TRANSFUSION Vol. 16
No. 4 ~~~
Scientific Articles
Use of a Low-Ionic-Strength Medium in Manual Tests for Antibody Detection H. C . MOOREA N D P. L. MOLLISON From the M R C Experimental Haematology Unit. St. Mary’s Hospital Medical School. London, England
to warnings of nonspecific agglutination a t NaCl concentrations below 0.2 g/dl (0.03 M)7 and partly due to the observation that at low ionic strength, due presumably to aggregation of IgG, com plemen t com pon en t s are bound to red blood cells,12leading to false positive results. l 1 Low and Me se te r g found that when red blood cells were suspended in a solution of sodium glycinate containing 0.03 M NaCI, T H E RATE OF ASSOCIATION of anti-D with buffered to pH 6.7 with phosphate, the inD-positive red blood cells can be increased cubation time of cells with antibody-contain1,000-fold by lowering the ionic strength of ing serum could be reduced to five minutes in the medium in which the reaction takes place performing the indirect antiglobulin test, and from 0.17 I to 0.03 I;’ see also Elliot, et ~ l . ~ false positive results were of negligible imIn detecting a wide variety of blood group portance. The present work was undertaken antibodies by the indirect antiglobulin test to investigate further the value of this the use of a low-ionic-strength medium method and to define as closely as possible allows the incubation time to be reduced the optimal conditions for enhancing results from one hour to five minute$ moreover the and avoiding false positive reactions. titer of a wide range of antibodies is in~ r e a s e d .Although ~.~ a low-ionic-strength meMaterials and Methods dium has been used in automated blood Low-Ionic-Strength Solution (LISS)’ grouping tests,8 there has been much hesiThe solution described by L ~ and w MesseterO tation in using this method in routine manas “LISS” consisted of: ual tests for the detection of blood group antibodies. Reluctance to use a lowsaline, 0.17 M, 180 ml phosphate buffer, 0.15 M, pH 6.7, 20 ml ionic-strength medium has been due partly and sodium glycinate, 0.3 M, pH 6.7, 800 ml. The present observations confirm the value of suspending red blood cells in a low-ionic-strength medium in the first stage of the indirect antiglobulin test; that is, during the period of incubation with antibody-containing serum. The main advantage of this procedure i s to shorten the time of incubation. I n this respect a low-ionic-strength medium appears to be superior to albumin as a suspending medium for the red blood cells. A further advantage is to increase the uptake of certain antibodies; this effect was pronounced with selected Rh antibodies believed to be of low affinity.
Received for publication August 20, 1975; accepted September 15, 1975.
In the present work, as sodium glycinate was not available commercially, the solution was pre-
29 1 Transfusion July-Aug. 1976
Volume 16 Number 4
29 2
MOORE A N D MOLLISON
pared as follows: Glycine, 18 g was dissolved in approximately 500 ml distilled water and NaOH, 1.0 M, was added by drops, with stirring, until the pH reached 6.7; (approximately 0.35 ml of 1.0 M NaOH was needed). Twenty ml phosphate buffer, 0.15 M, pH 6.7, (made by adding approximately equal volumes of 0.15 M N a 2 H P 0 4 and 0.15 M NaH,PO,) was added, followed by 1.79 g NaCl dissolved in approximately 100 ml distilled water. The mixture was then made up to one liter with distilled water. The conductivity of the final solution was found to be 3.7 mmho/cm a t 23 C. Sucrose Low-Ionic-Strength Solution This solution differed from "LISS" only in that 100 g sucrose was used in place of 18 g glycine. Red Blood Cell Suspension A concentration of 3 per cent washed red blood cells was used instead of the 5 per cent recommended by Low and M e s ~ e t e r .The ~ red blood cells were always washed twice in saline before being washed once in LISS and then suspended in LISS. A n tibody-Containing Sera One o r more examples of the following specificities were tested: a) in agglutination tests: anti-A, -B, -I, -i, -PI, -PP,Pk,-M, and -Lea; b) in indirect antiglobulin tests, using an antiIgC serum: anti-D, -E, -C", -c, -e, -K, -Fy', -Jk', -s, -Lub, -YP, -Csaand -Xga. The Rh antibodies tested included examples kindly supplied by Dr. B. E. Dodd. These antibodies had been selected on the basis that they reacted well with enzyme-treated red blood cells but only weakly or not a t all by the indirect antiglobulin t e ~ t . ~ . ~ A reference preparation (68/419) of anti-D was kindly made available to us by Dr. N. C . HughesJones. This preparation has a nominal value of 60 pg anti-D/ml and has been proposed as an international standard. I c) In indirect antiglobulin tests, using an antiPIEor, occasionally, an anti-filclA serum: anti-Lea, -Jka, Fy". Antiglobulin Sera The anti-IgG sera had been prepared in rabbits or goats by injecting purified IgG obtained by DEAE-cellulose chromatography. The sera had an optimal dilution of approximately one in 250 with Rh-sensitized cells; anti-PIEand anti-PIclA*
were used a t a dilution of one in 50. In a few cases, results were checked against a commercial antiglobulin reagent.** Indirect Antiglobulin Tests One-stage test. One drop of serum o r diluted serum was added to one drop of a 3 per cent suspension of red blood cells in a small plastic test tube and placed in a water bath a t 37 C. After varying periods of incubation the cells were washed four times. A f t e r discarding t h e supernatant, two drops of diluted antiglobulin serum were added and the tube spun immediately at low speed for one minute. The deposit was then gently resuspended and examined on a slide under the low power of a microscope. In tests with anti-complement-globulin sera, since the sera containing blood group antibodies had been stored a t 20 C for periods between one half and 20 years, they were diluted in fresh serum to provide active complement. One-stage test with albumin. One volume of a 3 per cent suspension of cells in 0.147 M NaCl was placed in a tube and centrifuged. After discarding the supernatant, the cells were resuspended in two volumes of 30 per cent albumin and three volumes of serum or diluted serum were then added. After incubation for periods between five and 30 minutes the cells were washed four times in normal saline; finally two drops of diluted antiglobulin were added to the button of cells, mixed, centrifuged and examined on a slide under the low power of a microscope. Two-stage test (modification of test described by Polley and Mollison") One volume of serum treated with four mg EDTA/ml was added to one volume of a 3 per cent suspension of red blood cells and incubated a t 37 C for five minutes. The cells were washed four times. After discarding the supernatant, one volume of fresh normal serum was added to the button of cells and the mixture incubated a t 37 C for five minutes. The cells were then washed four times, the supernatant discarded and two volumes of diluted anti-complement-globulin serum added. The mixture was then centrifuged and examined on a slide under the low power of a microscope. Agglutination of Enzyme-Treated Cells The method of Haber and Rosenfielde was used. Agghtination of Untreated Red Blood Cells One volume of serum was added to one volume of a 3 per cent suspension of red blood cells and
*Hoechst Pharmaceuticals, Frankfurt, West Germany.
