RU2264608C2 - Method of treating operational biopsy and sectional material for microscopic examination - Google Patents

Abstract

FIELD: pathohistology.

SUBSTANCE: samples are placed onto thread-like substrate with 0.5-cm intervals to form a bunch 10-12 cm long, which is loosely placed in container. The latter is successively passed through a series of vessels containing corresponding solutions, while being rotated around its axis to favor uniform treatment of material. Time and conditions are set in accordance with selected procedure. Treatment is commenced by dipping material into formalin at 34-37°C for 2 h followed by washing with running water during 4 h. Material is then dehydrated with 96% alcohol by way of passing container through five vessels: in the first one material is dehydrated for 5 h, in the following three for 3 h in each, and in the fifth vessel for 5 h. Tannage is carried out in alcohol-xylene solution for 1 h and then in xylene for 1 h, after which material is flooded with paraffin at 36°C for 1 h and at 56°C also for 1 h. Finally material is cut into block, which are pasted on wooden supports, cut on microtome, cuts are stained and subjected to microscopic examination.

EFFECT: reduced treatment time.

4 tbl

Description

The invention relates to medicine, namely to histopathology, in particular to the preparation of tissue samples for microscopic examination.

The micropreparation processing system has been around for almost 100 years with virtually no changes. The appearance of automatic processing, although it facilitated the work of laboratory assistants and significantly reduced the number of errors associated with the human factor (inattention, forgetfulness), but did not make significant changes to the technology and did not reduce the time for preparing the material for microscopy.

Various methods are used for processing surgical biopsy material, including those that allow obtaining results in a shorter time.

There is a fast method for processing surgical biopsy and sectional material with the filling of small pieces into celloidin. Celloidin is the best impregnating medium; it deforms tissues least of all. But just staying in celloidin and compaction takes 5-8 days. There is a shortened method of pouring into celloidin, but this method can be applied only to small pieces of tissue measuring 0.2 × 0.2 cm (V.G. Eliseeva, M.Ya. Subbotina, Yu.A. Afanasyeva, E.F. Kotovsky, Fundamentals of Histology and Histological Technique, Moscow: Medicine, 1967, pp. 143-144).

Stage of the method:

- cutting with a razor small pieces up to 2 mm,

- fixation (and dehydration at the same time) in two portions of 96% alcohol for 3 hours in each serving,

- pouring in 10% celloidin for 12 hours,

- gluing on wooden blocks,

- air drying,

- compaction in chloroform 30-60 minutes,

- cutting with a microtome and staining of sections for research.

The disadvantages of the method:

- celloidin is not produced in Russia and is a rare and expensive drug (earlier, old x-ray films were used in laboratories to obtain it, now it has become impossible, because x-ray films are not celloidine, but acetate);

- the celloidin blocks are softer than paraffin blocks, so the sections are not so thin and strong (the minimum thickness of the sections is 15 microns - 2 times thicker than with conventional pouring);

- the use for the study of only small pieces of tissue and the inability to use the method for a more informative microscopic examination of large pieces.

There is a method of quickly pouring small pieces into paraffin (G.A. Merkulov, a course of pathological histological technology, edition 4, Leningrad, Medgiz, 1961, p. 50).

This method makes it possible to reduce the entire process of processing the material to 10 hours due to the use of paraffin and chloroform at a temperature of 35-40 ° C. This method, as well as the method of pouring into celloidin, is used only for processing small pieces of tissue.

Stage of the method:

- cutting with a razor small pieces up to 2 mm,

- fixation (and dehydration at the same time) in two portions of 96% alcohol for 3 hours in each serving,

- placing the pieces in chloroform for 1-2 hours at a temperature of 35-40 ° C,

- placing the pieces in chloroform with paraffin for 30 minutes - 1 hour,

- pouring in 1 portion of paraffin at a temperature of 54-55 ° C for 1 hour and in a second portion of the same temperature for 30 minutes - 1 hour,

- cooling, cutting blocks, sticking to pads,

- cutting with a microtome and staining of sections for research.

The disadvantages of the method:

- time gain leads to a loss in completeness and reliability of the diagnosis due to the small size of the pieces;

- the negative impact of chloroform on the health of laboratory personnel.

