to obtain a ratio. If the level of activity is below a prescribed ratio, as determined empirically, the specimen need not receive further examination.
15 Claims, 2 Drawing Figures ge AlNlng aom b onic QuAy r PATIENT or RADIOACTIXL QLLMAIEBLAL ANALYSIS L coLi'iTimcELLs REMOVING F emsmc mg l EXTRANEOUS HOMOGENIZING i RE STE??? IV 5 BAD-[93mm w SPECIMEN & "'1 QUANTITY Ma ietta: g-1 sgg gsme UNDER SPECIMEN or CiL SjjcM FRQM ANALYSIS QL E M WITHDRAWING mew mot; 1 QUANTITY OF 15: ggggxggg' CELLS FOR NORMAL firm-N meets Mmmm QQNSIDERATIOQ To OBTAIN no REMOVING RINSING E m wig EXTRANEOUS Excess RADIOACTIVITY WMATW HOMDGENIZING PREDEI'ERMINED RADIOACTIV 9; EM FROM mm FEM MATERIAL BTNOQWL SAMPLE ML); OF NORMAL ROM NORMAL 11' CELLS CELLQUANTE! am oemmmc. ADDING QUANTITY A or RADIOACTIVE W DYE MATERIAL TO L couNTED CELLS RELATIONSHIP SHEET 2 0F 2 BETWEEN CELL CONCENTRATION AND DYE UPTAKE METHOD OF SCREENING TISSUE SPECIMENS FOR DIAGNOSTIC EXAMINATION The present invention relates to a method of screening an array of tissue specimens suspected of being diseased so that only the specimens which are clearly disease-free need not be subjected to detailed diagnostic examination. More particularly, the present invention relates to a specimen-screening process wherein dyes containing radioisotopes are employed so that the process can be performed in an automated manner.
At present, large numbers of tissue specimens which are suspected of being diseased are collected from patients each day and sent to a laboratory for diagnostic examination by a pathologist or cytologist. Such examination is usually performed by dyeing the tissue with a dye having an affinity for cells which have a particular disease and then examining the dyed cells under a microscope. This procedure is time consuming, and hence costly. Moreover, it must be performed by a highly skilled person. Since a large number of specimens which are examined are ultimately determined to be free of disease, it should be apparent that a process by which the clearly disease-free specimens are eliminated from further examination is highly desirable.
In U.S. Pat. No. 3,678,148 (owned by the assignee of the present application) there is disclosed a method for reducing the number of specimens which must be manually diagnosed. The patented procedure contemplates the dyeing of tissues with a radioactively-tagged dye, preferably when the tissue is mounted on a slide. This procedure substantially reduces the number of slides which require visual microscopic inspection but it does not reduce the requirement for mounting the samples on slides or the human time factor required to process the samples prior to the radioactive screening.
With the foregoing in mind, it is a primary object of the present invention to provide an improved method for screening from an array of tissue specimens suspected of disease only those specimens which possess sufficient indication of disease as to require detailed diagnostic examination.
It is another object of the present invention to provide a novel method for screening tissue specimens without requiring the mounting of specimens on slides prior to screening.
As a further object, the present invention provides a unique method by which the efficiency of the procedure for examining tissue specimens suspected of disease is increased.
It is a still further object of the present invention to provide a method whereby tissue specimens suspected of disease are capable of being screened in an automated process.
More specifically, in the present invention, specimens of tissue which are suspected of being diseased are subjected to a screening process to eliminate normal or disease-free specimens from the necessity of being subjected to a thorough diagnostic examination. The screening process includes the steps of removing extraneous cellular matter from a specimen of tissue, homogenizing the specimen, counting from the homogenized specimen a predetermined quantity of cells, treating the quantity of cells with a dye containing a radioactive substance, and removing the excess radioactive material from the cell-quantity. The next step includes solubilizing the cell-quantity and sensing the level of radioactivity of the cell'quantity. The level of radioactivity is compared with the corresponding level of a standard disease-free sample which had been subjected to the same process. The levels define a ratio, and those specimens which fall below the ratio are excluded from examination.
These and other objects, features and advantages of the present invention should become apparent from the following description and the accompanying drawings in which:
FIG. 1 is a block diagram of the process of the present invention and FIG. 2 is a chart graphically illustrating the relationship between the quantity of cells and the amount of uptake of a radioactive dye.
