CN106267422B

CN106267422B - Rh blood group incompatibility haemolysis disease therapeutic apparatus

Info

Publication number: CN106267422B
Application number: CN201610540876.4A
Authority: CN
Inventors: 翁炳焕; 李兰娟
Original assignee: Individual
Current assignee: Shu Lan Hangzhou Hospital Ltd; Womens Hospital of Zhejiang University School of Medicine
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2019-01-22
Anticipated expiration: 2036-07-01
Also published as: CN106267422A

Abstract

The present invention relates to the Rh blood group incompatibility haemolysis disease therapeutic apparatus of medical domain, it is characterized in that alternately washing the rhesus monkey erythrocytes containing the common Rh antigen of the mankind with the PB lysate that osmotic concentration is 25 and 35mmol/L, it is made and has dialysed hemoglobin but retained the antigenic ghost that can adsorb Rh antibody of Rh, it is fixed on the agarose of special concentration with specific proportion jointly with the anti-Rh antibody prepared with immunology, followed by assemble cylindrical adsorption device made of high-biocompatibility material, it is allowed to form antibody titer from high to low from sample introduction end to sample outlet end and agarose concentration but ghost from low to high is to be uniformly distributed, and nearly sample outlet end due to concentration is gradually high molecule micropore gradually the agarose of small special concentration and the mesh screen in absorber exit can the hematoclasis of effectively detention haemolysis patient produce Object is that one kind only removes pathogenic antibody, does not remove the treatment new method for the female tire Rh blood group incompatibility Hemolysis of blood plasma components having no toxic side effect.

Description

Rh blood group incompatibility haemolysis disease therapeutic apparatus

Technical field

The present invention relates to a kind of Rh blood group incompatibility haemolysis disease therapeutic apparatus for medical domain, are mainly used for female tire Rh blood group The removing of anti-fetal red blood cells antibody and red blood cell lysate in pregnant woman blood plasma is not conformed to.

Background technique

Fetus inherits the gene element of father and each half of mother, and fetal red blood cells can carry the antigen from male parent, The blood group for showing as fetus is different from parent.After the red blood cell of fetus enters the blood circulation of parent, the immune of parent is induced System generates antibody, and antibody enters fetal circulation system by placenta, in conjunction with fetal red blood cells, breaks fetal red blood cells It is bad, lead to female tire blood group incompatibility hemolytic disease of fetus an d neonate.

Human erythrocyte's blood group has 26 kinds, including the blood groups such as abo blood group, Rh blood group and MN, Lew, Kell and Fya, but The blood group of female tire blood group incompatibility Hemolysis can be caused with Rh blood group and abo blood group to be most common.Rh is rhesus macaque (Rhesus Macacus) two letters of the head of foreign language title.1940, the scientists such as blue moral stainer were in the erythrocyte immune with rhesus macaque When cavy and rabbit, the antibody (agglutinin) of anti-rhesus monkey erythrocytes is as a result generated in cavy and rabbit anteserum, then with containing this The serum of kind antibody is mixed with the red blood cell of people, and there are about 85% white red blood cells to be shown by this serum agglutination for discovery There is antigen identical with rhesus macaque, therefore referred to as Rh positive blood group on these white red blood cells；Separately there are about 15% white people Red blood cell not by this serum agglutination, referred to as Rh negative blood group, this blood group system is known as Rh blood group.Rh blood group antigens are It is determined by the allele of 3 pairs of close linkages on No. 1 chromosome, shares 6 kinds of antigens, i.e. C and c, D and d, E and e.Since D is anti- Original is found earliest, and antigenicity is most strong, therefore clinically all D antigen positive persons are known as the Rh positive, and no D antigen person is known as Rh yin Property.The antigenicity of Rh blood group antigens determines the severity of Hemolysis, and D antigen can cause serious Hemolysis, secondly anti-for E Original is again C, c and e antigen, and the antigenicity of d antigen is most weak, and anti-d antibody there is no to find at present.Han nationality and other big in China Part is national, and the people that the people of the Rh positive accounts for about 99%, Rh feminine gender only accounts for 1% or so, but in other ethnic groups, and Rh is negative People it is more, if Miao ethnic group be 12.3%, Tartar 15.8%.

Although the incidence that abo blood group does not conform to is very high, fetus haemolysis incidence is very low, even if haemolysis occurs, symptom is also Relatively light, few that nuclear icterus and oedema occurs, the gestational period is not necessarily to specially treated.And Rh blood group incompatibility is although rare, but it causes mother The severity extent of tire blood group incompatibility Hemolysis will overweight abo blood group and not conform to, so the diagnosis and prevention to Rh blood group incompatibility are extremely It is important.Rh blood group incompatibility generates the Rh antibody (the anti-D of IgG) of a large amount of anti-fetal red blood cells due to parent, into fetus body in after it is broken Bad a large amount of fetal red blood cells, make fetus severe anemia, anoxic, heart failure, hepar damnification, Hypoproteinemia, anasarca, chest Water, ascites even Intrauterine Fetal Death.In non-neonate, Rh blood group incompatibility haemolysis does not conform to compared with abo blood group there is morning jaundice time, most Early to occur after birth in 12 hours, majority appeared in 24 hours.Due to haemolysis generate a large amount of bilirubin cannot in time from Liver excludes, and aggravates icterus neonatorum, and a large amount of bilirubin penetrates into brain cell and causes nuclear icterus, often die of severe anemia, Heart failure, nuclear icterus, the death rate are very high.

Although Rh antibody passes through destruction fetus red blood cell after entering in fetus body can cause serious Hemolysis, certain Pregnant woman also can be by injecting Rh antibody in advance come Rh (D) positive red blood cell sensitization body of pre- anti-intrusion, and then it is molten to prevent Rh The generation of blood disease.Since current most countries have forbidden being carried out that D antigen is immune, and the source of the polyclonal anti-D of Ig is with volunteer It is extremely limited, and utilize the anti-D of Epstein-Barr virus conversion human B lymphocyte secretion Ig or Epstein-Barr virus conversion combination cell fusion preparation Ig Anti- D, because in vivo using EBV conversion bone-marrow-derived lymphocyte strain or hybridoma cell strain secretion the anti-D of Ig there are still much ask Topic, such as contained EBV have potential pathogenic；It may be with pathogen such as HIV and hepatitis virus；It is produced with hybridoma technology The anti-D albumen containing source of mouse of Ig easily causes allergic reaction or the oncogene with mouse, so by directly injecting Ig in vivo Anti- D is just restricted to prevent Rh Hemolysis.

