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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 notice of faculty disclosure in accordance with accme guidelines any individual in a position to influence and/or control the content of this ascp cme activity has disclosed all relevant financial relationships within the past 12 months with commercial interests that provide products and/or services related to the content of this cme activity the individual below has responded that she has no relevant financial relationships with commercial interests to disclose nanette c thomas mtascpsbb teleconferences spring 2010 copyright © 2010 by ascp 1 elutions elutions applications methods and clinical relevance nanette c thomas mtascpsbb c thomas irl director american red cross teleconferences spring 2010 copyright © 2010 by ascp 2 1

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 program objectives · discuss the applications of elution procedures · describe various elution methods using a historical perspective · determine optimal elution methods for given clinical situations · apply knowledge gained from elutions to aid in patient care teleconferences spring 2010 copyright © 2010 by ascp 3 factors affecting antigen-antibody complexes geometric fit or complementarity of shape is affected by and dependent on ­ van der waals forces develop from movement of electrons and produce a general attraction of molecules at short di ifllh distances ­ ionic or electrostatic bonds ­ hydrogen bonds more stable at low temp ­ hydrophobic bonds ­ ph teleconferences spring 2010 copyright © 2010 by ascp 4 the biochemistry of elutions · elution is the process whereby bound antibody is recovered from red cells · antigen-antibody complexes must be dissociated by one of the following mechanisms ­ change in thermodynamics ­ neutralizing or reversing forces of attraction ­ disturbing the structure of binding site teleconferences spring 2010 copyright © 2010 by ascp 5 2

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 elution methods from a historical perspective 1st generation ­ temperature changes 2nd generation ­ organic solvents g 3rd generation ­ ph changes teleconferences spring 2010 copyright © 2010 by ascp 6 elution methods 1st generation heat 1925 freeze-thaw freeze thaw 1947 lui rapid freeze 1976 sonication 1977 2nd generation 3rd generation ether 1956 cold-acid 1977 chloroform 1979 digitonin acid digitonin-acid 1977 xylene 1981 dichloromethane 1982 copyright © 2010 by ascp citric acid 1982 teleconferences spring 2010 7 elution controls last wash ­ test in parallel with eluate if nonreactive assures that antibody activity in eluate is recovered from a bound state on the red cells if reactive indicates cells reactive inadequate washing and invalidates test neutralization ­ anti-igg last wash check cells if positive no free antibody in last wash if negative test is invalid teleconferences spring 2010 copyright © 2010 by ascp 8 3

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 1st generation techniques principle ­ change in thermodynamics or temperature dissociates antigen antibody complexes advantages ­ quick and easy cheap ideal easy for igm antibodies disadvantages ­ most produce hemoglobin stained eluate not effective in recovering igg antibodies teleconferences spring 2010 copyright © 2010 by ascp 9 2nd generation techniques principle ­ solvents lower surface tension and reverse van der waal forces causing antigen-antibody dissociation advantages ­ excellent recovery of igg auto and allo antibodies disadvantages ­ flammable carcinogenic toxic gases time consuming teleconferences spring 2010 copyright © 2010 by ascp 10 3rd generation techniques principle ­ acidic solutions decrease ph and disrupt the ionic complementarity of antigen-antibody bonds g advantages ­all are sensitive not hazardous commercially available citric acid and cold-acid are fast disadvantages ­ costly possible false positives digitonin-acid technique is time consuming teleconferences spring 2010 copyright © 2010 by ascp 11 4

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 general applications of elutions 1 recover antibody bound to red blood cells for identification investigation of positive dat 2 in conjunction with adsorption to separate purify or concentrate antibodies 3 remove coating antibody from red blood cells for phenotyping with antiglobulin reagents teleconferences spring 2010 copyright © 2010 by ascp 12 recovery of bound antibody transfusion reaction hemolytic disease of the newborn autoimmune hemolytic anemia to be discussed later teleconferences spring 2010 copyright © 2010 by ascp 13 transfusion reaction · immune system detects foreign antigens and produces antibodyies · antibodies coat transfused cells · mi d fi ld agglutination dat and antigen mixed-field l ti ti d ti types · decreased survival of transfused cells teleconferences spring 2010 copyright © 2010 by ascp 14 5