Transfusion July-Aug. 1976
**Ortho Diagnostics, Raritan, N.Y.
Volume 16 Number 4
29 3
LOW- ION IC-STRENGT H M ED1 U M Table 1. Indirect Antiglobulin Tests (Anti-lgGI with Red Blood Cells Suspended in Saline and LlSS Respectively
Incubation Time (min)
Anti-D ( 1 in 100) Saline LlSS
1 5 10
2
%‘ 1
60
2 2
90
2
2 3 3% 3%
Anti-c Saline
0 % %
3% -
LlSS 1
2% 3
3
-
Anti-Xga Saline LlSS
0 %
1 1%
-
-
1% 1%
2 2
“In this Table and in Table 2, the numbers indicate the intensity of the agglutination reactions on an arbitrary scale of % to 4. (- = not done)
the mixture was left a t 37 C for five minutes before being lightly centrifuged. The button of cells was gently resuspended and examined on a slide under the low power of a microscope. Titration of Antisera Serial, doubling dilutions were made in a conventional manner. When comparing results with red blood cells suspended in saline and LlSS respectively, the “master dilution” method was employed so that exactly the same dilutions were used with cells suspended in saline and LISS respectively. Estimation of Drop Size When a drop of fluid is delivered into a plastic tube it is frequently noted that the drop is drawn towards the wall of the tube and that this apparently results in the delivery of drops of variable size. To investigate this point, 51Crwas added to serum and to a suspension of red blood cells in saline and single drops were delivered into plastic tubes which were then counted in a gamma counter. The effect of delivering drops with the tip of the pipette held close to the orifice of the tube and at least one inch above the tube was investigated, as was delivery into glass rather than plastic tubes.
Results Indirect Antiglobulin Tests with Anti-IgC With cells suspended in LISS, as compared with cells suspended in saline, positive reactions developed more rapidly and the antibody titer was about four-fold greater. As Table I shows, after one minute’s incubation positive results were obtained with cells in LISS in all three examples, but the results were negative with cells in saline in two out of the three examples. With cells in LlSS the reactions were maximal or almost maximal after ten minutes’ incubation. After 90 minutes’ incubation the reactions with cells incubated in saline were usually slightly weaker than those of cells incubated in LISS for only ten minutes. However, in some cases the maximum strength of the reaction in the two media was similar. No obvious difference was observed between the reactions given by the anti-IgG serum and the commercial “broad-spectrum” antiglobulin reagent. In tests with the reference preparation the lowest concentration of anti-D which could be detected in the indirect antiglobulin test after 15 minutes’ incubation using saline-suspended cells was 0.025 pg/ml and using cells in LISS was 0.005 pg/ml. The discrepancy between results obtained with cells suspended in saline and LISS respectively
Table 2. Indirect Antiglobulin Tests (anti-IgG) with a ) Cells Suspended in Saline; bl Cells Suspended in 30% Albumin and cl Cells Suspended in LlSS Incubation Time (mid ~~
~
0
30
2 3 3%
90
LISS
Saline
Anti-Csa Albumin
L I ss.
