CN101109752B - Method for detecting lymphocyte subgroup with mono-clone antibody SPA hematid rosette method - Google Patents
Method for detecting lymphocyte subgroup with mono-clone antibody SPA hematid rosette method Download PDFInfo
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Abstract
The invention relates to a method for inspecting lymphocytic subgroup by a single clone antibody SPA red cell garland way. The method comprises the following procedures: first the red cell garland reagent is solved, then the circumference blood leukomonocytes are separated; leukomonocyte suspension is prepared by a Henke balancing salt solution free of Ca and Mg containing 20% serum of a newly borne calf; meanwhile, the sensitized red cell suspension is made into a sensitized red cell application liquid by using a Henke balancing salt solution free of Ca and Mg containing 20% serum of a newlyborne calf; then the leukomonocyte suspension and the sensitized red cell application liquid are mixed by 1:1 V/V into a mixed suspension, which is centrifuged after placing still under 18-28 centigrade, then is placed still for 15-45 min under 4 centigrade; finally the mixed suspension is absorbed 15 or 20 times by a pipet having a sucking head, the absorbed suspension is made into cell smear, which is naturally dried, dyed and counted by a microscope with high magnification. Comparing with prior McAb-A-E method, the invention is of simplified detection process, shorter period, quantitative controlling, and can meet clinic detection.
Description
Technical field
The present invention relates to Medical Immunology, relate in particular to the method that a kind of monoclonal antibody SPA red blood cell garland method detects lymphocyte subgroup.
Background technology
Development along with molecular immunology, according to lymphocyte film surface cluster of differentiation antigen (cluster ofdifferentiation, CD) difference, T, B, NK lymphocyte can be divided into some subgroups, clinical labororatory is by the monoclonal antibody (McAb) of anti-differentiation antigen CD3, CD4, CD8, CD19, CD20, CD22, CD56, CD16 etc., peripheral blood lymphocyte is detected, judge total T cell, the complementary cell of T (TH), T inhibition/lethal cell (T according to its positive rate
s/ T
c) distribution and the variation of subgroup, bone-marrow-derived lymphocyte, NK cell etc., thereby judge the immunologic function of human body, reach diagnosis, the treatment of prevention and health care and disease, the purpose of prognosis (estimate according to the testing result doctor, estimate the development in future of certain disease).
At present. detection method commonly used has flow cytometer method, immunofluorescence technique, AP-AAP bridging enzyme immunoassay and SPA (staphylococcal protein A) garland method, wherein SPA garland method is divided red blood cell garland method and bacterium garland method, and red blood cell garland method and bacterium garland method are divided direct method and indirect method.
The direct method of SPA red blood cell garland method is because of using monoclonal antibody (McAb) and be monoclonal antibody SPA red blood cell garland method direct method, is called for short the McAb-A-E direct method.(compile by Department of Medical Administration of Ministry of Health of the People's Republic of China in " national clinical examination working specification " for this method, second edition, publishing house of Southeast China University, 1997:381-384 and first published 1991:320-322) in have summary to discuss, but do not have detailed method of operating, carry out concrete operations by the method for " lyophilized antibodies sensitized erythrocyte garland reagent operation instructions " regulation in the real work: (1) adds the pH value in lyophilized antibodies sensitized erythrocyte garland reagent be 7.0～7.4 no calcium, magnesium Han Keshi balanced salt solution (is called for short no calcium, magnesium Hank'S liquid) or phosphate buffer dissolving; (2) method is isolated peripheral blood lymphocyte with lymphocyte separation medium (isolated cell is with the centrifugal 20～30min of the rotating speed of 2000～2500rpm routinely; With no calcium, the washing of magnesium Hank'S liquid 2 times, each with the centrifugal 5～10min of the rotating speed of 1000～2000rpm; ), at last with the pH value be 7.0～7.4 contain that 20% newborn calf serum does not have calcium, magnesium Hank'S liquid is made into 5,000,000/mL; (3) test tube is 20 degree angles and tiltedly puts, hatch 45min in 37 ℃, cell suspension is taken out put in another test tube, centrifugal remove supernatant and recover commercial weight standby; (4) get a certain amount of sensitized erythrocyte suspension according to sample number to be checked, the centrifugal supernatant that goes suspends with the no calcium that contains 20% newborn calf serum, magnesium Hank'S liquid, returns to commercial weight; (5) lymphocyte suspension and sensitized erythrocyte suspension mixed in equal amounts (each 25 μ L), room temperature (22～25 ℃) is placed 10min down, and the centrifugal 10min of 500rpm continues under the room temperature to place 1 hour, and placed 2 hours or spent the night at 4 ℃; (6) after the suspension back adds 25 μ L Mei-Ge Shi dye liquor (May-Gruenwaid) room temperature dyeing 10min gently, the centrifugal supernatant that goes, Ji's nurse Sa dye liquor (Glemsa liquid) dyeing 30min (or single with the dyeing of Glemsa liquid) that adds 50 μ L again saw that the result all could on the same day or second day; (7) inspection as a result and judgement: get a suspension behind the mixing on slide and add a cover thin slice, check with high power lens; Sticking around the lymphocyte has three above red blood cells to be the garland positive cell, and the negative cell of person that do not stick the red blood cell sticks one, two red blood cell person and promptly removes and disregard, and amounts to several 100～200 lymphocytes, calculates the percent of rosette formation cell.
