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A rapid screening technique for lymphocytotoxic antibodies using tray-frozen lymphocytes
CRYOBIOLOGY
21, 480-485(1984)
A Rapid Screening
H. RUDER, Institute
Technique for Lymphocytotoxic Tray-Frozen Lymphocytes
G. OPELZ, of Immunology,
V. LENHARD, University
A. SCHAFER,
of Heidelhergv
Heidelberg,
Antibodies
Using
AND V. DANIEL West Germuny
A method whereby sera can be screened for the presence of lymphocytotoxic antibodies within 4 hr using lymphocytes frozen in microtest trays is described. The reactions of the sera of 48 hemodialysis patients against freshly prepared lymphocytes were compared with those against tube-frozen (384 reaction pairs) and tray-frozen (864 reaction pairs) cells. There was a better than 90% concordance, and only 3% of the reactions differed from negative to strongly positive or vice versa. Preliminary results indicate that the method is also suitable for B-cell (HLA-DK) antibody testing. Routine screening against 50- or lOO-cell panels can be accomplished more rapidly and more efficiently using tray-frozen lymphocytes. The data matrix for analysis of a serum’s HI>A specificity is greatly reduced in comparison to the conventional screening technique, rendering the method particularly suitable for microcomputer processing.
Screening of sera for lymphocytotoxic antibodies against a panel of random cell donors is a routine procedure in today’s HLA laboratory. Sera obtained from patients on dialysis awaiting renal transplantation, transplanted patients, recipients of frequent transfusions due to hematologic disorders, or multiparous women who are potential sources of HLA typing reagents are screened commonly against panels of 20-50 lymphocyte donors. If evidence for antibodies is obtained in the initial screening, further testing against a pane1 of 100 or more cells is carried out to define the antibodies’ HLA specificities. The standard procedure for antibody screening calls for the collection of an appropriate number of serum samples to fit the number of wells on a Terasaki microtest tray (usually 50 sera plus controls). The sera are added to trays which then are stored frozen. Usually, freshly isolated lymphocytes are added to the trays on days when the routine workload in the laboratory is light. With the exception of a few laboratories in which antibody screening is Received June 14, 1983; accepted January 4, 1984.
treated as a matter of priority, most laboratories require somewhere from 3 to 8 weeks to complete a screening against a 50cell panel. From a clinical viewpoint, this long time interval from the day of bleeding is unsatisfactory; often, the screening result arrives at a time when it is not anymore relevant for the patient’s clinical status. To speed up the process, many laboratories adopted the use of lymphocyte aliquots frozen at - 196°C in liquid nitrogen. Rather than having to rely on freshly isolated lymphocytes, a convenient number of frozen samples is thawed on a given day. Nevertheless, while the screening period can be shortened with the use of frozen cells, a 3to 4-week interval is still required in most laboratories, from the day the first serum is collected until the screening results are known. Because cytotoxic antibody screening can be utilized to its full potential only if the results can be made available rapidly, we developed a more efficient testing procedure. We describe herein a technique which allows screening against a 50- or even a IOO-cell panel within 4 hr. Prelimi480
001 l-2240184 $3.00 Copyright All nyht\
0 1984 by Academy Press, Ix. of reproduction m any form reserved.
LYMPHOCYTOTOXIC
ANTIBODY
nary data indicate that the technique can be adapted to testing for B-cell-specific (HLA-DR) antibodies. The new method also provides considerable advantages with respect to data management and computerization.
