a combined dye exclusion (trypan blue) and fluorochromatic technique for the microdroplet...

Tissue Antigens (1972), 2, 57-63

Published by Munksgaard, Copenhagen, Denmark No part may be reproduced by any process without written permission from the author(s)

A Combined Dye Exclusion (Trypan Blue) and

Fluorochromatic Technique for

the Microdroplet Lymp hocyto toxici ty Test

R. FONC* AND F. KISSMEYER-NIELSEN

Blood Bank and Tissue Typing Laboratory, University Hospital, Aarhus, Denmark

A combined dye exclusion (Trypan Blue) and fluorochromatic technique for lymphocytotoxicity test is described. The technique allows the test to be read either as a dye exclusion or a fluorochromatic assay as required, and is found to be useful for typing lymphocyte samples with a high background of killed cells present. I t is suggested that frozen lymphocytes, as well as samples which are shipped from one laboratory to another over a long distance and thus easily subjected to inadvertent damage during transit, should be labelled with Fluorescein Diacetate prior to their typing using the combined technique.

Received for publication 2 November, accepted 20 November 1971

The microdroplet lymphocytotoxicity test employing the dye exclusion principle has gained widespread acceptance for the de- tection of human leukocyte antigens in HL-A typing. This technique is reliable, rapid and relatively simple to perform. Moreover, the cytotoxic effect on the target lymphocytes can be assessed directly as the killed cells will take up dyes such as Trypan Blue making their recognition easy with the aid of an inverted or ordinary light microscope. Hence it is suitable for

typing freshly prepared or well-preserved froi.en lymphocytes. A drawback of this technique is that it does not readily distin- guish between the true or false positive reactions obtained with those lymphocyte samples which contain more than 20 to 30 % dead cells. The phenotypes deduced from the results of such reactions may be quite erroneous.

The fluorochromatic cytotoxic assay described by Bodmer et al. (1967) is a more elaborate procedure requiring labelling of

R.F.* Recipient of a study grant from Wellington Hospital, Wellington, New Zealand.

58 PONG AND KISSMEYER-NIEISEN

the target lymphocytes with a fluorescing material prior to testing. An indirect ap- proach is applied to asess the cytotoxic effects on the target cells by determining the number of living fluorescing cells remaining at the completion of the cytotoxic reaction. The dead cells having leaked out the accumulated fluorescein will not fluoresce when exposed to ultra-violet light. Although this technique requires a fluorescence-microscope and takes longer to perform owing to a rather prolonged incubation period at required room temperature, it is supposed to give a more precise typing result since the interpretation of the outcome of the cytotoxic test is not in- fluenced to any degree by the presence of the preexisting dead cells in the testing sample provided that these constitute not more than 75 "/. of the total cell count.

The present study was undertaken to explore the feasibility of combining dye exclusion and fluorochromasia and thus also the advantage offered from each of these into a single technique. The findings are presented in this paper.

Material and Methods Lymphocyte suspensions from 28 healthy blood donors were used in this study. The lymphocytes were isolated from freshly drawn venous blood by albumin flotation and were typed immediately by the lym- phocytotoxic microtechnique of Kissmeyer- Nielsen & Kjerbye (1967) using highly selected typing antisera (for a discussion of these antisera see Thorsby et al. 197 1 ) . The same lymphocyte suspensions were labelled with Fluorescein Diacetate (FDA), frozen and stored in liquid nitrogen a t - 196' C for up to 14 days before being retyped by the combined dye exclusion (Trypan Blue) and fluorochromatic technique with the same typing antisera.

A preliminary titration experiment using

six different antisera and the combined technique was carried out and the titres were compared with those obtained by using the dye exclusion method alone.

Ccll labelling and frcezing procedurczs The lymphocytes wcre labelled with FDA (Mann Research Laboratory, N.Y., U.S.A.) according to a modification (Douglas et al. 1971) of the original method of Rodmer ( 1967).