Clinically mainly there are pregnancy period plasma exchange, fetal transfusion, termination to the treatment of female tire blood group incompatibility Hemolysis at present Gestation and neonatal exchange transfusion treatment.Anti- D potency needs to consider intrauterine transfusion 32 or more, and fetal transfusion includes that fetus is intraperitoneal defeated Blood and the intravascular blood transfusion of fetus, all have certain risk, and none is proved effectively；Hyperbilirubinemia of newborn person can be used The drug therapies such as blue light illumination, intravenous injection of immunoglobulin, Blood exchanging therapy, Blood exchanging therapy are still that treatment newborn is molten when necessary The main method of blood disease；Anti- D potency is 64 or more during gestation, need to consider replacement therapy of blood plasma, pregnant woman blood plasma displacement be exactly The haemocyte and blood plasma that method separation pregnant woman is filtered in extracorporal circulatory system access, remove blood plasma and with the displacement liquid of equivalent (fresh ice Freeze blood plasma or 5% albumin) supplement feedback, each 2 000~3 000ml of replacement amount, 20~30ml/min of speed, treatment time 2~4h, every 1~3d are treated 1 time；Or pregnant woman's whole blood about 400ml is taken every time, its blood plasma about 300ml, supplement etc. are removed after low-temperature centrifugation Measure homotype fresh plasma, also defeated Autoerythrocyte (RBC).Plasma exchange can proportionally be reduced with the plasma volume being removed and be caused a disease The titre (potency) of antibody, so as to extend fetus in female intracorporal survival and growth and development time, terminal pregnancy can be postponed Time is the good selection that the pregnant woman of female tire ABO, Rh or other blood group incompatibilities prevents miscarriage in treatment in clinical early stage, has preferable Curative effect, also without other adverse reactions.But plasma exchange can only remove the partial antibody in blood, cannot stop the continuation of antibody Generate, the female tire blood group incompatibility Hemolysis occurred can not be reversed, need to carry out incessantly plasma exchange just it is effective, only fit Pregnant woman or spouse for fetus edema once to occur before gestation 20~22 weeks are the homozygote person of pathogenic antigens, especially While removing part pathogenic antibody, a large amount of blood plasma (multiple beneficial composition) is also eliminated together, although making with displacement liquid Supplement, but can not supply completely and be removed blood plasma and its various beneficials, and the displacement liquid measure substituted is big, expense is high Expensive, supplement allosome blood plasma easily causes the various side effects such as infectious disease and infusion reaction, and which limits the generally developments of plasma exchange. So there is an urgent need to a kind of only removal pathogenic antibody, not removing blood plasma (multiple beneficial composition), without using plasma exchange liquid Inexpensively, the replacement therapy of blood plasma new method of female tire blood group incompatibility Hemolysis safe, without side-effects.

Ag-Ab precipitation reaction was initially applied to research Liesegang's phenomenon in 1905 in gel, applied within 1932 In identification bacterium bacterial strain, nineteen forty-six Oudin carries out immunodiffusion in test tube, for analyzing antigen mixture, 1948 Elek and Ouchterlony establishes agar double diffusion test respectively, for identifying two or more antigens or antibody.Resist in gel The media gel agar or agarose of antigen-antibody precipitation reaction are a kind of polysaccharide bodies containing sulfate, and when high temperature can be dissolved in water, Gel is congealed into after cold, inside forms a kind of porous reticular structure, and aperture is very big, allows macromolecular substances (molecular weight Up to million or more) it passes freely through.The size in aperture further depends on agar concentration, and agar concentration is big, and aperture is relatively small, agar Concentration is small, and aperture is relatively large.The aperture of 1% agar gel is about 85nm, since agar or agarose have chemistry well Stability, water content is big after gel, and transparency is good, and convenient sources are easy to handle, therefore is a kind of good dispersive medium.Antigen Molecular weight with antibody generally all 200,000 hereinafter, be in free diffusing in gel substantially.When antigen and corresponding antibodies are through spreading When meeting in gel afterwards, forms antigen antibody complex and formed maximum compound if the two is appropriate in place's ratio of meeting Object.Since the molecular weight of compound increases, particle increases, thus does not continue to spread and generate precipitating, and this precipitating is just formed One " specific barrier ", all same antigen or antibody molecule cannot pass through, and the different molecule of property can lead to It crosses this barrier and continues to spread, until forming the compound of themselves.In this way, synantigen is not formed by precipitating respectively There is each position.Such reaction is known as agar gel diffusion, is at present with the routine of known antibodies detection unknown quantity corresponding antigens A certain amount of goat-anti people Ig antiserum ingredient is usually mixed in agar gel by laboratory diagnosis project, is made containing specificity The sero-fast agar plate of goat-anti people Ig is properly located to occur when serum to be checked is spread in agar plate in antigen and antibody ratios In conjunction with forming macroscopic white precipitate and no longer spread, in addition when female tire blood group incompatibility generates anti-fetal red blood cells antibody When, hematoclasis is caused in the presence of complement after antibody and erythrocyte binding, generates the destruction product of bigger molecule, it can be by certain The micropore institute detention of a little particular sizes.But so far there are no according to above-mentioned Mechanism Design treatment blood group incompatibility Hemolysis.

Summary of the invention

In order to solve the problems, such as to attack the treatment for the female tire Rh blood group incompatibility Hemolysis being unable to long, present inventors have proposed this hairs It is bright.

The invention aims to provide Rh blood group incompatibility haemolysis disease therapeutic apparatus；Another object is to provide for the system of therapeutic equipment Standby and application method.

The object of the present invention is achieved like this: the PB lysate for the PH7.4 that osmotic concentration is 25 and 35mmol/L is prepared, Alternately rhesus monkey erythrocytes of the washing containing the common Rh antigen of the mankind are made and have dialysed hemoglobin but retained antigenicity and can have The ghost of effect absorption Rh antibody immortalizes the B leaching that Rh antibody can be generated after Rh positive red blood cell sensitization separately with Epstein-Barr virus Bar cell, and by it with people and/or rat bone marrow tumour cell are fused becomes hybridoma bone-marrow-derived lymphocyte, prepare Rh antibody, then conduct Antigen-immunized animal prepares anti-Rh antibody, respectively with after 100 DEG C dissolve keep the temperature 42 DEG C 0.9%, 1.0%, 1.1%, 1.2%, 1.3% agarose is made into the application antibody of 1: 700,1: 500,1: 300,1: 200,1: 100 potency gradient, and is incorporated 95% ghost is successively taken 55~65ml to be added with entrance made of high-biocompatibility material from low to high by potency Spy has set in the hydrostatic column that can stop specific particle mesh screen, and make first to be added is cooled to after semi-solid gel just using antibody Then plus next time, be made make the gel in container antibody titer from high to low is formed from sample introduction end to sample outlet end and from as low as The high equally distributed absorber of agarose concentration and ghost, when the blood plasma separated in extracorporal circulatory system filters out absorber, The ghost and anti-Rh antibody that Rh antibody is fixed in adsorbent are combined into fixed compound, and the red blood cell being destroyed is broken Piece and it is in combination made of macromolecular immune complex by nearly absorber outlet end concentration is gradually high and molecular sieve gradually small agar Gel detention, remove fed back after the blood plasma of morbid substance is filtered out from absorber it is internal.

Rh antibody is the virulence factor of female tire Rh blood group incompatibility Hemolysis, and it is common that rhesus monkey erythrocytes contain human erythrocyte Rh antigen, be the immunising antigen of Rh antibody, and anti-Rh antibody is the immune antibody of Rh antibody, the two can be with Rh antibody Immune response occurs and forms compound, the present invention prepares PB lysate and be made with dialysing using rhesus monkey erythrocytes as raw material Except the hemoglobin for being harmful to human body but retains the antigenicity of absorption Rh antibody and do not adsorb the natural antibody of anti-A and anti-B Ghost, agarose medium is fixed on specific component jointly with the anti-Rh antibody prepared with immunology and absorption is made Agent can form fixed immune complex in conjunction with the Rh antibody in filtration blood plasma to be removed, and agar is solidifying Glue is formed by filter opening and reduces with increasing for agarose concentration, and the agarose concentration of nearly absorber entrance is low, and filter opening is just Greatly, be conducive to the combination of plasma perfusion and ghost and high titre anti-Rh antibody and Rh antibody, and the concentration in nearly exit is gradually Height, filter opening is just gradually small, be easy to detention Hemolysis erythrocyte destruction be formed by fragment and it is in combination made of macromolecular Immune complex, in absorber exit, the mesh screen that can stop particular size particle of setting also has the production of detention hematoclasis The effect of object forms the double barrier of specific immunity absorption and non-specific mechanical stop, special agar to morbid substance Semisolid is congealed into after sugared medium is cooling, inside forms the reticular structure of even porous, is conducive to free antigen and antibody and blood The uniform diffusion for starching composition, avoids the dead space of blood plasma liquid stream, increases ghost and anti-Rh antibody adsorbs the equal of Rh antibody Even property and surface area, enhance the effect of adsorbing therapy, and the blood plasma after having adsorbed Rh antibody is fed back in vivo after filtering out absorber, real Showed manually by Rh antibody from external treatment is transferred in vivo, be it is a kind of only remove pathogenic antibody, do not remove blood plasma and its more Female tire Rh blood group incompatibility Hemolysis cheap, safe, without side-effects without using plasma exchange liquid of kind beneficial is controlled Treat new method.