SCREENING
TECHNIQUE
481
right in a - 80°C freezer. The tubes were transferred the following day into the vapor phase of a liquid-nitrogen storage tank. Freezing of lymphocytes in Terasaki microtrst trays. Cells frozen in tubes were
thawed in a 50°C water bath and washed thrice. The cells were adjusted in medium MATERIALS AND METHODS RPM1 1640 containing 10% fetal calf serum For the purpose of this report, a compar- to a concentration of 4 x 103/pl. Five miison was made between the screening re- croliters of “freezing medium” (10% fetal sults of 48 serum samples tested on either calf serum and 14% Me$O in RPM1 1640) freshly isolated or tray-frozen lymphocytes was added to each well of a Terasaki mifrom 18 healthy donors (864 reaction pairs) crotest tray. Two microliters of the frozenand the screening results of 48 sera tested thawed lymphocyte suspension (8 x 10” on freshly prepared or tube-frozen lympho- cells) was added to each well using a 60cytes from 8 donors (384 reaction pairs). needle dispenser (Seromat, Greiner, NiirThat tray-frozen lymphocytes can be pre- tingen, West Germany). The trays were pared in larger series (i.e., 54 cells on a 60- placed onto copper plates (thickness 0.8 well tray) was demonstrated in subsequent mm) which had been precooled to - 80°C. Storage of frozen trays in a - 80°C freezer routine test series in our laboratory. Sera. Forty-eight sera obtained within 1 was tested for up to 3 months without noweek from 48 hemodialysis patients in our ticeable loss of cell viability. The freezer is local transplant waiting pool were tested. used for various routine purposes and The serum’s actual antibody status was un- opened several times per day. Whether known; however, the patients had been se- minor temperature fluctuations reduce the lected on the basis that about one-half of usable life span of trays during the period them had shown evidence of lymphocytobeyond 3 months is unknown. toxic antibodies in a screening performed 2 Preparution of truys for B-cell screening. months earlier. Thus, an informative rep- The techniques described above for tuberesentation of positive and negative sera and tray-freezing were used with the addicould be expected. In addition, two sera tional step of passing the freshly isolated containing HLA antibodies of known spec- lymphocytes through a nylon wool column. ificity were tested in three dilution steps. Cells (100 x 106)were added to a column For B-cell tray testing, 14 sera with known containing 350 mg nylon wool (LP-1 LeukoB-cell-specific (HLA-DR) antibodies were Pak, Fenwal, Morton Grove, Ind.) which used. had been preincubated with medium RPM1 Freezing of lymphocytes in tubes. Lym1640 containing 5% fetal calf serum at 37°C phocytes were isolated from heparinized for 1 hr, and rinsed with 20 ml warm (37°C) whole blood using the Ficoll-Hypaque gra- medium. The cell-loaded column was indient technique. The lymphocytes were ad- cubated at 37°C for 40 min and rinsed with justed in medium RPM1 1640 containing 40 ml of medium RPM1 1640 containing 5% 10% fetal calf serum to a concentration of fetal calf serum, which washed out the non20 X IO6 cells/ml, mixed with an equal adhering T cells. The nylon wool was revolume of “freezing medium” consisting of moved from the column, teased gently in a 10% fetal calf serum and 20% dimethyl sulf- plastic petri dish containing cold medium oxide (Me,SO) in RPM1 1640, dispensed at (RPM1 1640 with 10% fetal calf serum, room temperature into 0.4-ml plastic tubes 4”C), and incubated at 4°C for 10 min. Sub(Beckman, Rio Alto, Calif.), and placed up- sequently, the B cells were obtained by
482
RUDER
rinsing the nylon wool with medium. Approximately 10 x lo6 cells of a 80% pure B-cell preparation can be obtained from 100 X lo6 unseparated mononuclear cells. The isolated B cells can be tube-frozen at - 196°C in appropriate aliquots for the preparation of B-cell screening trays. Testing procedure. Freshly prepared and tube-frozen lymphocytes were tested using the standard microlymphocytotoxicity assay (1). For tray-frozen cells the technique was essentially the same, except that the test serum was added to each well of a microtest tray containing prefrozen lymphocytes. Prior to addition of the test serum, the trays were thawed by adding 20 ~1 of preheated (50°C) medium RPMI 1640 containing 10% fetal calf serum to each well. A simple mechanical 60-needle dispenser was built for this purpose in our local machine shop. The trays were centrifuged at 17OOgfor 30 set and another 40 l.~l of 37°C medium was added to each well, thereby flooding the trays. The medium was sucked off and mineral oil was dispensed into the wells to cover the cells. The trays were then ready for the addition of 2 ~1 test serum per well (plus negative and positive control sera). For B-cell testing, the incubation steps were altered to 60 min at 37°C for serum and cells, and 120 min at 22°C with complement. Scoring. Identical scoring was used for the three techniques (freshly prepared, tube-frozen, and tray-frozen lymphocytes): < 20% kill = negative ( -) 20-70% kill = positive ( +) > 70% kill = strongpositive ( + + ). For the distinction of “negative” from “positive” reactions, both + and + + were counted as “positive.” RESULTS
In the comparison of freshly prepared versus tray-frozen cells, 28 of the 48 patient sera were antibody-negative by at least one method. Twenty-one were negative by
ET AL.