According to the present invention, a process which is capable of being performed substantially entirely in automatic apparatus is provided for screening large quantities of tissue specimens to minimize the number of specimens which must be subjected to a detailed diagnostic examination. For this purpose, dyes carrying radioactive materials are employed to stain specimens suspected of disease, and the amount of radio-activity emitted by the specimen is sensed and compared with the level of radioactivity emitted by a standard sample of disease-free tissue. If the level of radioactivity emitted by the specimen is above a prescribed ratio relative to the level of the disease-free standard sample, the specimen is then subjected to a detailed diagnostic examination.
The method of the present invention is particularly suited for use in diagnosing tissue specimens which are obtained exfoliatively from human female reproductive organs such as the cervix, uterus or vagina. In conventional practice, such specimens are placed in a preservative pending their examination by a cytologist or pathologist, although a preservative is unnecessary if specimens are to be examined promptly (within 3 hours). A preferred liquid for containing a tissue specimen for prompt examination after collection is a physiologic saline solution (0.85 percent salt in distilled water) or Ringers solution. Other preferred preservatives include Papette and specially prepared solutions of benzene, methanol, ethanol, benzoic acid, sodium benzoate, ether and distilled water. Combinations of these solutions may be used in appropriately measured concentrations. It has been determined that specimens which have been contained in a preservative for as long as 18 months may be screened satisfactorily.
The process of "the present invention requires a specimen which is free from extraneous cellular material. To remove such extraneous material the specimen is subjected to an enzymatic treatment to remove extraneous material such as protein, nucleoprotein, and mucous. The treatment also separates the cells by causing lysis of the intercellular cement. Several conventional enzymatic treatments are available for this purpose. For instance, pepsin, trypsin, and hyaluronidase may be used satisfactorily simultaneously or sequentially. The incubation time, temperature and pH of the treatment solutions are properly controlled to avoid excessive cellular destruction. The optimum temperature to be used with these enzymes is 37 C; however, with certain other enzymes it may be necessary to vary the temperature as well as the pH of the treatment solution. It is noted that pepsin acts only in acid solutions, and trypsin acts in neutral or alkaline solutions.
In the event that the optimum pH for a particular enzyme has not been established, such pH can be deter-.
mined experimentally. For instance, a standard hemoglobin solution may be colormetrically measured for tyrosine release at various pH levels. The pH which produces the greatest quantity of tryosine is the optimum. Once the optimum pH is established, the optimum temperature and time may be similarly deter mined by reference to the tyrosine release curve, with the time being adjusted at the upsweep of the release curve while the plateau of the same is to be avoided.
After the extraneous material is removed, the specimen is homogenized to break up any large masses of tissue which may be present. Standard cell homogenizers have glass mortars and pestels of glass and/or teflon may be employed for this purpose. By homogenization, dispersion of the cells is meant. Cellular destruction is to be avoided.
After the specimen is homogenized and the cells dispersed, a predetermined quantity of cells is counted and removed from the mass of cells composing the specimen. For this purpose, conventional optical or electrical cell'counting apparatus is employed. In such apparatus, the flow of cells through an aperture produces an electronic pulse due to changes in resistance or light interference. The pulse is amplified in proportion to the quantity of cells passing through the aperture in any one instant. The amplified pulses are then fed into a multi-channel recorder which counts the total number of pulses (or cells) and which considers the size of the cells by recording voltage deviations which are proportional to the size of the cell particles. The spectrum of the voltage deviations is recorded in a multi-channel recorder for feeding into a computer unit.
Before the cells are counted the supernatant preservative solution in which the specimen is contained is changed to a solution which is of a standard electrical resistance. The specimen may be separated from its supernatant in a centrifuge. The sediment which is ob tained from centrifugation is rediluted in proper ionic strength so that the conductivity and/or optical properties of the cell-solution combination are standardized.
The type of standard solution which is employed depends on the cell-counting apparatus which is utilized. The viscosity of the solution must be accurately controlled to ensure a constant flow rate through the counting aperture. If an optical interference detector is employed, color interference as may be caused by hemoglobin contamination must be avoided. The hemoglobin contamination should be removed prior to suspension of the specimen in the counting fluid. In a resistance detector the ionic strength must be accurately controlled. Ion-free solutions may be used; however, when an ionic solution such as Isotone is used, the ionic strength must be precisely titrated or checked by determining its freezing point depression with an conventional osmometer. 1
After a predetermined quantity of cells is countedout and separated from the specimen, the remaining cells are withdrawn and preserved for future diagnostic consideration should such become necessary. In the event that the specimen does not contain a sufficient number of cells to provide enough for screening as well as subsequent examination the process cannot be continued, and another specimen must be obtained.
