BLOOD BANK PROCEDURES ABO Blood Group System In 1900 karl landstainer discovered the blood groups ABO

and classifieds human blood into A,B and O Groups. A fourth blood Group AB as discovered b! landstainer"s associates. #on $ecastello and sturli in 190%. &he four groups are determined b! the presence or absence of blood group antigens 'agglutinations( on the red cells and accordingl! an individual"s group A,B,AB, or O 'O is donates the absence of A or B antigens(. In addition, it has been sho n that corresponding to antigens A and B, these are naturall! occurring antibodies anti)A and B 'agglutinations( in the plasma*serum of individuall! hose red cells lack the corresponding antigen. Group +A" individuall! have anti)B, Group)B individuall! anti)A, Group +O" Individuall! have both anti)A and anti)B and Group AB individuall! have no agglutination in the plasma*,erum. &he A B O antigens and corresponding antibodies Antigen on RBC A B AB -one Antibody in Plasma Serum anti)B anti)A -on anti)A and anti)B Blood Group A B AB O

In 1991 von lungern .irs/feid sho ed that Group +A" could be divided into t o principal sub Groups A1 and A% on the basis of this the ABO s!stem is classified into si0 main Groups A1,A%,B A1,B1, A% B and O. AB! AN"#GENS$ A, B and . antigens are present not onl! on the red cells but are also idel! distributed through out the bod! tissues e0cept in the central nervous s!stem. A,B and . antigen cit! is determined b! specific sugars linked to the terminal portion of oligosaccharides 'short chain sugars( these are present on gl!coproteins or gl!colipids. In the red cell membrane both gl!colipids and gl!coprotein"s ith AB. activit! are present. In the plasma onl! gl!colipids in soluble form are found. 1ell membranes of endothelial and epithelial cells have both gl!colipids and gl!coproteins. Se%retor Status A, B and . ,ubstances are also found in the ,ecretions of 20 3 of the population the abilit! to secrete B and . substances is determined b! the presence of the secretor gene'se( in either the homo/!gous se se or hetero/!gous se se state, hich is inherited independentl! of the ABO and .h genes. -ormall! all secretors secrete ., in addition A and * or B sub stances.

S&o'ing Se%tor Status Blood Group O A B AB Oh ,ubstance secreted . A4. B4. A,B4. -il

,5B G6O57, O8 A and AB9 A and AB have divided into ,ubgroup A1, A%, A, B and A% B depending up on the reaction ith the e0tract of a lactin $olichos biflorus seeds or human Anti)A1 serum. Anti)A sera ver! seldom differentiate bet een A1 and A%. both human anti)A1 and lactin anti)A1 agglutinate A1 and A1B cells but not A% and A% B cells. %03 of persons ith A antigens in the A or AB group are A% 'or( A% B. It is not necessar! to classif! group A patients or donors as A1 or A% e0cept hen the individual serum contains anti)A1, anti)A1, occurs in the serum of 1)23 of A% group person and %%):;3 of A% B groups person. Anti)A1 courses discrepancies bet een ABO cells and serum test and ma! also causes crossmatch incompatibilities but is considered clinicall! significant if it reacts at :<=1. (ea) Subgroup o* A$ ,ub group eaker than A% occurs in fre>uentl! the! are characteri/ed b! the declining number of A antigen sites or red cells and reciprocal increased in . reactivit!. ?eaker variants of A are mainl! A:, Ag, Am and A. Intermediate. 1lassification of eak A subgroups based on. 1. $egree of agglutination b! anti)A, Anti)A1 and Anti)AB %. $egree of . reactivit! on the red cells. :. 7resence or absence of anti)A1 in the serum. @. 7resence of A and . substance in saline of secretors. Serologi%al Rea%tions o* A and B p&enotypes 6B1 6eactions of cells ith 7henot!pe Ano Anti ,erum A B@ B@ B@ B %mf )* )* ) ) ) B ) ) ) ) ) ) B@ )* ) AB B@ B@ B@ B%mg B1*B% )* B@ )*B )* . B,) B% B: B: B@ B@ ) B@ B@ 6eactions of serum against 6B1 ,aline of ,ecretors 1ontains A1 B@ ) B% ) ) ) ) ) ) A1 ) ) B% ) B@ B@ B@ A% ) ) ) ) )* ) B@ B@ B@ B B@ B@ B@ B@ B@ B@ ) ) ) O ) ) ) ) ) ) ) ) ) ,aline of ,ecretors 1ontains A4. A4. A4. A4. . A4. B4. . B4.

A1 A% A1 and % A: Ag Am B B% Bm

B to B @ means C agglutination of increasing strength ? means C eak agglutination ) no agglutination Df means C mi0ed field agglutination. Deans C occurrence of anti)A1 is variable.

(ea) Subgroups o* B ,ub groups of B are less common than subgroups of A and are onl! of theoretical value. &he! can be classified on the basis of the reactions sho n in &able. Bombay Group or O& P&enotype &he phenot!pe 'Oh( is characteri/ed b! the absence of A,B and . antigens on the red cells. &he serum of these persons contains anti)A1, anti)B and anti)., hich reacts ith all O blood groups, All these individuals are non)secretors of AB.. &he red cells and secretions in Bomba! group individuals track . as ell as A and B substances, irrespective of ABO genot!pe, i.e. individual of Oh phenot!pe ma! have normal A*B genes but the corresponding antigens are not e0pressed on red cells. &his t!pe of blood as first found in Bomba! hence its name. the fre>uenc! in India is around 19<E00. AN"#BOD#ES O+ ABO S,S"EAnti.A &he antibod! anti)A is found in group B and group O individuals and reacts ell ith A1 and A% cells but not as ell ith eaker subgroups of A. Anti.B &he antibod! anti)B is found in group A and O individuals, and reacts almost ith all B group cells but less effectivel! ith eaker variants of B group. Anti.AB An antibod! anti)AB is found in group O individuals. ,erum from O group individuals is particularl! useful in detecting some eak A and B antigens. Anti.A/ &his is found in 1)23 of A% and %% to :;3 of A% B individuals. It is usuall! active at room temperature or belo and is rarel! clinicall! significant, e0cept hen it reacts at :<=1. Anti.! Anti). ver! rarel! occurs as cold reactive agglutinin in individuals ith ver! lo levels of . antigens on their cells and have little clinical significance. .o ever, anti). found in Bomba! blood group 'Oh( is an alloantibod! and is clinicall! significant. It occurs as a heamol!sis and agglutinates cells at :< A B O Grouping$ PRAC"#CAL ASPEC"S O+ ABO GROUP#NG
(1) (2) (3) (4)

(5) (6) (7) (8) (9)

6outine ABO grouping must include both cells and serum testing as each test serves as check on the other. ABO grouping tests should be done at room temperature or lo erF testing at :<=c eakens reaction. Anti)sera used in the ABO grouping must be used as per the instruction of manufacturer. 1ontrols should al a!s be run ith respect to ABO grouping. Dost laboratories have >ualit! control of antisera once a da! in order to eliminate the need to run individual controls ever! time the reagents are used. &ubes, slides or micro plates should be labeled properl!. One should not rel! on the colored d!es to identif! the regent antiserum. ,erum should al a!s be added before adding cells and e0amine each tube after serum has been added to ensure that none has been missed. &ubes or slides should be clean and dr!. Optical aid should be used to e0amine reactions that appear negative b! the naked e!e. 6esults should be recorded immediatel! after observation.

.o ever. &his is so because the slide test is less sensitive than the tube test. 1learl! labeled samples of clotted blood in sterile plain tubes are best for ABO and 6h tests. . &he other disadvantage is that dr!ing of the reaction mi0ture can cause aggregation of cells that ma! be misinterpreted as agglutination.. 7ut one drop of anti)A serum and one drop of anti)B serum separatel! on the labeled plate or slide.lide test is not recommended as a routine test because it is not reliable for eakl! reactive antigens on cells and for serum t!ping tests ith lo titre anti)A and anti)B. Di0 the cells and reagent using a clean stick. %. . :. -o sign of heamol!sis should be there. Ideall! cell t!ping and serum t!ping should be performed separatel! b! different orkers ho check each other"s results b! a +call)back procedure". 7repare an appropriate %)."e%&ni0ues Blood . &ube sedimentation :. Slide testing It is used for emergenc! purpose ABO grouping for preliminar! test.93 of normal saline and mi0. . E.pread each mi0ture evenl! on the slide over an area of 1. &he sample can be stored at @=1and should be tested ithin @2 hours.It is advisable to ash the cells %) :times in saline . ?ash the cells thrice ith normal saline and make %). 6ock)rotate the slide or plate and leave the test for %min at room temperature '%0=)%@=1(. ABO "yping "e%&ni0ue ABO t!ping should be performed using the manufacturer"s directions.lide test %. allo ing eaker antigens*antibodies to be detected and because the contents can be protected from dr!ing and smaller amounts of reagents are re>uired. @. &he slide test ma! be performed on an! non)porous clean surface. &ests in hich discrepancies are identified. &hen rock again and look for agglutination. in smaller blood banks one trained orker is likel! to perform all tests and discrepancies normall! do not occur. centrifuge the blood sample at 1000):000 rpm for : min. If serum not completel! separated or become clear. Add drop of about %03 red cell suspension to each drop of t!ping serum. must be repeated. . One to t o milliliters of serum is pipetted into a pre)labeled tube 'identification double checked( for serum grouping and other tests. &ake about 1 ml of cells into pre)labelled tube 'identification double checked( and add 0. @. "ube testing &he tube techni>ue is recommended because it is eas! to perform and advantageous because the centrifugation involved enhances the reaction.)mm)diameter. :. -et&od 1. 6ecord the results.especiall! in case of cord blood or specimens in poor conditions . but it is usual to use glass or ceramic opa>ue tiles or microscopic slides. &here are three basic techni>ues9 1. .ample 1.3 cell suspension in normal saline.3 cell suspension in normal saline . Cell grouping -et&od 1.