both, 5 sera showed one positive reaction with either method, and 1 serum reacted with two tray-frozen cells (Table 1). Thus 27 of the 28 sera reacted with only 6 “false” reactions out of the 486 tested, a concordance of 99%. One serum gave 4 extra reactions with freshly prepared cells. Interestingly, this discrepancy could be traced clearly to carry over with the complement syringe from an adjacent well containing a strongly positive serum. Since this type of error cannot occur with the tray-frozen technique (only one patient’s serum is used per tray), an important source of “false positive” reactions is eliminated with this technique. Twenty of the 48 sera showed antibody reactivity when tested on both freshly isolated and tray-frozen target cells. Thereof, 5 sera gave no discrepancies at all in the number of positive reactions, 6 sera showed one more reaction with either method, and 5 sera gave two more reactions. If i 2 reacting cells is assumed as tolerance limit (about ? 10% reacting cell suspensions), 16 of the 20 sera (80%) fell within this limit (Table 1). Of the remaining 4 sera which gave 3 or more extra reactions, 3 reacted more often in the trayfrozen assay, suggesting that the method is somewhat more sensitive. That the extra reactions were not spurious false positive reactions is indicated by the fact that no excessive numbers of “false positives” were encountered with antibody-negative sera using the tray-frozen technique (Table 1). Again, the results of the one serum giving several extra reactions with freshly prepared cells were shown definitely to result from carryover from a strongly positive serum in an adjacent well. Of the entire series of 864 reactions, 785 (91%) were concordant; the correlation is improved further to 92% concordance if the 10 erroneous reactions due to carry over in the conventional screening technique are excluded. To evaluate whether discrepant reactions
LYMPHOCYTOTOXIC
ANTIBODY
483
SCREENING TECHNIQUE
TABLE I Sera of 48 Patients on Renal Transplant Waiting List Screened against Panel of 18 Cells No. additional reacting cell suspensions Freshly prepared
Tray-frozen
Total
Identical reactions
23
2
I
I
2
23
No. sera negative with one or both methods
28
21
0
I
2
3
0
I [’
No. sera positive with both methods
20
5
3
I
4
2
4
I”
Total
48
26
3
2
6
5
4
2“
” False positive due to carryover.
occurred more often with negative, weakly 18-cell panel, both with freshly prepared or reactive, or strongly reactive sera, compar- tray-frozen cells. Three dilutions were isons of reaction pairs were performed tested to evaluate whether weaker reac(Table 2). The reaction of freshly prepared tions would be more difficult to interpret lymphocytes was compared with that of with the tray-frozen technique. Table 3 tube-frozen or tray-frozen cells of the same shows that the two techniques gave essendonor. The two freezing methods yielded tially identical results, the tray-frozen virtually identical results. Ninety percent of method being slightly more sensitive. the reactions were the same as those obIn a preliminary experiment, we tested tained with freshly prepared cells, 7% whether tray-freezing was also suitable for showed a one-step discrepancy (- to + , testing of B-lymphocyte targets. Table 4 or + to + +), and only 3% differed from a shows the reactions of 14 sera with known negative to a strongly positive ( - to + +) HLA-DR specificity against 8 B-cell target reading or vice versa. cells prefrozen in microtest trays. Out of Two sera containing HLA antibodies of 112 reactions, 96 (86%) gave the expected known specificity were tested against the results. Only 16 reactions (14%) were disTABLE 2 Comparison of Reaction Pairs in Freshly Prepared, Tube-Frozen, or Tray-Frozen Antibody Screening Technique Tube-frozen cells %++ Freshly prepared cells %++ %+ %Identical (%) Discrepancies + + to + , or + to - (%) Discrepancies + + to - (%) No. of reaction pairs ’ Ten carryovers were excluded.
14.5 I.1 I.8
Tray-frozen cells
533 +
5%~
c/c+ +
%+
%-
1.3 I.1 2.1
1.3 I.9 73.9
11.9 0.9 I.5
0.7 0.8 3.0
I.8 I.8 77.6
89.5 7.4 3.1 384
90.3 6.4 3.3 854”
484
RUDER ET AL.
TABLE 3 Reactions of Freshly Prepared and Tray-Frozen Lymphocytes against Antisera of Known HLA Snecifitv Serum: Anti-HLA B7 + B27” Target cells (scores/correct scores) Serum dilution 1:l 1:2 I:4
B7+ B27+ 717 212 711 212 517 112
Other719 819 819
Serum dilution I:1 1:2 1:4
B7+ B27+ 717 212 617 212 617 212
Other819 919 Tray819 frozen
Freshly prepared
Serum: Anti-HLA Al0 + Al 1 + A~34~ Target cells (scores/correct scores) Serum dilution I:1 I:4 I:8
AlO+ All+ 414 Oil 314 Oil 114 oi I
Serum dilution 1:I I:4 I:8
AIO+ All + Aw34+ 414 l/l l/l 414 O/l l/l 214 Oil Oil
Aw34+ l/l ill Oil
Other12112 12,12 Freshly 12112 prepared Other12112 12112 Tray12112 frozen
‘I There were 7 HLA-B7-positive and 2 HLA-B27-positive cells in the 18-cell panel. ’ There were 4 HLA-Al0 cells, one HLA-All cell. and one HLA-Aw34 cell in the l8-cell panel.
crepant. Some of the “false” reactions may be due to the still imperfect characterization of the HLA-DR reagents. Generally, the observed reactions fitted the expected pattern. Thus, it appears likely that trayfrozen B lymphocytes will prove an effective means of testing for B-cell-specific (HLA-DR) antibodies.