Specific embodiment

Fig. 1 is the application schematic diagram of the Rh blood group incompatibility haemolysis disease therapeutic apparatus proposed according to the present invention.

Fig. 2 is the schematic diagram of internal structure of the plasma separator proposed according to the present invention.

Fig. 3 is the schematic diagram of internal structure of the absorber proposed according to the present invention.

In Fig. 1, one end of arterial blood line pipe (1) is connected with arteries, and the other end is through heparin pump (2) and blood pump (3) it is connected with plasma separator (4), plasma separator (4) absorption in parallel with two through blood plasma pump (6) and circulation line (7) Device (8), absorber (9) be connected, be then successively connected with circulation line (10), venous line (5), venous line (5) it is another End is connected with vein blood vessel.

In Fig. 2,1 is plasma separator, and 2 be plasma separator inner cavity, and 3 be the micropore on the tube wall of plasma separator inner cavity, 4 Being cannot be by the haemocyte of micropore (3), and 5 be the small molecule blood plasma components that can pass through micropore (3), and 6 be plasma separator exocoel, 7 be blood plasma outflux, and 8 be switchable valve.

In Fig. 3,1 is the Rh antibody moved down into after absorber, and 2 are fixed in the anti-Rh antibody in agar gel, and 3 are The anti-Rh antibody being fixed in agar gel combines the secure bond object formed after the Rh antibody moved down, and 4 are fixed in Rh positive Rhesus ghost in agar gel, 5 are fixed in the Rh positive Rhesus ghost in agar gel The secure bond object formed after the Rh antibody moved down is combined, 6 be the agar gel containing micropore, and 7 be by agar gel micropore The Rh antibody of detention.

Below with reference to Fig. 1, Fig. 2 and Fig. 3, preparation and application to Rh blood group incompatibility haemolysis disease therapeutic apparatus proposed by the present invention Embodiment be explained in detail.

One, the preparation of Rh (D) positive Rhesus ghost

1, primary red blood cell: not by the Rh (D) of the anti-A of ABO blood group system and anti-B serum agglutination positive " O " type Henghe Monkey red blood cells.

2, main agents: 30mmol/L PB buffer: solution A is 0.04mol/L Na₂HPO₄(Na₂HPO₄.12H₂O 14.328g is dissolved in 1000ml deionized water, and room temperature storage is spare)；Second liquid is 0.04mol/L NaH₂PO₄ (NaH₂PO₄.2H₂O 6.24g is dissolved in 1000ml deionized water, and room temperature storage is spare).Solution A 81.0ml and second liquid are taken respectively 19.0ml mixing 40mmol/L PB, then on the basis of 40mmol/L PB buffer plus deionized water dilution 0.75 30mmol/LPB buffer is become again, is made into 25 and 35mmol/L in proportion with method.Anti- D standard items and Rh (D) standard are red thin Born of the same parents are purchased from Guangzhou Lian Tai Bioisystech Co., Ltd.

3, preparation method

(1) the positive O-shaped rhesus monkey erythrocytes of the fresh Rh of sufficient amount are taken, respectively under conditions of 4 DEG C and 37 DEG C, with The centrifugal speed of 1500r/min × 5min is cleaned red blood cell 4~5 times with isometric physiological saline, to remove erythrocyte surface The abo blood group and Rh blood group and other immune antibodies that may adhere to or natural antibody.

(2) with 0.04mol/L Na₂HPO₄With 0.04mol/L NaH₂PO₄The osmotic concentration of preparation is 25 and 35mmol/L PH7.4 PB lysate, under 4 DEG C and 2500r/min × 10min of centrifugal condition, alternately and repeatedly Washed Red Blood Cells, pass through The alternating of osmotic concentration changes, and hemoglobin is promoted to give outside red blood cell and be removed.

(3) finally with physiological saline cleaning until supernatant is colourless, with the blood being harmful to the human body in red blood cell of dialysing completely Lactoferrin, precipitating are the ghost of the antigen containing D.

(4) detection of antigenicity: 1. by ghost and 37 DEG C of incubation 5min of anti-D standard items, the detection of centrifuging and taking supernatant Anti- D standard items potency reduces numerical value to determine the antigenicity of ghost.Specific method is: taking 10, test tube, respectively number 1 ~10, the 1st pipe is added ghost and precipitates 1.0ml, then other each pipes plus physiological saline 0.5ml inhale ghost from the 1st pipe Precipitating 0.5ml is added to the 2nd pipe, and 0.5ml to the 3rd is inhaled after mixing and is managed, and is diluted to the 10th pipe with same operation, finally pipe is sucked out 0.5ml is discarded, and every pipe adds anti-D standard items 0.5ml, 37 DEG C of incubation 5min to mix gently after centrifugation, is aggregated completely with appearance, is several Maximum dilution multiple without free cell represents the antigenicity of ghost, and extension rate is bigger, and antigenicity is stronger, adsorbs Rh The ability of antibody is stronger.Supernatant antibody titer can also be measured with Rh (D) normal erythrocytes to determine the antigen of ghost Property；2. measuring supernatant antibody titer with Rh (D) normal erythrocytes, anti-D standard items potency (known) subtracts supernatant humoral antibody effect Valence is the antigenicity (potency) of ghost.Supernatant antibody titer detection method are as follows: take 10 test tubes, respectively number 1~ 10, physiological saline 1ml is added in each pipe, takes supernatant 1ml to be added to removal 1ml after the 1st pipe mixes and goes to the 2nd pipe, equally to grasp It is diluted to the 10th pipe, finally pipe is sucked out 1ml liquid and discards, by the 40 μ l of serum diluted and 10 μ l of Rh (D) normal erythrocytes Mixing is incubated for 10 minutes, is centrifuged 5 minutes interpretation results, the agglutination pipe of maximum dilution multiple is antigenic (potency), and potency is answered 1: 1500 or more.

(5) content of hemoglobin detects: with hypotonic destruction ghost, with conventional chemical luminescence analysis or fluorescence analysis Method, the qualitative content of hemoglobin in quantitative detection ghost should be lower than the reference value lower bound of human normal plasma.

(6) ghost form: observing the form and integrality of ghost under the microscope, should be in circle shadow, no-reflection.

(7) for cellular morphology in circle shadow, no-reflection, highly sensitive routine hemoglobin qualitative detection is feminine gender, quantitative Detection is lower than the reference value lower bound of human normal plasma, adsorbs the ghost of 1: 1500 or more Rh antibody titer, leaves and takes spare.

