clinically significant unexpected antibodies are capable of causing hemolytic transfusion reactions secondary to accelerated destruction of a significant proportion of transfused red blood cells ( 1 ) . therefore ,
screening for unexpected antibodies should be part of all pretransfusion testing , with antibody identification in the event of a positive result . in the 1990s ,
the microcolumn gel technique was introduced for screening and identification of such unexpected antibodies ( 2 ) .
this method is not only easy to perform and economical of time but also easy to standardize and read , so it has become the most common technique in the blood bank laboratories of many countries ( 3 ) .
the two principal techniques for unexpected antibody screening and identification are the indirect antiglobulin and enzyme methods .
the most frequently used method is the indirect antiglobulin with gel ( liss / coombs ) , and the microcolumn assay technique using the liss / coombs gel test is the most popular for this purpose in korea ( 4 - 6 ) . in recent years
, the enzyme gel method ( nacl / enzyme ) has been added for antibody identification in a few hospitals in korea due to its higher and exact identification rate ( 7 ) .
however , the nacl / enzyme method is used only for antibody identification , so some unexpected antibodies could be missed in screening step . at present , there has been no study in korea of antibody screening and identification using these two methods .
the purpose of the present study was to compare the results of the liss / coombs and nacl / enzyme methods for screening and identifying unexpected antibodies and to evaluate the clinical usefulness of simultaneous testing by these two methods .
from may 2005 to april 2006 , unexpected antibody screening was performed on 15,014 samples using the liss / coombs and nacl / enzyme gel tests .
when unexpected antibodies were detected by either test , those antibodies were identified using both methods .
a 50 l sample of 0.8% screening or identification cell reagent and 25 l of patient serum were added to the microtube of each gel card .
after 15 min ' incubation at 37 , the card was centrifuged for 10 min , and the reactions for agglutination were examined macroscopically on an illuminated view box .
all tests were carried out using the diamed - id micro typing system ( diamed ag , cressier , morat , switzerland ) .
for the liss / coombs screening method , the liss / coombs card and two test reagents id - diacell i - ii ( diamed ag ) were used . for the nacl / enzyme screening method ,
the nacl / enzyme card and three test reagents diacell i - ii - iii p ( papainized ) ( diamed ag , i d ) were used .
when unexpected antibodies were detected by either test , those antibodies were identified using both methods . for the liss / coombs identification test
, the liss / coombs card and id - panel test reagent ( diamed ag ) were used . for the nacl / enzyme identification test
, the nacl / enzyme card and the id - panel p test reagent ( diamed ag ) were used .
an antibody screening result was defined as positive if one or both of the cell reagents agglutinated with the patient 's serum in the liss / coombs test , and if one or more of the three cell reagents agglutinated with the patient 's serum in the nacl / enzyme test . for antibody identification
, we interpreted each method as positive if one or more of the 11 cell reagents agglutinated .
when only one antibody was identified in the serum , we interpreted it as " identified " if all reactions in the 11 wells were consistent with the manufacturer 's identification table and as " unidentified " if the reactions in some wells were discordant with the table . when two or more antibodies were present , we interpreted them as " identified " if all antibodies were identified exactly with each method , as " partially identified " if at least one antibody was identified exactly with each method , and as " unidentified " if none of them was identified exactly .
if no agglutination reactions occurred in any of the 11 wells , we interpreted the result as " negative " .
from may 2005 to april 2006 , unexpected antibody screening was performed on 15,014 samples using the liss / coombs and nacl / enzyme gel tests .
when unexpected antibodies were detected by either test , those antibodies were identified using both methods .
a 50 l sample of 0.8% screening or identification cell reagent and 25 l of patient serum were added to the microtube of each gel card .
after 15 min ' incubation at 37 , the card was centrifuged for 10 min , and the reactions for agglutination were examined macroscopically on an illuminated view box .
all tests were carried out using the diamed - id micro typing system ( diamed ag , cressier , morat , switzerland ) .
for the liss / coombs screening method , the liss / coombs card and two test reagents id - diacell i - ii ( diamed ag ) were used . for the nacl / enzyme screening method ,
the nacl / enzyme card and three test reagents diacell i - ii - iii p ( papainized ) ( diamed ag , i d ) were used .
when unexpected antibodies were detected by either test , those antibodies were identified using both methods . for the liss / coombs identification test
, the liss / coombs card and id - panel test reagent ( diamed ag ) were used . for the nacl / enzyme identification test
, the nacl / enzyme card and the id - panel p test reagent ( diamed ag ) were used .