frozen cells are entirely acceptable. However, only freezing in trays provides the key advantage of giving a complete result within a few hours. Of course, some of the labor saved at the TABLE 4 14 Sera with Known HLA-DR Specificity Tested on Tray-Frozen B Lymphocytes HLA-OR
Phenotvoe
of
Tamet
Cell
DISCUSSION
The main advantage of using tray-frozen lymphocytes for cytotoxic antibody screening is that the laboratory can produce screening results much more rapidly. There is no need to wait until 50 sera have been accumulated in order to make testing on a 60-well tray economical. Even if a single serum is sent in on a given day, a frozen cell tray is simply thawed out and serum is added. No cells need to be isolated or adjusted at the time of testing. Thus, several sera can be screened against 50 cell donors (or 100 donors with the use of two different trays) by one technician within a few hours. Wood et al., using a two-step score, found a concordance rate of 94% comparing the reactions of freshly prepared lymphocytes with those frozen in tubes (4). We obtained very similar results, with both tube-frozen and tray-frozen cells. For practical purposes, the results obtained with
1.2
I,5
1,8
DR1
++
it
+
++
DR2
++
-
_
DR3
_
negati
HLA-DR
Of
Serum
2,4
3,5
ve..
4,-
6,7
_
_ .
++
_ +
OR3 DR3+7
Specificity
2;
%::
DR7
_
DR7
+
_
_ +
+
-
++ ++
DR4 DR4 DR415
++
+
DIE
it
-
++
+
DR5 DR5+6
-kt
il
++ -
-
+
+
++
++
+
-
it
.pJ
reaction
expected
positive
reaction
expected
q -
negative
reaction
observed
positive
reactjon
observed
+
stronglv
positive
observed
++
Reactions
as expected
86%
In=961
Reactions
not
14:
(n=16)
-
+
negative
as expected
it
++
++
LYMPHOCYTOTOXIC
ANTIBODY
time of testing must be invested in preparing the trays containing frozen cells. We are now routinely using trays containing frozen cells of 50 donors, which are prepared in series of 500 trays per day. About 20 technician-hours are necessary to produce one such set, starting from tube frozen cells. We are still bothered by the need to add a relatively large number of 8 x lo3 lymphocytes per well. This is necessary because a portion of the cells is lost during washing. Currently, we are trying to reduce the loss by developing a more efficient washing procedure. Nevertheless, since the cells are obtained in large volumes from healthy blood donors over a long period of time (liquid nitrogen-frozen cells can be stored for years), cell requirement has not been a significant problem. Well over 3000 sera were screened in our laboratory using the tray-freezing technique, convincing us that the method is both practical and reliable. At the time of preparing this manuscript we became aware of the paper by Nathan (2) who used a similar approach of freezing lymphocytes in microtest trays, albeit with less favorable results. The high background of IO-30% dead cells (2) would be unacceptable by current standards. DNase treatment improves the cell viability; however, it complicates the technique (3). Our trays show background values of 2-10% dead cells which allows the detection of even weak antibodies without expensive DNase treatment. We believe that several factors contribute to the improved cell viability obtained by us: (1) Tube-frozen and thawed cells must have a viability of ~95% before the cells can be accepted for tray freezing. (2) Thawing by adding 50°C medium gives optimum cell viability. (3) Placing the trays on precooled copper plates improves the freezing efficiency. (4) According to our preliminary results, the requirement of high cell viability prior to tray-freezing is parti-
SCREENING
TECHNIQUE
485
culary important with B cells. Four hundred Kunitz units DNase per well (DNase I, Sigma, St. Louis, MO.), as recommended by Sollmann (3), is useful for cells with poor viability in that it removes dead cells. Because DNase treatment is expensive, we have not used it in large test series. An important advantage of lymphocytotoxic antibody screening with cells frozen in trays lies in the relative ease of data handling and analysis. The number of records that must be kept on file for analysis is much smaller than with the conventional screening method. If, for example, 55 sera are screened against 100 cell donors, 55 x 100 = 5500 reactions must be recorded in the computer’s memory for the analysis of a given serum’s result obtained with the conventional method. In contrast, only 1 x 100 = 100 reactions must be stored with the tray-frozen panel. This is important particularly with respect to the use of microcomputer technology for HLA antibody specification. ACKNOWLEDGMENTS The authors acknowledge the excellent technical assistance of Sigrid Daffinger, Monika Renz, and Martina Kutsche, and the assistance of Walter Schmitt and Heinz Schwegler with building the dispenser for thawmedium. REFERENCES 1. Mittal, K. K., Mickey, M. R., Singal, D. P., and Terasaki, P. I. Serotyping for homotransplantion: XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 6, 913-927 (1968). 2. Nathan, P. Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing. Cryobiology 11, 305-927 (1974). 3. Sollmann, P. A., and Nathan, P. An improved method for preparing refrozen rethawed human lymphocytes on plates for microcytotoxicity studies. Cryobiology 16, 118- 124 (1979). 4. Wood. N.. Bashir. H., Gerally, J., Amos, D. B., and Yunis, E. J. A simple method of freezing and storing live lymphocytes. Tissue Antigens 2, 27-31 (1972).
