US3856930A - Method of screening tissue specimens for diagnostic examination - Google Patents

Method of screening tissue specimens for diagnostic examination Download PDF

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Publication number
US3856930A
US3856930A US00281168A US28116872A US3856930A US 3856930 A US3856930 A US 3856930A US 00281168 A US00281168 A US 00281168A US 28116872 A US28116872 A US 28116872A US 3856930 A US3856930 A US 3856930A
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United States
Prior art keywords
cells
predetermined quantity
level
specimen
process according
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US00281168A
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English (en)
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J Nodine
M Fletcher
J Waite
H Waite
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Bio Digital Sciences Inc
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Bio Digital Sciences Inc
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Priority to US00281168A priority Critical patent/US3856930A/en
Priority to CA178,297A priority patent/CA1011468A/en
Priority to GB3817673A priority patent/GB1447060A/en
Priority to IL42967A priority patent/IL42967A/en
Priority to DE2341061A priority patent/DE2341061C3/de
Priority to FR7329766A priority patent/FR2196719A5/fr
Priority to BR6249/73A priority patent/BR7306249D0/pt
Priority to JP48091643A priority patent/JPS4965891A/ja
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Publication of US3856930A publication Critical patent/US3856930A/en
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/60—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances involving radioactive labelled substances
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B07—SEPARATING SOLIDS FROM SOLIDS; SORTING
    • B07C—POSTAL SORTING; SORTING INDIVIDUAL ARTICLES, OR BULK MATERIAL FIT TO BE SORTED PIECE-MEAL, e.g. BY PICKING
    • B07C5/00—Sorting according to a characteristic or feature of the articles or material being sorted, e.g. by control effected by devices which detect or measure such characteristic or feature; Sorting by manually actuated devices, e.g. switches
    • B07C5/34—Sorting according to other particular properties
    • B07C5/346—Sorting according to other particular properties according to radioactive properties

Definitions

  • 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.
  • 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.
  • the present invention provides a unique method by which the efficiency of the procedure for examining tissue specimens suspected of disease is increased.
  • 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.
  • 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.
  • a process which is capable of being performed substantially entirely in automatic apparatus for screening large quantities of tissue specimens to minimize the number of specimens which must be subjected to a detailed diagnostic examination.
  • 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.
  • tissue specimens which are obtained exfoliatively from human female reproductive organs such as the cervix, uterus or vagina.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a predetermined quantity of cells is counted and removed from the mass of cells composing the specimen.
  • conventional optical or electrical cell'counting apparatus is employed.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • an appropriate pH 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.
  • 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.
  • 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.
  • radioactive dye material which is separated in the centrifuge is retained so as to avoid problems in the disposal of radioactive waste.
  • 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
  • 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.
  • the specimen may be passed through a liquid flow-through scintillation counter.
  • 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.
  • 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.
  • 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.
  • 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 No.4s and No.5s IS a. above 1.4 18
  • 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.
  • 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.
  • a process according to claim 1 including the step of homogenizing the predetermined quantity of cells before performing said counting step.
  • 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.
  • step of removing extraneous cellular material includes the step of treating the predetermined quantity of cells with cell-dispersing agents including enzymes.
  • said radioactive sensing step includes the step of solubilizing said predetermined quantity of cells before sensing the radi oactivity.
  • 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.
  • 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.
  • a process according to claim 11 wherein said female organs include cervices, uteri, and vaginae.
  • radioactive dye material includes tritiated Pyronin B (H- Pyronin B).
  • radioactive dye material includes tritiated methylene blue (H-methylene blue).
  • 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.

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US00281168A 1972-08-16 1972-08-16 Method of screening tissue specimens for diagnostic examination Expired - Lifetime US3856930A (en)

Priority Applications (8)

Application Number Priority Date Filing Date Title
US00281168A US3856930A (en) 1972-08-16 1972-08-16 Method of screening tissue specimens for diagnostic examination
CA178,297A CA1011468A (en) 1972-08-16 1973-08-08 Method of screening tissue specimens for diagnostic examination
IL42967A IL42967A (en) 1972-08-16 1973-08-13 A process for diagnosing specimens of cells for disease
GB3817673A GB1447060A (en) 1972-08-16 1973-08-13 Method of screening tissue specimens for diagnostic examination
DE2341061A DE2341061C3 (de) 1972-08-16 1973-08-14 Verfahren zur laufenden Klassierung von krankheitsverdachtigen Zellproben für Diagnostikprüfungen
FR7329766A FR2196719A5 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html) 1972-08-16 1973-08-14
BR6249/73A BR7306249D0 (pt) 1972-08-16 1973-08-14 Aperfeicoamento em processo para diagnosticar doencas em especimes de celula
JP48091643A JPS4965891A (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html) 1972-08-16 1973-08-15

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JP (1) JPS4965891A (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
BR (1) BR7306249D0 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
CA (1) CA1011468A (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
DE (1) DE2341061C3 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
FR (1) FR2196719A5 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
GB (1) GB1447060A (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)
IL (1) IL42967A (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3955559A (en) * 1974-08-13 1976-05-11 Velta Mikelevna Bramberga Method of cytological diagnosis of precancer conditions and cancer
US3957034A (en) * 1974-08-13 1976-05-18 Velta Mikelevna Bramberga Method of cytological diagnostication of precancer and cancer
US4459356A (en) * 1982-02-11 1984-07-10 Georgetown University Radioactive staining of gels to identify proteins
CN113732008A (zh) * 2021-08-13 2021-12-03 兰州大学第一医院 一种临床检验后废弃血标本再利用方法

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3673410A (en) * 1969-06-25 1972-06-27 John H Waite Method of examination of cell samples using a radioactively tagged dye
US3678148A (en) * 1969-06-26 1972-07-18 Information Utilization Corp Radioactive slide specimen analysis of and method of preparation

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3673410A (en) * 1969-06-25 1972-06-27 John H Waite Method of examination of cell samples using a radioactively tagged dye
US3678148A (en) * 1969-06-26 1972-07-18 Information Utilization Corp Radioactive slide specimen analysis of and method of preparation

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3955559A (en) * 1974-08-13 1976-05-11 Velta Mikelevna Bramberga Method of cytological diagnosis of precancer conditions and cancer
US3957034A (en) * 1974-08-13 1976-05-18 Velta Mikelevna Bramberga Method of cytological diagnostication of precancer and cancer
US4459356A (en) * 1982-02-11 1984-07-10 Georgetown University Radioactive staining of gels to identify proteins
CN113732008A (zh) * 2021-08-13 2021-12-03 兰州大学第一医院 一种临床检验后废弃血标本再利用方法

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CA1011468A (en) 1977-05-31
DE2341061B2 (de) 1979-06-07
GB1447060A (en) 1976-08-25
IL42967A0 (en) 1973-11-28
JPS4965891A (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html) 1974-06-26
DE2341061C3 (de) 1980-02-14
FR2196719A5 (GUID-C5D7CC26-194C-43D0-91A1-9AE8C70A9BFF.html) 1974-03-15
BR7306249D0 (pt) 1974-08-29
DE2341061A1 (de) 1974-03-07
IL42967A (en) 1976-02-29

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