Though the lyophilized antibodies sensitized erythrocyte garland reagent itself that the McAb-A-E direct method is adopted has high specificity, responsive, good stability, the term of validity is long, easy to use, the characteristics that only need ordinary optical microscope to detect, and experimental result can be preserved about two weeks, for over ten years by clinical labororatory and the widespread use of correlative study unit, but the McAb-A-E direct method as a kind of clinical detection method but because exist to detect step many, required specimen amount is more relatively, cycle is long, easily cause tested cellular damage and lose, indivedual immeasurable chemical control system indexs of important step, non-special garland is differentiated difficulty and is influenced defective such as count results, can't adapt to quick clinically, requirement accurately, make a lot of laboratories, particularly clinical labororatory accepts difficulty, has limited applying of this method.
Summary of the invention
Technical matters to be solved by this invention just provides that a kind of testing process is simplified, sense cycle is short, can carry out quantified controlling, satisfy the method that monoclonal antibody SPA red blood cell garland method that clinical detection requires detects lymphocyte subgroup.
For addressing the above problem, a kind of monoclonal antibody SPA red blood cell garland method of the present invention detects the method for lymphocyte subgroup, comprise the steps: that (1) adds 0.5mL pH value is 7.0～7.4 no calcium, magnesium Han Keshi balanced salt solution or phosphate buffer, is dissolved into sensitized erythrocyte and preserves liquid in lyophilized antibodies sensitized erythrocyte garland reagent;
(2) venous blood samples, adding the pH value with the volume ratio of 1:1～3 is 7.0～7.4 no calcium, magnesium Han Keshi balanced salt solution, behind the mixing, be added on the lymphocyte separation medium, the amount of lymphocyte separation medium and the volume ratio of dilute blood are 1:2～3, under 18～28 ℃ of temperature, with the centrifugal 15～25min of the rotating speed of 800～1200rpm; Take out mononuclearcell, and centrifugal to mononuclearcell, each with the centrifugal 6～10min of the rotating speed of 400～600rpm with no calcium, magnesium Han Keshi balanced salt solution washing back, abandon supernatant; Be that 7.0～7.4 no calcium, the magnesium Han Keshi balanced salt solutions that contain 20% newborn calf serum are made into 400～6,000,000/mL lymphocyte suspension with the pH value then;
(3) get sensitized erythrocyte and preserve liquid, to remove supernatant behind the centrifugal 6～10min of 400～600rpm rotating speed, with the pH value is that 7.0～7.4 no calcium that contain 20% newborn calf serum, magnesium Han Keshi balanced salt solutions suspend, and forms with the institute sensitized erythrocyte of getting and preserves the sensitized erythrocyte application liquid of liquid equivalent;
(4) lymphocyte suspension and sensitized erythrocyte are used liquid and are mixed into the volume ratio of 1:1 and mix suspension, place 10min under 18～28 ℃ of temperature; To leave standstill 15～45min at 4 ℃ behind the centrifugal 5min of 300～500rpm rotating speed;
(5) with the pipettor pressure-vaccum mixing suspension 20 of the band suction nozzle of 20 μ L or 25 μ L volumes or 15 times; Get and mix suspension pushing cell smear, dye with improvement Ji nurse Sa decoration method after the air dry, count with high power lens then: sticking around the lymphocyte has three above red blood cells to be the garland positive cell, and the negative cell of person that do not stick the red blood cell sticks one, two red blood cell person and removes and disregard; During counting, choose that zone, at least two places amounts to several 200 cells between the cell smear cephalocaudal 2/6～4/6, calculate the percent of rosette formation cell.
Venous blood in the described step (2) adopts heparin or disodium EDTA anti-coagulants to extract.