Two, the preparation of anti-Rh antibody

1, Rh feminine gender lymphocyte is prepared

(1) primary cell obtains: the purchase fresh concentration Rh feminine gender leucocyte 300ml in Zhejiang Province blood station；Separately buy fresh Rh Positive red blood cell suspension 300ml.(2) Rh feminine gender separation of lymphocytes prepares that (separating liquid is protected from light 4 degree of preservations, with preceding in 37 degree of water Heated in bath, entire separation process temperature should be controlled in 8-28 degree, influence disintegrate-quality too high or too low for temperature): sterile pumping The fresh concentration Rh feminine gender leucocyte of 20mL is taken, PBS liquid dilutes 3~5 times, 6mL is taken slowly to be folded with dropper along tube wall after mixing well Horizontal centrifugal in horizontal centrifuge (400r/min, 20 DEG C) is added in the 10mL centrifuge tube for be added 4mL lymphocyte separation medium 35min；It is divided into 3 layers after centrifugation in pipe, upper layer is blood plasma and PBS liquid, and lower layer is mainly red blood cell and granulocyte, and middle layer is lymph Cell separating liquid, having a white cloud and mist layer narrow band based on mononuclearcell in upper, middle layer interface is that single core is thin Born of the same parents (PBMC) are inserted into cloud and mist layer with capillary syring, draw PBMC and are placed in another 50mL centrifuge tube, are added 5 times with upper volume PBS It is centrifuged (300r/min, 20 DEG C) 10min, abandons supernatant, cell is resuspended in 50mLPBS, (350r/min, 20 DEG C) 15min is centrifuged, in abandoning Clearly, Buffer (PBS+0.5% newborn bovine serum+2mmol/LEDTA pH7.2) 2mL is added, cell is resuspended, predominantly Rh is negative Lymphocyte (T cell and B cell).

2, Rh feminine gender sensitized lymphocyte is prepared

Rh feminine gender lymphocyte and Rh positive red blood cell suspension are centrifuged 5min with 1000r/min, remove supernatant, regular growth Culture solution washs 1~2 time, by two kinds of sedimentation cell mixed in equal amounts, adds the regular growth culture solutions (RPMI-1640) of 5 times of amounts, and 37 DEG C, 5%CO2 is cultivated for 24 hours, is then washed centrifugation (1000r/min, 3min) with RPMI-1640 liquid and is added with removing cell debris Enter Tris-NH4Cl effect 5min, splitting erythrocyte, Quick spin (1000r/min, 3min), remove supernatant in cracking it is red Cell fragment, PBS washing centrifugation 3 times, RPMI-1640 washing centrifugation 1 time, to remove Tris-NH4Cl remaining in suspension, It is avoided to influence the survival of cell, at this point, mainly containing Rh feminine gender sensitized lymphocyte in suspension.

3, prepare the strain of Rh feminine gender sensitization B cell (Epstein-Barr virus converts Rh feminine gender sensitized lymphocyte)

Routinely Epstein-Barr virus Transformed Human Lymphocytes technology, takes Rh feminine gender sensitized lymphocyte, and adjusting concentration is 2x 10⁶Afterwards Suitable Epstein-Barr virus (EBV) stoste is added, is placed in 37 DEG C, 5%CO2 overnight incubation prepares the cause of Epstein-Barr virus conversion to be hybridized Quick lymphocyte.(1) 20% fetal calf serum (Gibco) { 56 DEG C inactivate 30 minutes }, 1.6 RPM1640 culture medium: reagent: are included ~1.8%HEPES, 1.2% glutamine, 1% penicillin and streptomysin；Cyclocyto enzyme A (CyA): 5ml (250mg)/bottle is used 1640 culture mediums are diluted to 0.2mg/ml, when use final concentration of 2ug/ml (0.5%)；Epstein-Barr virus (EBV) liquid: purchased from Chinese section Heredity institute of institute, is stored in subzero 80 degree, and when use takes out from refrigerator, and 37 degree melt rapidly, with the membrane filtration of 0.22um, It does not exceed 0.5~1 hour.(2) method: Rh feminine gender sensitized lymphocyte is taken, 6ml1640 full nutrient solution is added to be washed for the second time It washs, is centrifuged 1500 turns 15 minutes；Supernatant is abandoned, the full culture medium of 2ml 1640 is added and (before full culture medium is added, sets 37 degree of water-baths 10 Minute), Ciclosporin A (4%) and 1.2mlEBV liquid/part is then added, 25 square centimeters of culture is moved into after mixing well Bottle, sets 37 degree, in 5%CO2 incubator；If cell increases, slow or cell density is low or medium pH value is in acidity, is sucked out Half amount culture solution, carries out equivalent oil changing；It is transferred to when total amount reaches 14ml in 75ml culture bottle, every 2-3 weeks addition 5-10ml Fresh culture；Culture medium (the 1.6%1M HEPES buffer solution of 3ml Fresh is added within every 2-3 days；15% fetal calf serum (FBS)；1% penicillin and streptomysin；PRMI 1640 is supplied to 100%).7 days or so under the microscope, it is seen that lymphocyte is bright It is aobvious to increase, there is clustering phenomena；It about 6~8 weeks, when total amount reaches 45ml, sets in 50ml centrifuge tube, is centrifuged 1500 turns, 10 points Clock abandons supernatant, is made into that cell suspension is spare, and predominantly Rh feminine gender sensitization B cell strain [if to freeze, is added 3ml and freezes training It supports base (5% dimethyl sub-maple (dimethyl sufoxide), 95%FBS) to mix, at cell suspending liquid, (cell concentration is about 10⁵/ml).Cryopreservation tube packing, 1ml/ pipe, sets -20 DEG C of 2h, then set -70 DEG C of 2h, then freeze in -196 DEG C of liquid nitrogen (or immediately Moved into liquid nitrogen container after 80 degree, 1-2 hours under zero setting)].

4, screen the strain of Rh feminine gender effect B cell (Epstein-Barr virus that screening produces Rh antibody converts Rh feminine gender sensitized lymphocyte)

Effect B cell strain refers to the B cell strain that can generate Rh antibody, with antihuman globulin test or conventional ELISA method sieve Whether choosing produces Rh antibody lymphocyte: (1) Direct antiglobulin test: expressing on detection bone-marrow-derived lymphocyte strain (B cell strain) surface Rh antibody.Anti-humanglobulin serum and anti-D serum are bought, with brine 1 time and is made into 5% lymphocyte strain suspension, It takes 1 drop cell suspension and 2 to drip anti-humanglobulin serum mixing, sets low-speed centrifugal after room temperature 5min, gently mix, under naked eyes or mirror Observation, discovery lymphocyte strain agglutination person are that cell surface expression has Rh antibody (while it is outstanding to prepare unsensitized 5% lymphocyte Liquid adds anti-humanglobulin serum to do negative control；Anti- D serum is added to do positive control with Rh (D) positive red blood cell).(2) indirectly anti- Human immunoglobulin test: whether detection lymphocyte strain culture supernatant contains Rh antibody.With brine 2 times and it is made into The positive O-shaped red cell suspension of 5%Rh (D), respectively takes red cell suspension, lymphocyte strain culture supernatant and anti-humanglobulin serum 1 drop mixes, and sets low-speed centrifugal after room temperature 5min, gently mixes, and naked eyes or microscopic observation, discovery erythrocyte agglutination person are that lymph is thin Born of the same parents' strain culture supernatant contain Rh antibody (while using red cell suspension and anti-humanglobulin serum mixing tube as negative control, with Red cell suspension and anti-D serum mixing tube are positive control).Being filtered out with this direct or indirect antihuman globulin test can divide More efficient valence (compared with the positive is still tested after high magnification numbe dilution) B cell strain (hole) of Rh antibody is secreted, after resuming for 2~3 generations.

