WS/T 615-2018Estimation of radiation biological dose - analyzing premature chromosome condensation ring (English PDF)

4.1 Main instrument and reagent preparation

See Appendix A for the main instrument and reagent preparation.

4.2 Preparation of premature chromosome agglutination specimens

4.2.1 Sample collection

Human venous blood was extracted from 1 mL to 2 mL, and heparin anticoagulation tube was injected.

4.2.2 Sample inoculation and cell culture

Add 0.5 mL of heparin anticoagulated whole blood to 4.5 mL of whole medium (RPMI 1640) containing 20% fetal bovine serum, set 2 for each sample.

A parallel sample, cultured in a constant temperature incubator at 37 ° C for 46 h to 47 h, and then added to the final concentration of 50 nmol/L of Calyx spp.

(Calyculin A) or Okadaic acid with a final concentration of 500 nmol/L, continue to culture for 1 h to 2 h.

4.2.3 Harvesting cells

Blow the culture with a pipette, transfer to a 15 mL graduated centrifuge tube, centrifuge at 250 g for 10 min in a horizontal centrifuge, discard the supernatant, leaving approximately 1 mL.

The remaining precipitate.

4.2.4 Low permeability

Add 75 ° C pre-warmed 0.075 mol/L potassium chloride (KCl) to 8 mL ~ 10 mL per centrifuge tube, pipette evenly with a pipette, set 37 ° C Low permeability 10 min ~ 30 min.

4.2.5 Pre-fixation

Add 1 mL to 2 mL of freshly prepared fixative [methanol/glacial acetic acid = 3/1 (volume ratio)] to each centrifuge tube, gently pipette and mix with a pipette.

Evenly, centrifuge at 250 g for 10 min, discard the supernatant.

4.2.6 Fixed

Add 8 mL of fixative solution to each centrifuge tube, mix gently by pipetting, fix at room temperature for 20 min, centrifuge at 250 g for 10 min, and discard the supernatant.

4.2.7 Second fix

Add 8 mL of fixative, mix gently by pipetting, fix at room temperature for 15 min, centrifuge at 250 g for 10 min, discard most of the supernatant, and leave the supernatant is measured.

4.2.8 Preparation of cell suspension

The remaining supernatant and the precipitate were mixed by pipetting to obtain a cell suspension.

4.2.9 Production

Drop 2 to 3 drops of the cells onto the pre-cooled slide, over-fire for 2 s to 3 s, and dry naturally at room temperature.

4.2.10 Dyeing

Diluted Giemsa dye solution (Giemsa) to 8% solution in phosphate buffer pH 6.8, stained for 8 min to 10 min, rinse with tap water Dry it out.

5.1 Optical microscopic reading

Under the low magnification of the optical microscope (x10 times), find the appropriate premature chromosome agglutination splitting phase; then in the oil mirror (x100 times) the premature chromosome agglutination and division phase is adjusted to the middle of the field of view, and the number of precocious chromosome agglutination rings is counted.

5.2 Judging criteria and counting criteria for early maturing chromosome agglutination

The precocious chromosome agglutination ring is divided into a hollow ring and a solid ring. The hollow ring is a hollow circular or irregular annular structure. Solid ring is present the solid spherical structure is slightly dense and has a diameter larger than the transverse diameter of the early-maturing agglutination dyeing monomer. The solid ring is not counted during analysis. Precocious chromosome condensation See Appendix B for an example of a ring.

The split phase in which the two sister chromatids are lined up is called the G2/M premature chromosome agglutination and splitting phase, and the ring term appears in this split phase.

As a G2/M precocious chromosome agglutination ring. Two similar ring structures in the G2/M phase precocious chromosome agglutination and splitting phase Count 1 premature chromosome agglutination ring. The split phase in which the two chromatids are clearly separated is called the M/A premature chromosome agglutination and splitting phase.

The loop that appears in this split phase is called the M/A premature chromosome agglutination loop. Each ring structure in the M/A premature chromosome agglutination splitting phase, Count 1 premature chromosome agglutination loop.

5.3 Recording and saving of result data

When a premature chromosome agglutination loop is detected, a photograph is required. Record each early maturity chromosome agglutination split phase of the selected analysis in the early Mature chromosome agglutination loop analysis is recorded in the table. See Appendix C for the record of early maturity chromosome agglutination loop analysis.

