DE102004033905A1 - Cytological investigation method, especially for gynecological screening, e.g. in early diagnosis of cervical carcinoma, by determining nuclear chromatin content, optionally followed by other tests - Google Patents

Abstract

A cytological investigation method comprising: (a) determining the nuclear chromatin content of cells originating from a cell sample by conventional methods; (b) comparing the result with a theoretical value and making an evaluation (negative, uncertain or positive); and (c) carrying further investigations with another part of the cell sample, at least in the case of negative or uncertain evaluations in step (b), is new.

Description

The The present invention relates to an improved combined method for cytological screening, especially in gynecological Vorsorgezytologie.

By Classical screening cytology for the early detection of cancer of the cervical cervix (method after Papanicolau), the incidence of invasive cervical cancer was evident be lowered. additionally for cancer screening is with the method also the proof of other pathological changes possible, e.g. the detection of hormonal disorders or from infections.

In spite of the achievements achieved by using this classical method However, it turned out that in some cases not very reliable is. When screening with the method of Papnicolau is a certain Percentage of pathological findings not detected. So be (dependent on by the respective author) when using the classical method for squamous cell carcinoma up to 50% and at adenocarcinoma up to 80% of carcinomas diagnosed (see opinion on the early detection of carcinomas of Cervix, endometrium, vulva and vagina; Working Group Cancer Screening the German Cancer Society e.V. and the German Cancer Aid e.V. on 18.10.2000).

at The method of Papanicolau is a manual or semi-automatic method, in which the sample preparation and evaluation by certain persons, with individual evaluation steps be automated (e.g., by automatic image processing). Ultimately, however the quality of the test result partly due to the routine and training of the Personals affected. So far, one has therefore tried the odd false negative rate Findings through better staff training and modifications individual examination steps during sampling and sample preparation to lower. All these measures but have not been able to prevent that in some of the samples the test results of a subjective component subject to the use of the test personnel.

It Also known are methods that allow cell populations to be present can be vorgescreent of tumor cells or their precursors. In the gynecological Prevention cystology is already the so-called flow cytophotometry (hereinafter referred to as FCM). This can be Determine the core chromosome content of isolated cells in suspension. With increasing degree of dysplasia, the deviation from normal increases Kernchromatinehalt, so that with This method is relatively reliable Tumor cells and / or their precursors can be detected. These Although method has its reliability in numerous investigations proved, but could not prevail in the routine until today.

• 1. bei ausschließlicher Anwendung der FCM keine relevanten Zusatzbefunde, wie Aussagen über Hormonstörungen oder Infektionen zu erheben sind, und dass
• 2. auch mit dieser Methode nicht immer eindeutige Befunde erhoben werden, die anderweitig abgeklärt werden müssen.

Some reasons are that

• 1. in case of exclusive use of the FCM, no additional relevant findings, such as statements on hormone disorders or infections are to be collected, and that
• 2. Even with this method not always clear findings are collected, which must be clarified otherwise.

at the implementation the classical method described above fell to Papanicolau on that the hit rate with the findings also by pre-information decisively influenced can be. One study showed that at the check-up of a collective of false negatives in only 5% of the Cases the correct positive diagnosis has been made, as far as no continuing Information templates. However, the inspectors had the information that in these patients a pathological histological finding were present in 90% of cases the pathological cells detected.

By a so-called Vorscreenen and the consideration of the obtained here Results, the diagnostic certainty can be increased.

aim The present invention is the provision of a combined Method of cytological examination, in particular of cervical carcinoma, in which the hit rate of the findings in comparison to the previously applied Method can be significantly increased.

One Another advantage of the present invention is the provision a method of cytological examination, in particular of the Portio-cervical carcinoma, which is much easier to perform and its implementation at significantly lower costs possible is considered the classic procedure. The invention is based on the knowledge that with the inventive method a safe selection of samples without findings is possible and that only at the unclear and critical samples carrying out a more elaborate second Tests needed becomes.

In addition, the present invention will provide a method with which in cytological examination for easy and kosteni A prescreening of a patient collective after high-risk patients is possible.

• a) Bestimmen des Kernchromatingehalts von Zellen einer aus einer Zellprobe stammenden Zellsuspension in an sich bekannter Weise,
• b) Vergleich des in Schritt a) ermittelten Kernchromatingehalts mit einem Sollwertbereich für den Kernchromatingehalt und Erstellen einer Befundung (negativ, zweifelhaft, positiv)
• c) Durchführung weiterführender Untersuchungen mit einem weiteren Teil der gleichen Zellprobe, zumindest im Falle der Befundung zweifelhaft oder positiv in Schritt b).

The present invention relates to methods for cytological examination, in particular for gynecological screening cytology, and comprises the following measures:

• a) Determining the Kernchromatinehalts of cells of a cell sample derived from a cell suspension in a conventional manner,
• b) Comparison of the core chromosome content determined in step a) with a target value range for the core chromosome content and creation of a report (negative, doubtful, positive)
• c) carrying out further examinations with a further part of the same cell sample, at least in the case of findings dubious or positive in step b).

at the invention used Cells are in most cases cell material, which as part of cancer screening in gynecological screening for cervical carcinoma of the cervix is won. However, the method is similar for all cytological Examinations applicable.

These Cells can taken from the patient in any known manner, for example by Abstrichtechnik, brushes, cotton swabs or Spatula. examples for Collection techniques are e.g. in H.F. Nauth. Gynecological cytodiagnosis; 2002 Georg Thieme Verlag Stuttgart described.

