Unit 10 Identifiction of Unexpected Allontibodies A. Introduction 1. Unexpected llontibodies re ntibodies other thn nturlly occurring nti-a or -B. 2. Such ntibodies re found in some 0.3-2% of the popultion, depending upon the group of ptients or donor studied nd the sensitivity of the test methods. 3. Immuniztion to foreign RBC ntigens my result from:. pregnncy b. trnsfusion c. following deliberte injection with immunogenic mteril d. In some instnces the immunizing event is unknown. 4. Once n unexpected ntibody is detected in prentl or pretrnsfusion testing, its specificity should be determined nd its clinicl significnce ssessed. 5. A cliniclly significnt ntibody is one tht:. Shortens the nticipted survivl of trnsfused RBCs b. Hs been ssocited with HDFN. 6. The clinicl significnce vries.. Some ntibodies cuse destruction of incomptible RBCs within few hours or even minutes. b. Others decrese the nticipted survivl by only few dys. c. Documented experience with other exmples of the sme ntibody specificity cn be used in ssessing the reltive clinicl significnce of n ntibody. 7. Unfortuntely, for some ntibodies there re no dt nd decisions must be bsed on the premise tht n ntibody is not cliniclly significnt unless ctive in vitro t 37 C nd/or by the IAT. 8. Determining the specificity of ntibodies encountered in pretrnsfusion testing is importnt in ssessing the need to select ntigen negtive blood for trnsfusion. 9. Ptients with cliniclly significnt ntibodies should receive blood tht hs been found to lck the corresponding ntigen using potent ntiser. 10. In prentl testing, knowing the specificity nd immunoglobulin clss of n ntibody helps predict the likelihood of HDFN. 11. While it is not crucil to identify unexpected ntibodies in donor bloods, such testing is often undertken for the procurement of regent ntiser or teching smples. B. Generl Procedures 1. An dequte quntity of test serum nd RBCs is essentil to the resolution of ny serologicl problem. Either serum or plsm my be used. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 1
2. An EDTA-nticogulted blood smple is preferred for studies of utologous RBCs to void problems ssocited with the in vitro uptke of complement components by RBCs tht occurs when clotted blood smples re used. 3. Medicl History.. It is useful to know ptient's clinicl dignosis, number of pregnncies, trnsfusion history nd drug therpy. b. A recent trnsfusion my necessitte the use of procedures such s red cell seprtion techniques to determine the blood type of the utologous red cells. 4. It is pproprite to test the serum under investigtion t ll test phses t which ntibody ctivity ws initilly detected.. Additionl ntibodies my become pprent t different test phses. b. Rectivity of some ntibodies my be incresed by extending the incubtion time, lowering tempertures, incresing the serum to cell rtio or by using sensitive methods such s enzyme (ficin) techniques. 5. Advntges of using enzyme techniques re:. Enhnces rectivity of Rh ntibodies nd complement binding exmples of nti-le nd nti-jk. b. Dentures some blood group ntigens, especilly M, N, S, nd Fy, so if multiple ntibodies re present nd one hs specificity for MNS or Fy, it ids in identifiction of other ntibodies. 6. The serum under investigtion should be tested by the desired techniques with pnel of eight or more group O regent RBC smples of known blood group phenotype.. To be functionl, regent RBC pnel must mke it possible to identify with confidence the most frequently encountered, cliniclly significnt llontibodies. b. A distinct pttern of rectivity should be pprent for most exmples of single llontibodies. c. There must be sufficient RBC smples tht lck, nd sufficient RBC smples tht crry, the ntigens tht individuls frequently mke ntibodies to. 7. It is importnt to know how the serum under investigtion rects with the utologous RBCs (uto control) to determine whether llo-ntibody, uto-ntibody or both re present in the serum. C. Considertions in Interpreting Serologicl Results 1. Allontibodies of some blood group specificities frequently disply consistent serologic chrcteristics, it is importnt to look for chrcteristics such s:. Wht re the effects of temperture, suspending medium or enzymes on the rection with prticulr cell smple. b. Is there ny vrition in the strength of gglutintion observed mong rective RBC smples. c. Is hemolysis present. d. Is the uto-control positive or negtive. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 2