The reason for counting-out a predetermined quantity of cells is to enable the proper amount of radioactive dye to be added to the cell-quantity. For instance, it has been determined that there is a linear relationship between cell concentration and the uptake of dye and radioactive material. The linear relationship is illustrated in FIG. 2. Once the number of cells has been determined, the amount of dye to be added can be readily determined.
A sufficient amount of the radioactive material is added to the cells to ensure stoichiometric binding of dye to the cells. The radioactive material is added to the cells at an appropriate pH and for a sufficient period of time at a properly adjusted temperature. For instance, either H Pyronin B or H methylene blue each having a specific activity of 0.8 to 3.0 mc/mg may be used as a radioactive material in a concentration of l to 3 mg/IOO ml of solution. The solution is buffered at a pH which is in a range of 5.0 to about 7.0, and the temperature is maintained in a range of about 20 C to about 37C for about 5 to 10 minutes.
After the radioactive dye is added to the counted quantity of cells, the excess dye is removed, and the cells are rinsed. This may be readily effected by centrifugal separation of the cells from the dye. The cells may then be rinsed and the rinse supernatant centrifugally separated. Separation of the radioactive supernatant from the cells permits the radioactive material in the dye supernatant to be subsequently recovered, thereby minimizing disposal problems normally associated with the use of radioactive materials.
There are commercially available, automatic centrifuges which may be employed in the initial steps of removing extraneous cellular matter, in the steps preceding counting and/or sizing of the cells, as well as in the subsequent dyeing and rinsing steps. For instance, a centrifuge manufactured and sold by Ivan Sorvall, Inc., of Norwalk, Conn. has been employed satisfactorily. Preferably, the cuvettes which are employed in the Sorvall centrifuge are modified slightly to achieve an improved separation action. In the Sorvall separator, the steps which require separation, addition of supernatants, the removal of rinsing solutions, and the like, may be accomplished sequentially and completely au tomatically. For example, supernatants such as distilled water, ethyl alcohol, and/or mordants or bleaches as well as the radioactive dye material may be charged into storage vats in the separator for dispensation at the appropriate stage of the process. Furthermore, the supernatants may be drained into sumps for recycling. In this manner, a considerable amount of manual labor is conserved.
The dye solution may be preserved for day-to-day use by keeping it refrigerated; however, the addition of phenol or sodium benzoate is recommended to facilitate transfer and to stabilize the solution at the incubation temperature for cell-tagging which is about 37 C. The final solubilizer used may be any of a number of commercially available caustic solutions such as Liquosol. The solubilizer should only be applied in a sufficient amount to ensure cell destruction, since excess quantities result in increased quenching losses and a reduction in counting efficiency, in addition to being economically wasteful. If desired, larger amounts of a scintillation counting solution, such as Liquaphor may be used in the solubilization step prior to final transfer to the radiation-detection step.
The radioactive dye material which is separated in the centrifuge is retained so as to avoid problems in the disposal of radioactive waste. Thus, the average laboratory is capable of practicing the process of the present invention without meeting any special disposal requirements set by the Atomic Energy Commission. It is noted that once the excess radioactive materials are separated after the dye uptake step, further rinse solutions may be discarded through ordinary disposal channels with relatively insignificant quantities of radioactive materials being discarded.
The automated washing or rinsing steps may be effected in three to five different cycles with one or more different washing or rinsing solutions. Preferred washing and rinsing solutions may include deionized distilled water, alone or in combination with ethyl alcohol. It is desirable to wash and/or rinse the cells until the cellular binding component of the uptake becomes constant. In this manner, consistent results may be achieved. 7
After the cell specimens are treated with radioactive dye and properly rinsed, they are ready for the radiation sensing or detection step. For this purpose, the solubilizer is provided with appropriate fluors so that radiation can be sensed by the most sensitive radiation detection apparatus. The radioactivity of the cell specimens may be sensed by transferring the specimens to a vial which is carried on a continuous chain through a scintillation counter to yield an immediate discrete analysis. In the alternative, the specimen may be passed through a liquid flow-through scintillation counter. Although larger, chain-driven, thin-window, gamma detectors may be used in the final sensing step, it has been determined that currently available liquid scintillation detectors are more sensitive and adaptable. Nuclear simultaneously or within a reasonably short time period of the preparation of the specimen suspected of disease. It has been determined that the discrimination may be made with reasonable accuracy when the radioactivity level of the specimen is placed in the form of a ratio with the corresponding radioactivity level of the standard sample cells.
