CN111187807A

CN111187807A - In-situ hybridization dripping method

Info

Publication number: CN111187807A
Application number: CN201911424242.2A
Authority: CN
Inventors: 李珂; 高东艳; 王晓燕; 边巴央宗
Original assignee: SHANGHAI KINGMED MEDICAL DIAGNOSTICS INSTITUTE CO LTD
Current assignee: SHANGHAI KINGMED MEDICAL DIAGNOSTICS INSTITUTE CO LTD
Priority date: 2019-12-31
Filing date: 2019-12-31
Publication date: 2020-05-22

Abstract

The invention relates to an in-situ hybridization dripping method, which comprises the following steps: a) providing a carrier for bearing cells and a cell suspension sample mother solution to be dripped; b) dropping a first drop of cell suspension sample on the carrier c) dropping a next drop of cell suspension sample at the dropping position of the first drop of cell suspension sample after the first drop of cell suspension sample is basically spread uniformly on the carrier and the solvent of the first drop of cell suspension sample is basically volatilized completely; the operation is carried out until the dropwise addition of the nth cell suspension sample is completed, wherein n is an integer more than or equal to 2; wherein the dropping modes from the first drop of cell suspension sample to the nth drop of cell suspension sample are as follows: inserting a liquid suction nozzle of a liquid transfer device with a control button in a non-pressed state into the liquid level of the cell suspension sample mother liquid, dipping 0.5-2 microliter of sample, and discharging the sample onto the carrier.

Description

In-situ hybridization dripping method

Technical Field

The invention relates to the technical field of biological experiments, in particular to an in-situ hybridization dripping method.

Background

At present, when ISH samples of cells are prepared in an In Situ Hybridization (ISH) laboratory, a pipette-based dropping method and a common sample-adding gun dropping method are mainly used as dropping sheets. The pipette and slide method requires more cell suspension, after the cell suspension is blown and uniformly mixed, a 3ml pipette is used for sucking the suspension with a certain volume, one drop (about 50 mu l) or more drops are dropped on the anti-drop glass (1 item is detected), and the cell suspension is uniformly diffused and dried; the common sample adding gun dropping method is that a sample (more than or equal to 200 mu l) with less suspension is blown and uniformly mixed by a 200 mu l sample adding gun, a certain volume of suspension (about 50 mu l) is sucked and dropped on an anti-drop glass (about 10 mu l) for multiple times (1 item is detected), and the sample is uniformly diffused and dried.

There are commercially available multi-well slides, containing 10 or 12 individual wells, which are routinely used for the visualization of small fixed cell samples. Compared with the common standard microscope slide, the cell is limited in a relatively small surface area, so that the cell density is more suitable for FISH observation, and the probe consumption is saved due to the reduction of the cell number.

However, for some samples with very low abundance, such as plasma cell samples, after centrifugal enrichment, cell precipitates are invisible to naked eyes, and usually, only 1 drop of fixing solution (about 50 μ l) is added to prepare a suspension for a slide making pipette and a slide dropping method, so that more cell suspensions are required, cells can be distributed in the whole anti-drop slide, but the diffusion is not uniform, and the phenomena of few middle cells and cell aggregation at the periphery are easy to occur; the phenomenon of uneven distribution can be improved by a common sample adding gun dropping method because the volume of the absorbed suspension is small and easy to diffuse, and the volume of the adjustable liquid is covered in the same circle for many times. However, for a sample with too small amount of cells, a concentrated dropping piece with a smaller volume and a small amount of times needs to be selected. The two methods are not suitable for a sample with a small cell amount, the cell distribution is easy to be scattered, no signal exists in part of cell nucleuses in subsequent hybridization, and the countable number of cell nucleuses is less than 100, so that a report cannot be provided. In addition, the dispersion of cells also causes difficulty in reading the film by a technician, firstly, the effective cell nucleus count is less than 100, and the whole film is often moved to the visual field one by one to find the cell nucleus; and secondly, hybridization signals are regional, have different signal strengths and modes, and are generally required to count more cell nuclei for comprehensive interpretation.

In view of the above, the present invention is particularly proposed.

Disclosure of Invention

The invention relates to an in-situ hybridization dripping method, which aims at solving the problems that the pipette dripping method and the common sample adding gun dripping method are generally used for preparing samples rich in cells, and the two methods have no high requirement on the volume of the sucked suspension, the positions of the dripping positions cannot be completely consistent, the nucleus distribution is easy to be excessively dispersed, and the cell suspension dosage is large.