. 'spin tube method(. @. GRAD#NG O+ AGGLU"#NA"#ON REAC"#ON B @ . centrifuge the tubes at 1000 rpm for one minute after . B*. Observe the supernatant fluid for the presence of heamol!sis against ell lighted background. centrifuge after . E. 6ecord the results immediatel!.. but definite small clumps '10)1. @. '&able @. B 1 8ine granular appearance visuall!. B ) B*. Gentl! disperse the cell button and check for agglutination against a ell)lighted background. O ) ) ) ) B Interpretation 'blood group of test cells( AB A B O Oh B C AgglutinationF ) C -o agglutination . ? % to : cells sticking to gather per lo po er field. B*.eamol!sis Controls It is generall! unnecessar! to run controls each time ABO grouping is performedF ho ever. . -et&od 1. . All negative results must be e0amined under microscope. in urgent cases. B cell and O cell.. B : &hree or four individual clumps ith fe free cells. E.)10 min incubation at 6. C . reagent anti) sera and cells used should be controlled on a dail! basis as a part of routine >ualit! control programme. 2.)10 min incubation at 6.&. Add t o volumes 'drops( of anti)A in the tube labeled A t o volumes 'drops( of anti)B in tube labeled B and t o volumes 'drops( of anti) AB in tube labeled AB. ?here no agglutination is seen macroscopicall! e0amine the contents under microscope. in urgent cases. <. <. 7lace t o volumes 'drops( of the test serum in each tube. cells( per lo po er field. Serum grouping 5se a similar tube techni>ue to test patients"*donors" serum against saline suspensions of pooled %): samples of group A cells. %. B % Dan! fairl! large clumps ith man! free cells.&. Add one volume 'drop( of test cell suspension in each tube. Gentl! disperse cell button and see for agglutination. :. Di0 the contents of each tube b! gentle shaking and leave at 6. uneven distribution.. :. Di0 the contents of each tube b! gentle shaking and leave at room temperature '%0=)%@=1( for :0)E0 min.ingle clump of agglutination ith no free cells. Add one volume 'drop( of A cells to tube labeled A. ) B*.et up three ro s of clean test tubes and label them. 6ecord the results immediatel!. 'spin tube method(. one volume 'drop( of B cells to tube labeled B and one volume 'drop( of O cells to tube labeled O.. &able @.&. for :0)E0 min.%. . 2. ) All cells are free. Gabel three tubes A cell.( "able 123$ Re%ord o* t&e results 4ABO Grouping5 6eaction of test red cells ith Anti)A B B ) ) ) Anti)B B ) B ) ) Anti)AB B B B ) ) 6eaction of serum ith pooled cells A ) ) B*. Observe the supernatant fluid for the presence of heamol!sis against a ell)lighted background. B cells and O cells. B*.

1 ml of saline in to all 't o sets( test tubes. &hose sho ing agglutination are labeled as A1 and A1B and those not sho ing agglutination as A% and A%B.I 6ead results and record. at 1000 rpm for 1 min. Anti)A1 found as a naturall! occurring antibod! in the serum of A% and A% B individuals. . &o the third tube. Hight! per cent of group A and AB blood ill be agglutinated b! anti)A1 serum and are classified as A1 and A1B respectivel!. :.eamol!sis 'partial or total( must be interpreted as positive.)10 min.B . %.# 1. ) C -o agglutination JOccasionall! A% and A%B serum contains anti)A1 and thus gives reaction ith pooled A cells. ) or B*. Add 0. $eliver 0.%. %. :. as alread! described but the main division is into A1. Di0 the contents of each tube b! gentle shaking and leave at room temperature for :0)E0 min. @. -et&od 1.eamol!sis.uman anti)A1 'prepared b! absorption of group B anti)A serum ith A% cells(. Anti)A1 reagents can be obtained from three main sources9 1.1 ml or 1 volume from the dilution tube ith dilution of 191. A%. :. &o the first add 1 volume of %3 saline suspension of the donor"s * patient"s red cells. "#"RA"#ON O+ AN"#BOD#ES. 7lace 1 volume each of Anti)A1 reagent into three clean test tubes. ABO sub grouping &here are several sub groups of A.% and discard or save for further dilution if re>uired.1 ml or 1 volume from the mi0ture to tube : '19@ dilution(. In the tubes add 0. 1ontinue the same techni>ue. . according to the serum dilution. add 1 volume of kno n A% cells 'A1 and A% cells as positive and negative controls should al a!s be included(. A1 B and A% B. . Gabel a ro of test tubes. Di0 the contents of tube % ith a clean pipette and then transfer 0. &he lactin 'plant agglutinin( $olichos biflorus hich is specific for A1 antigen. D#LU"#ON -E"!OD 1. A% B. interpretation O ) ) ) ) A1 A%J A1 B A%BJ B to B@ C agglutination of increasing strength . . &hose negative ith anti)A1 are classified as A%. &o the second tube add 1 volume of kno n A cells.1 ml or 1 volume of serum to tubes 1 and % 'dilution 191 and 19%(. usuall! 191 through 191.DOUBLE D#LU"#ON S"EP.J B*.1 ml or 1 volume of saline into all tubes e0cept the first tube. %. In case of emergenc! centrifuge the tube after . %. Arrange t o sets of test tubes '10( in a test tube rack. "esting *or A/ and A6 6eaction of red cells ith anti ) sera Anti)A Anti)B Anti)AB Anti)A1 B@ ) B@ B@ B@ ) B@ ) B@ B@ B@ B@ B@ B@ B@ ) 6eaction of serum ith cells A B ) B*. ) ) ) or B*.J )*. Reading Gentl! shake the tube and e0amine.. C . through all dilutions and remove 0.

Lualit! assurances of reagents correct techni>ues. GROUP#NG O+ CORD OR #N+AN" BLOOD . &his helps to confirm antibod! specificit! and levels little to chance. Add 0. Obtain a fresh blood sample from the donor unit of patient to rule out discrepanc! due to contamination or un identification samples. 7referabl! pool the different cells sample. &he time for a clearl! visible reaction 'B1( and then for strong 'B@( reaction to occur is recorded ith the help of a stop atch. &he test is done b! mi0ing one drop of anti) serum ith one drop of 103 cell suspension on a slide or title and rocking gentl! at room temperature '6&(.$. A7#D#". .1% and discord or save for further dilution if re>uired.:. 1ord red blood cells should be ashed :)@ times ith saline to minimi/e error due to harton"s Kell!.E.erum grouping is not recommended in ne ) borns. Di0 the content of tube % ith a clean pipette and then transfer 0. incubate at room temperature for E0 min.and AI.1 ml from the dilution tube ill dilution of 19. 7erform direct agglutination test on the cells are coated ith antibod! as in . in describing the titre of the serum. &he agglutination titre is recorded as the reciprocal of the highest serum dilution in hich there is '?( agglutination. anti)AB and anti)A. Incubate in the appropriate manner according to the antibod! being tested. . <.1 ml or 1 volume of %). %. @.1 ml of serum to test tube 1 and % 'dilution 191 and 19%(. Gentl! dislodge the red cells and observe macroscopicall! for agglutination. B and O)group cells. as appropriate for individual problem. keep the tests at room temperature and at @=1 for :0)@. A%. Add 0. @. Group +O" cells and autologous cells should be used as controls to detect allo)agglutinins and auto)agglutinins. :.pecial precautions should be taken hile testing cord or ne )born infant blood since ABO antigens are not full! developed and allo)agglutinins are usuall! absent.1 ml or '1 volume( O$ the mi0ture to tube : '19@ dilution(. ?eak and negative reaction should be read microscopicall!. &hus a serum hich gives eak '?( agglutination at 19%. Repeat Preliminary Pro%edures 1.## E. In case of anti)A and anti)B.A. of A1.3 cell suspension. 6est the cells ith fresh and potent anti)A. anti)B. 1are full observations and interpretation of results resolve man! problems. 1ontinue the same techni>ue through all dilutions and remove 0.. .3 saline suspension of appropriate red cells to each tube. min and then interpret the results. 2. ?ash the cells :)@ times in normal saline to rule out roulea0 formation and prepare %).. 5se group O cord cells if anti)1 is suspected <. .peed and strength of agglutination is termed as avidit!. S"EP. SOL7#NG PROBLE-S O+ D#SCREPANC#ES Once a discrepanc! is detected in ABO cells and serum grouping repeat the test before additional investigation as carried out.E is said to have a titre of %. it is usual to ignore the diluting effect of the cells suspension. &est the serum against appropriate A1. or anti). A% and B cells. . E.

ii( . &ube sedimentation :. it should be tested ithin @2 hours. . .igh protein anti sera(. for a period of 1 !ear. anti)B. &he slide test is not recommended for routine tests because is it not reliable especiall! for eakl! reactive cells and it also has the disadvantage that driving of the reaction mi0ture can cause aggregation of the cells that ma! be misinterpreted as agglutination. Slide "esting &his ma! be used for emergenc! 6h '$( t!ping if centrifuge is not available. Blend of IgD anti)$ monoclonal and IgG anti)$ pol!clonal 'human anti)$( reagent. anti)AB.0Mg*l. . &he heteroh!bridoma cells. &he same method has been used to produce anti)A.R& 4D5 serum2 7otentiating substances such as albumin. &his contains macromolecular additives and gives rapid reliable results hen used in accordance ith manufactures directions. &hese antibodies are highl! specific and react e>uall! ell at %0=1 as ell as at :<=1 and are reliable for slide for rapid test tube techni>ue.S"EREAGEN"S +OR R& GROUP#NG &he different t!pes of anti)6h '$( sera available are9 /2 Poly%lonal &uman anti. &he t o kinds of saline active anti)6h '$( sera available are9 i( &raditional saline reagents are made from ra material containing predominantl! IgD antibodies hich agglutinate antigen positive cells suspended in saline. IgD anti)$ monoclonal reagent %. 62 Anti.aline)active anti)6h '$( sera prepared form IgG antibodies that have been chemicall! modified to convert them to agglutinate in saline medium. &hese are relativel! scarce due to non) availabilit! of ra material. 12 -ono%lonal antibodies &he first blood group specific human monoclonal antibodies ere produced from culture of l!mphoblastoid cells produced b! transformation of human B l!mphoc!tes ith Hpstein Barr #irus 'HB#(. 82 Anti. en/!me.lide test %. &here are three methods9 1. A.R& BLOOD GROUP S. etc.D sera for saline tube test. IgD and IgG anti)$ monoclonal reagent tube &he blood sample should be collected ith or ithout anticoagulant in a sterile tube and stored at @=1. In tissue culture.G serum are used to bring about agglutination ith human IgG anti)$. -o sign of heamol!sis should be there. &he t!pes of monoclonal anti)$ reagents are9 1.R& 4D5 serum of slide test or rapid tube test '. produce both IgD and IgG. appro0imatel! %0). R& Grouping Pro%edure 6h '$( t!ping should be performed according to the instructions given b! the manufacturers of the anti)sera or as described belo . B! taking l!mphoc!tes from 6h)immuni/ed donors both iGD and IgG antibodies have been obtained &he method as not successful because man! of cell lines die after a month Another approach has been made b! fusing l!moblastoid cells 'produced b! HB#)transformed B l!mphoc!tes( ith mouse m!eloma producing hetero)h!bridoma.