The present invention compared with prior art has the following advantages:
1, the present invention has reduced cellular damage by changing correlation parameter, the simplification treatment step that separates, handles cell, cellular morphology structure and natural form structure basically identical after having guaranteed to handle; By changing cell smear and dyeing counting method, can directly recognize the morphosis of cell at microscopically again.Identification and identification of cell, do not need lymphocyte is carried out purification process, thereby cancelled the lymphocytic operation steps of purifying in the former method, avoided the time-consuming complicated operations of former method, tested cell easy damaged and lose, defective that relative more, the non-specific cell garland of specimen amount is difficult to differentiate.
2, the present invention cancels the unnecessary condition and the step of antigen-antibody reaction in the former method, thereby has shortened the time of antigen-antibody reaction guaranteeing that testing result stablizes under the constant prerequisite.What immunology antigen-antibody reaction required time was general can finish about one hour.
3, owing in the former method " suspending gently " in the operating process do not carried out quantified controlling: firmly excessive during suspension, the garland red blood cell is scattered; Firmly not enough, then the dispersion of garland cell is inhomogeneous, agglomerate occurs, thereby the influence counting strengthens and detects error; The present invention is then by having stipulated the quantizating index that common lab generally can be used to this step, which kind of reaction tube no matter person and same operator adopt to operate according to the control criterion after quantizing in test each time to make the different operating, thereby reduced systematic error and accidental error, increased the stability, accuracy and the comparability that detect.
4, because the present invention passes through to change cell dyeing, method of counting, zone and the method chosen during to counting have been carried out standard, thereby have guaranteed the representativeness of counting, have reduced the caused testing result difference of human factor; After using the cell smear method simultaneously instead, can find some abnormal conditions beyond this test item:, juvenile cell original as occurring in the peripheral blood etc. can be examinee and clinician the diagnosis and treatment suggestion are provided.In addition, the cell smear of dyeing has and is easy to deposit check, carries interchange, is convenient to advantages such as mug.
5, because former method is to utilize adherent cell to have the motion characteristic of sticking at smooth glass, frosting, remove adherent cell, thereby reach the identification that do not influence the specific cell garland and the purpose of counting with the cultivation adherent method.The present invention then utilizes the morphosis feature of adherent cell itself, after the centrifugal speed parameter that changes the separating treatment cell, do not influencing as far as possible under the condition of the original cellular morphology of adherent cell, select best fixing means, dyeing liquor and colouring method for use, directly show the morphosis feature of adherent cell itself at microscopically, thereby reach the purpose of discriminating, discriminate between cells.
6, because the present invention has increased the disodium EDTA (EDTA-Na of present wide clinical application
2) anti-coagulants, the scope of used anti-coagulants is applicable to clinical more when having expanded former method collect specimen.
7, the present invention detects step by reducing, with sense cycle by original 7～8 hours (Time Calculation before CD3, CD4, the CD8 that finishes 1～3 part of sample detects and begin to count, do not comprise gate time) reduce to 2～3 hours, the shortening time reaches 5～6 hours, has satisfied the requirement of clinical fast detecting; Simultaneously owing to simplified operation steps, sample, reagent, equipment consumption etc. are reduced relatively, thereby on the basis that reduces cost, improved the stability that detects, thereby be easy to carry out applying of clinical detection in the laboratory of at different levels, all kinds of hospitals, research unit.
Embodiment
Embodiment 1 is at lyophilized antibodies sensitized erythrocyte garland reagent WuT
3, WuT
4, WuT
8Add 0.5mL pH value in (dried frozen aquatic products is the small amount of solid crystallization, dissolves behind the liquid feeding, does not influence volume) respectively and be 7.0 no calcium. magnesium Hank'S liquid, be dissolved into sensitized erythrocyte and preserve liquid.Cell suspension after the dissolving can add 0.1%NaN as toing many or too much for use
3, sealing can preserve for 2～4 weeks at 4～6 ℃.
Use anticoagulant heparin, extract patient venous blood 1mL on an empty stomach, add 1mL pH value and be 7.0 no calcium, magnesium Hank'S liquid mixing after, slowly be added on the 2mL lymphocyte separation medium with dropper, under 28 ℃, the hydro-extractor that with radius is 22cm is with the centrifugal 20min of the rotating speed of 800rpm.
Put into dropper sucking-off mononuclearcell that to be added with 4～6 times of amount pH values be 7.0 no calcium, another centrifuge tube of magnesium Hank'S liquid, inhale lymphocyte separation medium as far as possible less; Mixing, centrifuge speed are 400rpm, and centrifugal 10min abandons supernatant, are that 7.0 no calcium, magnesium Hank'S liquid are washed once with the pH value again.At last with the pH value be 7.0 the no calcium that contains 20% newborn calf serum, that magnesium Hank'S liquid is made into 4,000,000/mL lymphocyte suspension is standby.