21, 480-485(1984)
A Rapid Screening
H. RUDER, Institute
Technique for Lymphocytotoxic Tray-Frozen Lymphocytes
G. OPELZ, of Immunology,
V. LENHARD, University
A. SCHAFER,
of Heidelhergv
Heidelberg,
Antibodies
Using
AND V. DANIEL West Germuny
A method whereby sera can be screened for the presence of lymphocytotoxic antibodies within 4 hr using lymphocytes frozen in microtest trays is described. The reactions of the sera of 48 hemodialysis patients against freshly prepared lymphocytes were compared with those against tube-frozen (384 reaction pairs) and tray-frozen (864 reaction pairs) cells. There was a better than 90% concordance, and only 3% of the reactions differed from negative to strongly positive or vice versa. Preliminary results indicate that the method is also suitable for B-cell (HLA-DK) antibody testing. Routine screening against 50- or lOO-cell panels can be accomplished more rapidly and more efficiently using tray-frozen lymphocytes. The data matrix for analysis of a serum’s HI>A specificity is greatly reduced in comparison to the conventional screening technique, rendering the method particularly suitable for microcomputer processing.
Screening of sera for lymphocytotoxic antibodies against a panel of random cell donors is a routine procedure in today’s HLA laboratory. Sera obtained from patients on dialysis awaiting renal transplantation, transplanted patients, recipients of frequent transfusions due to hematologic disorders, or multiparous women who are potential sources of HLA typing reagents are screened commonly against panels of 20-50 lymphocyte donors. If evidence for antibodies is obtained in the initial screening, further testing against a pane1 of 100 or more cells is carried out to define the antibodies’ HLA specificities. The standard procedure for antibody screening calls for the collection of an appropriate number of serum samples to fit the number of wells on a Terasaki microtest tray (usually 50 sera plus controls). The sera are added to trays which then are stored frozen. Usually, freshly isolated lymphocytes are added to the trays on days when the routine workload in the laboratory is light. With the exception of a few laboratories in which antibody screening is Received June 14, 1983; accepted January 4, 1984.
treated as a matter of priority, most laboratories require somewhere from 3 to 8 weeks to complete a screening against a 50cell panel. From a clinical viewpoint, this long time interval from the day of bleeding is unsatisfactory; often, the screening result arrives at a time when it is not anymore relevant for the patient’s clinical status. To speed up the process, many laboratories adopted the use of lymphocyte aliquots frozen at - 196°C in liquid nitrogen. Rather than having to rely on freshly isolated lymphocytes, a convenient number of frozen samples is thawed on a given day. Nevertheless, while the screening period can be shortened with the use of frozen cells, a 3to 4-week interval is still required in most laboratories, from the day the first serum is collected until the screening results are known. Because cytotoxic antibody screening can be utilized to its full potential only if the results can be made available rapidly, we developed a more efficient testing procedure. We describe herein a technique which allows screening against a 50- or even a IOO-cell panel within 4 hr. Prelimi480
001 l-2240184 $3.00 Copyright All nyht\
0 1984 by Academy Press, Ix. of reproduction m any form reserved.
LYMPHOCYTOTOXIC
ANTIBODY
nary data indicate that the technique can be adapted to testing for B-cell-specific (HLA-DR) antibodies. The new method also provides considerable advantages with respect to data management and computerization.
SCREENING
TECHNIQUE
481
right in a - 80°C freezer. The tubes were transferred the following day into the vapor phase of a liquid-nitrogen storage tank. Freezing of lymphocytes in Terasaki microtrst trays. Cells frozen in tubes were
thawed in a 50°C water bath and washed thrice. The cells were adjusted in medium MATERIALS AND METHODS RPM1 1640 containing 10% fetal calf serum For the purpose of this report, a compar- to a concentration of 4 x 103/pl. Five miison was made between the screening re- croliters of “freezing medium” (10% fetal sults of 48 serum samples tested on either calf serum and 14% Me$O in RPM1 1640) freshly isolated or tray-frozen lymphocytes was added to each well of a Terasaki mifrom 18 healthy donors (864 reaction pairs) crotest tray. Two microliters of the frozenand the screening results of 48 sera tested thawed lymphocyte suspension (8 x 10” on freshly prepared or tube-frozen lympho- cells) was added to each well using a 60cytes from 8 donors (384 reaction pairs). needle dispenser (Seromat, Greiner, NiirThat tray-frozen lymphocytes can be pre- tingen, West Germany). The trays were pared in larger series (i.e., 54 cells on a 60- placed onto copper plates (thickness 0.8 well tray) was demonstrated in subsequent mm) which had been precooled to - 80°C. Storage of frozen trays in a - 80°C freezer routine test series in our laboratory. Sera. Forty-eight sera obtained within 1 was tested for up to 3 months without noweek from 48 hemodialysis patients in our ticeable loss of cell viability. The freezer is local transplant waiting pool were tested. used for various routine purposes and The serum’s actual antibody status was un- opened several times per day. Whether known; however, the patients had been se- minor temperature fluctuations reduce the lected on the basis that about one-half of usable life span of trays during the period them had shown evidence of lymphocytobeyond 3 months is unknown. toxic antibodies in a screening performed 2 Preparution of truys for B-cell screening. months