-
-
-
~
1 5 15
60
Anti-Jka Albumin
Saline
% %
0 0 1%
3 3% 3%
2
3 3 3 3% 3%
% % 1 1
-
%
1 1 1
-
1
2 2
2
-
294
MOORE AND MOLLISON
was greater when using certain selected Rh antisera. These sera had been selected (by Dr. B. E. Dodd) because they gave very poor reactions in the indirect antiglobulin test but strongly agglutinated enzyme-treated cells. With some of these antisera the indirect antiglobulin titer with cells in LISS was almost as high as the agglutination titer with enzyme-treated cells. The effect of adding albumin to the mixture of antibody-containing serum and cells was compared with that of omitting the albumin but suspending the cells in LISS. In all cases the degree of augmentation of the reaction by suspending the cells in LISS was far greater than that achieved by adding albumin to cells in saline. Table 2 shows an example. In case the combination of albumin and LlSS should prove advantageous, 30 per cent albumin was dialysed against LISS and then used for the resuspension of red blood cells. One volume of a 2 per cent suspension of cells in the dialysed albumin was incubated with an equal volume of serum and the cells tested in the usual way. This method proved to be very insensitive. Eflect of Washing Antibody-Coaled Cells in LISS When cells were washed in LISS rather than in saline before being tested with an antiglobulin serum, only a very slight enhancement of the reactions was observed; for example, in one series in which eight different antibodies were tested, the average “score” was 2.2 for cells washed in LlSS compared with 2.0 for cells washed in saline. Surprisingly, the effect of washing in LISS was greater with cells which had been suspended in saline during incubation with antibody, t h e average score being 1.9 for cells washed in LlSS compared with 1.3 for cells washed in saline. Indirect Antiglobulin Tests Using A n t i - 8 1or ~ Anti-PICIA
In one-stage tests with only five minutes’ incubation of the mixture of cells, antibody and complement, considerable enhancement of the reaction was observed with cells suspended in LISS rather than in saline. For example, in testing a sample of anti-Fya, with cells suspended in saline the titer of the antibody was two but with cells in LISS was 32. Similar results were observed in two-stage tests except that the reaction of cells in LISS incubated with relatively high concentrations of anti-Fya was apparently stronger than in the one-stage tests. Reactions with anti-Jka were similar. With one example, in a two-stage test the reaction was negative with saline-suspended cells, even when the cells were incubated with the antibody for one and a half hours in the first stageof the test. With cells in LlSS the serum had a titer of eight even
Transfusion July-Aug. 1976
when the period of incubation of cells with antibody was only five minutes. Agglutination Tests with Untreated Red Blood Cells Suspended in Saline or LISS In general, the same results were obtained with cells suspended in saline and LISS, titers being either identical or differing by not more than one doubling dilution. However, in testing examples of anti-M it was consistently noted that the antibody had a higher thermal range in tests with cells suspended in LISS. For example, serum D.S. was originally described by Cutbush and Mollison3 as agglutinating MM cells in saline at 34 C but not a t 37 C and M N cells at 31 C but not a t 34 C. Tests on a sample of serum stored since 1958 showed that it had retained the same characteristics. However, when the serum was tested with cells suspended in LISS, positive reactions were found with M M cells at 37 C and with M N cells a t 34 C . On the other hand, the thermal range of examples of anti-P,, -Lea, -I, and -i was no higher with cells in LISS than with cells in saline. Sizeof Drops Delivered from Pasteur Pipette When delivered into glass tubes o r when delivered into plastic tubes from a height of at least one inch, drops were usually within 10 per cent of the mean value. However, when delivered into plastic tubes with the tip of the pipette within a centimeter o r so of the orifice of the tube the volume delivered varied very widely. In one series in which drops were delivered into eight consecutive tubes, two drops were smaller than 6 jd and two greater than 14 ~ l . Conditions f o r the Development of False Positive Results In order to discover the concentration of NaCl at which false positive reactions would occur when fresh blood was added to an excess of solution containing NaCl and sodium glycinate, one volume of blood was added to 20 ml of LISS and to a range of solutions differing from LISS only in that the concentration of NaCl ranged from 0.045 M to 0.025 M (in LISS the concentration is approximately 0.03 M). After incubation a t 37 C for ten minutes the cells were washed and tested with various antiglobulin sera. Reactions were only doubtfully positive at 0.04 M NaCI, but at 0.035 M, positive results were regularly obtained with anti-&, anti-PlEand with anti-IgC. Evidently, when red blood cells suspended in LISS are mixed with an equal volume of serum, with an ionic strength equal to approximately 0.15 M NaCI, the final mixture must have an ionic strength equivalent to about 0.09 M. Thus even if two o r three drops of cells in LISS are inadvertently added to one drop of serum, there should
Volume 16 Number 4
LOW-IONIC-STRENGTH MEDIUM
be no risk of false positives due to too low an ionic
strength. I n order to check this conclusion, measured volumes of cells in LISS and of fresh serum were incubated together and tested with various antiglobulin reagents. Provided that no more than three volumes of cells in LISS were added to one volume of serum no significant agglutination by antiglobulin reagents was observed. On the other hand, when testing stored red blood cells it was noted that cells suspended in LISS tended
&,,.+ Similar reactions were observed cells in saline when the antiglobulin to give weak false positive reactions with anti-
with tested with same serum and it was concluded that the use of a really potent anti-plc serum is inadvisable in tests for antibody detection. False positive results were not a problem when using anti-IgC alone or when using a commercial “broad-spectrum” antiglobulin