2. Single llontibodies.. It is usully esy to recognize the specificity of single llo-ntibody tht yields cler-cut positive nd negtive rections with regent RBC smples. b. While the test phse t which serum rects is suggestive of specificity, it is importnt to remember tht exceptionl exmples will be encountered. c. The strength of observed rections my vry due to dosge ffect, vrition in the mount of ntigen on the cell, deteriortion of the ntigen during storge or the presence of multiple ntibodies. d. Although serum displys rection pttern indicting single ntibody, it is importnt to remember tht dditionl ntibodies my be present. 3. Specil considertions with Rh ntibodies.. If nti-e is identified in the serum of trnsfusion cndidte, the dditionl presence of nti-c should be considered. b. Determine the Rh phenotype of the ptient. Even when nti-c is not detectble, it is dvisble to select c-, E- (R1R 1) blood for trnsfusion to R1R 1 ptients with nti-e since nti-c is common cuse of delyed HTR. c. The reverse sitution cuses less of problem. It nti-c is identified the dditionl presence of nti-e my not be determined unless rre RzR1 RBCs re used. Also, lmost ll c- donor units will be E-. 4. Phenotype of utologous red cells.. Once n llo-ntibody hs been identified in serum it is necessry to test the ptient RBCs for the corresponding ntigen. b. When n llo-ntibody is present in the serum, the corresponding ntigen will be bsent on the utologous RBCs. 1) When the ptient types negtive for the ntigen to which the ntibody is directed, this provides dditionl confirmtion of ntibody specificity. 2) If the ptient types positive for the ntigen, this indictes misinterprettion of the ntibody work up. c. Antigen typing cnnot be performed on blood smples of recently trnsfused individuls unless it is done on pretrnsfusion smple or cell seprtion technique is performed (seprting ptient RBCs from trnsfused RBCs). 5. Probbility. For conclusive ntibody identifiction, there must be sufficient regent RBC smples tested tht lck, nd sufficient tht crry, the ntigen to which n ntibody ppers to disply specificity. b. There must be minimum of three cells which possess the ntigen tht rect nd three cells which lck the ntigen which do not rect. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 3
c. It is importnt to remember the limittions when ptient hs n ntibody ginst modertely high incidence ntigen such s e. It is necessry to test dditionl e- RBC smples before conclusively ssigning specificity, nd of equl importnce, identify possible "hidden" or "msked" ntibodies. D. Multiple ntibodies. 1. When serum contins two or more llontibodies, it my be difficult to interpret the results of serum studies using single pnel of regent RBCs. 2. Multiple llontibodies usully present in one or more of the following wys:. The observed pttern of rective nd nonrective tests does not fit tht of single ntibody. b. Rections of vrible strength re observed with the rective RBC smples tht cnnot be explined on the bsis of dosge. c. Different RBC smples rect t different test phses. d. Unexpected rections re obtined when ttempts re mde to confirm the specificity of suspected single ntibody. For exmple, if serum suspected of contining nti-e is found to rect with dditionl e- RBC smples, it is possible tht either nother ntibody is present, or the suspected ntibody is not relly nti-e. In such sitution ddition e- RBC smples must be tested. E. Antibodies to high incidence ntigens. 1. An llo-ntibody to high incidence ntigen should be suspected when ll regent RBC smples re rective, but the uto control is nonrective. 2. Once multiple ntibodies re ruled out these problems re often referred to n immunohemtology reference lbortory, which hve the necessry rre cells for performing such workups. 3. The ptient's siblings re often the best source of serologiclly comptible blood for ptients with ntibodies to high incidence ntigens. F. Antibodies to low incidence ntigens. 1. When serum smple rects only with RBCs from single donor unit the most likely possibilities to consider re: the unit is ABO incomptible, the donor cells hve positive DAT or re polygglutinble. 2. Rections between serum nd single donor or regent RBC smple my lso be cused by ntibodies to low incidence ntigens. 3. If RBCs known to crry low incidence ntigens re vilble, the serum my be tested ginst them. 4. It is inpproprite to dely trnsfusion while such studies re undertken, since finding comptible blood will not be problem. G. Anomlous serologicl rections. 1. Antibodies to vriety of drugs nd dditives cn cuse positive results in ntibody detection nd identifiction tests. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 4