Get 15 μ L sensitized erythrocytes and preserve liquid in 0.5mL love channel husband centrifuge tube, to remove supernatant behind the centrifugal 10min of 400rpm rotating speed, with the pH value is that 7.0 the no calcium that contains 20% newborn calf serum, magnesium Hank'S liquid suspend, and forms sensitized erythrocyte and uses liquid and also return to 15 μ L.
Getting lymphocyte suspension 15 μ L that the no calcium, magnesium Hank'S liquid of 20% newborn calf serum suspends adds the no calcium that contains 15 μ L20% newborn calf serums, sensitized erythrocyte that magnesium Hank'S liquid suspends and uses the 0.5mL of liquid and like in the channel husband centrifuge tube, abundant mixing, place 10min down at 28 ℃, to leave standstill 15min at 4 ℃ behind the centrifugal 5min of 300rpm rotating speed.
Pipettor 25 μ L volume pressure-vaccum mixing suspensions 15 times with the band suction nozzle; Get 10 μ L mixing suspensions and press blood sheet method pushing area 8～10cm
2Cell smear, after the air dry with the dyeing of improvement Ji nurse Sa decoration method, high power lens counting.Wherein improve Ji's nurse Sa decoration method and be meant dyeing liquor 3～5 is dripped off all standing cell smear, about 1min that dyes adds the pH value and is 6～10 dyeing of PBS (phosphate buffer), 2～5min of 6.4～6.8, and the water flushing gets final product.
Decision method: sticking around the lymphocyte has three above red blood cells to be the garland positive cell, and the negative cell of person that do not stick the red blood cell sticks one, two red blood cell person and removes and disregard; During counting, the zone of choosing between the cell smear cephalocaudal 2/6～4/6, at least two places amounts to several 200 cells, calculates the percent of rosette formation cell.
Testing result: CD3
+Be 80%, CD4
+Be 38%, CD8
+Be 46%, CD4
+/ CD8
+Be 0.83.
Embodiment 2 is at lyophilized antibodies sensitized erythrocyte garland reagent WuT
3, WuT
4, WuT
8In to add 0.5mL pH value respectively be 7.2 no calcium, magnesium Hank'S liquid, be dissolved into sensitized erythrocyte and preserve liquid.Cell suspension after the dissolving can add 0.1%NaN as toing many or too much for use
3, sealing can preserve for 2～4 weeks at 4～6 ℃.
With disodium EDTA (EDTA-Na
2) anti-freezing, extract patient venous blood 1.5mL on an empty stomach, add 3mL pH value and be 7.2 no calcium, magnesium Hank'S liquid mixing after, slowly be added on the 2.5mL lymphocyte separation medium with dropper, under 22 ℃, the hydro-extractor that with radius is 22cm is with the centrifugal 18min of the rotating speed of 1000rpm.
With dropper sucking-off mononuclearcell, putting into and being added with 4～6 times of amount pH values is 7.2 no calcium, another centrifuge tube of magnesium Hank'S liquid, inhales lymphocyte separation medium less as far as possible.Mixing, centrifuge speed are 500rpm, and centrifugal 8min abandons supernatant, are that 7.2 no calcium, magnesium Hank'S liquid are washed once with the pH value again.At last with the pH value be 7.2 the no calcium that contains 20% newborn calf serum, that magnesium Hank'S liquid is made into 6,000,000/mL lymphocyte suspension is standby.
Get 25 μ L sensitized erythrocytes and preserve liquid in 0.5mL love channel husband centrifuge tube, to remove supernatant behind the centrifugal 6min of 600rpm rotating speed, with the pH value is that 7.2 the no calcium that contains 20% newborn calf serum, magnesium Hank'S liquid suspend, and forms sensitized erythrocyte and uses liquid and also return to 25 μ L.
Getting the 0.5mL that lymphocyte suspension 15 μ L that the no calcium, magnesium Hank'S liquid of 20% newborn calf serum suspends add the no calcium that contains 15 μ L20% newborn calf serums, antibody sensitized red cell suspension that magnesium Hank'S liquid suspends likes in the channel husband centrifuge tube, abundant mixing, place 10min down at 22 ℃, to leave standstill 30min at 4 ℃ behind the centrifugal 5min of 400rPm rotating speed.
Pipettor 20 μ L volume pressure-vaccum mixing suspensions 20 times with the band suction nozzle; Get 10 μ L mixing suspensions and press blood sheet method pushing area 8～10cm
2Cell smear, with the dyeing of improvement Ji nurse Sa decoration method, high power lens is counted after the air dry.
