To Invent A Portable And Convenient ABO/Rh Blood Grouping Method In War Environment With Filter Paper

BackgroundAs a genetic trait manifested in antigen, blood group is classified on the basis of certain antigens in red blood cell surfaces. thirty kinds of blood group systems such as ABO, Rh, MNS,P etc, has been found and recognized by International Society of Blood Transfusion(ISBT).Among them, ABO and Rh is of the most importance blood group system. Found by Karl Landsteiner, ABO blood group systemis the earliest blood group system recognized by humankind. Rh blood group system is the most polymorphic system, which contains at least45antigens. The most importance five antigens in Rh blood group system are:D,E,C,c,e., and Antigen D has the best antigen city.Depending on whether Antigen D is shown on red blood cells, Rh blood group can be classified Rh positive and Rh negative.As an effective treatment method, blood transfusion has been widely used in clinical. Every year, approximately85million units blood is transfused into patients all over the world around. However, transfusion may have some kinds of risk. If blood transfused into patients is incompatible, acute hemolytic reaction may occur, causing shock and renal failure, and even death. About one patient in three may suffer hemolytic reaction if compatibility test was ignored before transfusion. Thus, rapid and correct identification of ABO/Rh blood group typing for donor and recipient is essential to the safety of blood transfusion.Classification of blood group is based on whether there are corresponding antigens on red blood cell membrane. According to Landsteiner’s law, when red blood cell membrane has certain antigens (such as A, B or D antigens), the serum mustn’t contain corresponding antibodies. Conversely, if certain antigens are lacked in red blood cell surface, presence of correspond antibodies must show in serum. Hence, most of the principles of ABO/Rh blood grouping methods are based on antigen-antibody agglutination.Slide, tube and micro gel are the most common methods used in clinical for ABO/Rh blood group typing. Though these methods are accurate, objective, and with high sensitivity and specificity, they also have some shortages:a. professional staffs are needed to operate; b. specific laboratory instruments are required; c. experimental conditions must be suitable; d. reaction time is long; e. more blood is used; f. require sample pre-treatment. Therefore, though meet the requirement of clinical and laboratory, traditional methods are unable to meet the requirement of ABO/Rh blood group typing in special situations, such as battlefield environment, severe natural disasters, and so on.Special circumstance usually refers to the followings:(1) the war. Compared with previous wars, modern warfare is higher in damage, faster in speed and larger in consumption, etc. Great changes in the military environment makes battlefield rescue show such new features as movable, dangerous, limited and so on. The new features of battlefield rescue require medical equipments have higher ability in quick respond and portable mobility. Military medical equipments must meet the following characteristics:a. small in size and light in weight; b. simple in operation; c. results are accurate and reliable; d. be able to visually read results in direct ways; d. no external power supply or other laboratory equipment; e. mobile and easy to carry.(2) Serious natural disasters. Earthquake, debris flow, tsunamis and other sudden natural disasters are unpredictable and devastating. Shortage in medical resources, in medical personnel and in basic medical equipment, is a major obstacle to rescue of the wounded.(3)Economic conditions behind developed countries or geographically isolated regions. People in these countries are not able to afford high medical costs as those in developed countries. They can only accept cheap basic health equipment, which however is in great short. According to World Health Organization (WHO), medical devices in less developed countries must meet the requirement of "ASSURED", namely:affordable, sensitive, specific, user-friendly, rapid and robust, equipment free and deliverable to end-users. Therefore, POCT (point of care test) method, which is cheap, portable, easy to operate and interpret, is essential for blood group detection of special circumstances.As a new trend for medical examination in21st, POCT gets more and more attention. Its main feature is "fast, side, convenient, easy", namely:with the most simple and economical method, non-professionals get the accurate results in the shortest possible time outside of traditional laboratory, in the draft of "POCT special permit document", U.S national Academy of Clinical Biochemistry(NACB) defined POCT as "near patient treatment unit, performed by clinical staff who didn’t accept clinical laboratory training or patients themselves(self-testing)".Many attempts have been made to achieve the goal of POCT for blood typing. Chinese