The accuracy of the discrimination may be observed by considering the following example. An array of 106 specimens was examined for disease by a pathologist, and each specimen was classified by a number in accordance with the standard pathological classification system. The specimens were subjected to the process of the present invention, and the level of radioactivity of each was compared with the corresponding level of a disease-free standard sample. The radioactivity level of the specimen was divided by the corresponding level of the standard sample to define a ratio. As may be seen in Table I, all specimens suspected of being diseased, Le, a classification of 4 or higher, had a radiation level ratio in excess of 1.4. All of the specimens which possessed a radiation level ratio below this had lower classification numbers, indicating an absence of disease. Thus, these specimens may be safely rejected from detailed diagnostic consideration. From Table II it may be observed that if the array constituted a daily collection of specimens, approximately 75 percent of the specimens could be eliminated immediately from detailed examination because they were below the prescribed ratio. Thus, only 25 percent of an array of specimens need be mounted on slides for detailed pathologic or cytologic examination.
Although the process of the present invention is particularly suited for use detecting the presence or absence of cellular cancer in female reproductive organs, the process TABLE 1 Frequency Distribution of Normalized Cervical Cell Sources.
l l l 2 l 2 l 2 l 3 2 l l l l l l l 2 l l 3 2 l 3 l l 2 2 2 3 5 2 l 2 l 3 2 2 4 2 l l 2 l l 4 2 4 3 l l I l 2 2 2 5 3 l 5 l l l l 2 2 l 4 2 3 4 5 l l l l l 2 l l l 2 5 2 2 5 5 2 l l l l l 2 l l l 4 5 5 5 2 5 2 5 0.6 0.8 L0 1.2 1.4 L6 L8 2.0 2.2 2.4 2.5 Ratio (Specific Activity of Unknown Specific Activity of Normal Control) Chicago, Packard and Beckman instrument companies TABLE II all manufacture suitable detectors.
After the cell specimen has passed the radiation sensing phase of the process a final determination as to the condition of the specimen may be made. For instance, it has been determined that a discrimination can be made between normal" and abnormal cells by comparing the radiation level of the spcimen cells with the radiation level of cells taken from a standard sample which has undergone the same preparation steps either STATISTICAL SUMMARY OF CERVICAL CELLS EVALUATED 1. Total Cases Evaluated 106 a. No.1's b. No.2s 30 c. No.3's 8 d. No.4s 6 e. No.5s l2 2. Total No.4s and No.5s IS a. above 1.4 18
TABLE ll-Continued STATISTICAL SUMMARY OF CERVICAL CELLS EVALUATED b. below 1.4 3. Total above 1.4 34
may be expanded to cover the detection of other diseases. Moreover, the process is capable of being automated so that a number of specimens may be processed simultaneously. It is noted that although some false positives i.e., specimens suspected of being diseased are later determined upon detailed diagnostic examination to be actually disease-free, it should be apparent that the process of the present invention enables 75 percent of true negatives, i.e., actually disease-free specimens to be rejected in an automated manner. The use of a normal standard sample of cells in each daily run reduces the possibility that variables such as changes in ambient temperature, pH of the solutions used, or manual manipulation time may adversely affect the reliability of the results. It is believed that with further work, the process may be made to make even finer discriminations, thereby reducing the number of false positives.
While a preferred process has been described in detail, various modifications, alterations and changes may be made without departing from the spirit and scope of the present invention, as defined in the appended claims.
1. In a process for diagnosing specimens of cells for disease employing the process known per se of staining a specimen of cells with a radioactively tagged dye,
rinsing excess radioactive dye material from the specimen of cells,
sensing the level of radioactivity emitted by the specimen of cells, comparing the radioactivity emitted by the specimen of cells relative to a standard of comparison ob tained by having measured the level of radioactivity emitted by a similar kind of specimen of cells known to be normal, and rejecting from detailed diagnosis consideration of that specimen of cells if its level of radioactivity is less than a predetermined level of activity, whereby specimens are screened prior to diagnostic examination to reduce the number of specimens to be examined, the improvement comprising,
prior to staining, counting-out a predetermined quantity of cells from a specimen to be diagnosed,
adding a predetermined quantity of radioactive dye material to the predetermined quantity of cells in relation to the number of cells counted, and
performing at least the step or steps involving radioactive measurement with the predetermined quantity of cells removed from supporting or surrounding matter in which staining was accomplished so that any supporting or surrounding matter which might have been subject to excess stain has been removed.