2. There re mny times tht rections will be obtined, nd ttempts to increse the strength of rectivity re unsuccessful.. After every ttempt to identify the specificity re unsuccessful, n interprettion of unble to determine ntibody specificity is mde. b. The ptient must be trnsfused with serologiclly (crossmtch) comptible blood. H. Selected Serologicl Procedures. 1. When pttern of rections fils to indicte specificity, or when the presence of n ntibody is suspected but cnnot be demonstrted, use of the following procedures my be helpful. 2. Enzyme techniques re very useful in ntibody identifiction studies.. Tretment of RBCs with proteolytic enzymes enhnces the rectivity of Rh, P, I, Lewis nd complement binding llontibodies such s nti-jk. b. Antigens of M, N, S nd Fy re depressed or destroyed. c. Enzyme techniques should be used whenever wekly rective ntibody my be n Rh ntibody or when ptient hs multiple ntibodies nd one of them is Fy. 3. Temperture reduction is useful for llontibodies (e.g., nti-m, -P1) tht rect better t cold tempertures. Specificity my become pprent t or below 22 C.. An uto-control is especilly importnt for tests t cold tempertures, becuse mny ser contin cold rective uto-ntibodies. b. Anti-I specificity is confirmed by testing the ptient serum with dult (I+) nd cord (i+) cells t 4 C. Positives with dult cells nd negtive with cord cells confirms nti-i specificity. 4. Incresing the serum to cell rtio increses the mount of ntibody in the test system which my increse the strength of rectivity of ntibodies present in low concentrtions.. Incresing to four drops is recommended, but n increse of 5-10 drops my be indicted nd extending incubtion time up to 60 minutes, mixing the solution periodiclly. b. It is importnt to remove the serum prior to wshing, s 3 wshes would not dequtely remove this volume of serum. c. NOTE: Be cutious when ttempting to increse the serum to cell rtio in LISS tests tht require equl volumes of serum nd cells. 5. Incresing incubtion time to 30-60 minutes my improve rectivity nd help clrify the observed pttern of rections. 6. Decresing the ph of the rection medium to 6.5 by the ddition of 0.2 N HCL to the ptient serum enhnces the rectivity of certin ntibodies, most notble, some exmples of nti-m. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 5
7. Some blood group ntibodies rect preferentilly in test systems utilizing Low Ionic Strength Slt (LISS) solutions.. LISS regent ccelerte ntibody uptke (IE, the first stte of the hemgglutintion rection tht involves ssocition of ntibody molecules to RBCs). b. A vriety of LISS procedures hve been described. 8. Use of thiol regents such s dithiothreitol (DTT) nd 2-mercptoethnol (2-ME) cleve inter-subunit disulfide bonds of IgM molecules.. The IgG molecules re reltively resistnt to such clevge, so tretment results in destruction of IgM but leves IgG intct ble to rect in-vitro. b. The pplictions of DTT nd 2-ME in immunohemtology include: 1) Determining the immunoglobulin clss of n ntibody, especilly if the ntibody hs the potentil of cusing HDFN. 2) Dissociting RBC gglutintes cused by IgM ntibodies (e.g., spontneous gglutintion of RBCs cused by potent cold rective uto-ntibodies). 3) Identifying specificities in mixture of IgM nd IgG ntibodies, prticulrly when n gglutinting IgM ntibody msks the presence of IgG ntibodies. 9. Prewrmed Technique I. Inhibition Tests. Sometimes cold uto-gglutinins my demonstrte high therml mplitude resulting in flse positive rections t 37C nd AHG. b. After confirmtion of the cold gglutinin it is cceptble to perform prewrmed ntibody screen nd crossmtch: 1) Plce RBCs to be tested in pproprite test tubes nd plce in 37C het block. 2) Prewrm tube of serum to 37C. 3) Add 3 to 4 drops of prewrmed serum to prewrmed cells nd incubte 1 hour. 4) Without removing tubes from het block dd sline tht hs been prewrmed to 37C. 5) Immeditely spin nd wsh 2 dditionl times with the wrm sline. 6) Perform the AHG procedure. c. The prewrmed technique cn confirm tht only cold uto-gglutinin is present or detect the presence of cliniclly significnt underlying llontibodies. 1. Some blood group ntigens exist in soluble form in such body fluids s sliv, urine or plsm.. These substnces re useful in ntibody identifiction studies, either to confirm ntibody specificity by inhibition or to neutrlize ntibodies tht msk the presence of concomitnt non-neutrlizble ntibodies. b. The following soluble blood group substnces cn be used in ntibody identifiction tests. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 6