Patent200320121646.2(Publication No.2,669,196), Chinese Patent200920222236.4(Publication No. CN201522495U) and Chinese Patent Application03215617.0(Publication No.2605574) disclosed a simple blood type identification card; Chinese Patent ZL200620112928.X (Publication No.2,901,313) disclosed a styling kit anti-ABO blood group;Blood detection method described in the above patents is simple to operate, easy to carry, accurate and reliable, but there are still shortcomings. firstly, they can only detect ABO blood group, unable to type ABO and Rh blood group simultaneously; secondly, they fail to read the typing results directly, which may raise the possibilities of false judgment caused by human errors. Thus, the objective of this project is to develop a POCT blood typing method for war field and other special circumstances. The method must satisfy the followings:detect ABO/Rh blood group simultaneously; cheap and portable; accurate and reliable results; results are intuitive and easy to judge; results can be read directly; simple to operate, non-professionals can perform it.As a commonly used laboratory tool, filter paper has attached more and more attention from researchers. It has great potential for the development of portable medical devices due to its unique advantages. The main advantages of filter paper are: cheap and available everywhere; biodegradable and easy to handle; lightweight, easy to carry; composed of fibers, so external energy is not needed because of capillary action in its holes. Capillary action is defined as a phenomenon of infiltrated liquid accelerates in capillary tube or a phenomenon of un-soaked liquid declined in capillary tube. The following phenomena shown in daily life are good examples of capillary action:thin layer chromatography, evapotranspiration, bath towel absorbent and paper absorbent. Made from fiber, filter paper contains lots of little holes inside, which helps the formation of capillary action. Based on the advantages mentioned above, we selected filter paper as our research object and intended to find a portable ABO/Rh blood group typing method which is suitable for special circumstances such as battlefield, severe natural disaster and so on, and to build foundation for further invention of portable ABO/Rh blood grouping card in battlefield. Chapterl Observation of filter paper based capillary action[Objective]To provide experimental basis for the development of filter paper based ABO/Rh blood grouping method by the means of observing capillary action in filter paper.[Methods]1Observation of capillary action on different kinds of filter papers:(1)Prepared six different domestic and foreign qualitative filter papers-rapid qualitative filter papers(101),medium-speed qualitative filter papers(102), slow speed qualitative filter paper(103),#2Whatman filter paper,#3Whatman filter paper, and#4Whatman filter paper.(2)cut all the mentioned filter papers into3cm X16cm strips.(3) Filled saline (2cm deep) and red inks (2drops) into a beaker with the volume of200ml.(4) Vertically placed the filter paper into the beaker and observe phenomena happed in the beaker.(5) Measured and record the height of infiltrated paper by liquid after10min.(6)repeated procedure (4) and procedure (5),20times respectively.2Observation of capillary action on anticoagulant vein blood spotted in filter paper.2.1Capillary action on anticoagulant vein blood without the add of serum antibody:(1)Prepared20cases of EDTA anticoagulant vein blood.(2) Prepared six different domestic and foreign qualitative filter papers-rapid qualitative filter papers(101),medium-speed qualitative filter papers(102), slow speed qualitative filter paper(103),#2Whatman filter paper,#3Whatman filter paper, and#4Whatman filter paper.(3)2cm above the lower,added10ul anticoagulant vein blood in the middle of six different kinds of filter paper (4) Vertically placed the filter paper into the beaker and observe phenomena happed in the beaker.(5) Measured and record the length of filter paper soaked by anticoagulant vein blood spotted. 2.2Capillary action on anticoagulant vein blood with the add of serum antibody:(1)selected20cases of anticoagulant vein blood (4cases of A+Rh+,1case of A+Rh-,5cases of B+Rh+,5cases of AB+Rh+,5cases of O+Rh+);(2) Prepared six different domestic and foreign qualitative filter papers-rapid qualitative filter papers(101),medium-speed qualitative filter papers(102), slow speed qualitative filter paper(103),#2Whatman filter paper,#3Whatman filter paper, and#4Whatman filter paper.(3)2cm above the lower edge,10ul of anticoagulant blood was spotted in the left(anti-A), middle(anti-B) and right side(anti-D) respectively.(4)serum anti-A,anti-B and anti-D was added in the corresponding site of each filter paper.(5)vertically placed the filter paper into a beaker(contained salving),and took it out10min later.(6)measured and record the length of filter paper soaked by anticoagulant blood.3Analysis of blood-soaked length and optical density in agglutinated and non-agglutinated:(1) Selected60cases of EDTA anticoagulant vein blood (type A and type B,30cases for each type).