JPH0420298A - Vice and method for pre-treating bone marrow - Google Patents

Abstract

PURPOSE:To preliminarily treat a specimen for the examination of none marrow image by a method not requiring a manual work and enabling the automation of the operation while preventing the danger of the infection of diseases, by treating the specimen with a protease at the pretreatment stage of the preparation of the bone marrow specimen and separating cells (blood cells) in the tissue solution of the bone marrow. CONSTITUTION:A sampling mechanism 9, a cleansing mechanism 9 and a stirring mechanism 10 are attached to a centrifuge comprising a plate 7 holding containers 6 onto the rotation shaft of a driving motor and a rotor portion 11 heated to a constant temperature to provide a preliminarily bone marrow image-treating device. The following processes are carried out with the provided device. A protease such as trypsin is dissolved in an isotonic buffer solution and the solution is controlled to the optimal pH for the enzyme. In the controlled solution, cells (blood cells) in a bone marrow tissue solution are separated into one, two or three cell fractions without the breakage of cell membranes for the preliminary treatment of the bone marrow image.

Description

[Detailed Description of the Invention] [Field of Application in Industry-1-] The present invention provides a processing method capable of separating cells (blood cells) in bone marrow tissue in a pretreatment stage for preparing a bone marrow image specimen, and a method using the processing. Regarding equipment.

[Conventional technology]

A bone marrow imaging test is a test performed to understand the pathology of diseases such as unexplained anemia and leukemia.

Puncture the posterior ilium and sternum and collect bone marrow fluid at 0.5 to 1 O.
Aspirate mρ and prepare a smear. Animals (e.g. Rat 1~
), cut off the tip of the femur and apply a syringe containing serum to wash out the bone fli $II lfa in the bone.

The collected tissue fluid is smeared manually or using an automated smear method. In the case of humans, it is usually performed using two methods: the wedge method and the coverslip method.

In the manual method, no anticoagulant is used and it must be smeared quickly before it clots. The fat in the tissue has faded and a smooth smear is very dangerous.

An automatic classification device called Sai-I-Sen-I~Rejuge is mainly used in animal experiments. It was originally used to prepare specimens for cytodiagnosis of body fluids.

The tissue fluid containing an anticoagulant is centrifugally washed with an isotonic buffer, etc., then placed in a centrifugation chamber, and a slide glass is attached to the outlet. A long and thin filter paper with a hole of 7 cn diameter is sandwiched between the centrifuge chamber and the crow, so that the holes in the filter paper make contact between the two. By centrifugation,
Since the liquid is adsorbed by the filter paper, the cells are collected in a dry state on the Sly I glass, creating a small circular smear surface.

The centrifugal washing described in 1- is performed in order to remove excess fat and fat from the bone marrow tissue fluid. Put tissue fluid containing anticoagulant into a Spitz tube, add an appropriate amount of isotonic buffer or serum,
Mix and place in the centrifuge. 1,500-2,5
Centrifuge at 00 rpm for 3-5 minutes and remove. ” - Pipette 1 and suck it up, then throw it away. Repeat this 2 to 3 times.

The prepared smear is stained, and 500 to 1000 cells (blood cells) are classified under a microscope. (End: (In the case of tree branches) O0 to 200 pieces) Even an experienced technician takes 20 to 30 minutes to test one sample, which is extremely tiring work.

[Problem to be solved by the invention]

The method used in the prior art described in I-1 is for specimens that can only be visually classified, with a collection of about 5 to 10 blood cells. Blood cells often break down at the end of the wedge method.

In automatic smearing θ ((Sai 1 Hesen 1 Herifuge)), centrifugal washing takes time and effort, cells (blood cells) are easily destroyed, and large immature cells (blood cells) tend to stick together. , smeared small P] shape is approximately 7 mm in diameter and the position is shifted, so it is not suitable for classification by an automatic classification device.

Peripheral blood specimens are classified by an automatic blood image classification device using a pattern recognition method based on the isomorphic parameters of the cells (blood cells).3 According to the attached document, the cells (blood cells) are It is known that even if they are not isolated, if there are 2 to 73, classification is possible, although the processing capacity will be reduced.

The purpose of the present invention is to separate about 2 to 3 cells (blood cells) in bone marrow tissue fluid by treatment with a proteolytic enzyme.

Another object of the invention is sampling in a centrifuge.

By adding a washing and stirring mechanism, centrifugal washing and enzyme treatment are automatically carried out in the same container, and the ratio of specific cells does not change, and the pre-treatment creates a cell suspension with less breakage. The goal is to provide equipment.

[Means to solve the problem]

In order to achieve this goal, a proteolytic enzyme was used, and bone marrow tissue fluid and an isotonic buffer solution of the enzyme were allowed to react in a container heated to a constant temperature, without destroying the blood cells. It is separated into two parts.