The method comprises the following steps:

a) providing a carrier for bearing cells and a cell suspension sample mother solution to be dripped;

b) dropping a first drop of the cell suspension sample onto the carrier;

c) after the first drop of cell suspension sample is basically spread uniformly on the carrier and the solvent is basically volatilized completely, dripping the next drop of cell suspension sample at the dripping position of the first drop of cell suspension sample; the operation is carried out until the dropwise addition of the nth cell suspension sample is completed, wherein n is an integer more than or equal to 2;

wherein the dropping modes from the first drop of cell suspension sample to the nth drop of cell suspension sample are as follows: inserting a liquid suction nozzle of a liquid transfer device with a control button in a non-pressed state into the liquid level of the cell suspension sample mother liquid, dipping 0.5-2 microliter of sample, and discharging the sample onto the carrier.

According to the invention, the liquid suction nozzle is used for dipping a trace sample and dripping the trace sample for multiple times, so that cells in a cell suspension sample can be enriched on the carrier, observation and statistics of the cells during subsequent slide reading are facilitated, the using amounts of reagents such as probes and confining liquid can be saved, the loss of the sample can be reduced to the greatest extent, and the detection efficiency and the detection rate can be effectively improved by a small amount of samples.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, and it is obvious that the drawings in the following description are some embodiments of the present invention, and other drawings can be obtained by those skilled in the art without creative efforts.

FIG. 1 is a schematic plan view of a hybridization apparatus according to an embodiment of the present invention, in which black dots are marks for indicating positions of labeled droppings on a table of the hybridization apparatus;

FIG. 2 is a schematic view of a hybridization apparatus according to an embodiment of the present invention after a slide is placed on the plane of the hybridization apparatus;

FIG. 3 is a schematic illustration of a 15ml centrifuge tube and a sample dipped therefrom as used in one embodiment of the present invention;

FIG. 4 is a schematic view of a slide after dropping a sample thereon in one embodiment of the present invention;

FIG. 5 is a schematic illustration of the distribution of nuclei in a slide dropped by the modified dispensing gun;

FIG. 6 is a schematic diagram showing the distribution of nuclei in a slide dropped by a pipette-dropping method or a common sample-adding gun-dropping method in the prior art.

Detailed Description

Reference will now be made in detail to embodiments of the invention, one or more examples of which are described below. Each example is provided by way of explanation, not limitation, of the invention. In fact, it will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing from the scope or spirit of the invention. For instance, features illustrated or described as part of one embodiment, can be used on another embodiment to yield a still further embodiment.

It is therefore intended that the present invention cover the modifications and variations of this invention provided they come within the scope of the appended claims and their equivalents. Other objects, features and aspects of the present invention are disclosed in or are apparent from the following detailed description. It is to be understood by one of ordinary skill in the art that the present discussion is a description of exemplary embodiments only, and is not intended as limiting the broader aspects of the present invention.

The invention relates to an in-situ hybridization dripping method, which comprises the following steps:

b) dropping a first drop of the cell suspension sample onto the carrier;

Here, the "control button is in a non-pressed state" means that the pipette is not in a liquid suction state (usually, first-stage pressing) or a liquid discharge state (usually, second-stage pressing).

Since the cell suspension sample is prepared by adding a redissolved solvent to centrifuged cells, and the preparation process usually requires blowing the solution to mix uniformly, the pipette and pipette tip used for preparing the solution are preferably the same as those used for dripping, and the pipette tip is not replaced midway, so as to reduce the loss of the sample.

In practice, the appropriate number of dips can be selected according to the type of cells to ensure acceptable detection accuracy. In some embodiments, n is an integer ≦ 20, such as 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19.

The invention is useful for enriching samples in a drip, and is particularly useful for enriching for a treasure sample, in some embodiments, the cell suspension sample has a density of cells of interest ≦ 12cells/μ l.

In some embodiments, the volume of the cell suspension sample stock solution is 100. mu.l or less, such as 80. mu.l or less, or 70. mu.l or less, or 60. mu.l or less, or about 50. mu.l or less.

The "cell of interest" is the cell that needs to be detected.

In order to obtain a quantity of 0.5-2 mul per dip without wasting the sample, the pipette tip is preferably inserted 1-3 mm below the surface of the cell suspension sample.

In some embodiments, the cell suspension sample has a volume of 80 μ l or less, or 70 μ l or less, or 60 μ l or less, or about 50 μ l.

In some embodiments, the cell of interest in the cell suspension sample is a plasma cell.

In some embodiments, the carrier has a cell loss prevention tablet adhered to the surface thereof.

In some embodiments, the cell loss prevention tablet is selected from the group consisting of poly-L-lysine, chrome alum-gelatin, and 3-aminopropyl-3-ethoxysilane (APES).

The cell anti-dropping tablet can effectively prevent the cell from being separated from the carrier in the operation process.

In some embodiments, the support is a planar support.

In some embodiments, the carrier has a concave aperture.

In some embodiments, the carrier is transparent.

In some embodiments, the carrier is a slide.