reagent. Discussion T h e present observations confirm the ~ l a i mthat ~ . ~the use of sodium glycinate with 0.03 M NaCl as a suspending medium for red blood cells in the indirect antiglobulin test enables the incubation period to be very greatly reduced. However, the present observations suggest that rather than using five minutes as previously s ~ g g e s t e d ,it~ might be wiser to use ten minutes since in several instances the reactions were found to be stronger at ten minutes than at five minutes (Table I). Previous authors7egseem to have dealt only with anti-IgG but our observations show that anti-PIE and anti-PIclAreactions a r e also usually optimal after only five minutes’ incubation, either in one stage tests in which fresh serum is incubated with cells in LISS or in the two-stage test in which EDTA-treated serum is first incubated with cells in LISS for five minutes and then (after washing) the cells are resuspended in saline and incubated with fresh serum for a further five minutes before being finally washed and tested with antiglobulin serum. The present observations show that some cold agglutinins can be detected at higher temperatures when reacted with red blood cells in LISS; only examples with specificity anti-M were found to behave in this way. Elliot, et ~ lpreviously . ~ noted that agglutina-
29 5
tion by anti-M but not by antibodies of the ABO and Lewis systems was enhanced by the use of a low-ionic strength medium. The findings with the selected Rh antibodies provided by Dr. B. E. Dodd, as examples which agglutinated enzyme-treated red blood cells but reacted very weakly or not at all in the indirect antiglobulin test, are of special interest. When these sera were tested against red blood cells in LISS the indirect antiglobulin titer was sometimes almost as high as the titer with enzyme-treated cells although, as described by Casey, et ~ 1 the . ~ reactions in the indirect antiglobulin test with saline-suspended cells were very weak. These observations strongly support the suggestion’ that these antibodies are of low affinity. It is important that equal volumes of cells in LISS and of serum should be incubated together. Although the addition of a considerable excess of LISS would be needed to-produce false positive reactions due to too low an ionic strength in the medium, the addition of an excess of serum, by raising the ionic strength, would diminish sensitivity. When using glass tubes it is satisfactory to deliver the serum and cells as drops from an ordinary Pasteur pipette. However, with plastic tubes which attract drops by electrostatic force the volume of drops varies too widely, as demonstrated in the present work. With such tubes it is therefore recommended that some method, such as the use of an Eppendorf pipette, which ensures delivery of a fixed volume (e.g. 50 PI) should be employed. It is worth emphasizing a few practical points in the performance of the test: 1) It is essential to wash red blood cells in saline before suspending them in LISS as otherwise false positive results may be obtained; 2) LISS should be stored at 4 C and, unless previously sterilized, should be discarded after a few days as it appears to be a good medium for the growth of microorganisms. It is convenient to autoclave aliquots of LISS so as to have a sterile supply of solution available for each day’s use. 3) When diluting anti-sera before testing, the diluent should be 8.5 g/1 NaCl buffered to pH 6.7. The same solution
MOORE A N D MOLLISON
should be used for washing cells prior to resuspending them in LISS. There would appear to be considerable advantages in using cells suspended in LISS in the routine performance of the indirect antiglobulin test. With short periods of incubation (e.g. 10 minutes) the method is more sensitive than that of suspending red blood cells in albumin and the sensitivity is on the average slightly greater than that achieved by prolonged incubation (90 minutes or more) with saline-suspended cells. With certain antibodies, presumably those of low affinity, the increased sensitivity using red blood cells in LISS compared with red blood cells in saline is very striking.
References Bangham, D. R., H. H. Gunson, and N. C. Hughes-Jones: To be published. 2. Casey, F. M., B. E. Dodd, and P. J. Lincoln: A study of the characteristics of certain Rh antibodies preferentially detectable by enzyme technique. Vox Sang. 23:493, 1972. 3. Cutbush, M., and P. L. Mollison: Relation between characteristics of blood-group antibodies in vifro and associated patterns of red cell destruction in v i v a Br. J. Haematol. 4:115, 1958. 4. Dodd, B. E., and D. A. Eeles: Rh antibodies detectable only by enzyme technique. Immunology I.
4:337, 1961. 5.
Elliot, M. E., Bossom, M. E. Dupuy, and S. P. Masouredis: Effect of ionic strength on the
6.
7.
8. 9.
Transfusion
July-Aug. 1976
serologic behavior of red cell isoantibodies. Vox Sang. 9:396, 1964. Haber, G, and R. E. Rosenfield: Ficin treated red cells for hemagglutination studies. In: P. H. Andresen-Papers in Dedication of his 60th Birthday, Munksgaard, Copenhagen, 1957, p 45. Hughes-Jones, N. C., B. Gardner, and R. Telford: The Effect of pH and ionic strength on the reaction between anti-D and erythrocytes. Immunology 7:72, 1964. Lalezari, P.: New method for detection of red blood cell antibodies. Transfusion 8:372, 1968. Low, B., and L. Messeter: Antiglobulin test in lowionic strength salt solution for rapid antibody screening and cross-matching. Vox Sang. 26:53,
1974. 10. Medical Research Council: Production of antibody
against purified yG-globulin in rabbits, goats, sheep and horses (Report of a Committee). Immunology 10:271, 1966. 11. Mollison, P. L.: Blood Transfusion in Clinical Medicine, 4th Ed. Oxford, Blackwell Scientific Publications, 1967, p. 439. and M. J. Polley: Uptakeofy-globulin and 12. -, complement by red cells exposed to serum a t low ionic strength. Nature 203:535, 1964. 13. Polley, M. J., and P. L. Mollison: The role of complement in the detection of blood group antibodies: Special reference to the antiglobulin test. Transfusion 1:9, 1961.
H. C . Moore, M.D., Baptist Memorial Hospital, 899 Madison Avenue, Memphis, Tennessee 38103. P. L. Mollison, M.D., M R C Experimental Haematology Unit, St. Mary’s Hospital Medical School, London W21PG, England.