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 transfusion reaction · antibodies in plasma are not always the same as antibodies in eluate · antibodies first coat transfused cells appear in plasma later ­ positive elution negative a/s within first 10 days of transfusion · elutions concentrate antibody so may be easier to id in eluate before plasma teleconferences spring 2010 copyright © 2010 by ascp 15 case study transfusion reaction 56 y/o female with a history of anti-e -k receives two units of e k red blood cells during hip replacement surgery 5 days ago post transfusion sample ­ anti-e -k auto control and dat with mixed-field agg noted g and c3 gg igg eluate ­ anti-jka 6 days later 11 days post transfusion ­ anti-jka in plasma a review of her history is remarkable for two previous pregnancies anamnestic secondary immune response teleconferences spring 2010 copyright © 2010 by ascp 16 hemolytic disease of the fetus and newborn · cord blood dat positive · jaundice increased bilirubin · usually mild benign clinical course y g · abo hdn ­ igg and rarely igm · non-abo hdn igg teleconferences spring 2010 copyright © 2010 by ascp 17 6

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 case study ­ abo hdn infant born to group o rh positive mother cord blood evaluation ­ group a rh positive positive dat igg heat eluate prepared from cord cells ­ nonreactive with reagent screening cells and group a and b reverse grouping cells teleconferences spring 2010 copyright © 2010 by ascp 18 case study ­ abo hdn acid eluate prepared from cord cells ­ anti-a,b identified clinical course ­ mild jaundice reversed by phototherapy cells eluate igg 0 0 0 1 1 last wash igg 0 0 0 0 0 19 sc i sc ii sc iii a cells b cells teleconferences spring 2010 copyright © 2010 by ascp adsorption/elution applications separate multiple specificities purify antibodies to use as typing sera confirmation of weak red blood cell antigens teleconferences spring 2010 copyright © 2010 by ascp 20 7

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 separate multiple antibody specificities most commonly used to adsorb out an antibody to a high incidence antigen to look for underlying common alloantibodies ­ adsorb multiple times onto phenotypically similar cells that are incompatible ­ test adsorbed sera for common alloantibodies ­ prepare eluate from first adsorbing cells and test against cells negative for high incidence antigen purification to use for typing sera teleconferences spring 2010 copyright © 2010 by ascp 21 case study ­ adsorption/elution/purification 69 y/o caucasian female with previously identified anti-vel group o rh positive c+e-c+e k f b jk b c e k fya-b jka+b only one group o vel cell to test need to exclude other common alloantibodies teleconferences spring 2010 copyright © 2010 by ascp 22 case study ­ adsorption/elution/purification rh-hr d 1 2 3 4 5 0 c 0 0 c 0 0 0 e 0 0 0 0 e kell f k k 0 duffy fya fyb kidd jka jkb lewis lea leb p p1 0 0 m 0 mn n 0 0 0 s s 0 0 0 0 velis 1 1 1 1 0 0 liss 37 1+h 1+h 1+h 1+h 0 0 igg 1 2 1 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 auto anti-fya excluded teleconferences spring 2010 copyright © 2010 by ascp 23 8