2. Lewis substnces. b. Le nd Le substnces re present in the sliv of persons with the pproprite Lewis phenotype, nd Lewis substnce cn be prepred from sliv. Most blood bnks use commercilly prepred Lewis substnce. b. Lewis substnce will neutrlize Lewis ntibodies in ptient specimen, llowing the detection of underlying, cliniclly significnt llontibodies. 1 3. P substnce 1. Soluble P substnce is present in hydtid cyst fluid s well s pigeon eggs. 1 1 b. P my msk the presence of underlying llontibodies. The ddition of P substnce to the ptient's 1 serum cuses neutrliztion of the nti-p, llowing the detection of underlying llontibodies. 4. Sd (Sid) substnce J. Titrtion. Sd blood group substnce is present in soluble form in vrious body fluids, with the most bundnt source being urine. b. The urine is dded to the ptient serum, cusing neutrliztion of the nti-sd. c. Aids in the detection of underlying llontibodies. 1. The titer of n ntibody is usully determined by testing seril two-fold dilutions of the serum ginst selected RBC smples. 2. Results re expressed s the reciprocl of the highest serum dilution tht cuses mcroscopic gglutintion. 3. Prentl studies.. When the ntibody is of specificity known to cuse HDFN or when the clinicl significnce of the ntibody is unknown, the results of titrtion studies, outcome of previous pregnncies re used to ssess the need for mniocentesis. b. Rising titers re indictive of ctive immuniztion of the mother. 4. Antibody identifiction. Some ntibodies cuse gglutintion of virtully ll regent RBC smples, but specificity is indicted by differences in the strength of rectivity with ech smple in titrtion studies. b. The titrtion procedure is not used very commonly for this purpose. 5. HTLA ntibodies.. HTLA ntibodies rect very wekly in the undiluted stte but, unlike most wekly rective ntibodies (e.g., nti-d with titer of 4), rect t high dilutions (e.g., 1 in 2000). b. Such ntibodies include nti-ch, -Rg, -Cs, -Yk, -Kn, -McC nd -JMH. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 7
K. Adsorption L. Elution c. When wek rections re observed in the IAT, titrtion studies my be used to estblish whether or not the rections re due to the presence of n HTLA ntibody. 1. Antibody cn be removed from serum by dsorption to RBCs crrying the corresponding ntigen.. The ntibody forms complex with RBC membrne-bound ntigens. b. When the serum nd RBCs re seprted, the ntibody remins ttched to the RBCs. c. Subsequent elution of the bound ntibody cn often give dditionl useful informtion. 2. Adsorption techniques re useful in situtions such s:. Removing uto-ntibody ctivity to permit detection of coexisting llontibodies. b. Removing unwnted ntibody from serum tht contins n ntibody suitble for regent use. c. Confirming the presence of ntigens on RBCs through their bility to remove specific serum ntibody. d. Confirming the specificity of n ntibody by showing tht it cn be bsorbed only to RBCs of prticulr blood group phenotype. e. Seprting multiple ntibodies present in single serum smple. 1. Elution techniques free ntibody molecules from sensitized RBCs so the recovered ntibody cn be tested. 2. A vriety of methods re employed with the primry objective being breking the bond between the ntigen nd the ntibody. 3. Elution techniques re primrily used for:. Identifiction of n ntibody coting bby's RBCs in the cse of HDFN. b. Identifiction of n ntibody cusing n cute or delyed hemolytic trnsfusion rection. c. Investigtion of positive DAT. d. Concentrtion nd purifiction of ntibodies, the detection of wekly expressed ntigens nd the identifiction of multiple ntibodies. e. Preprtion of ntibody-free intct RBCs for use in phenotyping or utobsorption. 4. Technicl fctors which influence the success of the elution techniques include:. Incorrect technique. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 8
b. Incomplete wshing 1) If cells re incompletely wshed contminting serum ntibody will cuse flse positive rections. 2) An liquot of sline from the lst wsh is sved nd tested in prllel. Positive rections with the lst wsh invlidtes the test. c. Binding of proteins to glss surfces. d. Dissocition of ntibody before elution. e. Instbility of elutes. M. Selection of Blood for Trnsfusion After Antibody Identifiction 1. After ntibody identifiction studies re complete determine the clinicl significnce of the ntibody. 2. If the ntibody is cliniclly significnt ntigen negtive donors must be found nd crossmtched for the ptient, Coomb s crossmtch must be done. 3. If the ntibody is not cliniclly significnt it is not necessry to provide ntigen negtive blood, but the donors must be comptible by the Coomb s crossmtch. MLAB 2431 Unit 10 Identifiction of Unexpected Allontibodies 9