an antibody screening result was defined as positive if one or both of the cell reagents agglutinated with the patient 's serum in the liss / coombs test , and if one or more of the three cell reagents agglutinated with the patient 's serum in the nacl / enzyme test . for antibody identification
, we interpreted each method as positive if one or more of the 11 cell reagents agglutinated .
when only one antibody was identified in the serum , we interpreted it as " identified " if all reactions in the 11 wells were consistent with the manufacturer 's identification table and as " unidentified " if the reactions in some wells were discordant with the table . when two or more antibodies were present , we interpreted them as " identified " if all antibodies were identified exactly with each method , as " partially identified " if at least one antibody was identified exactly with each method , and as " unidentified " if none of them was identified exactly .
if no agglutination reactions occurred in any of the 11 wells , we interpreted the result as " negative " .
unexpected antibodies were detected by at least one method in 234 of the 15,014 serum samples , including 25 autoantibody - containing samples ( 1.56% ) .
the positive screening rates of the liss / coombs , nacl / enzyme , and combined tests for unexpected alloantibodies were 0.48% ( n=72 ) , 1.29% ( n=194 ) , and 1.39% ( n=209 ) , respectively ( table 1 ) .
the positive rate of the combined methods was about threefold that of the liss / coombs method only , and highly discrepant results were seen between these methods . only 57 ( 27.3% ) of the total 209 alloantibody cases were positive by both methods , whereas 137 ( 65.6% ) and 15 ( 7.2% ) were positive only by the nacl / enzyme and the liss / coombs method , respectively . among the 57 samples showing positive in both screening tests ,
on the other hand , 11 samples were positive only by the nacl / enzyme identification test , and these antibodies were anti - e ( n=5 ) , anti - lea ( n=3 ) , anti - c+e ( n=1 ) , anti - e ( n=1 ) , and anti - e+lea ( n=1 ) ( table 2 ) .
one sample was negative in both identification tests . among the 15 samples having positive results only by the liss / coombs screening test , 7 samples were positive for the liss / coombs identification test only , 1 for the nacl / enzyme test only , and 2 for both tests . among the 137 samples having positive results only by the nacl / enzyme screening method , 102 samples were positive for the nacl / enzyme identification test only .
the 61 samples were finally identified as anti - c ( n=3 ) , anti - c ( n=1 ) , anti - c+k ( n=1 ) , anti - e ( n=25 ) , anti - e+c ( n=2 ) , anti - lea ( n=27 ) , and anti - leb ( n=2 ) .
but other 41 samples were " unidentified " in the nacl / enzyme identification test .
ten samples showed positive results in the liss / coombs method as well as the nacl / enzyme method , and these were finally identified as anti - d ( n=1 ) , anti - e ( n=1 ) , anti - e+c ( n=1 ) , or anti - lea ( n=4 ) , with 3 being unidentified .
according to the final identifications , anti - rhesus antibodies were the most common , and 78 antibodies were identified .
these antibodies were exactly " identified " in 20 cases and " partially identified " in 12 cases by the liss / coombs method and were exactly " identified " in 72 cases and " partially identified " in 6 cases by the nacl / enzyme method ( table 3 ) . of the 40 anti - lewis antibodies , all were identified by the nacl / enzyme method . in the liss / coombs method
the anti - fya , anti - fyb , and anti - xga antibodies were identified only by the liss / coombs method .
previously , screening for unexpected antibodies was performed with an indirect antiglobulin test or an enzyme test using a conventional tube method .
however , the recently introduced gel test has proved to be more sensitive and has many advantages ( 8 , 9 ) .
the use of liss has increased the number of antibodies detected , and clinically important antibodies have been found in increasing numbers .
in addition , the gel method is rapid , and interpretation of the results is easy . therefore , the gel test gained widespread usage throughout the world , including korea . in this study , we compared the results of the liss / coombs and nacl / enzyme tests using the gel method for screening and identifying unexpected antibodies and evaluated the clinical usefulness of simultaneous testing by two methods .
of the 15,014 patient samples tested , 0.48% had a positive reaction with the liss / coombs screening method .
this rate is similar to that in previous reports from korea ( 4 - 6 ) .
the positive rate of antibody screening increased to 1.29% with the nacl / enzyme method and to 1.39% using these two tests together .