No. 4 ~~~
Scientific Articles
Use of a Low-Ionic-Strength Medium in Manual Tests for Antibody Detection H. C . MOOREA N D P. L. MOLLISON From the M R C Experimental Haematology Unit. St. Mary’s Hospital Medical School. London, England
to warnings of nonspecific agglutination a t NaCl concentrations below 0.2 g/dl (0.03 M)7 and partly due to the observation that at low ionic strength, due presumably to aggregation of IgG, com plemen t com pon en t s are bound to red blood cells,12leading to false positive results. l 1 Low and Me se te r g found that when red blood cells were suspended in a solution of sodium glycinate containing 0.03 M NaCI, T H E RATE OF ASSOCIATION of anti-D with buffered to pH 6.7 with phosphate, the inD-positive red blood cells can be increased cubation time of cells with antibody-contain1,000-fold by lowering the ionic strength of ing serum could be reduced to five minutes in the medium in which the reaction takes place performing the indirect antiglobulin test, and from 0.17 I to 0.03 I;’ see also Elliot, et ~ l . ~ false positive results were of negligible imIn detecting a wide variety of blood group portance. The present work was undertaken antibodies by the indirect antiglobulin test to investigate further the value of this the use of a low-ionic-strength medium method and to define as closely as possible allows the incubation time to be reduced the optimal conditions for enhancing results from one hour to five minute$ moreover the and avoiding false positive reactions. titer of a wide range of antibodies is in~ r e a s e d .Although ~.~ a low-ionic-strength meMaterials and Methods dium has been used in automated blood Low-Ionic-Strength Solution (LISS)’ grouping tests,8 there has been much hesiThe solution described by L ~ and w MesseterO tation in using this method in routine manas “LISS” consisted of: ual tests for the detection of blood group antibodies. Reluctance to use a lowsaline, 0.17 M, 180 ml phosphate buffer, 0.15 M, pH 6.7, 20 ml ionic-strength medium has been due partly and sodium glycinate, 0.3 M, pH 6.7, 800 ml. The present observations confirm the value of suspending red blood cells in a low-ionic-strength medium in the first stage of the indirect antiglobulin test; that is, during the period of incubation with antibody-containing serum. The main advantage of this procedure i s to shorten the time of incubation. I n this respect a low-ionic-strength medium appears to be superior to albumin as a suspending medium for the red blood cells. A further advantage is to increase the uptake of certain antibodies; this effect was pronounced with selected Rh antibodies believed to be of low affinity.
Received for publication August 20, 1975; accepted September 15, 1975.
In the present work, as sodium glycinate was not available commercially, the solution was pre-
29 1 Transfusion July-Aug. 1976
Volume 16 Number 4
29 2
MOORE A N D MOLLISON
pared as follows: Glycine, 18 g was dissolved in approximately 500 ml distilled water and NaOH, 1.0 M, was added by drops, with stirring, until the pH reached 6.7; (approximately 0.35 ml of 1.0 M NaOH was needed). Twenty ml phosphate buffer, 0.15 M, pH 6.7, (made by adding approximately equal volumes of 0.15 M N a 2 H P 0 4 and 0.15 M NaH,PO,) was added, followed by 1.79 g NaCl dissolved in approximately 100 ml distilled water. The mixture was then made up to one liter with distilled water. The conductivity of the final solution was found to be 3.7 mmho/cm a t 23 C. Sucrose Low-Ionic-Strength Solution This solution differed from "LISS" only in that 100 g sucrose was used in place of 18 g glycine. Red Blood Cell Suspension A concentration of 3 per cent washed red blood cells was used instead of the 5 per cent recommended by Low and M e s ~ e t e r .The ~ red blood cells were always washed twice in saline before being washed once in LISS and then suspended in LISS. A n tibody-Containing Sera One o r more examples of the following specificities were tested: a) in agglutination tests: anti-A, -B, -I, -i, -PI, -PP,Pk,-M, and -Lea; b) in indirect antiglobulin tests, using an antiIgC serum: anti-D, -E, -C", -c, -e, -K, -Fy', -Jk', -s, -Lub, -YP, -Csaand -Xga. The Rh antibodies tested included examples kindly supplied by Dr. B. E. Dodd. These antibodies had been selected on the basis that they reacted well with enzyme-treated red blood cells but only weakly or not a t all by the indirect antiglobulin t e ~ t . ~ . ~ A reference preparation (68/419) of anti-D was kindly made available to us by Dr. N. C . HughesJones. This preparation has a nominal value of 60 pg anti-D/ml and has been proposed as an international standard. I c) In indirect antiglobulin tests, using an antiPIEor, occasionally, an anti-filclA serum: anti-Lea, -Jka, Fy". Antiglobulin Sera The anti-IgG sera had been prepared in rabbits or goats by injecting purified IgG obtained by DEAE-cellulose chromatography. The sera had an optimal dilution of approximately one in 250 with Rh-sensitized cells; anti-PIEand anti-PIclA*
were used a t a dilution of one in 50. In a few cases, results were checked against a commercial antiglobulin reagent.** Indirect Antiglobulin Tests One-stage test. One drop of serum o r diluted serum was added to one drop of a 3 per cent suspension of red blood cells in a small plastic test tube and placed in a water bath a t 37 C. After varying periods of incubation the cells were washed four times. A f t e r discarding t h e supernatant, two drops of diluted antiglobulin serum were added and the tube spun immediately at low speed for one minute. The deposit was then gently resuspended and examined on a slide under the low power of a microscope. In tests with anti-complement-globulin sera, since the sera containing blood group antibodies had been stored a t 20 C for periods between one half and 20 years, they were diluted in fresh serum to provide active complement. One-stage test with albumin. One volume of a 3 per cent suspension of cells in 0.147 M NaCl was placed in a tube and centrifuged. After discarding the supernatant, the cells were resuspended in two volumes of 30 per cent albumin and three volumes of serum or diluted serum were then added. After incubation for periods between five and 30 minutes the cells were washed four times in normal saline; finally two drops of diluted antiglobulin were added to the button of cells, mixed, centrifuged and examined on a slide under the low power of a microscope. Two-stage test (modification of test described by Polley and Mollison") One volume of serum treated with four mg EDTA/ml was added to one volume of a 3 per cent suspension of red blood cells and incubated a t 37 C for five minutes. The cells were washed four times. After discarding the supernatant, one volume of fresh normal serum was added to the button of cells and the mixture incubated a t 37 C for five minutes. The cells were then washed four times, the supernatant discarded and two volumes of diluted anti-complement-globulin serum added. The mixture was then centrifuged and examined on a slide under the low power of a microscope. Agglutination of Enzyme-Treated Cells The method of Haber and Rosenfielde was used. Agghtination of Untreated Red Blood Cells One volume of serum was added to one volume of a 3 per cent suspension of red blood cells and
*Hoechst Pharmaceuticals, Frankfurt, West Germany.