A 0.5 o/c FDA acetone stock solution was prepared by adding 2 ml of reagent grade acetone to 10 mg of FDA. This solution was placed in a tightly sealed, stoppered dark container and stored at -- 20' C until use.

For lymphocyte labelling the FD,4 acetone stock solution was dilutrd 1 .50 in I'ris balanced salt solution (I'ris-BBS) . One part of this diluted FDA solution was added to 10 Farts of a lymphocyte suspen- sion which had been isolated by albumin flotation technique, washed twice and resuspended in Tris-BBS to a concentration of 15X 10" cells per ml. The mixture was incubated in the dark a t room temperature for 30 min. After being labelled the cells were washed oncc in a large volume of Tris-BBS to remove the excessive FDA. They were then resuspended to the original concentration, i.e. 15X 106 per ml in Tris-BRS containing 3 YO W/V bovine serum albumin (BSA) (Bovine serum albumin fraction V, Sigma Chemical Co., St. Louis, Missouri, U.S.A.) and 10 % V/V Dimethyl sulphoxide (DMSO) (British Drug Houses Ltd., Poole, England). This medium was preparrd by dissolving 3 g BSA in 100 ml Tris-BBS and DMSO was then added in the proportion of 1 part of DMSO to 9 parts of Tris-RBS plus 3 "/o BSA.

Sixteen of our labelled lymphocyte samples were resuspended in a differmt medium consisting of equal volumes of pooled

59 MICKODROPI.ET 1.YMPtIOCYTOTOSICITY TEST

inactivated human AB serum and 20 % DMSO in Tris-BBS in order to evaluate the effectiveness of this mixture for pre- serving the viability of the lymphocytes during the freezing procedure.

The FDA-labelled cell suspensions were then distributed in 0.5 ml aliquots in Beck- man microfuge polypropylene tubes and cooled at the rate of 1’ C per minute using a Linde Biological controlling freezing unit (Model RF-SA, Union Carbide Co., U.S.A.). When the “heat of fusion” was reached at - 18’ C, a sustained flow of liquid nitrogen was let into the freezing chamber by turning on the override switch for 10 seconds. Thereafter, the tubes were frozen at the same rate to -50’ C when they were removed from the freezingcham- ber and placed immediately in a liquid nitrogm storage tank at - 196’ C.

Typing of the frozen cells by the combined technique The fro7en lymphocyte suspensions were thawed quickly to 40O C and washed once with Tris-BBS. The cells were resuspended to a concentration of 2X 103/ ,~11 in equal volumes of fresh human AB serum and rabbit serum (source of complement). The procedure for typing by the combined technique is exactly the same as that described by Kissmeyer-Nielsen & Kjerbye ( 1967) including the addition of 2 Trypan Blue solution to the reaction mixture at the end of the first incubation period, except in this case the target lymphocytes had been labelled with FDA. Therefore the tests could be read as for dye exclusion technique using an inverted microscope, or for fluorochromatic technique using a Leitz non- inverted fluorescence microscope (magnifi- cation 1OX 10) with or without a dry dark field condenser. For the purposes of comparison, all our tests were read twice by these two methods. I t was of course possible to employ the fluorescence microscope

alone for the two different ways of reading by turning on or off the fluorescence light source as required.

Scoring The score obtained for each cytotoxic reaction by the Trypan Blue method was de- signated as “Trypan Blue” score whilst that obtained by fluorochromasia was de- signated as “fluorescence” score.

For the “Trypan Blue” scoring system, a negative score represented less than 10 r/o killed cells present; 10 to 20 % dead cells were scored as +; 21 to 50 ”/o as ++; 51 to 7 5 % as +++; and 2 - 7 6 % as ++++.

The system proposed by Bodmer et al. (1967) with slight modification was used for the “fluorescence” scores. I n this system, scores were given for the degree of disappearance of the fluorescing lymphocytes when compared with the negative control on each test plate. A negative score represented no disappearance (or less than 10 % killed cells) ; 1, between 10 and 20 % disappearance; 2, between 21 and 50 ?h disappearance; 3, between 51 and 90 % disappearance; and 4, over 90 ”/o disappearance. A score of 2 or more indicated a positive reaction.