2. A process according to claim 1 including performing the identical steps to be performed on the predetermined quantity of cells on a disease-free standard normal sample consisting of the same quantity of cells as the predetermined quantity, and which in fact determines the size of the predetermined quantity, to afford a standard of comparison and using the radiation level of said disease-free sample as an actual standard of direct comparison.
3. A process according to claim 2 wherein said performing step of counting, adding, rinsing, and sensing of said standard normal sample is repeated periodically with respect to corresponding steps on said specimen.
4. A process according to claim 2 wherein the level of radioactivity emitted by the predetermined quantity of cells defines a ratio with the corresponding level of the standard normal sample, said ratio being determined by dividing the level of radioactivity of the predetermined quantity of cells by the level of radioactivity of the standard normal sample, and said prescribed level in said rejecting step being a ratio of at least 1.4.
5. A process according to claim 1 including the step of homogenizing the predetermined quantity of cells before performing said counting step.
6. A process according to claim 5 including the step of removing extraneous cellular material which may include protein and nucleoprotein from said predetermined quantity of cells.
7. A process according to claim 6 wherein said step of removing extraneous cellular material includes the step of treating the predetermined quantity of cells with cell-dispersing agents including enzymes.
8. A process according to claim 1 wherein said radioactive sensing step includes the step of solubilizing said predetermined quantity of cells before sensing the radi oactivity.
9. A process according to claim 8 wherein a fluid capable of emitting photons is employed in said solubilizing step.
10. A process according to claim 1 including the steps of withdrawing a quantity of specimen-cells prior to said counting step and preserving the same for further consideration.
11. A process according to claim 10 wherein said predetermined quantity of cells is obtained from one of the human female reproductive organs, and including the steps of subsequently diagnostically examining the withdrawn predetermined quantity of cells if the level of radioactivity in said rejecting step is above said pre scribed level.
12. A process according to claim 11 wherein said female organs include cervices, uteri, and vaginae.
13. A process according to claim 12 wherein said radioactive dye material includes tritiated Pyronin B (H- Pyronin B).
14. A process according to claim 12 wherein said radioactive dye material includes tritiated methylene blue (H-methylene blue).
15. A process according to claim 11 wherein said predetermined quantity of cells is contained in a solution and said radioactive dye material employed in said adding step includes a selected one of H-Pyronin B and H-methylene blue each having a specific activity of 0.8 to 3.0 mc/mg with said selected dye being buffered at a pH in a range of about 5.0 to about 7.0 at a temperature in a range of about 20 C to about 37 C for about 5 to IO-minutes and having a concentration in a range of about l to about 3 mg/l00 ml of solution.
UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent 3,856,930 Dated December 24, 1974 Inventor(s) John H. Nodine; John Herbert Waite; Martin J. Fletcher It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:
Column 3, line 15, "pestels" should be --pestles;
Column 4, line 10, after "radioactive" insert --dye--;
line ll, delete "binding" and insert -uptake-;
line 12, delete "to" (first occurrence" and insert --by-;
gigned and Emalcd this twenty-third Day Of September 1975 [SEAL] Arrest:
RUTH C. MASON C. MARSHALL DANN resting Officer (nmmissimu'r vflarvnls and Trademarks UNITED STATES PATENT OFFICE CERTIFICATE OF CORRECTION Patent N I 3 D t d Inventor(s) John H. Nodine; John Herbert Waite; Martin J. Fletcher It is certified that error appears in the above-identified patent and that said Letters Patent are hereby corrected as shown below:
Column 3, line 15, "pestels" should be pestles;
Column 4, line 10, after "radioactive" insert -dye-;
line 11, delete "binding" and insert -uptake; line 12, delete "to" (first occurrence" and insert -by-;
Signed and Sealed this twenty-third D 3y Of September I 9 75 [SEAL] Arrest:
RUTH C. MASON (f. MARSHALL DANN Mresting ()l'j icer (mnmissiuncr ujlarcnls and Trademarks