then it is most likel! the cells are coated ith immunoglobulin. .-et&od 1. iii( Incomplete Anticoagulated blood ma! clot on the heated slide.ome orkers routinel! perform the test in duplicate using antisera from t o different sources. foreign substance or another antiserum. iv( 6eagents ma! be identified incorrectl! resulting in the rong reagent being used in place of anti)6h '$(.3 cell suspension in plasma or serum in each tube. 7lace one drop of control medium or %%3 albumin on another labeled slide. . 7lace 1 drop of control diluting reagent or %%3 albumin in a tube labeled control. In such cases the cells are tested ith saline reactive antiserum. . Add 1 drop of %). . mm diameter. . Observe for agglutination. &he interpreted results ma! be false negative in the follo ing cases9 i( .erum factors can be ashed b! ashing the cells in normal saline. tilt gentl! and continuousl! for t o minutes. Gentl! re)suspend the cell button and observe for agglutination. 7lace both slides on a vie bo0 surface 'lighted(. 7lace 1 drop of anti)6h '$( serum in a tube labeled +test". Di0 the cell suspension and reagent. If there is agglutination in the control. "ube testing -et&od 1. #nterpretation A positive test has agglutination ith anti)6h '$( in the +test" and smooth suspension of the cells in the control. @. %. If after ashing the agglutination persists in control test.03 red cells suspended in plasma or serum on both the slides. the test results must be considered invalid and the test ith saline)reacting anti)$ must be performed. are read before time.03 cell suspension must be used.. %. hich normall! take t o full minutes to agglutinate. Di0 ell and keep at :<=1 for one hour 'sedimentation method(. &he interpreted result ma! be false positive in the follo ing cases9 i( $r!ing of reaction mi0ture ma! be confused ith agglutination. using a clean stick for each slide and spread the mi0ture evenl! on the slide over an area of 1. :. #nterpretation A( &he interpreted result ma! be false positive in the follo ing causes9 i( &he anti)6h '$( used ma! contain antibodies of other specificities in addition to anti)6h '$(. ii( A eak suspension of cells ma! agglutinate poorl!.. iii( Antisera and regents ma! be contaminated ith bacteria. @. 7lace one drop of reagent anti)6h '$( on a labeled slide. 7ut one drop of @0).aline suspended cells react poorl! or not at all. iii( ?eakl! active cells. ii( Immunological coating of the patient"s cells of factors in patient"s serum causing cellular aggregation can cause false agglutination in the control tube containing immunologic inert reagent. :.mall fibrin clots ma! give appearance of agglutination. iv( 7ol!)agglutinable red cells ma! cause false agglutination ith an! reagent containing human serum because the antibodies anti)& and anti)&n hich agglutinate these surface altered cells are present in most adult human sera. All negative results must be confirmed under microscope. ?hole blood from a severel! anemic patient ma! be concentrated b! centrifugation or b! removing plasma or serum. ii( . A negative test has a smooth suspension of cells in both the +test" and control. @0).

%. Controls i( ii( A negative control if supplied ith the test reagent. &he result is valid onl! if the results of controls are satisfactor!.I "esting *or D 1ells of lo )grade $ possess the $ antigen but e0pressed so eakl! that the! are not directl! agglutinated b! most anti)$ sera $ can be detected b! antiglobulin test. 7lace one drop of saline reactive anti)$ in a properl! labeled +test" tube.aline agglutinating anti)6h sera should be used hen the control of high protein anti)$ gives a positive test that persists even hen ashed red cells are used. 8ailure to properl! identif! reagents resulting in the rong reagent being used in place of anti)6h '$(.B( &he interpreted results ma! be false negative in the follo ing cases9 i( ii( iii( iv( Inadvertent failure to add the antiserum. Note$ A saline suspension of kno n 6h '$( positive 'O 61 6%( and 6h '$( negative 'Orr( cells should be run parallel ith the test as controls. 'IA&( . Anti. -ONOCLONAL AN"#. :. . 1ontrols should be put at the end and read first to ensure that the! ork in the shortest incubation period compared to the test sample. 1entrifuge usuall! 1000 rpm for 1 min. &he! are also not suitable for $J test as IgD antibod! generall! reacts poorl! in the indirect antiglobulin test. Di0 gentl! and incubate at :<=1 usuall! for 1. Gentl! re)suspend the cell button and observe for agglutination. &oo heav! cell suspension in the tube test ma! result in poor agglutination. Hnsure that the concentration of cells in the controls is comparable to that of the test cells. -et&ods Dethods for 6h '$( t!ping ith monoclonal regents are the same as described for anti)6h '$( sera for slide or rapid tube test. -egative result must be confirmed under microscope.):0 min. @.3 cell suspension of ell) ashed red cells ith normal saline. or saline)reacting anti)$ tube method. &hese sera are not suitable for the slide test. #nterpretation Agglutination in the test indicates that the red cells are 6h '$( positive.R& 4D5 REAGEN" &hese reagents ork e>uall! ell at %0=):<=1 and are reliable for emergenc! 6h '$( t!ping b! slide or immediate spin tube techni>ue besides the routine sedimentation tube techni>ue. Add one drop %). 1ells possessing eak e0pression of the $ antigen ma! not react ell in immediate spin)tube method..R& 4D5 sera *or saline test tube te%&ni0ue . &he controls for kno n 6h '$( positive cells 'O61 6%( and O 6h '$( negative cells 'Orr( should be put in parallel ith the test cells as in saline)reacting anti)$ controls. -et&od 1. 1ells giving a positive direct antiglobulin test can usuall! be tested ith saline)reacting anti)$ serum. Observe the controls before reading the test.

&he final volume of saline did not e0clusivel! dilute the A. #ND#REC" AN"#GLOBUL#N "ES" 4#A" #C"5 &his test is used to detect the presence of incomplete antibodies and complement binding antibodies in the serum after coating on red cells in vitro in. the reaction ma! be confirmed b! adding kno n IgG sensiti/ed cells. . If the test is negative. &he manufacturer"s package insert ill tell if the reagent can be used for $ testing. &he A. @. :. 9. . A positive result indicates the follo ing. 1. Gentl! re suspend the cell button and e0amine for agglutination. E. A.G serum.creening and detection of une0pected antibodies in serum. then sample is 6h '$( positive and there is no need to proceed ith antiglobulin phase of test.A( :. 1. 2. ash the cells :)@ times ith saline can decant the last ashing.. Control %ells *or A!G "ests Positi:e Control$ .All anti)$ sera.$-( %. &he red cells have been ade>uatel! ashed. especiall! anti)$ sera for saline tube test and IgD monoclonal sera are not suitable for testing as IgD react poorl! in the IA&. $iagnosis of auto immune haemol!tic anaemias 'AI.ensiti/ed O 6h '$( 7ositive cells. If there is strong agglutination of cells in +test" tube. 7ol!clonal anti)$ 'human IgG anti)$(F IgD and IgG anti)$ monoclonal reagent. Add 1)% drops of anti)globulin reagents '1oombs serum(. 10. 1entrifuge at 1000 rpm for 1 min. Investigation of drug induced red cell sensiti/ation. If no agglutination or doubtful reaction observed. <. -et&od 1. . @. Di0 and incubate both the tubes at :<=1 for 1. &ake one drop of appropriate control reagent in labeled tube.G reagent as added to the test tubes.3 of cell suspension to be tested to both the tubes. if the negative control test is positive no valid interpretation of $ test is made. and IgD anti)$ 'human IgG anti)$( can be used to detect $ b! IA&.ensiti/ed O 6h '$( -egative cells 5n sensiti/ed O 6h '$( 7ositive cells. $iagnosis of haemol!tic disease of ne born '. #nterpretation Agglutination in the +test" tube and none in +negative control" tube constitutes a positive test result and the blood is accordingl! labeled $N. :.):0 min.G reagents has remained active and not been neutrali/ed. re centrifuge and re)e0amine for agglutination. Add %). %. 1. %. @. AN"#GLOBUL#N 4COO-BS5 "ES" 9 D#REC" AN"#GLOBUL#N "ES" 4DA" DC"5 &his test is used to demonstrate the coating 'sensiti/ation( of red cells in vitro ith immune antibod! 'IgG( or the complement component 'generall! 1:( in. A best general control for the $A& * $1& and IA& * I1& is the addition of IgG sensiti/ed O6 h '$( 7ositive cells to an! A.G test that are non)reactive. 1ompatibilit! testing %. Investigation of haemol!tic transfusion reaction. Negati:e Control$ . Di0 gentl! and centrifuge at 1000 rpm for 1 min. &he presence of agglutination confirms the test result. $etection of red cell antigens not detected b! other techni>ues. :. &ake one drop of anti)6h '$( serum in a clean labeled +test" tube. 6esuspend the cell button gentl! and e0amine for agglutination and record the test result.