this result indicates that the nacl / enzyme method is sensitive in detecting unexpected antibody , especially rh antibodies . among the 137 samples showing positive results in nacl / enzyme screening only , 102 samples were also positive only with the nacl / enzyme identification method and 10 were positive with both the liss / coombs and the nacl / enzyme identification .
anti - rh ( n=35 ) , anti - le ( n=33 ) , and unidentified antibodies ( n=44 ) accounted for nacl / enzyme screening - only antibodies .
twenty - five samples showed negative results in both the liss / coombs and nacl / enzyme identification methods .
the decision about the clinical significance of nacl / enzyme screening - only positive results is difficult , and we should be careful in interpretation . in a few previous reports
, the authors stated that the enzyme method revealed a high proportion of nonspecific reactions with uncertain clinical value , and " enzyme - only " antibodies lack clinical significance , so the method is not employed routinely by many laboratories ( 10 , 11 ) . in our study , the unidentified antibodies ( 44/137 ) and negative results ( 25/137 ) in identification also accounted for a high proportion among the nacl / enzyme screening - only positive results .
these are considered nonspecific reactions , clinically insignificant , or both , although we could not evaluate individual patient data for clinical significance .
however , we can not completely accept the opinion that all nacl / enzyme screening - only positive results are insignificant .
a few reports about antibody screening in pregnant women showed different results that enzyme - enhanced methods often detect low concentrations of anti - rh antibodies not found by other methods ( 12 , 13 ) .
in addition , there are some cases of acute hemolytic transfusion reaction ( 14 ) or delayed transfusion reactions ( 11 , 15 ) caused by " enzyme - only " antibodies .
we found additional evidence supporting the importance of the enzyme method for antibody screening in this study . among the 137 samples that were nacl / enzyme - only positive
these antibodies were identified as anti - d ( n=1 ) , anti - e ( n=1 ) , anti - e+c ( n=1 ) , and anti - lea ( n=4 ) , with three unidentified results in the nacl / enzyme identification . however , these samples were only weakly reactive in liss / coombs identification , and most of them remained unidentified with the liss / coombs method .
these antibodies have a chance of being missed in screening if only liss / coombs methods were used in antibody screening .
thus , the liss / coombs method for screening could fail to detect some significant unexpected antibodies . for antibody identification , there are significant differences between the liss / coombs and nacl / enzyme methods ( table 3 ) .
some samples showed positive in the liss / coombs screening were positive only in the nacl / enzyme identification . and , it has the advantage of discriminating antibodies when mixed antibodies were present . in addition ,
11 samples were positive only in the nacl / enzyme identification , although these were positive in both screening methods .
however , the anti - fya and anti - xga antibodies were found on screening and were identified only by the liss / coombs method .
therefore , nacl / enzyme method should be used together with liss / coombs method . in the present study ,
the nacl / enzyme method showed about threefold higher detection rates , especially of rh antibodies in screening , and higher exact identification rates and discrimination power for mixed antibodies . in conclusion ,
simultaneous liss / coombs and nacl / enzyme testing is useful for antibody screening and identification because the nacl / enzyme method can detect and identify many significant antibodies that would be overlooked if only the liss / coombs method was used , although a high proportion of antibodies found in nacl / enzyme screening are insignificant and reflect nonspecific reactions .
 we evaluated the clinical usefulness of simultaneous liss / coombs and nacl / enzyme testing using the gel method for screening and identification of unexpected antibodies in 15,014 samples . 
 when unexpected antibodies were detected by either screening test , those antibodies were identified using both the liss / coombs and the nacl / enzyme gel test . 
 the positive screening rates of the liss / coombs , nacl / enzyme , and combined tests ( excluding 25 autoantibody cases ) were 0.48% , 1.29% , and 1.39% , respectively . among the 57 samples positive by both screening methods , the antibodies in 19.3% could be identified only by the nacl / enzyme method . among the 137 samples positive only by nacl / enzyme screening 
 , 74.5% showed positive results in antibody identification only by the nacl / enzyme test , although 7.3% were also positive in the liss / coombs test . 
 the nacl / enzyme method thus showed about threefold higher detection rates than the liss / coombs method , especially in screening for rh antibodies , and higher exact identification rates and discriminatory power for identifying mixed antibodies . 
 addition of the nacl / enzyme method to routine laboratory procedures may detect and identify considerable numbers of significant antibodies that might be missed if only the liss / coombs method is used . 
INTRODUCTION
MATERIALS AND METHODS
Performance of unexpected antibody detection
Interpretation of results
RESULTS
DISCUSSION