5, Rh feminine gender effect hybridoma (preparation Rh feminine gender effect B cell strain of hybridoma) is prepared

(1) culture medium and main agents: DMEM culture medium, HAT, HT Selective agar medium are purchased from Sigma company, top grade tire ox Serum (FBS) purchases Jinshi City on daytime ocean Hao biological products science and technology responsibility Co., Ltd；DMSO (-- methyl sulfoxide) it is that domestic analysis is pure Reagent.(2) myeloma cell prepares: fusion the last week takes out the myeloma cell (SP2/0) that a pipe freezes out of liquid nitrogen container, stands Be put into hot water thaw (using it is most be Sp2/0 cell strain, the cell strain growth and fusion efficiencies are good, itself is not secreted The highest growth scale of any heavy chain immunoglobulin or light chain, cell is 9 × 10⁵/ ml, doubling time are usually 10~15h； Selection homologous cell strain is considered in practical application relevant to human body, if Shanghai Fu Xiang Biotechnology Co., Ltd is to ATCC cell The NCI-H929 human myeloma cell strain that library is introduced).Appropriate complete culture solution is added after thawing, 1000r/m is centrifuged 3min；It repeats 1 time.Sediment is moved into Tissue Culture Flask, adds DMEM culture solution, sets CO2 incubator culture, once passed within 3~4 days Or expanding culture, fusion adjusts cell state in first 24 hours, guarantee that cellular morphology is good before merging, growth is vigorous.It is added suitable Basal medium is measured into centrifuge tube, 1000r/min centrifugation 5-10min after mixing is gently beaten, washes repeatedly cell 2 times.(3) Rh feminine gender effect B cell strain to be hybridized prepares: adjusting total cell number to 1 × 10 with basal medium⁸~2 × 10⁸Melt for cell It closes, blue dyeing phase-contrast microscopy is expected with platform, viable count should be higher than that 80% is qualified.(4) cell fusion: by Rh feminine gender The strain of effect B cell and myeloma cell are added in centrifuge tube with 5~10: 1 ratio, are mixed evenly, 1000r/min centrifugation 5min is discarded supernatant, and is gently beaten tube bottom to cell grainless and is precipitated, is repeated 2 times.The gently rotation preheating in 37 DEG C of water-baths The 50%PEG3000 of 1000 μ L of preheating is added drop-wise in fusion pipe in 60s along tube wall under aseptic condition by centrifuge tube after taking-up Gently rotating centrifugal pipe simultaneously, is also added drop-wise to centrifugation along tube wall in 3~5min for the basal medium of the 25mL of preheating later Guan Zhong, lightly rotating centrifugal pipe during addition are then allowed to stand in 37 DEG C of water-bath 10min, 1000r/min centrifugations 5min is discarded supernatant, and 50mL HAT culture medium is added.It is appropriate mix after be inoculated into 96 well culture plates, be placed in 37 DEG C, 5% It is cultivated in CO2 incubator.(5) hybridoma is cultivated: cell growth status in 96 well culture plates of observation, after 7~10 days only Hybridoma can grow division, discard HAT culture medium at this time, replace complete medium.Cell clone growth area reaches When 1/10 cell hole, culture supernatant is gone, the culture hole of the good hybridoma of growth conditions is selected, is marked under microscope thin Cell clone is drawn to the complete medium that has newly in the position of mark using sterile pipette tips in the position of born of the same parents' strain growth, size In culture hole, then successively doubling dilution to hole is counted below, and 37 DEG C, 5%CO2 incubator is interior to be cultivated one week or so, under microscope Cell growth status is observed, when cell clone is covered with to 1/10 or more hole floor space, cell or culture supernatant is taken to detect hybridization Oncocyte function.(6) screen Rh feminine gender effect hybridoma: Rh feminine gender effect hybridoma, which refers to, can generate Rh antibody Hybridoma, method produce the effect B cell strain of Rh antibody with screening, filter out energy with direct or indirect antihuman globulin test The Rh feminine gender effect hybridoma (clone) for secreting more efficient valence Rh antibody repeats next round dilution culture, repeats 2-3 Wheel, detection function are taken out after stablizing, are transferred to culture bottle mass propgation and [are such as intended to freeze and recover, then 12 hour adjustment before preservation Cell growth state takes one bottle of growth vigorous, the good cell of form, is made cell suspension after appropriate digestion, 1000r/min from Heart 5min, removes supernatant, and flicking tube bottom keeps cell loose, and the 9 parts of complete culture solutions and 1 part of DMSO of 4 DEG C of preservations are added, and packing is thin Cryopreservation tube is put into liquid nitrogen container after taking-up and saves backup by born of the same parents' cryopreservation tube, 1mL/ pipe, -70 DEG C of refrigerator overnights.Prepare before recovery 40 DEG C or so of hot water carefully takes out cryopreservation tube from liquid nitrogen, and being immediately placed in hot water uniformly to shake makes cell thaw, and solves It is centrifuged 5min in 1000r/min after jelly, opens cryopreservation tube under aseptic condition in superclean bench, by the cell after defrosting with completely Culture solution washed once, and is then centrifuged 5min in 1000r/min, discards supernatant, in case making to expand culture].(7) amplification Rh is negative Effect hybridoma: it moves into culture bottle, sets after Rh feminine gender effect hybridoma is gently resuspended using complete culture solution It 37 DEG C, cultivates in 5%CO2 incubator.Pass on amplification cultivation repeatedly according to a conventional method.

6, Rh antibody is prepared

(1) it separated in conventional manner from Rh feminine gender effect Hybridoma Cell Culture supernatant, purify Rh antibody.(2)Rh The preparation of antibody MAb mouse ascites: low-speed centrifugal collects the Rh feminine gender effect hybridoma after culture, cultivates by basis Base diluting cells number is 1 × 10⁷/ mL, mouse peritoneal inject 0.2mL/ only, mouse ascites production are observed after injection, to abdomen The obvious distension in portion rises, and punctures abdominal cavity with syringe needle, and centrifuge tube collects ascites, and acquisition finishes, and injects appropriate basis culture to mouse peritoneal Base is spaced 2~3 days, and same method takes ascites again, the ascites being collected into, and 10000r/min is centrifuged 5min, and Aspirate supernatant divides Dress, -20 DEG C of preservations.Contain a large amount of Rh antibody in supernatant, separates according to a conventional method, purifies Rh antibody, with antihuman globulin The potency of test or ELISA method measurement antibody.