Add one drop of IgG coated red cells to an! test that is non)reactive mi0. &he additional step should be never be substituted for the immediate reading because reaction due to IgG coating ma! become eaker after incubation. -ote9 &he clotted sample should be as fresh as possible 'not more than %@ hold( other ise the sample ma! be taken in H$&A to prevent the uptake of complement in vitro(.3 ashed cell suspension of O 6h '$( positive cells e>ual to the volume of diluted anti)$ serum. Di0. spin immediatel! at 100 rpm for 1 min. centrifuge at 1000 rpm for 1 min. <. . 1ells should sho B% agglutination 'if there is no agglutination the hole procedure in repeated b! taking less diluted anti)$ serum(. Add ..Preparation o* O R& 4D5 Positi:e Sensiti.. Add 1)% drop of pol! specific A.ed %ells Pro%edure 1. :. %. Di0. 7lace one drop of %). If there is no agglutination ash the cells three times ith a large volume of saline. 2.3 cell suspension to be tested in a clean labeled tube. 1entrifuge and read again. $A& is negative hen no agglutination is seen at either phase provided IgG coated red cells are added in step < of test. min. #N"ERPRE"A"#ON $A& is positive hen agglutination is observed either after immediate spin or after spin follo ing 6& incubation. <.3 suspension of sensiti/ed cells in saline.00 molecules of IgG per red cell but auto)immune haemol!tic anaemia has been reported ith an IgA coating belo this level. .elect a dilution of anti)6h '$( sera that costs the O 6h '$( 7ositive cells at :<=1 in vitro but does not agglutinate them one has to determine b! e0perience to hat e0tent anti)$ serum should be diluted to give sensiti/ed cells '-o agglutination(. Dake . E. Geave an apparentl! non)reactive test tube at room temperature for . @. $ecant the supernatant saline completel! ever! time. %. . incubate at :<=1 for :0 minutes. @. 1ompletel! decant the final supernatant ash. &hough this step is optional all manufacturers recommend it hen ma0imum sensitivit! for complement or IgG is desired. Di0 centrifuge at 1000 rpm for 1 min. Gook for the agglutination 'if there is agglutination the procedure is repeated b! taking more diluted anti)$ serum(.3 ashed sensiti/ed cells. A negative $A& does not necessaril! mean absence of coating globulin pol! specific reagents detect appro0imatel! . ?ash the red cells :)@ times in a large volume of saline care should be taken for ade>uate removal of the supernatant after each ash. Add 1 drop A. Gentl! shake the tube to dislodge the cell button and e0amine for agglutination using an optical aid and record the result. :. look for agglutination if a negative result 'no age( is obtained the test result is invalid and hole test should be repeated. 7lace t o or four drops of the test serum in a tube 'sample should be fresh for detecting complement)binding antibodies other ise fresh AB serum should be added to it(. #ND#REC" AGGLU"#NA"#ON "ES" or #ND#REC" COO-BS "ES" Pro%edure 1.G serum immediatel!. E. D#REC" AN"#GLOBUL#N "ES" 4DA" DC"5 Pro%edure 1. .G serum to 1 drop of the .

.%. 1entrifuge at 1000 rpm for 1 min. hile incubation at higher temperature ma! damage red cells or antibod! molecules #n%ubation time 8or saline or albumin techni>ues :0 min incubation at :<=1 is ade>uate to detect most of the clinicall! significant coating antibodies.3 suspension of ashed cells 'i. should be used. Gook for agglutination if a negative result 'no agglutination( is obtained the test result is in valid and the hole test should be repeated.. centrifuge at 1000 rpm for one minute..#A" #C" Of increase the rate of the antigen)antibod! reaction and reduces the incubation time ith out effecting sensitivit! for description of the action of bovine)albumin sec chapter on special method. 10. 8or some eak reactive in GI.RED CELL RA"#O Increasing the ratio of serum to red cells increases the degree of antibod! coating on red cells.. provided IgG coated cells are added in step % of IA& test. If no agglutination is seen ash three or four times in large amount of saline $ecant supernatant in each ash as completel! as possible.e. <. Note$ AU"O CON"ROL S!OULD BE KEP" (#"! "!E #A"2 #nterpretation IA& is positive hen agglutination is observed either after immediate spin or after spin follo ing 6& incubation. Add 1 drop of IgG coated red cells to an! test that is non)reactive. 2. Pro%edure A5 One stage met&od. incubation at lo er temperature decreases the rate of association bet een antigen and antibod!. H0amine for haemol!sis or agglutination using an optical aidF record results. Add one drop of @). +AC"ORS A++EC"#NG "!E SENS#". min. medium incubation time is 10)1. Di0 and incubate at :<=1 for :0)E0 min. H>ual volume of serum and %3 suspension of red cells in GI. SERU. Di0 and incubate for %0 min at :<=1 :. @. Agglutination at this stage indicates the presence of saline reacting antibodies. 11. BO7#NE ALBU-#N 466<5. min centrifuge and read again as in $A&. IA& is negative ho no agglutination is seen at either phase. Di0.. Suspending -edium &he sensitivit! of IA& can be increased ith the addition of %%3 BO#I-H AGB5DI. Geave an apparentl! non)reactive test at 6& for . E.3 cell suspension.Additi:e met&od 1. 7roceed further as in saline)IA& . 9. Gentl! shake the tube to dislodge the bottom and e0amine for agglutination using an optical aid. donors * patients red cells or screening red cells etc( :. O+ #A" "emperature Dost IgG antibod! and red cell reaction occurs optimall! at :<=1. 1entrifuge at 1000 rpm for 1 min . Add 1 O % drops of A.G serum. & o drops of albumin '%%3( are added in step % of saline)IA& %. A commonl! used ratio is % drops of serum to 1 drop of %).or H-PQDH or GI.

. 1entrifuge at 1000 rpm for 1)% at 1000 rpm for minute and e0amine for agglutination * haemol!sis E. E.)%0 min . Add 1 drop of %)@3 red cell suspension in the tube :. .Layering met&od 1. Di0 and incubate at :<=1 for 1.)%0 min . 1entrifuge at 1000 rpm for 1)% min E. 8irst t o steps are the same as in saline)IA& %. Danufacturers instructions should be follo ed. hich increase the chance of effective antigen antibod! collisions. Gentl! re suspend the cell button and observe for agglutination * haemol!sis. . 8or details see chapter on screening and identification for antibodies. .-E. ?ith out disturbing the cell button allo % drops of %%3 albumin to run do n the in side of the tube albumin ill form a la!er on the top of top of the cells R$o not mi0N. Di0 and incubate at :<=1 for minutes @. Gentl! re suspend the cell button and observe for agglutination * haemol!sis. %.B5 "'o stage met&od. Agglutination or haemol!sis is a positive result B5 "'o stage met&od 4Albumin layering5 1. 1onfirm all negative results under microscope.. Di0 and incubate at :<=1 for :0 min :. Agglutination or haemol!sis is a positive result. %. Add % drops of bovine albumin %%3 @. Add 1 drop of %)@3 red cell suspension in the tube :. Incubate further for 1. Besides the! reduce the negative charge on the red cell surface. Add %): drops serum to a labeled tube %. Add %): drops of serum to a labeled tube %. Add one drop of papin c!stein solution in step % of saline)IA& %.#A" Hn/!mes after the configuration of er!throc!te surface and increase the mobilit! of antigen and clustering of the antigenic site. Incubation at :<=1 for :0 min :. Danufacturers instructions should be follo ed.. 2. 1. Incubate of :<=1 for 10)%0 minutes <. -ote9 1. 7roceed further as in saline)IA& step)E &his procedure should be attached in R9th pageN EN=. Add one drop of %%3 bovine albumin along the side of the tube @. -ote9 1. Pro%edure Dostl! one stage method ith papain c!stein is conducted for determining antibodies b! en/!me)IA&.<3 as a stabili/er in other reagents speciall! those to be stored at @=1 -et&ods A5 One stage met&od 4Albumin used as an additi:e5 1. 7roceed further as step @ in saline)IA& -ote9 8or the preparation of papain)c!stein solution sec chapter on special methods. BO7#NE ALBU-#N SERU&his used in blood group serolog! as 'i( %%3 concentration as enhancer at agglutinationF and 'ii( 1oncentration of 1.

&o identif! the antibod! 'antibodies( and select blood that lacks the corresponding antigens 's( for cross)matching. Harl! detection of an antibod! ill give time to the pediatrician and the blood bank to prepare for intrauterine or e0change transfusion in the bab! or for an emergenc! transfusion to the mother.)%0 minutes and centrifuge at 1000 rpm for 1)% min. ii( $onors blood iii( Antenatal patients Patients re0uiring a trans*usion 6outing antibod! screening in recipient blood sample ill enable identifications of une0pected antibodies 'ies( and to find compatible blood.erum 1ell suspension % O @ 3 Bovine albumin %%3 1 O % drops 1 drop % drops Di0 and incubation :<=1 1. %.Bo:ine Albumin Serum . &o perform cross)matching using several other units an attempt to find units that fail to react ith the recipient"s serum. S%reening o* Antibodies &he candidates of antibod! screening are i( 7atients re>uiring a transfusion. &hese units are regarded as compatible and issued for transfusion. Gentl! re suspend the cell button and observe agglutination * haemol!sis. :. . If the une0pected antibodies are detected during screening test or compabilit! tests the technologist has t o choicesF 1. Donors Blood Hver! donor"s blood should be screened for antibodies. 1ontrol all negative result under m*s .tage)II . &hese second course of action is much less desirable than the first and should be taken in emergenc! onl!. Gentl! re suspends the cell button and observes agglutination.erum % O : drops % O @ cell suspension 1 drop Di0 at :<=1 incubate for :0 min 1entrifuge at 1000 rpm at 1 O % min ?ithout disturbing allo t o drops of %%3 albumin bovine inside of the test tube. Antenatal patients All antenatal patients should be screened for a antibodies besides anti 6h '$(.tage)I . other antibodies also hich could be problem for both the mother and the child. Incubate at :<=1 for 10 O %0 min. &he presence of antibodies in donor"s blood ma! cause a mild transfusion reaction of decrease of patient red cells. 72 SCREEN#NG AND #DEN"#+#CA"#ON O+ AN"#BOD#ES UNE>PEC"ED AN"#BOD#ES &hese antibodies are screened and detected in pre)transfusion testing of patient"s blood and during antenatal care in mother"s blood.

et up % tubes. . Di0 ell and incubate at :<=1 for 1 hour. @. . kidd. of course.. Add one drop of screening cells to each tube. /2 Saline test 1. should determine the full phenot!pe of individuals on staff and bleed them regularl!. 62 En. %..P#CAL PANEL SCREEN#NG CELLS $onor -o 1 % Geno &!pe 61 61 6% 6% 6h ' c $H e B)B)B )B)B) D-. &hus cross matching and antibod! screening both are ever! necessar!. <. Indirect antiglobulin test. . SELEC"#ON O+ SCREEN#NG CELLS 8or screening of antibodies a set of t o speciall! selected group O61 61 red cells are used. Add % drops of patients serum to each tube :. . 6esult should be scored and recorded.yme test Pro%edure 1. ". mm( %. . E. <.s. Hn/!me test O papain c!stein techni>ue :. 6esult should be scored and recorded. . Dan! antibodies can be detected more casil! if the antigens are homo/!gous on the red cells. :. &hese cells must carr! the main antigens of 6h. Add 1 drop of screening cells to each appropriate tube @. 1ell panels are commerciall! available in developed countriesF ho ever the! can also be prepared b! the individual laborator! institutions that prefer to prepare their o n panel.All the screening cells are group +O" it should be remembered that ABO in compabiilt! ill not be detected unless a cross)match is also carried up % tubes of 'si/e 100<. Di0 and incubate at 6& '%0)%. &his ill increase the chance of detecting the eak antibodies. An! haemol!sis must be noted as this indicates and positive result. E. &he presence of rare antigen should also be determined and noted. s BB) B)B) 7 71 B B Aell Ak BB )B $uff! 8!a 8!b B) BB Ge is Gea Geb )B B) Aidd Kka Kkb )B B) Gutheran Gua Gub )B )B &o maintain the optimum strength of the antigens the t o cells O61 61 and O6% 6% should be used separatel! 'and.hake the tubes gentl! and read microscopicall! negative readings should be checked microscopicall!. -et&ods *or S%reening o* antibodies &hree techni>ues are commonl! used9 1. duff!. 7 and Ge is 'and Gutherian is possible( blood group s!stems. 7lace % drops of patient"s serum in each tube. Add one drop of papin c!stein to each tube. 8or free/ing or red cells in gl!cerol see chapter on storage and preservation of blood. D-.aline test at room temperature %. in conKunction ith each other( and not pooled. kell. . particularl! of common group.hake the tubes gentl! and read microscopicall! negative readings should be scored and recorded..=1( for 1 hour. or free/e large donations from these individuals in gl!cerol. . D-.