Furthermore, after placing the container containing the tissue fluid in a centrifuge,
In order to automatically perform pretreatment, the centrifuge is equipped with a sampliner mechanism (reagent dispensing and supernatant removal) and a washing mechanism (nostle washing).
and a stirring mechanism added.

[For A1] The proteolytic enzyme used in the present invention is an enzyme that has an anti-inflammatory effect, and trypsin is used for necrotic tissue + l1g.
It has the function of melting things such as In addition, collagenase, S1-
1-knob I-kinase, α-chymo-1-lipsin, promelain, papain, etc., have similar functions. When a certain concentration of enzyme is mixed with an isotonic buffer solution adjusted to its optimum p I-1, heated to 30-37°C, and allowed to interact with bone marrow tissue fluid in a container, the enzyme removes excess fat in the tissue fluid. It melts the tissue and acts on the blood cells to separate them.

〔Example〕 Bone marrow tissue fluid is collected from humans or animals.

In the case of H1-, the doctor performs bone marrow aspiration and
Aspirate with an O syringe. In the case of small animals, at the time of autopsy, cut the femur and extrude it with a serum syringe.

First, prepare the enzyme treatment solution and isotonic buffer as follows.

[Enzyme treatment solution]

■ D) D3 S buffer solution ・ 20 D [也■
l-Lipsin 50 mQ (2) is prepared by adjusting the pH to 7 to 8 before use, and mixing 20 mQ (2) and 50 mQ (2).

[Isotonic buffer]

・Sulfur 1 helium chloride (NaCQ) 4,Q g / 500mQ ・Potassium chloride (KCQ, ) 0.2 g1500mQ ・Calcium chloride (Ca CQ 2) 0.28g1500mQ ・Sodium hydrogen phosphate (NaH2PO2・2H20)
39.0mg1500mQ ・Sodium hydrogen phosphate (N a zHP O4・12
H2O) 76.0g/500 m Q ・Lithium hydrogen carbonate (NaI-(CO2) 175m
g1500mff Fig. 1 shows the configuration of the device, and Fig. 2 shows the processing steps of the pretreatment device. Enzyme treatment solution 1 is placed in reagent container 3 by approximately 20 mQ and installed r1. Hemorrhoids 1 (buffer solution is approximately 50% in reagent container 2)
On+ Install Q insert. For cleaning water, use ion-exchanged water or distilled water at a ratio of 1:3 to approximately 500 liters).

The optimal rotation time for centrifugation and the optimal stirring time differ depending on the type of animal and the type of animal.
- Set the LCD panel 5 from '4.

The collected bone marrow tissue fluid is placed in a container 6 in which an anticoagulant (EDTA) has been dried and placed in a hole tough of a centrifugal mechanism to be fixed. When you press the 5TART key on the operation keyboard I-4,
The isotonic buffer solution is aspirated from the reagent container 2 by the dispenser 8 and discharged from the nostle 9 into the container 6. Stir for 5 to 0 seconds using a stirring bar (500-1.OOOrpm)
. After that, close the lids 71 and 12 of the centrifugal mechanism lid IJ, and
After centrifuging for 3 to 7 minutes at 500±300 rpm, 1, #1 is opened, the supernatant inside the container 6 is aspirated through the nostle 9, and discharged into the drainage tank] 3 outside the apparatus. Repeat washing a set number of times from the axiom of isotonic buffer to draining the supernatant.

Thereafter, the enzyme treatment solution is sucked from the reagent container 21, discharged from the nozzle 9 into the container 6, stirred for 5 to 10 seconds with the stirring rod 10, closed 71-12, and left for 5 to 10 minutes. After finishing the treatment, centrifuge at 500±30Q rpm for 3 to 7 minutes. Then, open the container 2 and aspirate the liquid 1 in the container 6. Furthermore, the isotonic buffer solution is aspirated from the reagent container 2 with the dispenser Δ8, discharged into the container 6, and stirred with the stirring bar 10.
Stir for 0 seconds (500-1.OOOrpm).

Stirring bar] O, nosle 9 dispenses washing water with dispenser 81, 5
suction and washing each sample in the washing tank 14. Cleaning tank 1
The water in the tank 4 is discharged to a drain tank 13 outside the device through a l-rain. The processing in the preprocessing device is now complete.

Figure 3 shows details of the centrifugal mechanism of the device. Drive motor 1-
7 is surrounded by a base 19 pasted with a heat insulating material 18, and a holder 7 for holding the container 6 is fixed on the motor shaft. High speed (1,
Since the motor rotates at a speed of 000 to 3,000 rpm, a spring 20 is provided to prevent vibration, and an air hole 21 is provided to prevent heat generation from the motor.

When 71 to 12 are closed, the controller 16 also controls the centrifugation for a set time. A catch tray 22 was added in case the sample accidentally spilled during work.