7, anti-Rh antibody is prepared

(1) experimental animal: immune is selected with experimental animal is the white goat of animal institute of Zhejiang University hybridization, 30 jin of weight The between twenty and fifty ewe two of health of left and right, is smeared at the back of animal with dyestuff before immune, makes specific label, using doing as everybody else does The feeding manner of stable breeding guarantees that sheep has to suitable movement daily, and it is left to move general half an hour at dusk every time for run duration The right side, is conducive to healthy and strong in this way, avoids sheep overfertilization, increases the immunity of body, and drinking-water is sufficient, and give suitable fine fodder, Green grass, corn, wheat bran, wheat, vitamin etc. keep its nutrition balanced.Often check experiment sheep health status.(2) it is immunized Source: mixing 0.1mL antigen before Rh antibody (Rh antibody, that is, anti-D serum, commercially available product) prepared by the present invention inoculation, 1.9mL without It is spare that immunogen emulsion is made in bacterium PBS, 2mL Freund adjuvant completely (or not exclusively).(3) goat is immune: two goats are chosen, Labeled as goat A, goat B, antigen inoculation.Immune position is front and back groin, and 2 points of every place's groin point are injected, a total of 8 A injection point, injection system are subcutaneous injection, and every goat per injection 4mL immunogene contains 250 μ g antigens；Each injection point Injecting immune original 0.5mL, containing about 32 μ g antigens.Preceding two goats are immunized to draw blood respectively 10mL, are labeled as 0dP1, -20 DEG C of preservations； Draw blood 10mL after 7 days immune, separates serum, is labeled as 7dP1, detects serum titer with ELISA, and -20 DEG C of remaining serum save, and 3 Start within~4 weeks to be immunized for second, immunogene is the sterile PBS+2mL incomplete Freund's adjuvant (IFA) of 0.1mL antigen+1.9mL, is exempted from Epidemic disease is drawn blood 10mL after 7 days, separates serum, is labeled as 7dP2, and ELISA detects serum titer, -20 DEG C of remaining serum preservations.Third Secondary immunization time is after 6~8 weeks, and immunogene is the sterile PBS+2mL incomplete Freund's adjuvant (IFA) of 0.1mL antigen+1.9mL, is exempted from Epidemic disease is drawn blood 10mL after 7 days, separates serum, is labeled as 7dP3, and ELISA detects serum titer, -20 DEG C of remaining serum preservations, if Serum titer does not reach 1: 10 at this time⁶More than, then it needs to be immunized again primary；If serum titer is up to 10⁶Do not have to then exempt from again above Epidemic disease, draw blood 50mL every other week later, separates serum, -20 DEG C save backup.(4) anti-Rh antibody serum preparation: generally exempt from every time It can be detected in sheep jugular vein blood collection within 7~10 days after epidemic disease, by assistant Baoding animal, keep standing position it, neck is cut After hair, sterile cotton balls cleaning disinfection, the blood sampling of jugular vein hand syringes is searched out, the fixation of syringe position is taken into 5~10mL of blood. Bioactivity is carried out after isolating serum.7~10 days after the third immunization, it once can use blood 30- after bioactivity is qualified 50mL aseptically separates serum, after the blood clotting in plate or triangular flask to be collected in, with sterile dropper in nothing After clot and bottle wall removing in collarium border (such as superclean bench), 37 DEG C are put into, 1~2h is put into 4 DEG C overnight after taking out, make Serum is sufficiently precipitated and (cannot freeze, otherwise generate haemolysis), separates serum through centrifugation, puts low temperature refrigerator preservation into, uses Before must dispense again and save backup after signing is qualified.(5) measurement of antiserum titre: antiserum titre is measured using ELISA Method, when coating are after sample is diluted to 1: 1000 concentration with coating buffer, and every hole adds 100 μ L on ELISA Plate, then puts It is put into aluminium box in 4 DEG C of refrigerators overnight, the next morning, which takes out, pats dry coating buffer, is washed three times with PBST, every minor tick Five minutes, ELISA Plate is patted dry with gauze for the last time, the confining liquid of 10% serum is added, every hole adds 100UL, is put into 37 DEG C, water 1~2h is bathed, then takes out again and pats dry confining liquid, wash 3 every minor tick of ELISA Plate five minutes with PBST, use gauze for the last time It pats dry, 100 hole μ L/ primary antibodies (1: 2000 dilution is diluted with the PBST of 4% cow's serum, is put into 37 DEG C, 1~2h of water-bath) is added, so It takes out again afterwards and pats dry primary antibody liquid, PBST washs ELISA Plate three times, and every minor tick 5 minutes pats dry enzyme mark with gauze for the last time Plate is added secondary antibody liquid (rabbit-anti sheep 1: 1000), and every hole adds 100 μ L, is put into 37 DEG C, 1~2h of water-bath, then take out and pat dry secondary antibody liquid 3 every minor tick of ELISA Plate is washed five minutes with PBST, is patted dry for the last time with gauze, every hole adds 50 μ L substrate developing solutions, black out Colour developing 10~after twenty minutes be added 2M 50 μ L of H2SO4 solution terminate reaction, after with microplate reader survey OD value (in half an hour).Or with Potency of the antihuman globulin test result positive of maximum dilution multiple as antibody.

The Rh antibody prepared by the present invention, that is, anti-D of Ig, anti-Rh antibody, that is, anti-Ig anti-D are same by technical solution of the present invention Ig anti-A, Ig anti-B, Ig anti-E, Ig anti-C and the anti-A of anti-Ig, the anti-B of anti-Ig, the anti-E of anti-Ig, the anti-C of anti-Ig can be prepared, goes forward side by side one Step preparation immunoadsorption therapy device.The present invention is after preparing Rh antibody, when carrying out the preparation of anti-Rh antibody, except through immune Goat preparation is outer, can also then take the immune spleen cell of mouse and myeloma cell fused, then by normal by the way that mouse is immunized Rule hybridoma technology prepares anti-Rh antibody.

Three, the preparation of adsorbent

1, the preparation of the adsorbent containing anti-Rh antibody

The prepared anti-Rh antibody for playing absorption Rh antibody is taken, antibody titer is detected, respectively after 100 DEG C dissolve 0.9%, 1.0%, 1.1%, 1.2%, the 1.3% agarose C1-4B physiological saline kept the temperature at 37~42 DEG C is made into 1: 700,1: 500, the application antibody of 1: 300,1: 200,1: 100 potency gradient, keeps the temperature spare at 37~42 DEG C.

2, the preparation of the adsorbent simultaneously containing anti-Rh antibody and Rh positive ghost

By the prepared rhesus macaque ghost for playing absorption Rh antibody, be respectively 1: 700 with anti-Rh antibody titer, 1: 500,1: 300,1: 200,1: 100 adsorbent is configured to contain anti-Rh antibody while 95%Rh positive blood shadow cell concentration With the adsorbent of Rh positive ghost, keep the temperature spare at 37~42 DEG C.

Four, the preparation of absorber

1, material

For the acrylate close with Human vascular endothelial etc high molecular material, it is desirable that good biocompatibility, no complement Activation and inflammatory reaction, can the change without leucocyte, blood platelet, blood oxygen pressure, complement C 3 C5a, pass through covalently, grafting, polymerization The methods of improve uniformity, the hydrophily of material surface, reduce influence to blood coagulation and oxidative stress.Add in absorber inner surface Hydrophilic gel generates CA/PMB30, CA/PMB80 and CA/PMB30-80 by controlling wet-spinning procedure, biofacies can be improved Capacitive.Certain anticoagulant substances are solidificated in carrier or absorber inner surface, blood clotting is can inhibit, reduces heparin dosage even reality Heparin, is such as aggregated on polyacrylonitrile-polyethyleneimine film, can reduce the allergic reaction of allergic constitution by existing no-rod tractor；It will Heparin covalent is integrated to polyether sulfone surface, can keep the mechanical property of polyether sulfone and improve the anticoagulant property of absorber inner surface Energy.The covalent immobilisation linoleic acid film on cellulose acetate film, or the linoleic acid for being covalently bound to polyacrylic acid is grafted onto PS membrane Surface can have better histocompatbility and anticoagulant effect.

2, specification

The hydrostatic column that the bottom diameter for being made 50mm × 60mm is small, top diameter is big, or rectangular, infundibulate is made, volume about 200 ~300ml, top and the bottom entrance are equipped with cell screen clothes, and top diameter sieve mesh number is 500 mesh, and bottom diameter sieve mesh number is 50 mesh, liquid It is 200 aim cell strainers that mesh number, which is arranged, in body exit, constitutes the second defence line for preventing cell fragment from entering circulation, liquid It is equipped with buffer area between inlet and outlet and mesh screen, is conducive to the stability of system circulation.

3, method

55~65ml is successively taken to contain the suction of anti-Rh antibody and Rh positive ghost simultaneously from low to high by antibody titer In attached dose of addition hydrostatic column, it is cooled to the adsorbent being first added after semi-solid gel and just then adds next time, being made makes Gel in container forms antibody titer from high to low and agarose concentration and blood from low to high from sample introduction end to sample outlet end The equally distributed absorber of shadow cell, when the blood plasma separated in extracorporal circulatory system filters out absorber, Rh antibody is fixed on absorption Ghost and anti-Rh antibody in agent are combined into fixed compound, the red cell debris that is destroyed and it is in combination made of Macromolecular immune complex by nearly absorber outlet end concentration is gradually high and molecular sieve gradually small special agar gel and absorber goes out Mouthful the detention of sieve institute, remove after the blood plasma of morbid substance is filtered out from absorber and feed back in vivo, avoid pregnant woman Rh anti-to reach Body enters fetal blood and mitigates the therapeutic purposes of the state of an illness.