(2)Detected the ABO blood group of60cases with the method mentioned above(method2).(3)Measured the length of filter paper soaked by anticoagulant vein blood.(4)Pictured results with digital camera,and then analyzed mean density and accumulative density in agglutinated and non-agglutinated with software Image-Pro Plus6.0.Methods for the use of image-pro plus are as follows:①open the photo with software Image-Pro Plus6.0.②optical density switch: measure-calibration-intensity-new-sd. opt density-system.③select AOI. Irregular-magicward-trace.④AOI switch:measure-count/size-edit convert AOI to objects.⑤select items:measure-count/size-measure-select measures-density(mean)/IOD-measure-ok.⑥check results:measure-count/size-measurement data.(4) analyzed data with spss13.0.[Results] 1. Observation of capillary action on different kinds of filter papers:Filter papers were infiltrated by saline, which makes red stripes on papers. Length of red strips was different among six kinds of filter papers. Red stripe on#4Whatman was the longest (151.55±5.21,mm) while domestic slow qualitative filter paper was the shortest (80.1±4.82). Length difference of red stripes on three kinds of Whatman filter papers was statistically significant (p<0.05); Length difference of red stripes on domestic three kinds of filter papers was statistically different(p<0.05); Length difference of red stripes on six different filter papers was statistically different(p<0.05).2Observation of capillary action on anticoagulant vein blood spotted in filter paper.2.1Capillary action on anticoagulant vein blood without add of serum antibody: anticoagulant vein blood wicked along with saline and made the formation of red strips on papers. Length of red strips was different among six kinds of filter papers. Red stripe on#4Whatman was the longest (50.90±5.20,mm) while domestic slow qualitative filter paper was the shortest (3.35±1.42). Length difference of red stripes on three kinds of Whatman filter papers was statistically significant (p<0.05);Length difference of red stripes on domestic three kinds of filter papers was statistically different(p<0.05); Length difference of red stripes on six different filter papers was statistically different(p<0.05).2.2Capillary action on anticoagulant vein blood without add of serum antibody: Two phenomena were seen on filter papers:(1) non-agglutinated blood wicked along with saline in filter papers, red color in reaction zone faded; red stripe was formed in filter papers.(2)agglutinated blood did not wick along with saline in filter papers, red color in reaction zone unchanged, no red stripe was formed.2.3. Analysis of blood-soaked length and optical density in agglutinated and non-agglutinate:(1) Phenomena in60cases were consistent with those results mentioned above:non-agglutinated blood wicked along with saline in filter papers, red color in reaction zone faded; red stripe was formed in filter papers; agglutinated blood did not wick along with saline in filter papers, red color in reaction zone unchanged, no red stripe was formed.(2)Length of red strip on agglutinated side was (0.26±0.44)mm while non-agglutinated side was (49.32±4.78)mm, length difference between them was statistically significant(p<0.01);mean density on reaction zone of agglutinated side was0.60±0.06while non-agglutinated side was0.41±0.04.mean density difference between them was statistically significant(p<0.01); accumulative density on reaction zone of agglutinated was (39779.23±6437.36) while non-agglutinated was (23556.38±3593.99), difference between them was statistically significant(p<0.01).[Conclusion]Six different types of filter paper have capillary action. capillary action on#4Whatman filter paper was the most obvious while domestic qualitative filter paper was the weakest.Two phenomena were shown in antigen-antibody action zone:non-agglutinated blood wicked along with saline in filter papers, red color in reaction zone faded; red stripe was formed in filter papers; agglutinated blood did not wick along with saline in filter papers, red color in reaction zone unchanged, no red stripe was formed.Capillary action on filter paper can be used to type ABO/Rh blood group system. Chapter2To analyze influence factor of filter paper based ABO/Rh blood grouping method[Objective]To analyze influence of the following factors on filter paper based ABO/Rh blood grouping method:filter paper, antibody, reaction time, ratio of antigen/antibody.[Methods]1Influence of antigen/antibody:(1) Prepared50cases of anticoagulant blood (Type A and Type B, each25cases);(2)#4Whatman filter paper and domestic antibody were used to detect50cases of anticoagulant blood (A and B blood group, each had25cases).each case was detected three times with the following serum/blood ration:10ul/5ul,10ul/10ul,10ul/20ul.(3)Length of red strips was measured with rulers after lOmin).