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 case study ­ adsorption/elution/purification · adsorb plasma three times onto untreated group o e k fya jkb cells · test adsorbed plasma for other alloantibodies · prepare eluate from first adsorbing cells ­ confirm specificity ­ freeze for future use teleconferences spring 2010 copyright © 2010 by ascp 24 case study ­ adsorbed plasma rh-hr d 1 2 c 0 c 0 e 0 e 0 kell f k 0 0 0 0 k duffy fya fyb kidd jka jkb lewis lea leb p p1 0 m 0 mn n 0 s 0 0 s is 0 0 liss 37 0 0 igg 0 1 0 0 0 0 0 3 0 0 0 0 0 0 0 0 0 0 0 0 4 0 0 0 0 0 0 0 0 0 0 1 anti-jkb is excluded additional cells are tested and confirm the presence of anti-e and ­k in the adsorbed plasma teleconferences spring 2010 copyright © 2010 by ascp 25 case study ­ elution from 1st adsorption eluate-igg i ii iii auto vel group o vel group a vel group b vel 2 2 2 0 0 0 0 last wash igg 0 0 0 0 0 0 0 purification freeze eluate as anti-vel suitable for all abo groups teleconferences spring 2010 copyright © 2010 by ascp 26 9

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 separate multiple antibody specificities anti-c -d -g · ant-c and ­d specificities present · virtually all c cells are g d cells are g anti g · why do we care if anti-g is present ­ rr patient receives c+d blood ­ rr patient receives d platelets ­ pregnancy and administration of rhig · usually tested in a referral lab r r cells teleconferences spring 2010 copyright © 2010 by ascp 27 separate multiple antibody specificities anti-c -d -g 1 adsorb patient s plasma to completion onto r r c+g+d cells 2 test adsorbed plasma for presence of anti-d 3 prepare eluate from first r r adsorbing cells could contain anti-c and/or ­g adsorb this anti c g eluate to completion onto r2r2 or roro d+g+c cells 4 test adsorbed eluate for presence of anti-c 5 prepare eluate from first r2r2 or roro adsorbing cells and test for anti-g if anti-g present will look like anti-c and ­d teleconferences spring 2010 copyright © 2010 by ascp 28 case study anti-c -d and/or -g plasma adsorbed to completion onto r r cells check for anti-d rh-hr dcceef kell k k duffy fya fyb kidd jka jkb lewis lea leb p p1 mn mnssr r ads cells is 0 0 0 0 0 0 liss 37 0 0 0 0 0 0 igg 0 2 2 2 0 0 1 0 0 0 0 2 0 0 0 3 0 0 0 4 0 0 5 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 anti-d present if pregnancy not a candidate for rhig teleconferences spring 2010 copyright © 2010 by ascp 29 10

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 case study anti-c -d and/or -g eluate prepared from r r adsorbing cells anti-c and/or ­g adsorbed to completion with r2r2 cells to remove anti-g if present test for anti-c rh-hr dcceef kell k k duffy fya fyb kidd jka jkb lewis lea leb p p1 m mn nssr2r2 ads cells eluate igg 0 0 0 0 0 0 1 0 0 0 2 0 0 0 3 0 0 4 0 0 5 0 0 0 6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 anti-c not present teleconferences spring 2010 copyright © 2010 by ascp 30 case study anti-c -d and/or -g eluate prepared from r2r2 adsorbing cells test for anti-g rh-hr dcceef 1 0 0 0 2 0 0 0 3 0 0 4 0 0 5 0 0 0 6 0 0 0 kell k 0 0 0 0 0 k 0 duffy fya fyb kidd jka jkb lewis lea leb p p1 0 0 m 0 mn n 0 0 0 s 0 0 0 s 0 0 eluate igg 1 1 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 looks like anti-c and ­d anti-g sample contains anti-d and anti-g sample does not contain anti-c teleconferences spring 2010 copyright © 2010 by ascp 31 confirmation of weak antigens · · · · · · abo subgroups u and vel negative vs variants para bombay y dominant lua-b antigen suppression largely replaced by dna molecular testing teleconferences spring 2010 copyright © 2010 by ascp 32 11