$. #ND#REC" AN"#GLOBUL#N "ES" Pro%edure 1.. D. 1. Gua Gub . O red cells and A% cells 'identified(. :. <.istor! of transfusion or pregnanc!.s. Gea Geb. A. Kka Kkb. see chapter on special methods. %.etup % tubes %. &his acts as control for AG&. 9. 2. 7 and Gutheran blood group s!stem namel!. Repeat Preliminary "est 1. B. E. 10. Dedical histor!)diagnosis. &hese cells must carr! the ma0imum antigens of 6h. $uff!. %. 1. 6esult of previous testing. Gentl! shake the tube to dislodge the cell button and e0amine for agglutination * haemol!sis macroscopicall! negative readings should be checked microscopicall!. #DEN"#+#CA"#ON Before beginning the antibod! 'ies( identification. $rug therap! 'including 6h immunoglobulin( @. If there is no agglutination or haemol!sis. s. kell. direct A. &hese are usuall!2 O 10 groups +O" cells from different donors. . H. e. ABO cells and serum grouping should be carried out using anti)A. Add 1 O % drops of antiglobulin serum to each tube. it is use full to revie the follo ing. -et&od o* antibody dete%tion &he serum should be tested against panel cells '2 O 10( patient"s o n cell 'auto control( and t o cord cells. Add 1 drop of screening cells to each tube. Ge is. :. If patients is group A perform A sub t!ping using anti)A 'icctin( :. 8or more details sec chapter on agglutination '1oombs( test AN"#BOD. kidd. H0amine for agglutination and for haemol!sis and record the result. 7. anti)ABF A. Add 1 drop of IgG anti)$ coated cells in non)reactive tests.. Di0 and centrifuge at 1000 rpm for 1 min and e0amine for agglutination. . 7erform 6h '$( t!ping @. .. . anti)B. ?ash the contents of both tubes : O @ times in large volume of saline $ecand each ash the completel! as possible. 8!a 8!b. @.G test '$A&( Sele%tion o* panel o* %ells &hese panel cells are commerciall! available though the! can be prepared in the individual laborator!. 7lace %)@ drops of patient"s serum in each tube. D-. c. 1. k. Di0 and incubate at :<=1 for :0)E0 min. &hree techni>ues are commonl! used.aline test at room temperature .Note$ An! haemol!sis must be noted as this indicates agglutinations for preparation of papain c!stein solution.-. If a negative result no agglutination( is obtained the result is in valied and hole test should be repeated. Di0 ell and centrifuge at 1000 rpm for 1 min. up appropriate number of 100<. E. 6esult should be scored and recorded. anti)7. /2 Saline test Purpose$ &he saline test at 6& is used to identif! IgD agglutinations. . H0amine for agglutination and haemol!sis record the result.%. auto control. 6esult should be a cored and recorded. anti)-. &his techni>ue serves to enhance the reaction of 6ah Ge is and kidd antibodies.=( for 1 hour . Di0 and incubate at 6& '%0)%. <. mm tubes. 'for pond cells. :. Di0 ell and incubate at :<=1 for one hour. An! haemol!sis must be noticed as this indicates agglutinations man! le is antibodies produce haemol!sis in saline hen fresh serum is used. 7lace % drops of patient"s serum in each tube. Hn/!mes ma! eaken or inactivate certain antigens like D. :. -egative reading should be checked microscopicall!. It auto agglutination is due to cold agglutinings. E. t o group O cord cell suspension Sone 6h '$( B one 6h '$( ) I to each appropriate tubes. @.-E "ES" Purpose$ Hn/!me tests are useful for the identification of arm reacting 'IgG or complement binding( allo)and auto)antibodies. %. . anti Geb. . &here ma! be anti)I. namel! anti)D. 7lace % O @ drops of patient"s serum in each tube..hake the tube gentl! and read macroscopicall! negative results should be checked microscopicall!. . 7lace % drops of patient"s serum in each tube. Add 1 drop of papain c!stein solution to each tube.hake the tube gentl! and read macroscopicall!. @. etc. if the serum fails to react against the cord cells. Indirect antiglobulin test. patient"s ashed cell suspension for the auto)control and % group O cord ashed cell suspension TO 6h '$( B. patient"s cell suspension for the auto control and % group +O" cord cell suspension 'O6h '$( B O6h '$()( to each appropriate tubes. anti)I.. Add 1 drop of panel cell. -et&od$ Saline.-. patient"s cell suspension for auto control. .etup the appropriate number of '100<. Di0 and incubate at :<=1 for :0)E0 min. O 6h '$( )U to each appropriate tubes. Hn/!me test. anti).AG" Pro%edure 1. @.. Add 1 drop of 7anel cell. -et&od 1.( test tube for panel up appropriate number of tubes. anti)Gea. t o cord cell( %. Pro%edure$ 1. #ND#REC" AN"#GLOBUL#N "ES" Purpose$ &he indirect AG& is done to detect arm reacting IgG and complement binding allo)and auto) antibodies. reaction ill commonl! be negative at :<=1. 8!a and 8!b. .. <. auto control. EN=. Note$ An! haemol!sis must be noted as this indicates agglutination. :.. . Note$ &he above procedure can be performed at a lo er temperature '@=1( hich tends to enhance the reaction of man! cold antibodies. % group +O" cord cells( %. Add one drop of panel cell. .

&his acts as control for AG&. 10. H0ample 9. 11. If there is no agglutination or haemol!sis. ash the contents of each tube : to @ times in large volume of normal salineF decant ash as completel! as possible. <. Add 1 drop of IgG anti)$ coated cells in non)reactive tests9 Di0 and centrifuge at 1000 rpm for 1 min and e0amine for agglutination. B! hat techni>ues do reactions occurV 8or identification cross out all the antigenic determinates occurring on the panel cells that did not react ith test serum beginning ith the first cell and proceeding to the end of the panel see e0amples 9. Add 1 O % drops of antiglobulin serum to each tube. &he result of the three techni>ues must be looked at individuall! and then as hole. Gentl! shake the tube to discord the cell button and e0amine for agglutination and * or haemol!sis macroscopicall! negative reading should be checked microscopicall!. If negative result 'no agglutination( is obtained the result is invalid and hole tests should be repeated.% &able .E.1 &able.1 and 9. Note$ 8or more defails see chapter on antigloblulin '1oomb"s( test. 2. 9.% H0ample O 9. 1entrifuge at 1000 rpm for 1 min. #nterpretation o* result &o define the antibod! 'ies( that has been found. Di0 ell.

&his techni>ue also serves to detect maKor ABO grouping error.o ever in certain situation because of the non)availabilit! of group specific blood group O blood is selected for an A or B patient or A*B blood for an AB patient.aline techni>ue is designed to detect IgD antibod! 'ies( that react optimall! 6& '%%=1( or lo er in recipient"s serum against donor"s cells. &he donor"s red cells must be taken from the tube attached to the bag. &he methods used must include those that ill demonstrate IgD and IgG antibodies compatibilit!. &he recipient serum or plasma must be cross)matched ith donor"s red cells before hole blood or red cells are used for transfusion this is maKor cross match must be done e0cept hen the demand is ver! urgent and there is no time for cross)matching.7# CO-PAB#L#". . "ES"#NG 4CROSS -A"C!#NG5 1ompabilit! tests are done to ensure that particular unit of blood ma! be safet! transfused to a patient. .aline techni>ue is in ade>uate as a compabilit! test because clinicall! significant IgG antibodies are not detected b! this method. . SAL#NE "EC!N#?UE +OR #g.CO-PA"#B#L#". -ormall! group specific blood 'ABO and 6h '$( group( as that of the patient is selected. Saline met&od demonstrate ABO %ompability .

. 7erform A. 7ut % drops of patient"s serum in a labeled tube %. %. 8or details see chapter on A. 1entrifuge at 1000 rpm for 1 min in spin method centrifugation is optimal in sedimentation method.-E "EC!N#?UE 8or method see chapter special methods ALBU-#N "EC!N#?UE 8or method see chapter on special method.. :. 7ut % drops of patient"s serum in labeled tube. &he maKorit! of incomplete antibodies is IgG and is detected b! A.G test. 2. 7atient"s ABO and 6h '$( &esting . 1entrifuge and check for agglutination)if there is no agglutination tests in valid. It a consistenc! high standard of performance of indirect antiglobulin test is not available in an! laborator!. #nterpretation$ . Gabel 1 tube for each donor sample to be tested. ?ash the cells three times ith normal saline. 1entrifuge at 1000 rpm for 1 min. another techni>ue 'en/!me*albumin( capable of detecting antibodies reactive at :<=1 ma! be in reported as an alternative. E.G test.aemol!sis or agglutination at an! stage indicates in compabilit!.&uman globulin test 4#A"5 *or #gG %ompabtibility -et&od 1. Add IgG coated red cells to negative A. :. . Add 1 drop of %)@ drops saline suspended red cells of the donor to the tube.. EN=. 6ead the result observe for haemol!sis and agglutination. #nterpretation Agglutination or haemol!sis indicates a positive result 'incompatible( Note$ Immediate spin test is acceptable but the incubation improves sensitivit! of the test if the recipient has eakl! reactive anti)A or anti)B or if the donor"s red cells have eak e0pressions of an antigen eg A% B red cells.G test. "ES" +OR #gG AN"#BOD#ES Anti.-et&od 1. <.G test. CO-PA"#B#L#". E.!uman Globulin "est Indirect anti)human globulin test 'IA&( is the most important and idel! serologicall! procedure in modern blood banking to test the compatibilit! bet een recipients serum and donor"s cells. Di0 and incubate for . #ndire%t anti. @. @. check for haemol!sis*agglutination. PRO+OR-A +OR CROSS -A"C! 1. Add 1 drop of %)@3 saline suspended red cells of donor. Incubate for :0 O E0 min at :<=1.)10 min 'spin method( or incubate for :0)E0 min 'sedimentation method( at 6&.

