CN100410368C

CN100410368C - Pretreating method for chromosome nuclear type analysis lymphocyte culture specimen

Info

Publication number: CN100410368C
Application number: CNB2005100858831A
Authority: CN
Inventors: 翁炳焕
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-07-19
Filing date: 2005-07-19
Publication date: 2008-08-13
Anticipated expiration: 2025-07-19
Also published as: CN1900273A

Abstract

The present invention is the pre-treatment process of lymphocyte culture specimen for chromosome nuclear type analysis in medical field. The process includes conventional drawing peripheral blood or fetus umbilical cord blood specimen, mixing with anticoagulant in special abacterial lymphocyte culture specimen pre-treating tube for chromosome nuclear type analysis, centrifuging at 3000 rpm for 10 min, sucking the upper layer white precipitate as the lymphocyte layer on the surface of the erythrocyte precipitate, and inoculating for culturing. Or, the process includes sucking certain amount of heparin to blood taking syringe, drawing blood specimen, mixing blood with heparin anticoagulant, turning the syringe at 37 deg.c for 3-5 hr for separating out the lymphocyte through natural depositing, eliminating the blood plasma in the upper layer and inoculating the precipitate as the lymphocyte layer on the surface of the erythrocyte precipitate for culturing.

Description

Pretreating method for chromosome nuclear type analysis lymphocyte culture specimen

The present invention relates to pretreating method for chromosome nuclear type analysis lymphocyte culture specimen, be mainly used in lymphocyte relative separation and purification before the chromosome nuclear type analysis lymphocyte cultivation is done in each medical institutions cytogenetics laboratory or antenatal diagnosis laboratory.

Karyomit(e) is the genetic material in the nucleus, after lymphocyte in the blood is cultivated under certain condition, through results, film-making, the trysinization poststaining shows band, can examine under a microscope karyomit(e), the mankind have 23 pairs of karyomit(e)s, wherein 22 pairs is euchromosome, 1 pair of sex chromosome for the decision gender, be loaded with the gene of genetic information on the karyomit(e), chromosome number increases or reduces, or karyomit(e) has disappearance, textural anomalies such as transposition claim chromosomal disorder, corresponding symptoms can appear, normally whether chromosome number and structure, karyomit(e) length in the karyomit(e) sheet of making after needing lymphocyte to cultivate, shape, just can debate not come out under the good situation such as degree of scatter and dyeing, if cultivate, methods such as inoculation are bad will to influence the lymphocyte growth cycle, or obtains less lymphocyte in mid-term and all can influence result's accuracy even can't diagnose chromosomal disorder.Present chromosome karyotype analysis, the used sample of blood lymphocytes cultural method is to extract peripheral blood or fetal cord blood, with the heparin is the antithrombotics anti-freezing, drip quantitative anticoagulated whole blood then and in substratum, do the lymphocyte cultivation, academia generally acknowledges, red corpuscle in antithrombotics heparin and the whole blood and some composition in the blood plasma can suppress lymphocyte growth, thereby influence result's accuracy and working efficiency.

In order to address the above problem, thereby the urografic acid methylglucamine salt solution that academia once had suggestion to use the different concns different specific weight is made gradient density centrifugal method isolated lymphocytes, but agents useful for same costliness, operation is tired, easily pollute, be not suitable for clinically, still adopt classical anticoagulant heparin Whole blood lymphocyte direct inoculation culture method clinically, the problems referred to above still exist.

The objective of the invention is to provide pretreating method for chromosome nuclear type analysis lymphocyte culture specimen, and its energy is easy, relative separation goes out lymphocyte for cultivating apace, removes the composition that is unfavorable for the lymphocyte incubation growth in heparin, red corpuscle and the blood plasma.

The object of the present invention is achieved like this: aseptic technique method routinely extracts quantitative peripheral blood or fetal cord blood preparation, putting into about 0.5 centimetre-1.0 centimetres aseptic special dyeing body nuclear type analysis lymphocyte of 120 centimetres, diameter that is about that contains quantitative anticoagulant heparin agent immediately cultivates pretreated tube and makes blood and the antithrombotics mixing, centrifugal 10 minutes of 3000rpm removes and draws or do not remove upper plasma behind the upper plasma directly to draw the linen throw out in upper strata on red erythroprecipitin surface be the buffy coat inoculation culture.Or according to the anti-freezing ability of heparin draw quantitative heparin to the blood sampling syringe in, require to extract quantitative blood then routinely, after rotating needle tubing mixing blood and antithrombotics, needle point upwards was inverted 3 hours-5 hours under 37 ℃ condition, with natural sinking method isolated lymphocytes, after squeezing out upper plasma, be the throw out of erythrocyte surface that lymphocyte is inoculated in the substratum and cultivates again.

The present invention is antithrombotics with the heparin, and concentration is 125IU/mL, but 0.1mL heparin anti-freezing 1mL whole blood.