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 case study ­ investigation of abo subgroup forward grouping antisera a 0 b 0 a,d 0 d 3 rh cont 0 forward type ­ group o reverse grouping cells a1 0 a2 0 b 4 reverse type ­ group a teleconferences spring 2010 copyright © 2010 by ascp 33 case study ­ investigation of abo subgroup forward grouping ­ additional testing ­ 15 rt incubation ­ 15 4oc incubation ­ enzyme pretreatment of autologous cells include auto control ­ anti-h ulex europaeus 4 all results consistent with group o teleconferences spring 2010 copyright © 2010 by ascp 34 case study ­ investigation of abo subgroup reverse grouping ­ additional testing to enhance isoagglutinins ­ 15 rt incubation include auto control ­ 15 4oc incubation include auto control ­ all results consistent with group a results suggest a possible subgroup of a next step ­ adsorption/elution with anti-a and/or saliva studies teleconferences spring 2010 copyright © 2010 by ascp 35 12

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 case study ­ investigation of abo subgroup adsorption/elution works better with polyclonal sources of antisera ­ expired research ­ plasma or serum from group b general method ­ incubate cells and antisera at 4oc ­ heat or freeze-thaw elution ­ test at all phases teleconferences spring 2010 copyright © 2010 by ascp 36 case study ­ investigation of abo subgroup lot #1 is rt a1 4oc agt is rt a2 4oc agt is rt b 4oc agt is rt o 4oc agt eluate last wash lot #2 eluate last wash lot #3 eluate last wash 0 1 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 a1 0 1 1 1 0 0 0 0 0 0 a2 0 1 1 1 0 0 0 0 0 0 b 0 0 0 0 0 0 0 0 o 0 0 0 0 a1 0 1 1 1 0 0 0 0 0 0 a2 0 1 1 1 0 0 0 0 0 0 b 0 0 0 0 0 0 0 0 o 0 0 0 0 a antigen sites are present probable ael teleconferences spring 2010 copyright © 2010 by ascp 37 confirmation of weak antigens same process can be used to detect weakened or variant antigens of other blood groups ex u vel lu kell system ­ adsorption temperature ­ elution method ­ phases of testing time consuming labor intensive dependent on availability of antisera molecular teleconferences spring 2010 copyright © 2010 by ascp 38 13

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 obtaining dat negative cells for phenotyping · cells may be coated with igm or igg · must leave red cell antigens intact · methods to disperse igm agglutinates ­ wash cells with warm saline 37 ­ 45oc ­ exposure to 56oc heat for three minutes ­ thiol reagents to cleave inter-chain disulphide bonds teleconferences spring 2010 copyright © 2010 by ascp 39 obtaining dat negative cells for phenotyping · methods to disperse igg antibodies ­ chloroquine diphosphate cdp splits immune complexes and inhibits antigenantibody reactions ­ edta-glycine acid ega dissociates antibody by lowering ph inactivates kell system antigens · ega more efficient than cdp in removing igg for warm reactive autoantibodies teleconferences spring 2010 copyright © 2010 by ascp 40 autoimmune hemolytic anemia elutions serve multiple purposes 1 removal of bound antibody to obtain dat negative cells for phenotyping 2 2 removal of bound antibody to prepare cells for autoadsorption 3 reactivity of eluate prepared from autologous cells with dat negative cells confirms presence of autoagglutinin teleconferences spring 2010 copyright © 2010 by ascp 41 14

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elutions applications methods and clinical relevance nanette c thomas mtascpsbb 3/9/2010 case study waiha 77 y/o male with myelodysplasia newly diagnosed admitted for shortness of breath and dizziness h/h ­ 6.4/19 three units of packed cells ordered for transfusion teleconferences spring 2010 copyright © 2010 by ascp 42 case study waiha initial testing at hospital ­ a positive ­ a/s and autocontrol 3 in gel ­ dat positive with ps and igg 2 nonreactive with anti-c3 ­ gel panel panreactive 3 no history of prior transfusions sent to referral laboratory for further testing teleconferences spring 2010 copyright © 2010 by ascp 43 case study waiha referral laboratory testing plan · obtain extended phenotype · obtain dat negative cells ­ ­ phenotype for antiglobulin reactive antigens test plasma and eluate to assess presence of warm autoagglutinin · if warm autoagglutinin present perform autoadsorptions to exclude underlying alloantibodies teleconferences spring 2010 copyright © 2010 by ascp 44 15

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