Transfusion July-Aug. 1976
**Ortho Diagnostics, Raritan, N.Y.
Volume 16 Number 4
29 3
LOW- ION IC-STRENGT H M ED1 U M Table 1. Indirect Antiglobulin Tests (Anti-lgGI with Red Blood Cells Suspended in Saline and LlSS Respectively
Incubation Time (min)
Anti-D ( 1 in 100) Saline LlSS
1 5 10
2
%‘ 1
60
2 2
90
2
2 3 3% 3%
Anti-c Saline
0 % %
3% -
LlSS 1
2% 3
3
-
Anti-Xga Saline LlSS
0 %
1 1%
-
-
1% 1%
2 2
“In this Table and in Table 2, the numbers indicate the intensity of the agglutination reactions on an arbitrary scale of % to 4. (- = not done)
the mixture was left a t 37 C for five minutes before being lightly centrifuged. The button of cells was gently resuspended and examined on a slide under the low power of a microscope. Titration of Antisera Serial, doubling dilutions were made in a conventional manner. When comparing results with red blood cells suspended in saline and LlSS respectively, the “master dilution” method was employed so that exactly the same dilutions were used with cells suspended in saline and LISS respectively. Estimation of Drop Size When a drop of fluid is delivered into a plastic tube it is frequently noted that the drop is drawn towards the wall of the tube and that this apparently results in the delivery of drops of variable size. To investigate this point, 51Crwas added to serum and to a suspension of red blood cells in saline and single drops were delivered into plastic tubes which were then counted in a gamma counter. The effect of delivering drops with the tip of the pipette held close to the orifice of the tube and at least one inch above the tube was investigated, as was delivery into glass rather than plastic tubes.
Results Indirect Antiglobulin Tests with Anti-IgC With cells suspended in LISS, as compared with cells suspended in saline, positive reactions developed more rapidly and the antibody titer was about four-fold greater. As Table I shows, after one minute’s incubation positive results were obtained with cells in LISS in all three examples, but the results were negative with cells in saline in two out of the three examples. With cells in LlSS the reactions were maximal or almost maximal after ten minutes’ incubation. After 90 minutes’ incubation the reactions with cells incubated in saline were usually slightly weaker than those of cells incubated in LISS for only ten minutes. However, in some cases the maximum strength of the reaction in the two media was similar. No obvious difference was observed between the reactions given by the anti-IgG serum and the commercial “broad-spectrum” antiglobulin reagent. In tests with the reference preparation the lowest concentration of anti-D which could be detected in the indirect antiglobulin test after 15 minutes’ incubation using saline-suspended cells was 0.025 pg/ml and using cells in LISS was 0.005 pg/ml. The discrepancy between results obtained with cells suspended in saline and LISS respectively
Table 2. Indirect Antiglobulin Tests (anti-IgG) with a ) Cells Suspended in Saline; bl Cells Suspended in 30% Albumin and cl Cells Suspended in LlSS Incubation Time (mid ~~
~
0
30
2 3 3%
90
LISS
Saline
Anti-Csa Albumin
L I ss.