earlier. Thus, an informative rep- The techniques described above for tuberesentation of positive and negative sera and tray-freezing were used with the addicould be expected. In addition, two sera tional step of passing the freshly isolated containing HLA antibodies of known spec- lymphocytes through a nylon wool column. ificity were tested in three dilution steps. Cells (100 x 106)were added to a column For B-cell tray testing, 14 sera with known containing 350 mg nylon wool (LP-1 LeukoB-cell-specific (HLA-DR) antibodies were Pak, Fenwal, Morton Grove, Ind.) which used. had been preincubated with medium RPM1 Freezing of lymphocytes in tubes. Lym1640 containing 5% fetal calf serum at 37°C phocytes were isolated from heparinized for 1 hr, and rinsed with 20 ml warm (37°C) whole blood using the Ficoll-Hypaque gra- medium. The cell-loaded column was indient technique. The lymphocytes were ad- cubated at 37°C for 40 min and rinsed with justed in medium RPM1 1640 containing 40 ml of medium RPM1 1640 containing 5% 10% fetal calf serum to a concentration of fetal calf serum, which washed out the non20 X IO6 cells/ml, mixed with an equal adhering T cells. The nylon wool was revolume of “freezing medium” consisting of moved from the column, teased gently in a 10% fetal calf serum and 20% dimethyl sulf- plastic petri dish containing cold medium oxide (Me,SO) in RPM1 1640, dispensed at (RPM1 1640 with 10% fetal calf serum, room temperature into 0.4-ml plastic tubes 4”C), and incubated at 4°C for 10 min. Sub(Beckman, Rio Alto, Calif.), and placed up- sequently, the B cells were obtained by
482
RUDER
rinsing the nylon wool with medium. Approximately 10 x lo6 cells of a 80% pure B-cell preparation can be obtained from 100 X lo6 unseparated mononuclear cells. The isolated B cells can be tube-frozen at - 196°C in appropriate aliquots for the preparation of B-cell screening trays. Testing procedure. Freshly prepared and tube-frozen lymphocytes were tested using the standard microlymphocytotoxicity assay (1). For tray-frozen cells the technique was essentially the same, except that the test serum was added to each well of a microtest tray containing prefrozen lymphocytes. Prior to addition of the test serum, the trays were thawed by adding 20 ~1 of preheated (50°C) medium RPMI 1640 containing 10% fetal calf serum to each well. A simple mechanical 60-needle dispenser was built for this purpose in our local machine shop. The trays were centrifuged at 17OOgfor 30 set and another 40 l.~l of 37°C medium was added to each well, thereby flooding the trays. The medium was sucked off and mineral oil was dispensed into the wells to cover the cells. The trays were then ready for the addition of 2 ~1 test serum per well (plus negative and positive control sera). For B-cell testing, the incubation steps were altered to 60 min at 37°C for serum and cells, and 120 min at 22°C with complement. Scoring. Identical scoring was used for the three techniques (freshly prepared, tube-frozen, and tray-frozen lymphocytes): < 20% kill = negative ( -) 20-70% kill = positive ( +) > 70% kill = strongpositive ( + + ). For the distinction of “negative” from “positive” reactions, both + and + + were counted as “positive.” RESULTS
In the comparison of freshly prepared versus tray-frozen cells, 28 of the 48 patient sera were antibody-negative by at least one method. Twenty-one were negative by
ET AL.
both, 5 sera showed one positive reaction with either method, and 1 serum reacted with two tray-frozen cells (Table 1). Thus 27 of the 28 sera reacted with only 6 “false” reactions out of the 486 tested, a concordance of 99%. One serum gave 4 extra reactions with freshly prepared cells. Interestingly, this discrepancy could be traced clearly to carry over with the complement syringe from an adjacent well containing a strongly positive serum. Since this type of error cannot occur with the tray-frozen technique (only one patient’s serum is used per tray), an important source of “false positive” reactions is eliminated with this technique. Twenty of the 48 sera showed antibody reactivity when tested on both freshly isolated and tray-frozen target cells. Thereof, 5 sera gave no discrepancies at all in the number of positive reactions, 6 sera showed one more reaction with either method, and 5 sera gave two more reactions. If i 2 reacting cells is assumed as tolerance limit (about ? 10% reacting cell suspensions), 16 of the 20 sera (80%) fell within this limit (Table 1). Of the remaining 4 sera which gave 3 or more extra reactions, 3 reacted more often in the trayfrozen assay, suggesting that the method is somewhat more sensitive. That the extra reactions were not spurious false positive reactions is indicated by the fact that no excessive numbers of “false positives” were encountered with antibody-negative sera using the tray-frozen technique (Table 1). Again, the results of the one serum giving several extra reactions with freshly prepared cells were shown definitely to result from carryover from a strongly positive serum in an adjacent well. Of the entire series of 864 reactions, 785 (91%) were concordant; the correlation is improved further to 92% concordance if the 10 erroneous reactions due to carry over in the conventional screening technique are excluded. To evaluate whether discrepant reactions
LYMPHOCYTOTOXIC
ANTIBODY
483
SCREENING TECHNIQUE
TABLE I Sera of 48 Patients on Renal Transplant Waiting List Screened against Panel of 18 Cells No. additional reacting cell suspensions Freshly prepared
Tray-frozen
Total
Identical reactions
23
2
I
I
2
23
No. sera negative with one or both methods
28
21
0
I
2
3
0
I [’
No. sera positive with both methods
20
5
3
I
4
2
4
I”
Total
48
26
3
2
6
5
4
2“
” False positive due to carryover.