R es u 1 t s CompariJon between Trypan Blue and fluorescmce scoring It was observed that in the combined dyc exclusion (Trypan Blue) and fluorochromatic technique the addition of 2 “/o Try- pan Blue solution to the final reaction mixture of the cytotoxicity test system did not interfere with the ability of the viable FDA-labelled lymphocytes to fluoresce under ultra-violet light. Thus in the present investigation we were able to read and score all the tests first using the Trypan

60 FONG AND hISSMEYl3R-NIELSEN

Table 1 Comparison between the results obtained b-v j luoro- chromatic technique on 8 samples of frozen lynz- phocytes and the results by dye exclusion (Tsypnn Blue) technique on the same cell preparations before

they were frozen

Frozen cells (fluorochromatic

technique)

- + 1

346 1 Fresh cells -t (dye exclusion -- technique)

I 439

Blue method as for our routine practice in typing and again as fluorochromatic assays by fluorescence microscopy. The results of the individual tests as recorded by these two methods were then compared.

The positive reaction of 8 out of the total 28 frozen FDA-labelled lymphocyte samples were not determined with any cer- tainty by the Trypan Blue method after they had reacted with the panel of 54 antisera. This was due to the fact that each sample contained a high background of stained cells ranging from 50-75 YO or more ZIA revealed in the negative control. However, it wa5 possible to score these same tests when they were visualiied by fluorescence microscopy. A comparison of the overall results from these 8 samples obtained by fluorochromatic method and that obtained by Trypan Blue method before the same cell preparations were froren is given in Table 1. As can be seen, there is good general agreement between the two methods of recording indicating also the usefulness of the combined technique in that if a given lpmphocytotoxicity test can- not be read using the conventional Trypan Blue method it can still be scored using the fluorochromatic technique. There were 4 discrepancies recorded. Three were from

one single cell sample which had very few viable cells present as shown in the negative control (less than 20 “/o were viable as demonstrated with Trypan Blue). The

Table 2 Comparison of the overall results f rom 20 frozen FDA-fabelled lymphocyte samples when each was read by the Trypan Blue and the jluorochromatic

techniques

Trypan Blue I method -

Fluorochromatic method

I

177 0 1 902

10x0

Table .3

Correlation betzceen the “Trypan Blue” scores and the “jluorescence” scores as recorded by Trypan Blue and jluorochromatic methods for all the positive reactions f rom 20 samples of frozen lymphocytes

treated by the combined technique

Fluor- Trypan Blue score escence

score

- - - - 0 -

2 ~ ~

- 2 12 -

~ 3 - 11 56 23

4 1 -- 3 70 -

Trypan Blue scoses less than 10:; killed cells

i- between 10 and 200; killed cells +- 4- between 21 and SO”/; killed cells 4- 4 - + between 51 and 757; killed cells + + + + over 757:, killed cells

Fluorescence scores 0 less than lo‘:;, killed cells 1 between 10 and 2076 killed cells 2 between 21 and SOYo killed cells 3 between 51 and 90:/, killed cells 4 over 90q(, killed cells

MICRODROPLET 1,YMPHOCYTOTOXICITY TEST 61

fourth was from a second sample with an oligospecific antiserum giving a Trypan Blue negative fluorescence positive pattern.

No difficulty was experienced in reading the reactions from the remaining 20 samples either with the Trypan Blue or the fluorochromatic methods as the background killed cells present in each of these was low enough (5 "/o to 15 %) allowing the reader to score the tests accurately. Table 2 shows a direct comparison of the overall results obtained by these two methods and a correlation of the two types of scores recorded for all the positive reactions is given in Table 3. These data again sug- gest an excellent agreement between the two methods for reading and recording the results of the same tests. Furthermore the score assigned to the strength of an individual positive reaction by one method is closely correlated to that of the other. The only discrepancy was from one cell sample which, as mentioned, gave a Trypan Blue negative, fluorescence positive pattern with the same oligospecific antiserum. Apart from this no other discrepancies were de- tected when these results were compared with those obtained with Trypan Blue before the same cell preparations were frozen.