-
-
-
~
1 5 15
60
Anti-Jka Albumin
Saline
% %
0 0 1%
3 3% 3%
2
3 3 3 3% 3%
% % 1 1
-
%
1 1 1
-
1
2 2
2
-
294
MOORE AND MOLLISON
was greater when using certain selected Rh antisera. These sera had been selected (by Dr. B. E. Dodd) because they gave very poor reactions in the indirect antiglobulin test but strongly agglutinated enzyme-treated cells. With some of these antisera the indirect antiglobulin titer with cells in LISS was almost as high as the agglutination titer with enzyme-treated cells. The effect of adding albumin to the mixture of antibody-containing serum and cells was compared with that of omitting the albumin but suspending the cells in LISS. In all cases the degree of augmentation of the reaction by suspending the cells in LISS was far greater than that achieved by adding albumin to cells in saline. Table 2 shows an example. In case the combination of albumin and LlSS should prove advantageous, 30 per cent albumin was dialysed against LISS and then used for the resuspension of red blood cells. One volume of a 2 per cent suspension of cells in the dialysed albumin was incubated with an equal volume of serum and the cells tested in the usual way. This method proved to be very insensitive. Eflect of Washing Antibody-Coaled Cells in LISS When cells were washed in LISS rather than in saline before being tested with an antiglobulin serum, only a very slight enhancement of the reactions was observed; for example, in one series in which eight different antibodies were tested, the average “score” was 2.2 for cells washed in LlSS compared with 2.0 for cells washed in saline. Surprisingly, the effect of washing in LISS was greater with cells which had been suspended in saline during incubation with antibody, t h e average score being 1.9 for cells washed in LlSS compared with 1.3 for cells washed in saline. Indirect Antiglobulin Tests Using A n t i - 8 1or ~ Anti-PICIA
In one-stage tests with only five minutes’ incubation of the mixture of cells, antibody and complement, considerable enhancement of the reaction was observed with cells suspended in LISS rather than in saline. For example, in testing a sample of anti-Fya, with cells suspended in saline the titer of the antibody was two but with cells in LISS was 32. Similar results were observed in two-stage tests except that the reaction of cells in LISS incubated with relatively high concentrations of anti-Fya was apparently stronger than in the one-stage tests. Reactions with anti-Jka were similar. With one example, in a two-stage test the reaction was negative with saline-suspended cells, even when the cells were incubated with the antibody for one and a half hours in the first stageof the test. With cells in LlSS the serum had a titer of eight even
Transfusion July-Aug. 1976
when the period of incubation of cells with antibody was only five minutes. Agglutination Tests with Untreated Red Blood Cells Suspended in Saline or LISS In general, the same results were obtained with cells suspended in saline and LISS, titers being either identical or differing by not more than one doubling dilution. However, in testing examples of anti-M it was consistently noted that the antibody had a higher thermal range in tests with cells suspended in LISS. For example, serum D.S. was originally described by Cutbush and Mollison3 as agglutinating MM cells in saline at 34 C but not a t 37 C and M N cells at 31 C but not a t 34 C. Tests on a sample of serum stored since 1958 showed that it had retained the same characteristics. However, when the serum was tested with cells suspended in LISS, positive reactions were found with M M cells at 37 C and with M N cells a t 34 C . On the other hand, the thermal range of examples of anti-P,, -Lea, -I, and -i was no higher with cells in LISS than with cells in saline. Sizeof Drops Delivered from Pasteur Pipette When delivered into glass tubes o r when delivered into plastic tubes from a height of at least one inch, drops were usually within 10 per cent of the mean value. However, when delivered into plastic tubes with the tip of the pipette within a centimeter o r so of the orifice of the tube the volume delivered varied very widely. In one series in which drops were delivered into eight consecutive tubes, two drops were smaller than 6 jd and two greater than 14 ~ l . Conditions f o r the Development of False Positive Results In order to discover the concentration of NaCl at which false positive reactions would occur when fresh blood was added to an excess of solution containing NaCl and sodium glycinate, one volume of blood was added to 20 ml of LISS and to a range of solutions differing from LISS only in that the concentration of NaCl ranged from 0.045 M to 0.025 M (in LISS the concentration is approximately 0.03 M). After incubation a t 37 C for ten minutes the cells were washed and tested with various antiglobulin sera. Reactions were only doubtfully positive at 0.04 M NaCI, but at 0.035 M, positive results were regularly obtained with anti-&, anti-PlEand with anti-IgC. Evidently, when red blood cells suspended in LISS are mixed with an equal volume of serum, with an ionic strength equal to approximately 0.15 M NaCI, the final mixture must have an ionic strength equivalent to about 0.09 M. Thus even if two o r three drops of cells in LISS are inadvertently added to one drop of serum, there should
Volume 16 Number 4
LOW-IONIC-STRENGTH MEDIUM
be no risk of false positives due to too low an ionic
strength. I n order to check this conclusion, measured volumes of cells in LISS and of fresh serum were incubated together and tested with various antiglobulin reagents. Provided that no more than three volumes of cells in LISS were added to one volume of serum no significant agglutination by antiglobulin reagents was observed. On the other hand, when testing stored red blood cells it was noted that cells suspended in LISS tended
&,,.+ Similar reactions were observed cells in saline when the antiglobulin to give weak false positive reactions with anti-
with tested with same serum and it was concluded that the use of a really potent anti-plc serum is inadvisable in tests for antibody detection. False positive results were not a problem when using anti-IgC alone or when using a commercial “broad-spectrum” antiglobulin