Improvement Ji's nurse Sa decoration method and decision method are with embodiment 1.
Testing result: CD3
+Be 64%, CD4
+Be 43%, CD8
+Be 33%, CD4
+/ CD8
+Be 1.30.
Embodiment 3 is at lyophilized antibodies sensitized erythrocyte garland reagent WuT
3, WuT
4, WuT
8In to add 0.5mL pH value respectively be 7.4 no calcium, magnesium Hank'S liquid, be dissolved into sensitized erythrocyte and preserve liquid.Cell suspension after the dissolving can add 0.1%NaN as toing many or too much for use
3, sealing can preserve for 2～4 weeks at 4～6 ℃.
Use anticoagulant heparin, extract patient venous blood 2mL on an empty stomach, add 4mL pH value and be 7.4 no calcium, magnesium Hank'S liquid mixing after, slowly be added on the 3mL lymphocyte separation medium with dropper, under 18 ℃, the hydro-extractor that with radius is 22cm is with the centrifugal 25min of the rotating speed of 1200rpm.
With dropper sucking-off mononuclearcell, putting into and being added with 4～6 times of amount pH values is 7.4 no calcium, another centrifuge tube of magnesium Hank'S liquid, inhales lymphocyte separation medium less as far as possible.Mixing, centrifuge speed are 600rpm, and centrifugal 6min abandons supernatant, are that 7.4 no calcium, magnesium Hank'S liquid are washed once with the pH value again.At last with the pH value be 7.4 the no calcium that contains 20% newborn calf serum, that magnesium Hank'S liquid is made into 5,000,000/mL lymphocyte suspension is standby.
Get 20 μ L sensitized erythrocytes and preserve liquid in 0.5mL love channel husband centrifuge tube, to remove supernatant behind the centrifugal 10min of 500rpm rotating speed, with the pH value is that 7.4 the no calcium that contains 20% newborn calf serum, magnesium Hank'S liquid suspend, and forms sensitized erythrocyte and uses liquid and also return to 20 μ L.
Getting lymphocyte suspension 20 μ L that the no calcium, magnesium Hank'S liquid of 20% newborn calf serum suspends adds the no calcium that contains 20 μ L20% newborn calf serums, sensitized erythrocyte that magnesium Hank'S liquid suspends and uses the 0.5mL of liquid and like in the channel husband centrifuge tube, abundant mixing, place 10min down at 18 ℃, to leave standstill 45min at 4 ℃ behind the centrifugal 5min of 500rpm rotating speed.
Pipettor 25 μ L volume pressure-vaccum mixing suspensions 15 times with the band suction nozzle; Get 10 μ L mixing suspensions and press blood sheet method pushing area 8～10cm
2Cell smear, with the dyeing of improvement Ji nurse Sa decoration method, high power lens is counted after the air dry.
Improvement Ji's nurse Sa decoration method and decision method are with embodiment 1.
Testing result: CD3
+Be 77%, CD4
+Be 40%, CD8
+Be 30%, CD4
+/ CD8
+Be 1.33.
Embodiment 4 is at lyophilized antibodies sensitized erythrocyte garland reagent WuT
3, WuT
4, WuT
8In to add 0.5mL pH value respectively be 7.4 phosphate buffer, be dissolved into sensitized erythrocyte and preserve liquid.Cell suspension after the dissolving can add 0.1%NaN as toing many or too much for use
3, sealing can preserve for 2～4 weeks at 4～6 ℃.
Use EDTA-Na
2Anti-freezing, extract patient venous blood 1.5mL on an empty stomach, add 2mL pH value and be 7.0 no calcium, magnesium Hank'S liquid mixing after, slowly be added on the 2.5mL lymphocyte separation medium with dropper, under 20 ℃, the hydro-extractor that with radius is 22cm is with the centrifugal 15min of the rotating speed of 1200rpm.
With dropper sucking-off mononuclearcell, putting into and being added with 4～6 times of amount pH values is 7.0 no calcium, another centrifuge tube of magnesium Hank'S liquid, inhales lymphocyte separation medium less as far as possible.Mixing, centrifuge speed are 400rpm, and centrifugal 10min abandons supernatant, are that 7.0 no calcium, magnesium Hank'S liquid are washed once with the pH value again.At last with the pH value be 7.0 the no calcium that contains 20% newborn calf serum, that magnesium Hank'S liquid is made into 4,500,000/mL lymphocyte suspension is standby.