GO? IO-I1 .aline test at 6& Giss)IA& test at :<=1 1ompatible 'Qes*-o( -ote9 A positive result 'agglutination and or haemol!sis indicates in compabilit!.&6H-G&. #ssue o* blood 1.1ell Grouping )A ) A1 ) B ) AB 6h '$( test )$ . Antibod! screening on patient cell. -ormal saline techni>ue for IgD %. 1ross matching 6outine test for cross match 1. $onor"s ABO and 6h '$( group . min test( 1.aline test at 6& IA& or en/!me albumin test at :<=1 1ompabilit! Qes*-o Note$ A positive result 'agglutination and or haemol!sis indicates in compatibilit!. :. 'Giss( for cross)match '1. $A& in patient"s cells @. Giss)IA& $onor"s -o . 7atients ABO and 6h '$( Group Interpretation of cross matching test $ate and time of issue Identification of the person performing the tests. 1ross match result should be sent along ith the blood to be issued. -ormal saline techni>ue for IgD %. &he cross match report form must include.erum Grouping A cells B cells ABO Group 4 6h '$( O cells %. IA& or en/!me * albumin techni>ue for IgG. $onor"s -umber . Gabel or tag is securel! attached to the unit of blood it should contain9 i( ii( $onor"s blood identification number. %. i( ii( iii( iv( v( vi( vii( viii( $onor"s blood identification number $onor ABO and 6h '$( group H0pir! date 7atients name and identifications number etc.

epatitis B -A-B rarel! . ?B1. 7roto/oal infection . 7latelets antigens or plasma proteins. $evelopment of antiplatelet antibod! usuall! 7G NON #--UNOLOG#CAL E++EC"S #mmediate E**e%ts 8ever ith shock 1ongestive heart failure .I#( .aemol!sis ith s!mptoms Usual Etiology Bacterial contamination #olume over load 7h!sical destruction of blood due to free/ing or overheating mi0ing non)isotonic solutions ith red cells. Hngraftment of transfused functional leucoc!tes.iii( iv( v( 7atients name and identification number 7atients ABO and 6h '$( groups. :. and Interpretation of cross)matching test.epatitis AI$.!philis Dono nucleosis like effects Usual Etiology Dultiple transfusion . .taiamal parasites &reponoma polida 1!tomegalo virus Hpstein Barr virus 'HB#( Immune complications of blood transfusion ma! be caused b! red cell leucoc!te or platelet incompabilit! or allergic reactions to plasma components. 1heck the blood e0pir! date to avoid issuing out dated blood.aemol!sis ith s!mptoms 8ebric non haemol!tic reaction Anaph!la0is 5rticaria -on cardiac pulmonar! oedema Delayed E**e%ts Alloimmunisation of 6B1. .epatitis)A 6etrovirus in donors blood '.aemol!sis Graft)vs)host discat 7ost transfusion purpura Usual Etiology 6ed cells incompabilit! Antibod! to leucoc!te antigens Antibod! to IgA Antibod! to leucoc!tes or complement activation Antibod! to leucoc!tes or complement activation Usual Etiology 7rimar! alloimmunisation due to e0posure to antigens of donor origin. -ote9 If blood issued before compabilit! test are completed label should clearl! state uncross matched blood. In spect the unit to make certain it does not have abnormal colour or appearance. Anaemestic or secondar! response of antibod! to red cell antigens. 7##2 AD7ERSE E++EC" O+ BLOOD "RANS+US#ON Ad:erse e**e%ts o* "rans*usion #mmunologi%al e**e%ts #mmediate E**e%ts . At the time of issue of blood observe the follo ing9 i( ii( iii( 6echeck the identification of the patient and donor blood. Delayed E**e%ts Iron .

8!a s!stems. chill B!ring sensation at the site of transfusion 7ain in chest. It is not serious as donor antibodies are diluted.H It the reaction is not checked there ma! be haemoglobinaemia .top transfusion and inform the patient"s ph!sicians.& .RED CELLS #NCO-PA"#B#L#". hich is usuall. iii( ?eak antibodies not detected b! routine tests.O1A 7.aemorrhage .A. B! the donor antibodies. &his usuall! results from the indiscriminate use of group +O" blood hich has to been screened to ascertain the anti)A and anti)B titre and haemol!sis.( in recipient. recipient sample( ii( 1onfusion in the identit! of patients at the time of collection of sample or at the time of transfusion. . All phases ma! not occur in patients.H 8ever. 1. nearl! al a!s due to 6h incompabilit! and sometimes due to A. In some cases e0travascular haemol!sis dela!ed for 5p to t o eeks or more after transfusion. associated ith in compabilit! in ABO s!stem and e0travascular haemol!sis. lumbar region of back . 7O. . 62 "e%&ni%al Errors i( Hrror in blood grouping and cross)matching.A.A.hock %.H 6enal failure Oliguria Anuria 5raemia @. ii( Incompatibilit! not detected in cross)matching due to improper method. 6H1O#H6Q 7. Causes o* &aemolyti% trans*usion rea%tions /2 Clari%al Errors i( in ade>uate or incorrect labeling of blood 'pilot tube.. #n %ase o* suspe%ted trans*usion rea%tion. blood container. %. iv( $estruction of recipient red cells. Aeep the intravenous line open ith infusion of normal saline or other suitable intravenous infusion.t&e trans*usional must ta)e t&e *ollo'ing a%tions 1. &he manifestations of red cell in compabilit! include acute intravascular haemol!sis. A-56I1 7. . After some da!s the tubular functions return and diuresis ma! have effect on blood urea and creatinine. Signs and symptoms o* &aemolyti% trans*usion rea%tion &here are @ phases of reactions. In the beginning urine is glomerular filtrate ith a lo concentration of urea. It occurs hen donor blood has antibodies against antigen '. &here ma! be e0cessive loss of potassium. :.!per bilirubinaemia . .H 7atient phases large amount of urine. It occurs in the transfusion of group +O" blood to group A1 group B 'or( group AB recipients. 6echeck all labels. forms and identit! of the patient to determine if the patient received the correct blood or component.A.aemoglobinurea $isseminated intravascular co agglutination :. A.O1A 7...

and infre>uentl! Brucellosis. . Bacteriological smear and culture of donor"s blood is done. the administrative set attached and all related labels and form. blood from the bag or from a segment of the table still attached to the unit.erpes etc. If nothing abnormal is found in the above findings.@. #nterpretation o* Laboratory +inding 1. s!philis. . @. 7reserve the urine passed after transfusion to check the presence of haemoglobin produced b! the l!sis of red cells. %. 7ollio. to check an! error in ABO and 6h '$(. ii( If antibod! coated donor)in compatible cells are not immediatel! destro!ed the direct AG& on the post reaction sample ill be positive. @. Laboratory in:estigations 1. iii( If irregular antibodies is detected on screening a( identif! antibodies b! panel of cells b! saline en/!mes and indirect AG& techni>ue Auto) control and t o cord cells 'one $ 6h B. 1heck identit! of the patient donor blood and all relative and all relevant papers to ensure that there as no clerical error. en/!mes albumin and indirect antiglobulin test techni>ues. . &he direct AG& ill be negative. If an! findings is positive or doubtful or the patients clinical condition strongl! suggest a haemol!tic reaction the follo ing investigations are arranted. :. ii( Qello or bro n discolouration in samples dra n @)10 hours after the transfusion indicates increased bilirubin. IgA is tested in recipient blood in case of anaph!lactic compabilit! is done. HB#. $irect antiglobulin test i( If direct AG& is negative the red cells are not coated ith IgG antibod! there is no in compabilit!. care full! dra n to avoid mechanical haemol!sis. %.end the fresh blood sample of the patient. :. 6epeat ABO 6h '$( testing in the patients pre)and post transfusion blood sample. 1ompare the patients are and transfusion specimen for the colour of serum of plasma. iv( In non)immunologic reactions the direct AG& ill be negative. en/!me and indirect AG& techni>ue.( on donors red cells corresponding to the implicated antibod!'ies( iv( In case of the multiple transfusion donors O donor compabilit! is done. O+ "RANS+US#ON &he main diseases transmitted through blood are hepatitis. ii( 6epeat antibod! screedning in patients pre and post transfusion samples and in blood from the bag or segment of tube still attached to the unit ith O6161 and O6% 6% screening cells b! saline. it indicates that there has not been an acute haemol!tic reaction. . iii( If the patients blood sample is dra n after several hours of the suspected reaction the antibod! coated donor)in compatible cells are destro!ed. &o0oplasmaosis and some other viral infections like 1D#. to the having remaining blood or component. malaria and AI$. 7### SCREEN#NG +OR SU#"AB#L#". one $ 6h ) ( are also included. i( 7ink or red discolouration in post)transfusion sample indicates the presence of free haemoglobin due to the destruction of red cells. b( Identif! the antigen '. i( 6epeat the cross)match testing both pre and post transfusion samples of the patients against the sample of blood from the bag or segment of tube still attached to the unit b! saline..