In some embodiments, the carrier is placed on a platform, the platform is provided with a mark, and the carrier is placed above the mark, and the mark is used for indicating the dropping positions of the first drop of the cell suspension sample to the nth drop of the cell suspension on the carrier.

In actual operation, because the single dropping amount is very low, the liquid dropped on the carrier is nearly transparent, and when the solvent is basically completely volatilized, an operator is difficult to position the dropping position of a subsequent sample, and at the moment, a mark can be preferably set to ensure the accuracy of operation.

In some embodiments, the platform is a hybridization meter platform.

In some embodiments, the support is heated to 34 ℃ to 40 ℃ prior to or at the time of the dropwise addition.

The operation of the table top of the hybridization instrument accords with the operation situation of the experiment, and the table top of the hybridization instrument can provide heat to accelerate the volatilization of the solvent, so that the operation time is shortened, and the sample diffusion is more uniform.

In some embodiments, the vector is divided into a plurality of hybridization regions, and step c) is performed after all regions have been subjected to step b), and step d) is performed after all regions have been subjected to step c).

Multiple hybridization regions can be used for different detection purposes. When the sheet is dripped, after the first dripping is finished in each area, the second and subsequent times of dripping are carried out, so that the waiting time can be reduced, and the operation efficiency is increased.

In some embodiments, the pipette tip is a 100 μ l or 200 μ l pipette tip. The specification of the pipette tip can be selected according to actual needs, and the length of the pipette tip is moderate, so that the pipette tip can be used for sucking cell suspension samples in various centrifuge tubes.

In some embodiments, the pipette tip has a filter cartridge.

In some embodiments, the solvent of the cell suspension sample is a cell fixative.

In some embodiments, the effective component in the cell fixing solution is selected from any one or more of ethanol, methanol, formaldehyde, acetone, and paraformaldehyde.

In some embodiments, the cell fixative is Carnoy's fixative.

The target of in situ hybridization may be DNA or RNA. According to the difference of the probes used in the in situ hybridization or the difference of the principles, the invention does not limit the kind of in situ hybridization to which the method is specifically applied, and the preferred type of in situ hybridization can be Chromogenic In Situ Hybridization (CISH) or Fluorescent In Situ Hybridization (FISH);

further, the fluorescence in situ hybridization method can be RNA FISH using branched DNA signal amplification, a multicolor fluorescence in situ hybridization technique, a chromatin fiber fluorescence in situ hybridization technique, and the like.

Embodiments of the present invention will be described in detail with reference to examples.

Examples

This example provides a FISH spot method for enriched cells of plasma cells.

In this example, 6 package items of multiple myeloma (Tp53, RB1, 1q21, IGH/CCND1, IGH/FGFR3, IGH/MAF) from Zhongshan Hospital were mainly examined. After plasma cell enrichment, the sample with small cell amount is added with 1 drop of fixative (about 50 mul) to prepare suspension, the blank of the anti-drop slide is marked with items and experiment numbers, and the positions of the mark points on the surface of the hybridization instrument are generally enabled to be right, left and right, respectively with a proper distance; the slides were placed on a hybridization apparatus (37 ℃) in order to achieve uniform diffusion of the suspension dropped onto the slide and faster evaporation of the fixative.

The pipettor used in this example was a sample application gun.

And blowing and beating the uniformly mixed suspension by using a 200 mu l sample adding gun, inserting the gun head into the position which is about 2mm below the liquid level without pressing a button at the top end, taking out the centrifuge tube, beating a small amount of suspension (about 1 mu l) carried in the top end of the gun head on a glass slide, and sequentially dripping 6 items. Since the total volume of the suspension is small, approximately 8 drops per item can be made, thus forming approximately a small circle with a diameter of approximately 0.8cm, and the nuclei are concentrated and uniformly distributed in the small circle, and the subsequent probe hybridization and the technician counting can be performed intensively on the area.

The specific operation is as follows:

the method comprises the following steps: the position of the hybridization instrument surface marking point, one item marking point, two items marking two points, and moderate distance, as shown in figure 1;

step two: the blank of the anti-shedding slide is marked with items and experiment numbers and is placed on a hybridization instrument, as shown in figure 2;

step three: blowing and beating the mixed suspension by using a 200-microliter pipettor, then inserting the gun head into the liquid level for about 2mm without pressing a button at the top end, and taking out the centrifuge tube as shown in figure 3;

step four: a small amount of suspension (about 1 mu l) carried in the tip of the pipette head is fully applied to the slide according to the positions of the spots to form a small circle with the diameter of about 0.8cm, 6 items are sequentially applied according to Tp53, RB1, 1q21, IGH/CCND1, IGH/FGFR3 and IGH/MAF, the process is repeated for 4-8 times, and the number of application is increased according to the number of cell deposits on the surface of the slide actually observed (note that one drop is applied to each item in sequence and repeated overlapping is carried out instead of applying one item first and then continuing applying one item, so as to ensure that the cell amount on each item is approximately the same), as shown in FIG. 4;

step five: after the slide is dried, the slide is placed under a microscope with a common light source for observation, and the distribution condition of cell nuclei in a visual field is shown in figure 5; for comparison, the nuclear distribution of the slide glass dropped by the prior art pipette-dropping method or the ordinary sample-adding gun-dropping method is shown in FIG. 6. The pictures are for reference only, the actual distribution can only be seen under a microscope (magnification 100 ×), the slide only has a trace of the suspension.