Get 25 μ L sensitized erythrocytes and preserve liquid in 0.5mL love channel husband centrifuge tube, to remove supernatant behind the centrifugal 6min of 600rpm rotating speed, with the pH value is that 7.0 the no calcium that contains 20% newborn calf serum, magnesium Hank'S liquid suspend, and forms sensitized erythrocyte and uses liquid and also return to 25 μ L.
Getting lymphocyte suspension 25 μ L that the no calcium, magnesium Hank'S liquid of 20% newborn calf serum suspends adds the no calcium that contains 25 μ L20% newborn calf serums, sensitized erythrocyte that magnesium Hank'S liquid suspends and uses the 0.5mL of liquid and like in the channel husband centrifuge tube, abundant mixing, place 10min down at 20 ℃, to leave standstill 40min at 4 ℃ behind the centrifugal 5min of 300rPm rotating speed.
Pipettor 20 μ L volume pressure-vaccum mixing suspensions 20 times with the band suction nozzle; Get 10 μ L mixing suspensions and press blood sheet method pushing area 8～10cm
2Cell smear, with the dyeing of improvement Ji nurse Sa decoration method, high power lens is counted after the air dry.
Improvement Ji's nurse Sa decoration method and decision method are with embodiment 1.
Testing result: CD3
+Be 55%, CD4
+Be 38%, CD8
+Be 14%, CD4
+/ CD8
+Be 2.79.
Embodiment 5 is at lyophilized antibodies sensitized erythrocyte garland reagent WuT
3, WuT
4, WuT
8In to add 0.5mL pH value respectively be 7.2 phosphate buffer, be dissolved into sensitized erythrocyte and preserve liquid.Cell suspension after the dissolving can add 0.1%NaN as toing many or too much for use
3, sealing can preserve for 2～4 weeks at 4～6 ℃.
Use anticoagulant heparin, extract patient venous blood 2mL on an empty stomach, add 3mL pH value and be 7.2 no calcium, magnesium Hank'S liquid mixing after, slowly be added on the 3mL lymphocyte separation medium with dropper, under 28 ℃, the hydro-extractor that with radius is 22cm is with the centrifugal 20min of the rotating speed of 900rpm.
With dropper sucking-off mononuclearcell, putting into and being added with 4～6 times of amount pH values is 7.2 no calcium, another centrifuge tube of magnesium Hank'S liquid, inhales lymphocyte separation medium less as far as possible.Mixing, centrifuge speed are 600rpm, and centrifugal 8min abandons supernatant, are that 7.2 no calcium, magnesium Hank'S liquid are washed once with the pH value again.At last with the pH value be 7.2 the no calcium that contains 20% newborn calf serum, that magnesium Hank'S liquid is made into 4,500,000/mL lymphocyte suspension is standby.
Get 25 μ L sensitized erythrocytes and preserve liquid in 0.5mL love channel husband centrifuge tube, to remove supernatant behind the centrifugal 8min of 600rpm rotating speed, with the pH value is that 7.2 the no calcium that contains 20% newborn calf serum, magnesium Hank'S liquid suspend, and forms sensitized erythrocyte and uses liquid and also return to 25 μ L.
Getting lymphocyte suspension 25 μ L that the no calcium, magnesium Hank'S liquid of 20% newborn calf serum suspends adds the no calcium that contains 25 μ L20% newborn calf serums, sensitized erythrocyte that magnesium Hank'S liquid suspends and uses the 0.5mL of liquid and like in the channel husband centrifuge tube, abundant mixing, place 10min down at 28 ℃, to leave standstill 20min at 4 ℃ behind the centrifugal 5min of 400rpm rotating speed.
Pipettor 25 μ L volume pressure-vaccum mixing suspensions 15 times with the band suction nozzle; Get 10 μ L mixing suspensions and press blood sheet method pushing area 8～10cm
2Cell smear, with the dyeing of improvement Ji nurse Sa decoration method, high power lens is counted after the air dry.
Improvement Ji's nurse Sa decoration method and decision method are with embodiment 1.
Testing result: CD3
+Be 45%, CD4
+Be 14%, CD8
+Be 38%, CD4
+/ CD8
+Be 0.37.
Embodiment 6 adds 0.5mL pH value in lyophilized antibodies sensitized erythrocyte garland reagent WuB be 7.0 phosphate buffer, is dissolved into sensitized erythrocyte and preserves liquid; Cell suspension after the dissolving can add 0.1%NaN as toing many or too much for use
3, sealing can preserve for 2～4 weeks at 4～6 ℃.