To Extraction, a portion of the recovered cell material is streaked on a microscope slide and conserved or fixed in known manner (e.g. Introduction into an approximately 80% alcoholic solution, by spray fixation), the alcoholic fixation is most preferred.

The remaining removed cells are in a conventional manner preserved or fixed in suspension. Examples of this are introduction the cells in alcoholic fixative and / or pretreatment of Cells after fixation with detergents or lipophilic substances, being a primary one Alcohol fixation is usually used.

The Determination of the Kernchromatinehalts in step a) can by all suitable for it Procedure done. Examples are the FCM or chromatin measurement on the smeared cell material. In the second case, the cells become on the slide specially dyed and the nuclear chromium content is measured manually or automatically.

Prefers the determination after step a) is carried out by flow cytophotometry. examples for this method is described in H.M. Shapiro; Practical Flow Cytometry, Fourth Edition, Wiley-Liss, Hoboken, New Jersey.

at The core chromosome content is analyzed using the core chromium content of healthy, normal, non-pathologically altered cells as a reference (value is referred to as 2c or euploid). Pathologically altered cells show deviations of the Kernchromatinehalts of this comparative size. Of the Kernchromatinehalt of these cells is larger than the normal cells (greater 2c). The cells become, as far as the Kernchromatinehalt not a straight multiple (not 4c or 8c) of 2c is designated as aneuploid. The presence of an aneuploid core chromium content, in particular greater than 4c, is an indication of the presence of pathologically altered Cells, further clarification require. The extent of Deviations of the nuclear chromosome content pathologically changed Cells of 2c are in different cell groups and tumor cell populations (e.g., cervical squamous epithelium, pleural mesothelial cells, adenocarcinoma cells the lungs) are pronounced to varying degrees, but essentially similar.

When further Investigations in step c), all methods are considered, the to a specification of the result obtained from step b) can serve.

there it can be classic dyeing and Methods of Analysis, Screen of the Classic Smear, Mono Layer Preparation, immunohistochemical methods, HPV determination, MIB1 mitotic rate p16 Protein and / or molecular pathology and molecular biology HPV methods of determination.

Preferably this is the secondary one Investigation in step c) of the method according to Papanicolau cancer screening Portio-cervical carcinoma or those described above Statement.

The inventive combination The examination methods are also useful when first a Determination of Zellhrromatinehalts done, as by the Asservierung From cell material follow-up examinations can be made.

The inventive combination of examination methods has a number of advantages:
With the better detection rate, the rate of false negative findings can be lowered.

In studies on early detection of cancer, the majority of the samples, about 90%, can be done by a simple determination of the core chromosome content. This can be in the Early cancer detection, especially in automated implementation of the method, achieve a significant cost reduction.

By the asservation of the cell material is also after performing Step a) in case of conspicuous Findings and / or at the request of the doctor or the patient still cell material (e.g., as a conventional swab or as a cell suspension) for the above listed secondary Examinations available. At continuing Question can be carried out immediately an additional examination. By using additional Investigations can further increase the diagnostic reliability. The patient does not have to for re-commissioned another specimen and examined it become. This more rational approach leads to a further and cost savings.

The The following example describes the process according to the invention without limiting this.

process Description

In a preserving fluid present cells, the u.a. won for gynecological cancer screening were used as cell suspension in a FCM (flow DNA cytophotometer (Flow CytoMetry) on the content of nuclear chromatin measured and analyzed.

The Analysis was carried out according to the scheme described above.

to Analysis became one in the device integrated program that uses as a result a so-called Histogram provided.

at pure screening for cancer or precancerous lesions after this examination step a judgment (negative, doubtful, positive).

revealed the determination of the content of Kemchromatin in a sample an inconspicuous Chromatinmuster, so pure cancer screening was the study at this point completed.

revealed the determination of the nuclear chromosome content in a sample is a conspicuous or dubious chromatin pattern, or was a continuing clinical Issue (e.g., inflammatory Change, Hormone status), a further examination of the residual material was carried out for clarification the still open questions.

The Further investigation was carried out on other cells of the already asserved Sample. As examination methods comb - depending on the respective Question - for example classic dyeing and analysis methods, immunohistochemical methods or even molecular-pathological and molecular biological methods into consideration.

To Existence of all test results became a final finding created.

Claims (6)

Method for cytological examination comprising the measures: a) Determining the nuclear chromosome content of cells from a cell sample originating cell suspension in a conventional manner, b) Comparison of the determined in step a) Kernchromatinehalts with a setpoint range for the Kernchromatinehalt and creating a report (negative, doubtful, positive) c) implementation further leading Investigations with another part of the cell sample, at least in the case of finding doubtful or positive in step b). Method according to claim 1, characterized in that determining the nuclear chromosome content by flow cytophotometry he follows. Method according to claim 1, characterized in that that as a continuing Examination of a manual and / or mechanical evaluation of the Cytological preparations prepared from the removed cells are carried out. Method according to claim 3, characterized that as a continuing Investigation of early cancer detection of cervical carcinoma after Papanicolau. Method according to claim 1, characterized in that that as a continuing Examination of a classical dyeing and analysis method, a screen of classical smear, one Mono layer preparation, immunohistochemical methods, HPV determination, MIB1 mitotic rate p16 Protein and / or molecular pathological and molecular biology HPV Determination methods are used. Method according to claim 1, characterized in that that the cytological examination is a gynecological examination Prevention Cytology is.