occurred more often with negative, weakly 18-cell panel, both with freshly prepared or reactive, or strongly reactive sera, compar- tray-frozen cells. Three dilutions were isons of reaction pairs were performed tested to evaluate whether weaker reac(Table 2). The reaction of freshly prepared tions would be more difficult to interpret lymphocytes was compared with that of with the tray-frozen technique. Table 3 tube-frozen or tray-frozen cells of the same shows that the two techniques gave essendonor. The two freezing methods yielded tially identical results, the tray-frozen virtually identical results. Ninety percent of method being slightly more sensitive. the reactions were the same as those obIn a preliminary experiment, we tested tained with freshly prepared cells, 7% whether tray-freezing was also suitable for showed a one-step discrepancy (- to + , testing of B-lymphocyte targets. Table 4 or + to + +), and only 3% differed from a shows the reactions of 14 sera with known negative to a strongly positive ( - to + +) HLA-DR specificity against 8 B-cell target reading or vice versa. cells prefrozen in microtest trays. Out of Two sera containing HLA antibodies of 112 reactions, 96 (86%) gave the expected known specificity were tested against the results. Only 16 reactions (14%) were disTABLE 2 Comparison of Reaction Pairs in Freshly Prepared, Tube-Frozen, or Tray-Frozen Antibody Screening Technique Tube-frozen cells %++ Freshly prepared cells %++ %+ %Identical (%) Discrepancies + + to + , or + to - (%) Discrepancies + + to - (%) No. of reaction pairs ’ Ten carryovers were excluded.
14.5 I.1 I.8
Tray-frozen cells
533 +
5%~
c/c+ +
%+
%-
1.3 I.1 2.1
1.3 I.9 73.9
11.9 0.9 I.5
0.7 0.8 3.0
I.8 I.8 77.6
89.5 7.4 3.1 384
90.3 6.4 3.3 854”
484
RUDER ET AL.
TABLE 3 Reactions of Freshly Prepared and Tray-Frozen Lymphocytes against Antisera of Known HLA Snecifitv Serum: Anti-HLA B7 + B27” Target cells (scores/correct scores) Serum dilution 1:l 1:2 I:4
B7+ B27+ 717 212 711 212 517 112
Other719 819 819
Serum dilution I:1 1:2 1:4
B7+ B27+ 717 212 617 212 617 212
Other819 919 Tray819 frozen
Freshly prepared
Serum: Anti-HLA Al0 + Al 1 + A~34~ Target cells (scores/correct scores) Serum dilution I:1 I:4 I:8
AlO+ All+ 414 Oil 314 Oil 114 oi I
Serum dilution 1:I I:4 I:8
AIO+ All + Aw34+ 414 l/l l/l 414 O/l l/l 214 Oil Oil
Aw34+ l/l ill Oil
Other12112 12,12 Freshly 12112 prepared Other12112 12112 Tray12112 frozen
‘I There were 7 HLA-B7-positive and 2 HLA-B27-positive cells in the 18-cell panel. ’ There were 4 HLA-Al0 cells, one HLA-All cell. and one HLA-Aw34 cell in the l8-cell panel.
crepant. Some of the “false” reactions may be due to the still imperfect characterization of the HLA-DR reagents. Generally, the observed reactions fitted the expected pattern. Thus, it appears likely that trayfrozen B lymphocytes will prove an effective means of testing for B-cell-specific (HLA-DR) antibodies.