Use anticoagulant heparin, extract patient venous blood 2mL on an empty stomach, add 2.5mL pH value and be 7.4 no calcium, magnesium Hank'S liquid mixing after, slowly be added on the 3mL lymphocyte separation medium with dropper, under 26 ℃, the hydro-extractor that with radius is 22cm is with the centrifugal 15min of the rotating speed of 1000rpm.
With dropper sucking-off mononuclearcell, putting into and being added with 4～6 times of amount pH values is 7.4 no calcium, another centrifuge tube of magnesium Hank'S liquid, inhales lymphocyte separation medium less as far as possible.Mixing, centrifuge speed are 600rpm, and centrifugal 8min abandons supernatant, are that 7.4 no calcium, magnesium Hank'S liquid are washed once with the pH value again.At last with the pH value be 7.4 the no calcium that contains 20% newborn calf serum, that magnesium Hank'S liquid is made into 5,500,000/mL lymphocyte suspension is standby.
Get 25 μ L sensitized erythrocytes and preserve liquid in 0.5mL love channel husband centrifuge tube, to remove supernatant behind the centrifugal 8min of 600rpm rotating speed, with the pH value is that 7.4 the no calcium that contains 20% newborn calf serum, magnesium Hank'S liquid suspend, and forms sensitized erythrocyte and uses liquid and also return to 25 μ L.
Getting the 0.5mL that lymphocyte suspension 25 μ L that the no calcium, magnesium Hank'S liquid of 20% newborn calf serum suspends add the no calcium that contains 25 μ L20% newborn calf serums, antibody sensitized red cell suspension that magnesium Hank'S liquid suspends likes in the channel husband centrifuge tube, abundant mixing, place 10min down at 26 ℃, to leave standstill 45min at 4 ℃ behind the centrifugal 5min of 500rpm rotating speed.
Pipettor 20 μ L volume pressure-vaccum mixing suspensions 20 times with the band suction nozzle; Get 10 μ L mixing suspensions and press blood sheet method pushing area 8～10cm
2Cell smear, with the dyeing of improvement Ji nurse Sa decoration method, high power lens is counted after the air dry.
Improvement Ji's nurse Sa decoration method and decision method are with embodiment 1.
Testing result: the B cell is 9%
Embodiment 7 is at lyophilized antibodies sensitized erythrocyte garland reagent WuB, WuT
3, WuT
4, WuT
8In to add 0.5mL pH value respectively be 7.0 phosphate buffer, be dissolved into sensitized erythrocyte and preserve liquid; Cell suspension after the dissolving can add 0.1%NaN as toing many or too much for use
3, sealing can preserve for 2～4 weeks at 4～6 ℃.
Use anticoagulant heparin, extract patient venous blood 1mL on an empty stomach, add 3mL pH value and be 7.4 no calcium, magnesium Hank'S liquid mixing after, slowly be added on the 3mL lymphocyte separation medium with dropper, under 26 ℃, the hydro-extractor that with radius is 22cm is with the centrifugal 15min of the rotating speed of 1000rpm.
With dropper sucking-off mononuclearcell, putting into and being added with 4～6 times of amount pH values is 7.4 no calcium, another centrifuge tube of magnesium Hank'S liquid, inhales lymphocyte separation medium less as far as possible.Mixing, centrifuge speed are 600rpm, and centrifugal 8min abandons supernatant, are that 7.4 no calcium, magnesium Hank'S liquid are washed once with the pH value again.At last with the pH value be 7.4 the no calcium that contains 20% newborn calf serum, that magnesium Hank'S liquid is made into 5,500,000/mL lymphocyte suspension is standby.
Get 25 μ L sensitized erythrocytes and preserve liquid in 0.5mL love channel husband centrifuge tube, to remove supernatant behind the centrifugal 8min of 600rpm rotating speed, with the pH value is that 7.4 the no calcium that contains 20% newborn calf serum, magnesium Hank'S liquid suspend, and forms sensitized erythrocyte and uses liquid and also return to 25 μ L.
Getting the 0.5mL that lymphocyte suspension 25 μ L that the no calcium, magnesium Hank'S liquid of 20% newborn calf serum suspends add the no calcium that contains 25 μ L20% newborn calf serums, antibody sensitized red cell suspension that magnesium Hank'S liquid suspends likes in the channel husband centrifuge tube, abundant mixing, place 10min down at 26 ℃, to leave standstill 45min at 4 ℃ behind the centrifugal 5min of 300rpm rotating speed.
Pipettor 20 μ L volume pressure-vaccum mixing suspensions 20 times with the band suction nozzle; Get 10 μ L mixing suspensions and press blood sheet method pushing area 8～10cm
2Cell smear, with the dyeing of improvement Ji nurse Sa decoration method, high power lens is counted after the air dry.