(4) Analyzed data with spss13.0.2Influence of Filter paper:(1)Prepared type A and type B blood (25cases for each);(2) each case was tested with the following filter paper separately:rapid qualitative filter papers(101),medium-speed qualitative filter papers(102), slow speed qualitative filter paper(103),#2Whatman filter paper,#3Whatman filter paper, and#4Whatman filter paper(domestic antibody, antigen/antibody=1:1).(3)10min later, measured and record the length of red strips on filter papers.(4) Analyzed data with spss13.0.3Influence of Antibodies:(1) Prepared type A and type B blood (25cases for each).(2) each case was detected with domestic and foreign antibody respectively(#4whatman filter paper, antigen/antibody=1:1).(3)10min later, measured and record the length of red strips on filter papers.(4) Analyzed data with spssl3.0.4Influence of Reaction time:(1) Prepared type A and type B blood (25cases for each).(2)Each case was detected with#4whatman filter paper (domestic antibody, antigen/antibody=1:1).(3)10min later, measured and record the length of red strips on filter papers.(4) Analyzed data with spssl3.0.[Results] 1Influence of antigen/antibody:Among three ratios, there was no significant difference in the length of red strips on agglutinated (p=0.88), neither was there on non-agglutinated (p=0.64).For each ratio, length of red strips between agglutinated and non-agglutinated was significantly different (p<0.01)2Influence of Filter paper:length of red strips on non-agglutinated was significantly different (p<0.01) among six filter papers. Red strips of non-agglutinated on#4Whatman filter paper was the longest(49.64±4.6,mm),while domestic slow quantitative filter paper was the shortest(2.02±1.56)3Influence of Antibodies:Length of red strips between agglutinated and non-agglutinated was significantly different (p<0.01).There was no significant difference (p=0.56) in the length of red strips of non-agglutinated side between domestic and foreign antibodies, neither was on the agglutinated side (p=0.77).4Influence of Reaction time:red strip non-agglutinated side was shown in the first minute (5.08±1.28,mm),in the third minute, red strip length was longer than2cm(22.66±2.64,mm).red strip difference among each minute was significantly different (p<0.01).[Conclusion]Antibody/antigen ratio and antibody had no influence on the results of filter paper based blood grouping method. Length of red strip on non-agglutinated was associated with filter paper and reaction time. Chapter3Optimization and clinical validation of filter paper-based ABO/Rh blood grouping method.[Objective]To optimize and clinically verify filter paper-based ABO/Rh blood grouping method.[Methods]1Optimization of filter paper-based ABO/Rh blood grouping method:(1) Prepared6cm×16cm#4Whatman filter paper and wrote patient’s name on the upper of right side.(2)4cm above the edge of filter paper, red character "A"、"B"、"+" was wrote with red pencil on the left(anti-A),middle(anti-B) and right (anti-D) side respectively.(3)2cm lower the red character, volume of10ul plastic burette was used to add a drop of anticoagulant vein blood on each side respectively.(4)A drop of anti-A, anti-B, and-D serum was added on the left, middle and right side correspondingly.(5)Vertically placed the filter paper on a beaker which contain a little save in.(6)10min later, took the filter paper out and red result.2. Clinical validation of filter paper-based ABO/Rh blood grouping method:①Randomly selected400cases anticoagulant vein blood.②One person tested the ABO/Rh blood group of400cases with slide method as followings:①Prepared a clean slide and wrote "anti-A","anti-B" and "anti-D" on the left, middle and right side respectively.②Added a drop of serum anti-A, anti-B and anti-D on the corresponding place.③Added a drop of anticoagulant vein blood on serum antibodies.④Shook the slide to ensure antigen and antibody react completely.⑤Observed and record results. If a red dot or platelet aggregation was seen,result is positive.(3) The other one tested the ABO/Rh blood group of400cases with filter paper based method as mentioned above(see method1).(4) Compared the results of slide method and filter paper based method.[Results]1Optimization of filter paper-based ABO/Rh blood grouping method:Red characters on non-agglutinated side were covered by red stripes while characters on agglutinated side were still visible; blood grouping results can be read directly by uncovered red characters.2. Clinical validation of filter paper-based ABO/Rh blood grouping method:400cases’ test results between slide method and filter paper based method were completely consistent (Group A:110cases, Group B:114cases, GroupAB:49cases, Group O:127cases. Rh+:400cases,Rh-:0cases).(Conclusion]With the advantage of portable、rapid and accurate, optimized filter paper-based ABO/Rh blood grouping method is able to be used for battlefield.Filter paper based ABO/Rh blood grouping method has the same sensitivity、 specificity and accuracy as traditional blood grouping method.