6.1 Sample Collection

Human venous blood was extracted from 6 mL to 8 mL, and heparin anticoagulation tube was injected.

6.2 Blood donor requirements

2 to 3 healthy adults, non-radiologists, both men and women, aged 18 to 60 years old, no acute or chronic disease, no shots within six months History of contact with chemical poisons, no history of viral infection in the past month.

6.3 Irradiation conditions

The sample is uniformly irradiated; the X or gamma ray irradiation dose ranges from 4 Gy to 20 Gy; and the irradiation dose rate is selected from 0.3 Gy/min to 1 Gy/min.

The irradiation dose point was 6 to 8 (evenly distributed); irradiation was performed at 37 °C. After the irradiation, the sample was placed in a 37 ° C incubator for 2 h, After the cell culture.

6.4 Preparation, analysis and recording of premature chromosome agglutination specimens

Preparation, analysis and recording of premature chromosome agglutination specimens were carried out in accordance with the methods described in Chapters 4 through 5.

6.5 Data Processing

The number of cells per chromosome in the precocious chromosome agglutination ring between the two groups was determined by U-test of Poisson distribution, and the number of cells in the precocious chromosome agglutination ring between groups the chi-square test was used to compare the relationship between the number of cells in the precocious chromosome agglutination ring and the dose of radiation, and the regression equation was fitted.

The hypothesis test uses a chi-square test.

6.6 Dose-effect curve fitting

Dose-effect between X- or gamma-ray induced premature chromosome agglutination ring and irradiated dose according to 4.3 of GB/T 28236-2011 it is advisable to fit the quadratic polynomial and fit according to equation (1).

Y = c D D2 (1)

In the formula.

Y -- the number of cells per chromosome of the precocious chromosome agglutination ring;

c -- constant term, the number of cells per cell number of the precocious chromosome agglutination ring (PCC loop/split phase);

-- dose first term factor;

D -- the dose of radiation, in units of Gy;

-- The dose quadratic coefficient.

6.7 Establishing a dose-response curve

Dose-response curves should be established in laboratories performing dose estimation; conditional laboratories can establish different radiation types and different dose rates Dose-effect curve.

7.1 Preparation of premature chromosome agglutination specimens

The method for preparing premature chromosome agglutination specimens for the estimated dose sample should be the same as the method for establishing the dose-response curve.

7.2 Analysis of the number of agglutination and division of precocious chromosomes

The.200 premature chromosome agglutination and splitting phase can be analyzed first, and the obtained premature chromosome agglutination ring per cell number p is substituted into the formula (2).

The number of split phases that need to be analyzed.

n = (1 ) 96.04p

In the formula.

n -- the number of split phases that need to be analyzed;

p -- the number of cells per chromosome of the precocious chromosome agglutination;

96.04 -- with a 20% tolerance, 1.96)-(1 pp = p × 20%, the constant obtained from the 95% confidence interval calculation.

7.3 Calculation of the number of cells per cell and the 95% confidence interval of the early maturing chromosome agglutination loop the calculation of the number of cells per cell of the early maturing chromosome agglutination ring is calculated according to formula (3).

In the formula.

p -- the number of cells per cell detected in the precocious chromosome agglutination loop;

x -- the number of early maturing chromosomes detected;

n -- Premature chromosome agglutination split phase number.

The 95% confidence interval for detecting the number of cells in the precocious chromosome agglutination loop is calculated according to formula (4).

95%CI = p +/- 1.96Sp (4) in the formula.

95% CI - 95% confidence interval;

p -- the number of cells per cell detected in the precocious chromosome agglutination loop;

Sp -- The standard error per cell number of the precocious chromosome agglutination loop, calculated according to formula (5).

In the formula.

Sp -- the standard error per cell number of precocious chromosome agglutination rings;

x -- the number of early maturing chromosomes detected;

n -- Premature chromosome agglutination split phase number.

7.4 Estimation of exposure dose

Substituting the number of cells of the precocious chromosome agglutination ring obtained in the sample to be estimated and the upper and lower limits of the 95% confidence interval into the established agent the dose-effect curve estimates the exposure dose. See Appendix D for an example of biological dose estimation for accident victims.