Phenotypes The phenotypes of all but 3 of the 28 frozen samples as deduced from the results recorded by the fluorochromatic method were identical to those obtained when the corresponding samples were typed before freezing by the Trypan Blue method. In 2 of these frozen samples the discordance was due to the presence of an extra fluorescence positive result recorded with the one oligospecific antiserum which, besides HL- A13 and FJH (=W27) , also determined the low frequency antigen 407x (Kiss- meyer-Nielsen et al. 1970). The explana- tion for this is uncertain. However, it was interesting to note that when these two

samples were typed with Trypan Blue when they were freshly prepared, one was found to possess two HL-A antigens (HL- A2, RR ( = WlO) ) and the other to possess three (HL-A2, Li (=>W19) , HL-A12). Therefore whether this extra antigen should be included in their phenotypes was debatable. The remaining discordant result was from a cell sample which had very few viable cells present and in which 3 discrepant reactions were recorded. Errors in interpretation of these discrepant reactions were the obvious causes for the discordance.

l i trat ions The titre obtained for each of the 6 antisera tested against a sample of FDA-labelled lymphocytes with known phenotype was shown to be similar regardless of whether the results were recorded by Try- pan Blue or fluorochromasia, or whether the target cells used were fresh or frozen. A typical example of such results is given in Table 4.

Comparison of the t w o media used for cell freezing All the 8 frozen cell samples which had 50 "/o or more background killed cells present were frozen in the medium of Tris- BBS containing 3 % bovine serum albumin and 10 "/o DMSO. Of the remaining 20 samples which had 5-15 yo killed cells in each, 4 were frozen in the same medium while the remainder were frozen in a medium containing equal volumes of inactivated AB serum and 20 70 DMSO in Tris- BBS. When the cytotoxic reactions from these last 16 samples were read under ultra- violet light, no excessive non-specific background fluorescence was observed. In fact, with the addition of 2 "/o Trypan Blue in the reaction mixture the fluorescence of the surviving lymphocytes appeared ac- centuated.

62 FONG AND KISSMEYER-NIELSEN

Table 4 Comparison of titre obtained from an antiserum by Trypan Blue and Fluorochromatic methods (The fresh

and frozen lymphocytes used are from the same cell preparation and all labelled with F D A )

Serum dilutions

1:l I 1:2 I 1:4 1 1 :8 I 1:16 I 1:32 I 1:64

Trypan Blue

Fluorescence score 4 4 3 2 0 0 0

~ .. + + + - I - t - k t f + + + + Fresh FDA- labelled lymphocytes

Trypan Blue

Fluorescence - l i l l I i t i r k S + FDA-labelled ScOre

frozen

4 4 3 2 0 0 0

The scoring system is the same as shown in Table 3.

Discussion The close correlation between the results of the lymphocytotoxicity tests obtained in- dependently by the Trypan Blue and the fluorochromatic methods on the same lymphocyte samples shown in the present investigation is in good agreement with that reported by Bodmer et al. (1967). There- fore when the lymphocytotoxicity tests are performed using the combined technique as described, they can be read and scored equally satisfactorily and conveniently by one or the other, or both, of these two methods using either an inverted or an ordinary microscope equipFed with a fluorescence light source. The addition of 2 % Trypan Blue solution to the final reaction mixture in this technique not only shows no interference with the fluorescence of the FDA-labelled lymphocytes, but also provides an excellent dark field for visuali- zation of the fluorescing cells, thus elimin- ating the need of a special dark field condenser for the microscope. Since most of the required incubation time is needed for the action of complement, the reaction rate of which is strongly temFerature depen- dent, we found that incubation of the FDA-labelled lymphocytes, antisera and complement together at 3 7 O C, as indicated

in the combined technique, enabled us to shorten total incubation period to 1 hour compared to the 2 to 3 hours required by the regular fluorochromatic method when incubation takes place at room trmpei a- ture. Providing the lymphocytes are well labelled with FDA beforehand, any spon- taneous 10s; of fluorescence due to incubation at 37' C is not readily observed with human eyes within a short period of time, hence the tests can be read satisfactorily within 3 to 4 hours after the end of the incubation Feriod. Reyond this, however, the fluorescence will have faded markedly unless the plates are kept in a 4' C refri- gerator where they can bc stored up to 24 hours before readinq