Five, the preparation of blood separator

1, preparation principle: according to the size of visible component in blood of human body (cell): normocyte is about 7 microns of (μ It m), is the discoid cell of concave-concave；Leucocyte is divided into 5 kinds, and about 12 μm of neutrophil leucocyte, eosinophil is more bigger, basophilic Property granulocyte and neutrophil leucocyte it is close, 6-8 μm of small lymphocyte, approximate with red blood cell, monocyte maximum, about 15-20 μm. Blood platelet is disc, and 1~4 micron to 7~8 microns of diameter is differed, and the platelet mean diameter of people is 2-4 microns, thickness 0.5~ 1.5 micron.

2, it the material of blood separator and requirement: with absorber of the invention, selects poly-vinegar non-woven fabrics, acetate fiber, take off Rouge cotton etc., it is desirable that good biocompatibility, permeability height, hardly activating complement do not cause inflammatory reaction and leucocyte, blood small The change of plate, blood oxygen pressure, C3a, C5a.

3, the type and spec of blood separator: plasma separator according to the present invention can be bought from businessman, or committee Hold in the palm businessman's preparation.Hollow fibre type filter made of plasma separator high molecular polymer, the hole can permit blood plasma filtration, but It can stop all cell components.Hollow-fiber film diameter is 270~370 μm, and film thickness is 50 μm, and aperture is 0.2~0.6 μ M, fibre length are 13.5~26 μm.

Six, the component of Rh blood group incompatibility haemolysis disease therapeutic apparatus

1, key member

(1) washed corpuscles and blood plasma blood separator: are used for.

(2) plasorber: including anti-Rh antibody, rhesus macaque ghost and agar gel medium, anti-for removing Rh The compound etc. that body, RBC fragment, Rh antibody and RBC fragment are formed.

2, additional member: including blood pump, heparin pump, sound pulse pressure and air monitering, temperature control system, off gas system, The parts such as monitored conductivity system, ultrafiltration monitoring and leakage blood monitoring form.

(1) blood pump (Blood Pump): for pushing blood circulation to maintain going on smoothly for Blood index treatment, usually Blood pump part often has rotary test speed function, and to monitor the blood circumstance of patient, therefore blood pump runner and groove spacing are set It is accurate, and need often adjustment, the case where according to bloody path pump line, spacing is generally set as 3.2~3.3mm, can not be too loose, Otherwise it is inaccurate to will cause blood flow detection；Also can not be too tight, it otherwise will cause pipe breakage.

(2) heparin pump (Heparin Pump): heparin pump is equivalent to the micro-injection pump clinically applied, to continue to Injecting heparin in sieving pipeline (patient blood) contacts with air since the blood of patient recycles in vitro, is easy to happen blood coagulation Phenomenon anticoagulative can be occurred using heparin pump.

(3) sound pulse pressure monitors: the main stopping state to dynamic monitoring blood separator micropore of arterial blood pressure monitoring, separately Outside to monitor extracorporal circulatory system thrombus, solidification and the variation of pressure.When blood flow deficiency, angiosthenia will be reduced；When have blood coagulation, When thrombosis, especially separator blockage of the micro orifice, angiosthenia will be increased；Vein pressure monitoring is used to monitor pipeline blood reflux Pressure, when separator blockage of the micro orifice, blood coagulation, thrombosis, blood flow is insufficient and venous return syringe needle falls off when, vein pressure It will decline, if bloody path return pipe distortion blocking or reflux syringe needle block, vein pressure will be increased.

(4) air monitering (Air Detector): the air bubble for monitoring blood pathway generally uses ultrasonic listening Principle, in order to avoid patient occur air embolism and be arranged.When having monitored air bubble, detection system can drive it is dynamic, Vein bloody path folder carrys out blocking blood flow, prevents dangerous generation.

In short, Import computer regulates and controls and is made the people of operation on the basis of key member of the present invention and additional member Property, the personalization for the treatment of, the safety of design and modularization, automatic monitoring and regulation, liquid crystal display, voluntarily judgement is warned Report the therapeutic equipment of the micro computers such as reason and ring off signal processing.

Seven, the operating method of Rh blood group incompatibility haemolysis disease therapeutic apparatus

1, it installs: with each portions such as sterile working connecting components, including plasma separator, absorber and each circulation line.

2, it is vented: with sterile saline filling liquid separator, absorber and each circulation line, excluding separator, absorber And its gas, bubble in circulating line, it goes through, confirmation after gas, bubble without using.

3, lead to liquid: arterial blood line pipe 1 being connected into arteries, goes through again be vented whether complete, liquid in operation Whether stream is unobstructed, and flow liquid in pipe is avoided to pollute.

4, anticoagulant: to be injected from heparin pump into liquid stream anti-coagulants (heparin), be for the first time 2500 ∪ or 20~30 ∪/kg.

5, start: arterial blood line pipe (1) is connected to the arteries of curer, venous line (5) are connected curer's Vein blood vessel, then opens blood pump, and blood flow is 100~150ml/min, in Fig. 1, when arterial blood is through arterial blood line pipe (1) enter plasma separator (4), the blood plasma separated reaches absorber through circulation line (7) under the action of blood plasma pump (6) (8), wait be full of blood plasma, about 10 minutes, blood plasma is begun releasing, is flowed out through circulation line (10), it is synchronous that blood is perfused to absorber (9) Slurry, when the blood plasma in absorber (8) has nearly flowed, starts again at perfusion blood plasma, and absorber (9) begins releasing blood plasma at this time, Two absorbers (8) in parallel, absorbers (9) are alternately.In Fig. 2, when blood to be separated enters plasma separator (1) When inner cavity (2), the effect through valve (8) can enter the outer of separator by the small molecule blood plasma and its composition (5) of micropore (3) Chamber (6) is then flowed out through plasma outlet port (7), and cannot be flowed out by the haemocyte (4) of micropore (3) through valve (8).In Fig. 3, When Rh antibody (1) enters absorber, the anti-Rh antibody (2) being fixed in agar gel respectively is combined into fixed conjugate (3), the rhesus macaque blood shadow thin (4) being fixed in agar gel is combined into fixed conjugate (5), and the red blood cell being destroyed is broken Piece and it is in combination made of macromolecular antigen antibody complex cannot pass through the net of gel pore and adsorber enclosure exit It sieves by detention, the haemocyte that purified blood plasma is separated with plasma separator is fed back after converging.So until being previously set Plasma circulation amount (usually 9L), treatment just end, if entire therapeutic process is controlled by computer, and can detect at any time Working condition, automation more convenient using meeting and safety.

Eight, the verifying of therapeutic equipment effect

1, the verifying of ghost absorption Rh antibody efficacy

The effect of to understand therapeutic equipment, the present invention devise easy test method test rhesus macaque ghost absorption Rh The effect of antibody.Take prepared rhesus macaque ghost, be added to after 100 DEG C dissolve keep the temperature it is spare at 37~42 DEG C In 1.0% agarose C1-4B, it is configured to the adsorbent of 95% ghost concentration；Take 2.5 × 300mm Wei Shi erythrocyte sedimentation rate of sterilizing Pipe 9, the adsorbent of 95% ghost concentration is drawn to 200mm scale, becomes semisolid after adsorbent is cooling；Shopping center Blood station fresh frozen plasma 200mL, (the anti-D type serum dry powder standard items of people, it is limited that Guangzhou joins safe biotechnology to another purchase Rh antibody Company), 1: 200,1: 300,1: 600 antibody titer is made into fresh plasma, routinely (the reference of Rh (anti-D) titre detection method Specification), further detection confirms whether above-mentioned prepared antibody titer is consistent, (Rh) antibody (titre) before referred to as filtering, so Antibody is injected separately into the upper end blank pipe of 3 blood sedimentation tubes before respectively taking 10ml to filter afterwards, and ghost is contained in blood sedimentation tube lower layer to be flowed through Adsorbent after, collect efflux, (Rh) antibody after referred to as filtering, routinely Rh (anti-D) titre detection method confirms titre, then divides Antibody after filter is not made to filter out for the 2nd time in the adsorbent containing ghost, is so repeated 3 times filtration and antibody titer inspection It surveys, as a result (table 1) illustrates, after Rh antibody filters simple absorber, most of Rh antibody is by accordingly containing ghost Adsorbent absorption, after the 1st time, the 2nd time, the 3rd filtration, the average titer of Rh antibody is reduced to filter from 1: 367 before filter respectively Afterwards 1: 183,1: 58,1: 29, illustrate the increase with filtration number, Rh antibody can be adsorbed constantly by ghost and be removed, Female tire blood group incompatibility Hemolysis is treated to achieve the purpose that reduction pregnant woman (newborn) blood plasma Rh antibody titer.