Closest to the claimed one is the classical method of pouring material into paraffin (G.A. Merkulov, Pathological Histological Technique Course, vol. 4, Leningrad, Medgiz, 1961, p. 49). This method consists of the following steps.

1. Preparation for processing. The material for histological examination can be of two types: biopsy (intravital, from the organs and tissues of patients) and sectional (from the organs and tissues of the dead). Pieces of tissue are taken for examination from 1 × 1 cm to 0.2 × 0.2 cm in size. The material is processed using the AT-4 apparatus (a universal machine for histological tissue processing, manufactured by the Zhdanov Plant of Technological Equipment). Pieces of tissue 1 × 1 cm in size are placed in lattices of 5 pieces each and placed in containers containing no more than 10 lattices (total 50 pieces for one wiring in the AT-4 apparatus). The container sequentially passes through a series of vessels containing the corresponding liquids, while it rotates around an axis, which contributes to a more uniform processing of the material. Time and temperature conditions are set before starting the machine in accordance with the selected method by means of a control mechanism. During the diagnosis, the number of pieces of biopsy material should not exceed 3, and for surgical material - 10, on average 30% of the biopsy material and 70% of the surgical material are usually processed per transaction, that is, on average, material from 11 patients is examined.

2. Immersion of tissue pieces in 20% formalin at a temperature of 18 ° C for long-term preservation of the shape of the tissue or organ without reducing the volume - 24 hours.

3. Rinsing with running water to wash formalin - 8 hours.

4. Dehydration of waterlogged pieces in a series of ascending alcohols (concentration from 50 ° C to 100 ° C).

5. Tanning of the investigated material. Due to the fact that after alcohol the pieces become brittle, xylene is tanned to give them elasticity. For a smoother effect, alcohol-xylene is first used, and then pure xylene.

6. Seal. An elastic piece cannot be finely cut on a microtome, so it must be sealed by passing through paraffin baths.

7. Gluing onto pads, cutting on a microtome and coloring of preparations.

The disadvantages of the method:

- the small capacity of the AG-4 apparatus is limited by the volume and capacity of the container, with a large workload of the laboratory, an additional purchase of automatic machines is required;

- heating formalin only to room temperature 18 ° C reduces the intensity and completeness of tissue impregnation;

- additional fixation in formalin alcohol lengthens the study, although the quality is not significantly affected;

- a long stay of tissues in formalin requires the same long washing of tissues in running water, as there remains a fixation artifact - a formalin pigment that interferes with diagnosis;

- dehydration in a series of alcohols and pouring in paraffin occur with a great investment of time;

- the main disadvantage of this method is the duration of the processing of the material.

The disadvantages of the technology lead to a lower result in duration and productivity:

- often patients have to expect a test result of at least 7 days (the treatment itself takes 5 days and the doctor’s morphological study 2 days);

- a long diagnostic process can lead to a later start of differentiated therapy;

- waiting not only delays the start of treatment, but also increases the patient’s stay in the surgical department;

- uncertainty and a state of expectation - severe emotional stress for patients and their relatives.

The objective of the invention is to reduce the processing time of surgical biopsy and sectional material for microscopic examination without loss of quality of the samples and accelerate the morphological diagnosis.

This object is achieved in that the samples for processing are placed on a carrier in the form of a thread, while the samples are placed on a thread with an interval of 0.5 cm with the formation of a garland 10-12 cm long, which is freely placed in a container. The container sequentially passes through a series of vessels containing appropriate solutions, while it rotates around an axis, which contributes to a more uniform processing of the material. Time and temperature conditions are set before starting the machine in accordance with the selected method by means of a control mechanism. Processing begins by immersion in formalin at a temperature of 34-37 ° C for 2 hours, then washed with running water for 4 hours. Dehydrated with alcohol at a concentration of 96%, sequentially holding the container in five containers: in the first container, dehydrate for five hours, then in three containers for 3 hours in each. In the fifth tank, dehydrate for five hours. Tanning is carried out in an alcohol-xylene solution for 1 hour and in xylene for 1 hour, pour paraffin at a temperature of 36 ° C for 1 hour and at a temperature of 56 ° C for 1 hour. Then blocks are cut out, glued onto wooden blocks, sections are made on a microtome, stained and sent for microscopic examination.