The major advantage of the combined technique is for typing lymphocyte samples of dubious viability. Such samples are not infrequently encountered when they are sent from laboratories overseas and are un- doubtedly due to unexpected delays in shipment and/or storage at inappropriate temperatures during transit. Whether they have been freshly prepared or well froyen prior to mailing, they often show marked deterioration in quality upon arrival. Typ- ing these samples using the Trypan Blue method alone, as is the routine practice in

MICRODROPLET 1.YMPHOCYTOTOXICITY TEST 63

many laboratories including our own, may or may not give any results because of the possibility of having large numbers of stained cells in the background making scoring difficult and inaccurate. If, however, these samples had been labelled with FDA prior to typing, satisfactory and usually accurate results can be obtained simply by switching over the reading of the cytotoxicity tests from Trypan Blue to fluorochromasia providing that no less than 25 % of the total cell population in each of these samples was still viable. I t is therefore suggested that as a routine all the frozen as well as the shipped lymphocyte samples should be FDA-labelled prior to typing (not a la- borious procedure), and that the first reading of the lymphocytotoxicity test should be with Trypan Blue on an inverted microscope. If this proves to be unsatisfactory owing to high background of killed cells, a second reading of the te;t using a fluorescence microscope is instituted. Such pro- cedures are also helpful in studying other lymphocyte samples of limited supplies, for example, retyping frozen samples from ca- daveric kidney donors for retrospective evaluation purposes.

Although we found that the viability of the lymphocytes were better preserved when they were frozen in the medium containing equal parts of pooled inactivated AB serum and 20 % DMSO in Tris-BBS, than in Tris-BBS containing 3 % bovine serum albumin and 10 % DMSO, no meaningful comparison can he made re-

garding the superiority of one medium over the other since our experience in cell freezing is limited. However, it seems reasonable to indicate that the former medium can be used as effectively as the latter with human AB serum serving as an alternative stand- ard protein source for protecting the cell membranes during freezing.

References Bodruer, W., Tripp, M. & Bodmer, J. (1967)

Application of a fluorochromatic cytotoxicity assay to human leukocyte typing. Histocom- patibility Testing 1967, pp. 341-349. Munks- gaard, Copenhagen.

Douglas, K. S., Perkins, H. A., Cochrum, K. CIr Kountz, S. L. (1971) Comparison of the HL-A phenotypes of lymphocytes and kidney cells determined by the Fluorochromasia Cyto- toxicity Assay. I . clin. Invest. 50, 271-281.

Kissmeyer-Nielsen, F. & Kjerbye, K. E. (1967) Lyniphocytotoxic micro-technique purification of lyrnphocytes by flotation. Histocompatibility Testing 2967, pp. 381-383. Munksgaard, Co- penhagen.

Kissnieyer-Nielsen, F., Staub Nielsen, L., Lind- holm, A, , Sandberg, L., Svejgaard, A. & Thorsby, E. (1970) The HL-A system in re- lation to human transplantations. Histocom- pati6ility Testing 1970, pp. 105-135. Munks- gaard, Copenhagen.

‘Thursby, E., Engeset, A., & Lie, S. 0. (1971) HL-A antigens and suspectibility to diseases. Tissue Antigens 1, 147-152.

address: I?. Fang Blood Bank and Tissue Typing Laboratory tiniversity Hospital Aarhus Denniark

a combined dye exclusion (trypan blue) and fluorochromatic technique for the microdroplet...

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