Titre testing result (1/x) before and after adsorbent of 1 Rh antibody of the table filtration containing ghost

2, the verifying of anti-Rh antibody absorption Rh antibody efficacy

The effect of to understand therapeutic equipment, the present invention devise easy test method and test anti-Rh antibody absorption Rh antibody Effect.Prepared anti-Rh antibody is taken, is added to and keeps the temperature after 100 DEG C dissolve in 37~42 DEG C of 1.0% spare agarose C1- In 4B, titre is 1: 300~500 after mixing；2.5 × 300mm Westergren's blood sedimentation tube 9 of sterilizing are taken, draw 1.0% agar respectively Sugared C1-4B solution is to 200mm scale, and agarose becomes semisolid after cooling；Shopping center blood station fresh frozen plasma 200mL, Another purchase Rh antibody (the anti-D type serum dry powder standard items of people, Guangzhou Lian Tai Bioisystech Co., Ltd), is matched with fresh frozen plasma At 1: 128,1: 256,1: 512 antibody titer, routinely Rh (anti-D) titre detection method (reference book), detection confirmation are anti- Body titre, (Rh) antibody before referred to as filtering, antibody is injected separately into the upper end blank pipe of 3 blood sedimentation tubes before then respectively taking 10ml to filter, wait flow After outflow, efflux is collected through the 1.0% agarose C1-4B containing anti-Rh antibody of blood sedimentation tube lower layer and out of blood sedimentation tube, is claimed For (Rh) antibody after filter, routinely Rh (anti-D) titre detection method confirms titre, then passes through antibody after filter resist containing anti-Rh respectively 1.0% agarose blood sedimentation tube of body filters out, and is so repeated 3 times filtration and antibody titer detection, as a result (table 1) illustrates, Rh antibody After filtering simple adsorbent, part Rh antibody is adsorbed by corresponding anti-Rh antibody, after the 1st time, the 2nd time, the 3rd filtration, The average titer of Rh antibody be reduced to filter from 1: 298 before filter respectively after 1: 149,1: 48,1: 17, illustrate with filtering number Increase, Rh antibody can be removed constantly, to reach the mesh for reducing Rh antibody titer and treating female tire blood group incompatibility Hemolysis 's.

Titre testing result (1/ before and after 1.0% agarose simple adsorbent of 1 Rh antibody of the table filtration containing anti-Rh antibody x)

In short, above-mentioned easy confirmatory experiment shows the Rh antibody to dissociate in blood plasma, it can be (permanent by adsorbent of the invention River monkey ghost and anti-Rh antibody) absorption removing, it was demonstrated that the Rh blood constituted using blood separator and absorber as critical component Type does not conform to haemolysis disease therapeutic apparatus with significant therapeutic efficiency.

Claims

1. a kind of Rh blood group incompatibility haemolysis disease therapeutic apparatus for medical domain, which is characterized in that including by plasma separator, suction The extracorporeal circulation apparatus that adnexa and circulation line are constituted, the plasma separator are hollow fibre filter membrane, hollow fibre filter membrane Diameter is 270~370 μm, and film thickness is 50 μm, and aperture is 0.2~0.6 μm, and fibre length is 13.5~26 μm, and absorber exists Mesh screen is arranged in exit, adsorbent is filled with inside absorber, the adsorbent is formulated in ghost and anti-Rh antibody Agarose solution and be made, it is red thin that the ghost alternately washs rhesus macaque with the PB lysate of 25mmol/L and 35mmol/L Born of the same parents and be made, so that the sample introduction end of absorber to sample outlet end is formed antibody titer from high to low and agarose concentration from low to high But ghost is to be uniformly distributed, so that ghost and anti-Rh antibody be made to adsorb Rh antibody jointly and gel pore and absorber The common detention red blood cell lysate of mesh screen.

2. Rh blood group incompatibility haemolysis disease therapeutic apparatus according to claim 1, which is characterized in that the ghost and anti-Rh Antibody is formulated in agarose solution and refers to that being uniformly formulated in the anti-Rh that potency is 1: 100~700 with the ghost of 95% content resists Then body is formulated in agarose solution.

3. Rh blood group incompatibility haemolysis disease therapeutic apparatus according to claim 1, which is characterized in that from the sample introduction end of absorber to The anti-Rh antibody titer of sample outlet end is successively with 1: 700,1: 500,1: 300,1: 200,1: 100 point 5 layers.

4. Rh blood group incompatibility haemolysis disease therapeutic apparatus according to claim 1, which is characterized in that from the sample introduction end of absorber to The agarose concentration of sample outlet end is successively with 0.9%, 1.0%, 1.1%, 1.2%, 1.3% point 5 layers.

5. Rh blood group incompatibility haemolysis disease therapeutic apparatus according to claim 1, which is characterized in that the PB lysate by 81.0ml Na containing 0.04mol/L₂HPO₄Solution A and 19.0ml NaH containing 0.04mol/L₂PO₄Second liquid be made into 40mmol/L's 25mmol/L and 35mmol/L are diluted to deionized water after PB.

6. Rh blood group incompatibility haemolysis disease therapeutic apparatus according to claim 1, which is characterized in that the shell of the absorber holds Product is 200~300ml, and top and the bottom are equipped with cell screen clothes, and top diameter cell screen clothes mesh number is 500 mesh, bottom diameter cell screen clothes mesh number For 50 mesh, it is 200 aim cell strainers that mesh number, which is arranged, in liquid outlet.

7. the preparation method of the adsorbent in Rh blood group incompatibility haemolysis disease therapeutic apparatus according to claim 1, feature exist In, washed Rh positive Rhesus red blood cell 2 times under conditions of 4 DEG C and 37 DEG C respectively with isometric physiological saline, then with infiltration The PB lysate that concentration is the PH7.4 of 25mmol/L and 35mmol/L alternately washs, finally with physiological saline under conditions of 4 DEG C Cleaning, until supernatant is colourless, precipitating is separately anti-with anti-Rh obtained as thoroughly except the ghost of the antigen containing D of hemoglobin Body is made into 1: 700,1: 500,1: 300,1: 200,1: 100 respectively with 0.9%, 1.0%, 1.1%, 1.2%, 1.3% agarose Apply antibody, be then incorporated the Rh positive Rhesus ghost of 95% content adsorbent.

8. -6 any Rh blood group incompatibility haemolysis disease therapeutic apparatus answering in preparation extracorporal circulatory system branch according to claim 1 With, which is characterized in that one end of extracorporal circulatory system branch routing arterial blood line pipe (1) is through heparin pump (2) and blood pump (3) and blood It starches separator (4) to be connected, plasma separator (4) absorber in parallel with two through blood plasma pump (6) and first circulation pipeline (7) (8,9) it is connected, is then successively connected with second circulation pipeline (10), venous line (5).