 Novelty of invention

1. Samples of tissue for processing are placed on a carrier in the form of a thread, while the samples are strung on a thread with an interval of 0.5 cm, forming garlands 10-12 cm long, which are freely placed in the container.

2. Formalin treatment is carried out at a temperature of 34-37 ° C for 2 hours, because this temperature corresponds to body temperature and is physiological for human tissues (protein coagulation occurs at a higher temperature - 40-41 ° C).

3. Rinsing with running water for 4 hours, because this period is enough to remove formalin from samples that were processed in the first stage in a shorter time - 2 hours.

4. Dehydrate with alcohol with a concentration of 96% sequentially in five containers: in the first container for five hours, then in three containers for 3 hours in each and in the fifth container - 5 hours. Such a concentration of alcohols gives high-quality dehydration with the least time.

5. Tanning in an alcohol-xylene solution for 1 hour and in xylene for 1 hour, which allows to obtain a good tissue densification.

6. Pour with paraffin at a temperature of 36 ° C for 1 hour and at a temperature of 56 ° C for 1 hour, which reduces the processing time and allows you to get high-quality sections with a thickness of 8 microns.

The set of essential features of the invention allows to obtain a new technical result.

1. Reducing the processing time of surgical biopsy and sectional material to obtain samples without compromising their quality.

2. Earlier issuance of samples for morphological analysis, due to which the patient’s hospital stay is reduced.

3. Earlier initiation of differentiated treatment.

Over the course of 1.5 years, we conducted a series of exploratory studies in which the duration, temperature conditions, concentration of solutions, and the number of pieces of tissue to be processed varied. In this way, possible errors were directionally modeled, characteristic artifacts were studied for specific violations of the technology. In this case, the quality of the preparations obtained was carefully analyzed. In preliminary experiments, about 10 thousand micropreparations were studied, as a result of which such processing times were selected that were necessary and sufficient to maintain the quality of the preparations.

The method is as follows.

Small pieces of tissue 0.2 × 0.2 cm are placed in solutions in gauze bags. The bags are fixed to the threads. The study of large pieces makes it possible to more accurately diagnose, for processing, samples are placed on a carrier in the form of a thread, while samples and bags with samples are placed at intervals of 0.5 cm with the formation of a garland 10-12 cm long, which is freely placed in the container. The thread is held in the center of the sample.

Posting to solutions and staining is performed using the AT-4 apparatus (a universal machine for histological processing and dyeing of tissues). The container with pieces of tissue placed on the thread, sequentially passes through a series of vessels containing the appropriate solutions, while it rotates around the axis, which contributes to a more uniform processing of the material.

Formalin treatment is carried out at a temperature of 34-37 ° C for 2 hours, then washed with running water for 4 hours. Dehydrated with alcohol at a concentration of 96% successively in five containers: in the first tank, dehydrated with alcohol at a concentration of 96% for five hours, then in three containers of 3 hours each, in the fifth container - 5 hours. Tanning in an alcohol-xylene solution for 1 hour and in xylene for 1 hour, pour paraffin at a temperature of 36 ° C for 1 hour and at a temperature of 56 ° C for 1 hour. Cool, cut out blocks, place them on wooden blocks, make sections on a microtome, stain them for microscopic histological examination.

Detailed data on the differences of the prototype (classical method) and the proposed method are given in table 1.

Table 1
Comparison of methods for processing surgical biopsy and sectional material No. Prototype The proposed method Kolich. pieces Time (hour) Conditions Kolich. pieces Time (hour) 1. The volume of processed material fifty 225 2. Formalin 20% at a temperature of 18 ° C 24 At a temperature of 34-37 ° C 2 3. Formalin alcohol 24 - 4. Rinsing in running water 8 4 4. Alcohol 86% 24 - 5. Alcohol 96% 24 5 6. Alcohol 96% - 3 7. Alcohol 96% - 3 8. Alcohol 96% - 3 9. Alcohol 96% - 5 10. Xylene alcohol 3 1 eleven. Xylene 6 1 12. Paraffin xylene 3 1 thirteen. Paraffin 36 ° С 2,5 1 14. Paraffin 56 ° С 1,5 1 Total 50 pieces (11 patients) 120 hours 225 pieces (50 patients) 30 hours

As can be seen from table 1, the material processing step on the AT-4 apparatus is reduced by 4 times, and the amount of processed material per load at the same time increases by almost 5 times, that is, in general, productivity increases by 20 times.