( Dade up to 1000 ml of $*?. • Gram e>uivalent eight9 eight in gram of substance hich ill produce or react ith * more of h!drogen ion. • 7ercentage solution9 the percentage of a solution gives the eight or volume of solute in 100 units of total solution.1.#>2 SPEC#AL -E"!ODS #NS"RUC"#ON +OR PREPARA"#ON O+ SOLU"#ON &he definitions.% 7O@ Dolecular eight of A.1. Dolecular eight of -ao.% 7O@ %.%O Dolecular eight of -a .F so grame e>uivalent eight and gram molecular eight are same.% 7O@ C :9B%B:1BE@C1:Eg 5p to 100 ml of distilled ater %. • -ormal solution9 a one normal 'ID( solution contains one gram)e>uivalent eight of solute in a liter of solution. calculations and methods given belo are based upon elementar! chemistr!. dissociates ith one mole . ?eight* eight ' * ( giving gram of solute in 100 gm of solution #olume*volume 'v*v( giving milliliters of solute in 100 ml of solution ?eight*volume ' *v( giving grams of solution in 100 ml of solution H0amples9 Atomic eights 'rounded to hole numbers( .E g 0. • Dolar solution9 a one molar 'ID( solution contains one mole of solute in a titre of solution.-ao.C1F OC1EF -aC%:F 7C:1F .E C %:. C %:B1B1E B @0 g 1.@ g Of solution -a .F AC:9 Dolecular eight 1( -a .. -acl)9 gms $*? mark to 1 liter p.-ao.olution9 1 D A. Normal Solution 1.1.aline for use in blood group serolog! should have -acl 9 gms*G $*?.% 7O@ %.%O C %:B%B:1BE@B:E C 1.93 of -acl re>uired 0.% 7O@ C :9B%B:1BE@ C 1:E Dolar .%O re>uires 0. -a . gram)molecular eight9 eight in grams of a substance is e>ual to the molecular eight of a substance.0 P&osp&ate bu**er #SO osmati% 7repare t o stock solutions .C:%F 1lC:.01. .. Per%ent Solution 0.9 gm solute made up to 100 ml of $*?. Normal Saline . O 2.%O made up to 1000 ml of $*?.% 7O@ %.% 7o@ C %:B%B:1BE@C1%0 %( A. 0.m -a .%7O@ %. &he solvent is generall! distilled ater unless other ise indicated. one mole of -ao. re>uires 10@0 C @0 g of solution '-ao. DE+#N#"#ON • Dole.

%.A( 0.elect filter of avelength .i and . #nterpretation .I1-et&od 1. . ml Solution. 8resh capillar! blood can also be used if added immediatel! to reagent solution. C.A :% ml %@ ml 12 ml 1: ml 9..1. Add %0 Yl of blood to .@ <..)10 min to ensure the completion of the reaction.@.b.'c!anmethaemoglobin(.: g*G 7repare orking buffer solutions of the desired p.)%0 min to arm up before use. &he reagent should be clear and pale !ello in colour hen measured as blank in a photometric calorimeter at a ave length of . &his solution of . b! mi0ing appropriate volumes of the t o solutions.aas( 1ml and saponic %12 'alcoac Inc( 1 ml p. ml &riton X)100 '6ohm and . . Reagent 4Diluent5 Dodified $rabkin"s reagent 7otassium ferric!anide O %00 mg 7otassium c!anide O ..0 mg 7otassium dih!drogen phosphate O 1@0 mg -omidet 7@0 'shell chemical co( 1 ml $*? O 1 liter Other non ionic detergents hich can be used in place of nomidet include stero0 . &he .AN-E"!E-OGLOB#N 4!#CN5 -E"!OD &he basis of the method is dilution of blood in a solution containing potassium c!anide and potassium ferric!anide.< Solution. &he absorbance of the solution is them measured in photoelectric calorimeter at a ave length of .et the colorimeter at /ero against blank .I1.0 O <. :.@0 nm.H '. @.. .% <. %. p! <.D -a.@0 nm.@ g*G %1. itch on the photoelectric calorimeter and ait for 1. the absorbance must read /ero.0 <.is read! to be compared ith standard. . &he unused solution should be discarded at the end of the da! on hich the ampule is opened to avoid contamination. . should be <. Deasure the absorbance value of the test solution prepared as in step %.tandard solutions are available commerciall!.standard solution is used for direct comparison ith blood hich is also converted to .D -a% .1. A fe e0amples are. Deasurement can be carried on blood hich has been stored at @=1.I1.%7O@ %. 8resh blood or free flo ing capillar! blood added to an! solid anticoagulant '1 mg H$&A 1 ml( can be used.NA-E"!AE-OGLOB#N 4!#CN5 S"ANDARD SOLU"#ON .arleco( 0.bco are all converted to . ml of diluent stopper the tube containing the solution and invert it several times. nm 'or ith a light green filter( Blood Sample 1.7o@ %:. Allo to stand at 6& for . ml C.B E2 ml <E ml 2% ml 2< ml 90.E <.I1.@0 nm W 1.%O O B( 0.

1entrifuge at 1000 rpm for 1)% min. $o not mi0. Albumin increases the dielectric constant of the medium and thus reduces to /eta potential %. Hspeciall! those to be stored at @= Use o* Albumin in Blood Group Serology 1.. of 1)< 3 as a stabili/er in other reagents. @.Stage met&od 4Albumin used as an additi:e5 1. Danufactures instruction should be follo ed. Albumin is used as an additive to the serum cell mi0ture or can be la!ered on the cell button. &he reagent should be discarded. :. 1onfirm all negative results under microscope. <. Add %): drops serum to a labeled tube.. ?ith out disturbing the cell button allo % drop of %%3 albumin to do n the inside of the tube.)%0 min .b3 'or mg*dl( %. 1entrifuge at 1000 rpm for 1)% min E. &he addition techni>ue takes less time and is more fre>uentl! used but the la!ering method is more sensitive for detecting IgG antibodies. -et&ods A5 One. 6ecord the absorbance value directl! in the calorimeter calibrated for direct reading of . E. Add 1 drop of %)@ 3 red cells suspension in the tube :.. g*dl of . If the calorimeter is not meant for taking direct reading of haemoglobin g*dl record the absorbance reading and haemoglobin can be calculated from the follo ing formula. .1. . Note$ 1. @. Incubate at :<=1 for 10)%0 min. 1onfirm all negative results under microscope. $ue to this effect the electrical repulsion bet een the red blood cells is less and cells agglutinate. Gentl! re)suspend the cell button and observe for agglutination*haemol!sis.. &he mi0ture of blood and reagent should be clear turbidit! is due to contamination and give false result. 2. Add 1 drop of %)@3 red cell suspension in the tube :. Bo:ine Albumin Serum &his is used in blood group serolog! as i( %%3 conc. B5 "'o stage met&od 4Albumin layering5 1.( in hich albumin is dissolve 'mostl! %%3 albumin is used in serolog! as :0 3 albumin has the tendenc! to cause roulea0(. Add % drops of bovine albumin %%3 @. Blood should not be clotted. Danufactures instructions should be follo ed. of std g*dl Pre%autions 1. Di0 and incubate at :<=1 for :0 min. %. . 7ipette should be accurate to take %0 Yl of blood. :. Add %): drops serum to a labeled tube %. . Agglutination or haemol!sis is a positive result %.tandard solution should be discarded at the end of da! on hich ampoule in opened.b C O$ of the test * O$ of the std 0 conc. <.. as enhancer of agglutinations and ii( 1onc. @. Albumin ill form a la!er on the top of the cells. if it becomes turbid. . Agglutination 'or( haemol!sis is positive result %. -o it is believed that the effect of albumin in enhancing the agglutination is actuall! due to the potentiating effect of the lo ionic strength saline 'GI. %. Note$ 1. Gentl! re)suspend the cell button and observe for agglutination 'or( haemol!sis. Di0 and incubate at :<=1 for 1.

&he clear supernatant is slightl! less active than the original suspension.-ES &he action of en/!mes on red cells potentiates agglutination in at least % a!s. &he increased agglutinabilit! of en/!me treated red cells is much more. and $uff!.cl. -et&ods &he % stage techni>ue is used for detection and the one stage techni>ue issued for detection and identification of IgG antibodies and for cross matching also the t o stage techni>ues is more sensitive than the one stage techni>ue. Note$ Auto control should be kept. Incubate at :<=1 for 1. the activit! at red cells ith antibodies. 8irst it removes. .yme solution a( 7reparation of stock solution i( 7apain is dissolve in 0.0< g*l( %.3 -ormal saline.% 7O@ '9. #i/9 6h)Chr. <. Preparation o* 'or)ing solution 1.@E g*l( to 1 volume of D*1. c( 8or use 100 ml of the stock solution is added 109 volumes of D*1. phosphate buffer.: prepared b! adding : volumes of D*1.2.uspend 1 g of papain 'B$.0( in 100 ml of 0. ?ith IgG antibodies than ith IgD antibodies en/!mes enhance.. Dake %)@ 3 cells suspension in normal saline.:.):0 min. a( &ake % volumes of serum in a test tube b( Add 1 volume of papainsed red cells c( Incubate at :<=1 for :0 min. '/eta potential( and allo ing the cells.2.7O@ '9. iii( 8icin is dissolve in phosphate buffer saline p. iv( Bromaline is dissolve in phosphate buffer saline p. Pro%edure 46. en/!me ma! potentional the agglutination b! removing structure hich stericall! interfere ith the access of antibod! molecule. :.1 ml of : times ashed cells in a test tube. .*merck 19:. d( see for agglutination '6ead microscopicall!(..EN=. In one stage techni>ue proteal!tic effect of en/!mes inhibited b! the serum the method in hich one volume of serum is placed in a test tube one volume of en/!me solution 'lo ( is la!ered carefull! on top of the serum mi0ing bet een en/!mes and serum. le is and 7 s!stem certain antigen a re)destro!ed or modified b! en/!mes vi/ DF -.. Add 0.yme te%&ni0ue Preparation o* en. <.3 -acl 'this solution ma! be kept for several months at @=1 although this solution is best kept at)%0=1(. .% ml of papain orking solution. All stock solutions are prepared as 13 strength ie 1 gram is dissolved in 100 ml diluent. ii( &r!psin is dissolve in -*%0 . it is a stock solution. p. &ake 0. . O D O A. -a% . &he tube one incubated for 1 hour and the cells then e0amined for agglutination. Before use 1 volume of stock solution is diluted ith 9 voloumes of diluent hich in case of papain is sorensens phosphate buffer p.ialic acid from the red cell surface and thus reduces the surface charge. "'o stage en. &o cone close to one another second.stage test using papain5 1. .a. 7repare a papain solution as follo s. a( b( c( d( 7re treatment of red blood cells. a( . b( If desired this suspension can be centrifuge dafter storage for %@ hours at @=1 ith occasional agglutination. kidd.