Improvement Ji's nurse Sa decoration method and decision method are with embodiment 1.
Testing result: the B cell is 9%, CD3
+Be 55%, CD4
+Be 44%, CD8
+Be 30%, CD4
+/ CD8
+Be 1.47.
Embodiment 8 gets 30 routine samples, adopts method of the present invention and former McAb-A-E direct method to compare test respectively, and testing result is as follows:
Through paired t-test method statistical analysis, its result is as follows with SPSS10.0 version statistical software:
Two groups of CD3 compare: t=-0.672, P=0.507
Two groups of CD4 compare: t=0.793, P=0.434
Two groups of CD8 compare: t=-0.192, P=0.849
Two groups of CD4/CD8 compare: t=0.753, P=0.458
Four groups of results' P value is all greater than 0.05 (P〉0.05), and difference does not have conspicuousness.Be that method of the present invention and former McAb-A-E direct method testing result difference do not have conspicuousness.
Claims (2)
1. monoclonal antibody SPA red blood cell garland method detects the method for lymphocyte subgroup, comprises the steps:
(1) adding 0.5mL pH value in lyophilized antibodies sensitized erythrocyte garland reagent is 7.0～7.4 no calcium, magnesium Han Keshi balanced salt solution or phosphate buffer, is dissolved into sensitized erythrocyte preservation liquid;
(2) venous blood samples, volume ratio adding pH value with 1: 1～3 is 7.0～7.4 no calcium, magnesium Han Keshi balanced salt solution, behind the mixing, be added on the lymphocyte separation medium, the amount of lymphocyte separation medium and the volume ratio of dilute blood are 1: 2～3, under 18～28 ℃ of temperature, with the centrifugal 15～25min of the rotating speed of 800～1200rpm; Take out mononuclearcell, and centrifugal to mononuclearcell, each with the centrifugal 6～10min of the rotating speed of 400～600rpm with no calcium, magnesium Han Keshi balanced salt solution washing back, abandon supernatant; Be that 7.0～7.4 no calcium, the magnesium Han Keshi balanced salt solutions that contain 20% newborn calf serum are made into 400～6,000,000/mL lymphocyte suspension with the pH value then;
(3) get sensitized erythrocyte and preserve liquid, to remove supernatant behind the centrifugal 6～10min of 400～600rpm rotating speed, with the pH value is that 7.0～7.4 no calcium that contain 20% newborn calf serum, magnesium Han Keshi balanced salt solutions suspend, and forms with the institute sensitized erythrocyte of getting and preserves the sensitized erythrocyte application liquid of liquid equivalent;
(4) lymphocyte suspension and sensitized erythrocyte are used liquid and are mixed into 1: 1 volume ratio and mix suspension, place 10min under 18～28 ℃ of temperature; To leave standstill 15～45min at 4 ℃ behind the centrifugal 5min of 300～500rpm rotating speed;
(5) with the pipettor pressure-vaccum mixing suspension 20 of the band suction nozzle of 20 μ L or 25 μ L volumes or 15 times; Get 10 μ L mixing suspensions and press blood sheet method pushing area 8～10cm
2Cell smear, with the dyeing of improvement Ji nurse Sa decoration method, wherein improve Ji's nurse Sa decoration method and be meant dyeing liquor 3～5 is dripped off all standing cell smear that about 1min dyes after the air dry, add the pH value and be 6～10 dyeing of phosphate buffer, 2～5min of 6.4～6.8, the water flushing gets final product; Count with high power lens then: sticking around the lymphocyte has three above red blood cells to be the garland positive cell, and the negative cell of person that do not stick the red blood cell sticks one, two red blood cell person and removes and disregard; During counting, choose that zone, at least two places amounts to several 200 cells between the cell smear cephalocaudal 2/6～4/6, calculate the percent of rosette formation cell.
2. monoclonal antibody SPA red blood cell garland method as claimed in claim 1 detects the method for lymphocyte subgroup, it is characterized in that: the venous blood in the described step (2) adopts heparin or disodium EDTA anti-coagulants to extract.
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| 李代渝等.红细胞花环法在急淋白血病免疫分型中的应用.《上海医学检验杂志》.1993,第8卷(第1期),13-14. * |
| 杨萍等.单疱性角膜炎患者细胞免疫状态的研究——以Wu系列McAb测定外周血T细胞亚群.《眼科研究》.1991,第9卷(第1期),8-11. * |
| 肖宗清等.葡萄球菌免疫球蛋白花环法检测人外周血T淋巴细胞亚群的初步探讨.《上海免疫学杂志》.1988,第8卷(第3期),211-213. * |
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