Since the throughput of the apparatus is significantly increased, in order to maintain good quality of the samples with such an increase in the amount of the studied material, there is a need for a more frequent change of solutions: once every 3 days instead of 1 time per week according to the prototype method (classical method).

table 2
The dependence of the quality of drugs on various temperature conditions of fixation Processing stage Drug quality Fixation in 20% formalin at different temperatures 18-20 ° C 25-28 ° C 35-37 ° C 38-41 ° C Good in 24 hours Good in 10 hours Good after 2 hours Samples are unsatisfactory (sharp edge compaction of the pieces without a sufficient study of the middle) Additional fixation in formalin alcohol Not significantly affected Not significantly affected Not significantly affected Not significantly affected Table 3
The dependence of the quality of the preparations on the duration of washing in running water Rinsing in running water 1 hour 2 hours 4 hours 8 ocloc'k Poor (formalin pigment left) Poor (formalin pigment left) Good Good

We conducted a series of experiments on the dehydration of drugs under different conditions: different alcohol concentrations and different exposure times. Experiments have shown that dehydration in 86% alcohol allows one to obtain high-quality drugs only after exposure for at least 24 hours, followed by treatment with 96% alcohol for 48 hours and xylene alcohol for 3 hours, as the classical method recommends. If you start dehydration with a more concentrated 96% alcohol, you can significantly reduce the time of dehydration while maintaining good quality drugs

Table 4
The dependence of the quality of drugs on the time of pouring in paraffin Conditions Drug quality Prototype Experiment The proposed method Xylene 6 o'clock 30 minutes 1 hour Good Unsatisfactory. The pieces are pulled apart because they are not densely packed. Good Paraffin xylene 3 hours 30 minutes 1 hour Good Unsatisfactory. The periphery is sealed, the center is not sealed. Good Paraffin 36 ° C 2.5 hours 30 minutes 1 hour Good Unsatisfactory Good Paraffin 56 ° С 1,5 hour 30 minutes 1 hour Good Unsatisfactory. The center of the piece does not have time to soak in paraffin Good

When processing biopsy material was inaccurate, in particular, fixation in 20% formalin for 2 hours at a temperature of 40-41 ° C led to a sharp edge compaction of the pieces without sufficient study of the middle. After pouring into paraffin, the center of the block was soft, loose and fibrous, like cotton wool. When slicing on a microtome, slices were obtained of uneven thickness, torn. Dyes were perceived unevenly by the tissues, which made the histological picture fuzzy.

With insufficient washing (less than 2 hours) after fixation, brown lumps appeared in micropreparations - a formalin pigment, which could imitate an old hemorrhage or the presence of a pigment tumor. Therefore, the development of the proposed method was done very carefully on a large number of tissue samples.

EXAMPLE.

In one day from medical institutions delivered to the study:

1. Scrapes from the uterine cavity from 11 patients, each of which was divided into 5 blocks (55 pieces in total).

2. 10 gastrobiopsies on average, 3 pieces for each (a total of 30 pieces).

3. 3 removed gallbladders, 5 pieces each (total 15 pieces).

4. 2 vermiform processes of 5 pieces each (total 10 pieces)

5. 3 facial papillomas, 4 pieces each (12 pieces in total)

6. 5 cases of sectional material after the autopsy of the deceased, 20 blocks each (100 pieces)

A total of 222 pieces of tissue from 29 patients and 5 dead.

Before fixation, pieces of not more than 1 × 1 × 0.5 cm were cut from surgical, biopsy and sectional material and strung on cotton threads 10-12 cm long with an interval of 0.5 cm, but not more than 20-24 pieces per garland, which is dictated by the height of the glasses with the solution. Scrapes and slices of gastrobiopsies of 0.2 × 0.2 cm in size are tied in gauze bags and also hemmed with garlands. There are 12 such garlands; they are freely placed in a container and immersed in glasses, in which they are freely washed by solutions.

Fixation was carried out with 20% formalin at a temperature of 36 ° C for 2 hours. Washed with running water for 4 hours. After that, the pieces had the texture of dense rubber both along the edges and in the center.