frozen cells are entirely acceptable. However, only freezing in trays provides the key advantage of giving a complete result within a few hours. Of course, some of the labor saved at the TABLE 4 14 Sera with Known HLA-DR Specificity Tested on Tray-Frozen B Lymphocytes HLA-OR
Phenotvoe
of
Tamet
Cell
DISCUSSION
The main advantage of using tray-frozen lymphocytes for cytotoxic antibody screening is that the laboratory can produce screening results much more rapidly. There is no need to wait until 50 sera have been accumulated in order to make testing on a 60-well tray economical. Even if a single serum is sent in on a given day, a frozen cell tray is simply thawed out and serum is added. No cells need to be isolated or adjusted at the time of testing. Thus, several sera can be screened against 50 cell donors (or 100 donors with the use of two different trays) by one technician within a few hours. Wood et al., using a two-step score, found a concordance rate of 94% comparing the reactions of freshly prepared lymphocytes with those frozen in tubes (4). We obtained very similar results, with both tube-frozen and tray-frozen cells. For practical purposes, the results obtained with
1.2
I,5
1,8
DR1
++
it
+
++
DR2
++
-
_
DR3
_
negati
HLA-DR
Of
Serum
2,4
3,5
ve..
4,-
6,7
_
_ .
++
_ +
OR3 DR3+7
Specificity
2;
%::
DR7
_
DR7
+
_
_ +
+
-
++ ++
DR4 DR4 DR415
++
+
DIE
it
-
++
+
DR5 DR5+6
-kt
il
++ -
-
+
+
++
++
+
-
it
.pJ
reaction
expected
positive
reaction
expected
q -
negative
reaction
observed
positive
reactjon
observed
+
stronglv
positive
observed
++
Reactions
as expected
86%
In=961
Reactions
not
14:
(n=16)
-
+
negative
as expected
it
++
++
LYMPHOCYTOTOXIC
ANTIBODY
time of testing must be invested in preparing the trays containing frozen cells. We are now routinely using trays containing frozen cells of 50 donors, which are prepared in series of 500 trays per day. About 20 technician-hours are necessary to produce one such set, starting from tube frozen cells. We are still bothered by the need to add a relatively large number of 8 x lo3 lymphocytes per well. This is necessary because a portion of the cells is lost during washing. Currently, we are trying to reduce the loss by developing a more efficient washing procedure. Nevertheless, since the cells are obtained in large volumes from healthy blood donors over a long period of time (liquid nitrogen-frozen cells can be stored for years), cell requirement has not been a significant problem. Well over 3000 sera were screened in our laboratory using the tray-freezing technique, convincing us that the method is both practical and reliable. At the time of preparing this manuscript we became aware of the paper by Nathan (2) who used a similar approach of freezing lymphocytes in microtest trays, albeit with less favorable results. The high background of IO-30% dead cells (2) would be unacceptable by current standards. DNase treatment improves the cell viability; however, it complicates the technique (3). Our trays show background values of 2-10% dead cells which allows the detection of even weak antibodies without expensive DNase treatment. We believe that several factors contribute to the improved cell viability obtained by us: (1) Tube-frozen and thawed cells must have a viability of ~95% before the cells can be accepted for tray freezing. (2) Thawing by adding 50°C medium gives optimum cell viability. (3) Placing the trays on precooled copper plates improves the freezing efficiency. (4) According to our preliminary results, the requirement of high cell viability prior to tray-freezing is parti-
SCREENING
TECHNIQUE
485
culary important with B cells. Four hundred Kunitz units DNase per well (DNase I, Sigma, St. Louis, MO.), as recommended by Sollmann (3), is useful for cells with poor viability in that it removes dead cells. Because DNase treatment is expensive, we have not used it in large test series. An important advantage of lymphocytotoxic antibody screening with cells frozen in trays lies in the relative ease of data handling and analysis. The number of records that must be kept on file for analysis is much smaller than with the conventional screening method. If, for example, 55 sera are screened against 100 cell donors, 55 x 100 = 5500 reactions must be recorded in the computer’s memory for the analysis of a given serum’s result obtained with the conventional method. In contrast, only 1 x 100 = 100 reactions must be stored with the tray-frozen panel. This is important particularly with respect to the use of microcomputer technology for HLA antibody specification. ACKNOWLEDGMENTS The authors acknowledge the excellent technical assistance of Sigrid Daffinger, Monika Renz, and Martina Kutsche, and the assistance of Walter Schmitt and Heinz Schwegler with building the dispenser for thawmedium. REFERENCES 1. Mittal, K. K., Mickey, M. R., Singal, D. P., and Terasaki, P. I. Serotyping for homotransplantion: XVIII. Refinement of microdroplet lymphocyte cytotoxicity test. Transplantation 6, 913-927 (1968). 2. Nathan, P. Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing. Cryobiology 11, 305-927 (1974). 3. Sollmann, P. A., and Nathan, P. An improved method for preparing refrozen rethawed human lymphocytes on plates for microcytotoxicity studies. Cryobiology 16, 118- 124 (1979). 4. Wood. N.. Bashir. H., Gerally, J., Amos, D. B., and Yunis, E. J. A simple method of freezing and storing live lymphocytes. Tissue Antigens 2, 27-31 (1972).