Further, the entire container 6 is heated to 30 to 37° C. by the heater 23, and controlled to be maintained at an appropriate temperature by the humidity sensor 24.

After the treatment in the pretreatment device is completed, the container 6 is taken out and the slides 1 to 1 are smeared and stained using the existing centrifugal smear devices ′, 9′. The prepared specimen is automatically classified as a bone marrow image using a blood image automatic classification device using a pattern recognition method.

The proteolytic enzyme used can be substituted with collagenase in addition to 1-lipsin. Also, to help with cell notarization,
There is also a method of adding a chemical agent to the enzyme solution, E1) i''A (ethylene cyamine thiol-oxalic acid).

Suitable enzyme concentrations are 0.2-0.5% for I-lipsin and 0.02-0.1% for collagenase. There are several types of silk combinations of buffer solution, enzyme, and 1':D i''A, and examples are given below.

(Example A) i) Isotonic buffer solution 20 m (1 1-lium chloride (NclCQ) 4, , Oy, / 500 rn Q Potassium chloride (KCQ) 0.2 g / 500 m Q.

Lucium chloride (cacQz) 0.28g1500mQ Hydrogen non-acid 1~lium (N a HzP 04 ・2
1-TzO) 39mg1500mM 1-lium hydrogen phosphate (N a 2I (P 04・1
21-TzO) 76 mg / 500 m Q 1-lium hydrogen carbonate (NaHCO3) 175 mg /
500m Q.

■ Collagenase/iomg (0.05%) (3) 1
~ Ribsin inhibitor 1 mg (Example B) 1) P B S buffer i1-11-2O (see p, 9) (?) 1 Helipsin 50 mg t'3) E D T A -4, 6 mg Concentration of enzyme If the concentration is increased or the treatment time is prolonged, it will also act on the separated cells (blood cells) and destroy the cell membrane, so it is necessary to pay attention to the concentration and time. 1- Since it is configured as explained above, it produces the effects described below.

If the i'+ii treatment [-culm;+= operation is carried out, dispensing, stirring, centrifugation and -n will be required in addition to the time required for dispensing, stirring, and the process, and the risk of infection will increase. According to the present invention, by putting the collected bone marrow tissue fluid into a container and pumping it into the cellar 1 in the device, 7 cells/1k can be prepared in 15 to 20 minutes, and a specimen that can be automatically classified can be provided.

Figure 4 shows the cell distribution diagram of the enzyme-treated specimen and the untreated specimen. In unprocessed specimens, the cells (blood cells) are approximately 5-10
However, when enzyme treatment is performed, cells (1r+t
l"R) -1-3 Pu degree d', i: 'J, IL
(1) Automatic classification is now possible with a 110 liquid image automatic classification device for peripheral blood. The number of samples processed is 5 cells in peripheral blood.
The time is doubled, so it is not much, but it is about one-third of the time required for visual classification. However, the device can also be operated at night, which has the effect of reducing technician fatigue.

Table 1 shows the results of myeloid imaging of untreated specimens and enzyme-treated specimens. Good results were obtained without any change in the abundance ratio of specific cells (blood cells).

Table 1 Classification results Number of classification: 1000 pieces/piece

[Brief explanation of the drawing]

Fig. 1 is a three-dimensional diagram of an apparatus according to an embodiment of the present invention, Fig. 2 is a treatment process diagram of the pretreatment apparatus, Fig. 3 is a diagram of the centrifugal mechanism of the apparatus, and Fig. 4 is an untreated specimen and an enzyme-treated specimen. FIG. ] Enzyme treatment solution, 2. Reagent container (isotonic pre-relaxation solution), 3.
Reagent container (washing water), 6 container, 7 holder, 9]・
・Nozzle (sampling mechanism), 1o Stirring bar, 14・
Cleaning vortex, 7 Motor, 8 Insulation, 23 Heater,
24 ・Temperature sensor, 4 (3・Control unit.

Claims (1)

[Scope of Claims] 1. A bone marrow image preprocessing method, which comprises separating cells (blood cells) in bone marrow tissue fluid by treatment with a proteolytic enzyme in the preprocessing step of preparing a bone marrow image specimen. 2. The pretreatment according to claim 1 involves dissolving a proteolytic enzyme such as trypsin in an isotonic buffer solution and adjusting the optimum pH of the enzyme.
1. A method for preprocessing bone marrow images, which comprises separating cells (blood cells) in bone marrow tissues into 1 to 3 cells by the action of enzymes in a solution adjusted to 1 to 3 cells without destroying cell membranes. 3. In a centrifuge consisting of a plate that holds a container on the rotating shaft of a drive motor and a rotor heated to a constant temperature, by adding a sampling and washing mechanism and a stirring mechanism, bone marrow tissue fluid can be washed with enzymes. A bone marrow image preprocessing device characterized in that processing can be performed in the same container.