0 ml .@ 0. 6educes electro static carrier surrounding red cells and antibod! molecules. Di0.%7O@ 1 #olume @ #olumes for p. LO( #ON#C S"RENG"! SAL" SOLU"#ON 4L#SS5 In normal saline -aB and cl) ions form ionic clouds around oppositel! charged sites on antigen and antibod! molecules respectivel! and partiall! neutriali/ed them. Incubate at :<=1 for 1 hour . "&e e**e%t o* L#SS solution 1.7O@ A.1 @. 'german( is added in %.% if p.One. .%( at the time of preparing papain c!stein reagent.7O@. . 2..@ Preparation o* papain %ystein reagent 1. <. <. min.o ever all antibodies are not e>uall! response to GI.0( is dissolving in %. E. '10 grams of -ao. :.? 8ilter and keep them separatel! in sterile condition in refrigeration. &ake 1 volume serum in a test tube %. -a%. in . :. &hus the rate and degree of antibod! uptake is increased % to @ folds in comparison ith normal saline.%( i( 7hosphate buffer stock solution D*1.tore in small ali>uot O at %0=1. $eposit or filter through hatman"s filter -o. . E. -a% . B! adding . AdKust p. Incubate at :<=1 for 1 hour. Di0 papin solution and c!stein solution and add phosphate buffer 'p. g of 1!stein ..2. E.%(. E.@ volume 9.*Derck for agglutination 'read microscopicall!( Note$ Auto control should be put. . @. E.0 ml of phosphate. . separate he supernatant and discard. 'p. Increases the rate of antibod! uptake % so @ folds compared ith normal saline and also increases the total amount of antibod! taken up.? D*1. A.stage En. is less it can be increased.E volumes for p..%O( if p.( to make the volume 1000 ml.%O 11.%( %. .0EE gm B 1000 ml $. ml of phosphate buffer 'p. 10 grams of papain 'B$. grind if necessar!. -et&ods 1. Add 1 volume of %3 cell suspension @. is more it is discarded.% @ volumes 1 volume for p. solution 'anti)A and anti)B usuall! remain un effected.. 1entrifuge the solution after 1.yme te%&ni0ue 7reparation of 7apain 1!stein reagents 7reparation of phosphate buffer 'p. . Add 1 volume of papain c!stein reagent. E. ?hen the ionic strength of the reaction mi0ture is reduced the number of ions available to form ionic clouds around cells or protein molecules is decreated and the! unit more effectivel! it result in an in)created at B reaction bet een the oppositel! charged antibod! molecules and red cells. to E.2EE gm B 1000 ml $. %. ii( 7reparation of orking solution 'p.% 7O@ 9.

@.. iu*ml( should give a B*BB reaction ith 61 red cells b! the routine GI. 1. &he solution is made up to 1 liter ith distilled ater. $ispense into 100 ml amounts.odium gl!cinate p.. D 7hosphate buffer p. of sodium a/ide)0..00 g for 1. min in emergenc!.< ) %0 ml 0. %.and e0amine for agglutination using optical aid and record the result. ?ash there cells one in GI. 1%. $etection of antibod! ith lo e>uilibroium constant Incubation period is reduced. E. Grade and record results. p. 1. <. ?uality Control A5 Non. look for agglutination if a negative result 'no agglutination( is obtained the test result is invalid and fe hole test should be repeated. &his e0amination should be carried out in paralled test using the previous batch of GI. 11. E. mi0 thoroughl! E. should be ithin the range E.GI.olution described b! lo and messeter consists of 0. &itre of antibod! increases.Serologi%al 1. Preparation o* L#SS Solution GI. 1entrifuge e0amines the supernatant for haemol!sis resuspend the cells and observe for agglutination. :.. .< ith 1. <. E. Osmolarit! %<0)%2. @. solution is as follo s. Alternativel! a lo conc. $etection of antibod! increases... . E. Incubate at :<=1 for 1. .00 ml of distilled ater 'sodium gl!cinate is not available commerciall!(. It is sterili/ed b! seit/ filtration or autoclaving for storage at @=1 stored at %0=1 to avoid pacterial gro th.:. mmol... 12 g of gl!cine is dissolve in about .)A-G test.1< D .aline ) 120 ml 0.<9 g of -acl dissolve in 100 ml of distilled ater is added to the solution. AdKust p. min in routine and . is adKusted to E. 2.. to E.E):.( p.0:D. min centrifuge and read again as in $A&.-ao.)E. Serologi%al A eak IgG anti)$ '0. &he p.E.1 g*l ma! be added. . Add 1)% drops of A.2.< b! drop ise addition of 1 ..%. E. %0 ml of phosphate buffer '0.. .< ) 200 ml &he procedure of preparation of 1 liter of 0.: D .< as added to the gl!cine solution. suspended cell in a tube..1.s( 10. ?ash the red cells t ice in normal saline %.< mmol*cm at %:=1 :. Add 1 drop of IgG coated red cells to an! test that is non)reactive mi0 centrifuge at 1000 rpm for 1 min.. :. &ake e>ual volume of serum and GI.#A"5 1. %. 1entrifuge at 1000 rpm for 1 min '. ?ash the cells : times in large amount of normal saline decant supernatant the each ash as completel! as possible..G serum 9.1. Gentl! shake the tube to dislodge the solution . -et&od 4L#SS. Dake %):3 cell suspension GI. conductivit! should be :. @.-ao. Geave atapparentl! non)reactive test at room temp for .

. in used should be discarded after @2 hours. should be used at ambient temp as could GI. %. straight from @=1 storage increases un anted cold antibod! reaction. 8or routine ork GI. @. Pre%autions 1. E. $ivided the cells into t o ali>uots. ?ash the remaining red cells for a minimum of . times ith a large volume of salin '10 ml or more( save the supernatant of the fifth as to test for free antibod!.. 1entrifuge the mi0ture and discard the supernatant antisera. <. E. After the final ash centrifuge the cell so that the! are tightl! pached and removed as much of saline as possible. Add serum e>ual to the volume to packed cells in one ali>uot. Bottle of GI. Add an e>ual volume of saline to the ashed and packed cells and mi0 . . . Antibod! screen and cross matching is done on re>uest at the time of operation. If not repeat the procedure using another ali>uot of packed red cells al a!s test to ensure that the antibod! that is to remain in the serum is sufficientl! reach before continuing ith repeat absorption.AN"#BOD. to them traces of residual serum ill results in non)specific uptake of auto logous serum complement :. And shall lack the antigen corresponding to another antibod! if an! that is not to be absorbed shall lack the antigen corresponding to another antibod! if an! that is not to be absorbed i. %.#nterpretation It is same as in $A& or IA& . ?ash 1 ml of cells to be tested at least : times ith saline $iscard the supernatant after last ash. Incubate at the optional temp at hich antibod! to be absorbed ill react to :0 O E0 mm mi0ing occasionall!.. ?ash a large volume of red cells to be used in the absorption. ABSORP"#ON "EC!N#?UE Appli%ation &o remove an un anted antibod! from serum for the purpose of antibod! identification or to prepare reagent antisera -et&od 1. 1entrifuge and recover the serum.. Uses o* L#SS 1. . :. %. suspend %):3 cell should be used. ABSORP"#ON AND ELU"#ON @*or 'ea) sub groups o* A or BA 1. @. : times in normal saline these cells must possess the antigen corresponding to the antibod! that is to be absorbed.e remain in the serum. %. It is useful in emergenc! cross)matching due to sort incubation time.. Add 1 ml of anti)A1 to red cells is eak variant of a is suspected or 1 ml of anti)B if eak variant of B is suspected..creening identification and >uantitation of antibod!..aemol!sis or agglutination is a positive result. useful in electine surger! patient serum is kept after doing blood group @. &est the serum to see that absorption is complete. &he to be should be shake gentl! to see agglutination . H>ual volume of serum and GI. ALL. Di0 the cells ith antisera and incubate at room temperature for one hour. 6ed cell should be ashed % in normal saline to free there from serum before adding GI. :.. @. :.

E=1 ater bath for ten min and mi0 the red cell saline mi0ture at least one during this period. !aemolysin test &he haemol!sin test detects incomplete IgG.3 saline suspension of ashed A cells to tube A and % drops of B cells suspension to tube B..aemol!sin test issued to determine the abilit! of anti)A and anti)B Kin group O subKects to cause the haemo!sin of cells and therefore to determine their safet! hen group O is transfused out cross matching or hen group is give to an individual of an other blood group. . . 7lace % drop of fresh serum not more than %@ hold in each of he tubes labeled A and B. Alternati:e -et&od 1 volume of . . "esting o* ELU"E 1.core the degree of l!sis according to the intensit! of the supernant colour 'pink ro red( and cell button si/e ashed packed red cells. If anti)B as used test the elute against : sample of B cells and : group fo O cells at room temp at :<=1 and ith antiglobulins serum. test the elute against three different samples A1 cells and : group O cells at room temp at :<=1 and ith antiglobulin serum. :. ABO antibodies b! their abilit! to haemol!se appropriate red cells in the presence of complement. 1entrifuge and e0amine the supernatant against a ell lighted hite back ground to detect haemol!sis. Di0 gentl! and incubate of :<=1 for % hours @. if seru is un avoid b! more than %@ hour old add an e>ual volume of fresh. 2. If the fifth saline ash material is reactive ith A and B cells the results of the test made on elute are not valid because it indicates that active antibod! as present in the medium un attached to the cells being tested. :. Note$ use of eaker cell suspension or large amount of serum ill increase incidence of haemol!tic activit!. Hlute the absorbed antibod! b! placing the tube at .uman AB serum free from l!sine as a source of complement hich is important to reaction. it is not describe. &est the fifth saline as in the same manner to sho that ashing has removed all antibod! not bound to the cells. %. #nterpretation If the elute agglutinates or reacts ith antiglobulin testing ith specific A or B cells and does not react ith O cells tested have active A or B antigen or their surface capable of binding ith specific antibod!.<. 1entrifuge and remove the cherr! colored elute and discard the cells. If anti)A as used. &o include substantial amount of such antibodies in grouping sera because the! can complete ith the complete 'IgD( antibodies and ma! inhibit agglutination 'pro/on effect( . . -et&od 1.03 suspension of ashed A1 or B cells in suspended in 9 volumes of fresh test anti)A or anti)AB sera the mi0ture is incubated at :<=1 for % hours then centrifuge and the supernatant is inspected for haemol!sis. If the elute also reacts ith O cells it indicates non)specific reactivit! in elute and the results are not valid. Add % drops of fresh %). %.

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