reagent. Discussion T h e present observations confirm the ~ l a i mthat ~ . ~the use of sodium glycinate with 0.03 M NaCl as a suspending medium for red blood cells in the indirect antiglobulin test enables the incubation period to be very greatly reduced. However, the present observations suggest that rather than using five minutes as previously s ~ g g e s t e d ,it~ might be wiser to use ten minutes since in several instances the reactions were found to be stronger at ten minutes than at five minutes (Table I). Previous authors7egseem to have dealt only with anti-IgG but our observations show that anti-PIE and anti-PIclAreactions a r e also usually optimal after only five minutes’ incubation, either in one stage tests in which fresh serum is incubated with cells in LISS or in the two-stage test in which EDTA-treated serum is first incubated with cells in LISS for five minutes and then (after washing) the cells are resuspended in saline and incubated with fresh serum for a further five minutes before being finally washed and tested with antiglobulin serum. The present observations show that some cold agglutinins can be detected at higher temperatures when reacted with red blood cells in LISS; only examples with specificity anti-M were found to behave in this way. Elliot, et ~ lpreviously . ~ noted that agglutina-
29 5
tion by anti-M but not by antibodies of the ABO and Lewis systems was enhanced by the use of a low-ionic strength medium. The findings with the selected Rh antibodies provided by Dr. B. E. Dodd, as examples which agglutinated enzyme-treated red blood cells but reacted very weakly or not at all in the indirect antiglobulin test, are of special interest. When these sera were tested against red blood cells in LISS the indirect antiglobulin titer was sometimes almost as high as the titer with enzyme-treated cells although, as described by Casey, et ~ 1 the . ~ reactions in the indirect antiglobulin test with saline-suspended cells were very weak. These observations strongly support the suggestion’ that these antibodies are of low affinity. It is important that equal volumes of cells in LISS and of serum should be incubated together. Although the addition of a considerable excess of LISS would be needed to-produce false positive reactions due to too low an ionic strength in the medium, the addition of an excess of serum, by raising the ionic strength, would diminish sensitivity. When using glass tubes it is satisfactory to deliver the serum and cells as drops from an ordinary Pasteur pipette. However, with plastic tubes which attract drops by electrostatic force the volume of drops varies too widely, as demonstrated in the present work. With such tubes it is therefore recommended that some method, such as the use of an Eppendorf pipette, which ensures delivery of a fixed volume (e.g. 50 PI) should be employed. It is worth emphasizing a few practical points in the performance of the test: 1) It is essential to wash red blood cells in saline before suspending them in LISS as otherwise false positive results may be obtained; 2) LISS should be stored at 4 C and, unless previously sterilized, should be discarded after a few days as it appears to be a good medium for the growth of microorganisms. It is convenient to autoclave aliquots of LISS so as to have a sterile supply of solution available for each day’s use. 3) When diluting anti-sera before testing, the diluent should be 8.5 g/1 NaCl buffered to pH 6.7. The same solution
MOORE A N D MOLLISON
should be used for washing cells prior to resuspending them in LISS. There would appear to be considerable advantages in using cells suspended in LISS in the routine performance of the indirect antiglobulin test. With short periods of incubation (e.g. 10 minutes) the method is more sensitive than that of suspending red blood cells in albumin and the sensitivity is on the average slightly greater than that achieved by prolonged incubation (90 minutes or more) with saline-suspended cells. With certain antibodies, presumably those of low affinity, the increased sensitivity using red blood cells in LISS compared with red blood cells in saline is very striking.
References Bangham, D. R., H. H. Gunson, and N. C. Hughes-Jones: To be published. 2. Casey, F. M., B. E. Dodd, and P. J. Lincoln: A study of the characteristics of certain Rh antibodies preferentially detectable by enzyme technique. Vox Sang. 23:493, 1972. 3. Cutbush, M., and P. L. Mollison: Relation between characteristics of blood-group antibodies in vifro and associated patterns of red cell destruction in v i v a Br. J. Haematol. 4:115, 1958. 4. Dodd, B. E., and D. A. Eeles: Rh antibodies detectable only by enzyme technique. Immunology I.
4:337, 1961. 5.
Elliot, M. E., Bossom, M. E. Dupuy, and S. P. Masouredis: Effect of ionic strength on the
6.
7.
8. 9.
Transfusion
July-Aug. 1976
serologic behavior of red cell isoantibodies. Vox Sang. 9:396, 1964. Haber, G, and R. E. Rosenfield: Ficin treated red cells for hemagglutination studies. In: P. H. Andresen-Papers in Dedication of his 60th Birthday, Munksgaard, Copenhagen, 1957, p 45. Hughes-Jones, N. C., B. Gardner, and R. Telford: The Effect of pH and ionic strength on the reaction between anti-D and erythrocytes. Immunology 7:72, 1964. Lalezari, P.: New method for detection of red blood cell antibodies. Transfusion 8:372, 1968. Low, B., and L. Messeter: Antiglobulin test in lowionic strength salt solution for rapid antibody screening and cross-matching. Vox Sang. 26:53,
1974. 10. Medical Research Council: Production of antibody
against purified yG-globulin in rabbits, goats, sheep and horses (Report of a Committee). Immunology 10:271, 1966. 11. Mollison, P. L.: Blood Transfusion in Clinical Medicine, 4th Ed. Oxford, Blackwell Scientific Publications, 1967, p. 439. and M. J. Polley: Uptakeofy-globulin and 12. -, complement by red cells exposed to serum a t low ionic strength. Nature 203:535, 1964. 13. Polley, M. J., and P. L. Mollison: The role of complement in the detection of blood group antibodies: Special reference to the antiglobulin test. Transfusion 1:9, 1961.
H. C . Moore, M.D., Baptist Memorial Hospital, 899 Madison Avenue, Memphis, Tennessee 38103. P. L. Mollison, M.D., M R C Experimental Haematology Unit, St. Mary’s Hospital Medical School, London W21PG, England.