Blood taking needle of the present invention, the available system needle tubing of moulding is put into centrifuge tube immediately after the blood sampling, and longer as blood inversion time in needle tubing, it is better then to use the glass needle tubing.

Fig. 1 is that the aseptic special dyeing body caryogram that proposes according to the present invention divides the folding lymphocyte to cultivate the sectional view of sample pretreated tube.

Below in conjunction with Fig. 1 lymphocyte of the present invention being cultivated the sample pretreated tube is explained in detail.

As shown in Figure 1, the length that lymphocyte is cultivated sample pretreated tube 3 is about 120 centimetres, and diameter is about 0.5 centimetre-1.0 centimetres.Scale 1 is for putting into the mark that reaches behind the antithrombotics heparin, scale 2 is for putting into the mark that reaches behind the blood, scale 1 and scale 2 have specific volume ratio, ratio in the anticoagulant heparin 1.0mL whole blood of 0.1mL125IU/mL concentration is set, pretreated tube lid 4 and pretreated tube 3 are joined with screw thread, pretreated tube 3 and pretreated tube lid 4 is the raw material manufacturing with the rigid plastics, pretreated tube lid 4 can also rubber be the raw material manufacturing, after the tight pretreated tube 3 of pretreated tube lid 4 lids, be sealed state in the pretreated tube 3, can prevent effectively that extraneous bacterium from entering and making the sample pollution.

The concrete pretreating method for chromosome nuclear type analysis lymphocyte culture specimen of the present invention is: putting into concentration in aseptic special dyeing body nuclear type analysis lymphocyte culture specimen pretreated tube is the anticoagulant heparin agent 0.1mL (reaching scale mark 1) of 125IU/mL, 50 ℃ to 100 ℃ dry for standby.Extracting examinee's peripheric venous blood or Cord blood 1mL then puts in the sample pretreated tube and (reaches scale 2 marks), shake mixing gently, centrifugal 10 minutes then with 3000rpm, this moment, the upper strata was a blood plasma, the lower sediment thing is a red corpuscle, erythrocyte surface one deck is canescence for containing lymphocytic leukocytic cream, draws this leukocytic cream throw out and cultivates as lymphocyte.Or elder generation's inhaled concentration in the blood drawing syringe is 125IU/mL anticoagulant heparin agent 0.1mL, extract peripheral blood 1mL, after the rotation needle tubing makes blood and antithrombotics mixing, syringe needle upwards is upside down in 37 ℃ of incubators 1 hour-5 hours then, cell is sunk naturally, use aseptic syringe needle again instead, extrude the upper plasma that leans on syringe needle one end in the needle tubing, when treating that the upper strata throw out promptly contains lymphocytic leukocytic cream and partly enters syringe needle, promptly syringe needle is inserted through the bottle cap of disinfectant culturing bottle in advance, should make this moment syringe needle upwards but do not touch nutrient solution in the culturing bottle as far as possible, the throw out in needle tubing upper strata and the syringe needle is pushed in the nutrient solution, make lymphocyte and cultivate.The microorganism that so just can remove immune factor in red corpuscle, heparin and the blood plasma, antimicrobial substance and may exist etc. is unfavorable for the material of lymphocyte growth, and concentrate relatively comparatively easily and isolated more lymphocyte for cultivating, thereby improved the effect that lymphocyte is cultivated, be fit to the processing of clinical big sample.

Claims

1. pretreating method for chromosome nuclear type analysis lymphocyte culture specimen that is used for medical field, its principal character is to extract quantitative peripheral blood or fetal cord blood preparation routinely with aseptic technique, put into be about 120 centimetres that contain quantitative anticoagulant heparin agent immediately, in the about 0.5 centimetre aseptic special dyeing body nuclear type analysis lymphocyte culture specimen pretreated tube of diameter, and make blood and antithrombotics mixing, centrifugal 10 minutes of 3000rpm, remove and draw or do not remove upper plasma behind the upper plasma directly to draw the upper strata pale precipitation thing on red erythroprecipitin surface be that buffy coat is inoculated in the substratum and cultivates, or according to the anti-freezing ability of heparin draw quantitative heparin to the blood sampling syringe in, require to extract quantitative blood then routinely, after rotating needle tubing mixing blood and antithrombotics, needle point upwards be upside down in 37 ℃ 3 hours-5 hours, with natural sinking method isolated lymphocytes, after squeezing out upper plasma, be the throw out of erythrocyte surface that lymphocyte is inoculated in the substratum and cultivates again.

2. pretreating method for chromosome nuclear type analysis lymphocyte culture specimen according to claim 1, its principal character are that aseptic special dyeing body nuclear type analysis lymphocyte culture specimen pretreated tube is that raw material is made with plastics or rubber.

3. pretreating method for chromosome nuclear type analysis lymphocyte culture specimen according to claim 1, its principal character are that used antithrombotics is the heparin of 125IU/mL.