For dehydration, containers with the material for processing were carried out through 5 containers with alcohol at a concentration of 96% (in the first tank for 5 hours, in the second, third and fourth tanks for 3 hours, in the fifth - 5 hours). Good dehydration in alcohols allowed to reduce the time of soaking the pieces with xylene, paraffin-xylene and paraffin at a temperature of 36 ° C and 56 ° C up to 1 hour. After paraffin impregnation, the pieces when piercing with a thin needle had a dense consistency both at the edges and in the center, which indicates the uniformity of the compaction process. Then the pieces were not glued on wooden cubes, respectively labeled and began to be called blocks. When cutting blocks on a microtome, the slices were translucent (thickness not more than 10 microns), the microtomes were twisted with chips on the knife, and when trying to straighten them on a glass slide, they did not tear, indicating a sufficient piece density combined with plasticity. When staining sections, alkaline dyes stained only the nuclei, and acidic stains - all other tissue structures, staining was uniform and contrast, which made it possible to conduct a qualitative study during light microscopy.

Ready-made preparations for diagnosis were received by the doctor after 2 days. The histopathological report was delivered to medical institutions for 3-4 days, and in rare cases, the need for additional stains or consultations to clarify the diagnosis - on 5-6 days.

Such reduced terms for establishing a final diagnosis led to an earlier start of treatment.

1. Of the 11 patients in the gynecological department (examination of scrapings from the uterine cavity), malignant tumors were detected in two cases, which allowed women to be sent to the oncology dispensary in a short time and to begin treatment earlier. It is known that the expectation of a scraping result is always accompanied by a high level of anxiety, for 9 patients this period was significantly reduced.

2. In the study of 10 gastrobiopsies, one patient was also referred to the oncology dispensary, and in 9 earlier specific treatment was started, which led to a reduction in the hospital stay of patients.

3. In the study of 3 gallbladders and 2 appendix processes, inflammation was confirmed, which determined the management tactics in the postoperative period.

4. In the study of tumor processes on the face 1 was defined as malignant, the patient was immediately sent to an oncology dispensary, 2 as malformations of the skin that do not require treatment after removal.

Thus, when applying the proposed method for processing surgical biopsy and sectional material, 29 patients received diagnostic pathological assistance in one cycle, 4 of them were sent to the oncology dispensary for specific therapy, and 25 targeted treatment was started on average 4 days earlier than with the classical method material processing, which could give a possible savings of 120 bed days (25 × 4 = 120).

Reducing the timing of execution of autopsy protocols from 14 to 7 days is important for the timely submission of statistical information on mortality to health facilities at the end of the month, quarter, and especially the year.

The proposed method is used in the Novokuznetsk Cancer Dispensary, in the State Medical and Prophylactic Institution "Municipal Pathological Bureau with a Histological and Cytological Laboratory", in the Pathological Department of the hospital No. 1 of Osinniki, Kemerovo Region, in the histological laboratory of the 1st maternity hospital in St. Petersburg.

Thus, the proposed method allows, without compromising the quality of histological preparations and any additional costs, to reduce the processing time of surgical biopsy and sectional material, accelerate morphological diagnosis, reduce the patient’s stay in the hospital, reduce the period of emotional stress for the patient, begin differentiated treatment earlier.

Claims (1)

A method for processing surgical biopsy and sectional material for microscopic examination, including processing samples with various solutions: formalin when heated, washing with water, dehydration with alcohol, tanning in xylene, pouring paraffin, cooling and cutting blocks, gluing them onto blocks, making slices on a microtome and staining the drug, while the samples are placed on a carrier, loaded into a container, sequentially moved through containers with various solutions, characterized in that the processing of forms they carry out aline at a temperature of 34-37 ° C for 2 hours, washed with running water for 4 hours, dehydrated with alcohol with a concentration of 96% in series in five containers: in the first container for 5 hours, then in three containers of 3 hours in each and in the fifth container 5 hours, tanning in an alcohol-xylene solution for 1 hour and in xylene for 1 hour, pour paraffin at 36 ° С for 1 hour and at 56 ° С for 1 hour, processing samples are placed on a carrier in the form of a thread with an interval of 0 5 cm with the formation of a garland 10-12 cm long, which is freely placed in a container.