The comparison of the technical effects of the comparative example and the present example is shown in table 1:

TABLE 1

By the method, the cells in the cell suspension sample can be enriched on the carrier. In the prior art, when a sample adding gun is used for dropping a sample, the operation is relatively extensive, and the cell suspension is generally dropped on a carrier at one time, so that the spreading area of a sample on the carrier is too large; in the invention, the cell nuclei are uniformly and intensively distributed in a circle with the diameter of about 0.8cm by dripping the cell nuclei at the same position for multiple times, so that the cell density in unit area is increased, the cell distribution is more uniform, the follow-up observation and statistics of the cells are facilitated, and the use amount of hybridization reagents such as probes and the like can be saved (the comparison of the two is shown in table 2).

TABLE 2

The methods provided by the present invention are particularly suitable for the instillation of low abundance samples of the deposited cells, such as the plasma cells mentioned in the examples. In actual detection, in situ hybridization of plasma cells is usually used for diagnosis of Multiple Myeloma (MM), however, part of MM bone marrow has a low proportion of tumor cells, and detection results are easily interfered by normal cell signals, and the international myeloma working group stipulates that all MM should be enriched or sorted by using magnetic beads (MACS) for ISH detection to improve detection rate. The plasma cell enrichment specimen has strict requirements, and if the specimen is in a poor state or is stored for too long, the enrichment effect is affected. These objective conditions lead to the occurrence of bone marrow dry-out or low plasma cell proliferation index per se in patients and the ineligibility of the samples to be tested, which results in the very low number of plasma cells obtained by enrichment and difficulty in reaching the counting requirement of the laboratory (the lowest 100 cell nuclei with signals in most blood items have different detection items), but due to the clinical complexity, the laboratory detection often has a high rate of single regression.

Such a sample is prepared from a cell suspension sample with a low content, the density of target cells is much lower than that of a common sample, and it is usually clinically necessary to perform a plurality of tests on one sample (for example, 6 different tests mentioned in the examples), so that a small amount of samples are divided into several separate tests. When a liquid flows under the action of an external force, the flow of the liquid is restrained by the existence of molecular cohesive force, so that internal friction force is generated along the interface of the liquid, and the characteristic is called the viscosity of the liquid. By utilizing the viscosity, the invention can furthest reduce the loss of the sample by dipping a trace sample by the liquid suction nozzle and dripping the trace sample for a plurality of times, and can realize that a small amount of samples can effectively improve the detection efficiency and effectively reduce the rate of single drop in detection by the enrichment effect.

In addition, the method has low requirement on the operation skill of technicians, can effectively solve the problem of the sample drip through manpower, reduces the cost and the technical threshold compared with a porous slide, and is more beneficial to the popularization of the industry.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. An in situ hybridization spotting method, comprising:

b) dropping a first drop of the cell suspension sample onto the carrier;

2. The in situ hybridization spotting method of claim 1 wherein n is an integer no greater than 20.

3. The in situ hybridization droplet method of claim 1, wherein the cell suspension sample has a density of cells of interest of 12cells/μ l or less.

4. The in situ hybridization pipetting method of claim 1, wherein the pipette tip is inserted 1mm to 3mm below the surface of the cell suspension sample to dip the sample.

5. The in situ hybridization dripper method of claim 1, wherein the cell of interest in said cell suspension sample is a plasma cell.

6. The in situ hybridization dripping method according to claim 1 wherein a cell loss preventing tablet is attached to the surface of the carrier.

7. The in situ hybridization droplet method of claim 1, wherein the carrier is placed on a platform, the platform is provided with a mark, and the carrier is placed above the mark, and the mark is used for indicating the dripping positions of the first droplet of the cell suspension sample to the nth droplet of the cell suspension on the carrier.

8. The in situ hybridization droplet method according to any one of claims 1 to 7, wherein the carrier is heated to 34 ℃ to 40 ℃ before or during the droplet.

9. The in situ hybridization spotting method of any of claims 1-7 wherein the carrier is divided into a plurality of hybridization zones, and step c) is performed after all zones have been subjected to step b), and step d) is performed after all zones have been subjected to step c).

10. The in situ hybridization dripping method according to any one of claims 1 to 7, wherein the in situ hybridization is chromogenic in situ hybridization or fluorescent in situ hybridization.