The Effects of Red Blood Cell Suspending Media on Hemagglutination and the Antiglobulin Test J. M. FITZSIMMONS A N D P. A. MOREL From the Irwin Memorial Blood Bank of the Sun Francisco Medical Society. San Francisco, California
Initial experimeptal work with low ionic strength salt solution (LISS) indicated that problems in reactivity arose due to the extreme lability of the solution after a short period of storage. We have now found that the addition of one gram of sodium azide per liter of LISS abolishes the storage problems and raises the osmolality of the solution only slightly (from 297 to 327 mosmlkg). Test red blood cells were washed three times in normal saline. The last wash was completely removed and a two per cent cell suspension in each of the media being studied was prepared. REPORTSfrom several laboratories have Fresh cell suspensions were made each day. described the advantages of performing A semi-automatic pipette was used to insure hemagglutination tests in low ionic strength delivery of relatively fixed volumes of 0.1 ml medial .4.6.Y or in the presence of polymserum and cell suspension which were mixed and incubated as described below. Initial tests erized albumin s o l ~ t i o n s . ~We * ~ have * ~ rewith two volumes of serum and one volume of a cently completed a comparative evaluation four per cent cell suspension prepared in LISS of antibody detection and identification indicated that sensitivity was enhanced only methods using four different suspending when at least equal volumes of a two per cent media for the test cells to determine which cell suspension were mixed with the serum. These findings are consistent with the concluof these offered maximum sensitivity in sions of Moore and Mollison.6 the shortest incubation time without loss of In test systems containing either normal saline specificity. or LISS, the mixtures were incubated at RT (20 to 25 C) for 15 minutes and at 37 C for 30 Methods minutes. The mixtures containing either of the albumin solutions were incubated at 37 C only. Red blood cell suspending media were Each of the tests was read for agglutination normal saline, conventional 22% bovine albumin after each incubation period and the antiand polymerized albumin (Ortho Diagnostics), globulin test was performed after the 37 C and low ionic strength salt solution (LISS) prepared by a modification of Low and M e ~ s e t e r . ~ incubation. Hemagglutination scores as described by Marsh5 were derived from titrations To prepaie the 0.03M Low Ionic Strength Salt of antisera diluted in normal group AB serum. Solution (327 mosdkg) place in one liter volumetric flask 1.75 g NaCI, 18.00 g glycine, and 20.00 ml phosphate buffer. Add distilled water Results to the mark and adiust the OH to 6.7 with NaOH. Add 1 g sodium azide. Store at 4 C. The Sera fro,,, Normal Blood Donors phosphate buffer consists of 11.3 ml 0.15M As a negative control population, sera from KH,PO, and 8.7 ml 0.15M Na,HPO,. 170 normal random blood donors were screened by all four methods (Table 1). Positive antibody detection tests were noted with six of Received for publication December 4, 1977; accepted January 29, 1978. these sera, five at RT and one by the antiEvaluation of the effects of four suspending media for test cells in antibody detection and identification is reported. The media under investigation are normal saline, low ionic strength salt solution, 22% bovine albumin and polymerizedalbumin. Hemagglutinationat 37 C was enhanced in the test systems employing polymerized albumin. With shortened incubation times, the antiglobulin test was enhanced in tests against red blood cells suspended in the low ionic strength salt solution. When incubation times were extended to 30 minutes, all suspending media were apparently equally effective.
0041-1 132/79/0100/0081 $00.75 0 J . B. Lippincott C o . Tran\fusion January-February I979
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Table 1. Antibody Studies of Normal Donors and Patients Normal Blood Donors ~
Patient Referrals
~~
Number tested Number negative Number false positives
170 164
in LISS (RT) Enhancement by LISS-AGT
6 0
. 0 anti-IH anti-M
anti-c (2) anti-Lea (1) anti-H (1) (1) 0
(1)
(1) anti-Fyb (1) anti-c anti3ka (2) anti-Bga (2)
globulin test. Of the five cold agglutinins, one agglutinated only the cells suspended in LISS; the specificity of this agglutinin was autoanti-IH.The other four cold antibodies agglutinated cells suspended in either normal saline or the low ionic strength salt solution. Two of these cold agglutinins proved to be autoanti-1 and the other two anti-M. One of these anti-M antibodies exhibited dosage against test cells suspended in normal saline, but strongly agglutinated all M positive test cells in low ionic strength salt solution, heterozygous as well as homozygous M cells. The one antiglobulinreactive donor serum was weakly positive by all four of the test methods, but could only be Table 2. Room Temperature Hemagglutination Scores
a n t i 4 #l a n t i 4 #2 a n t i 4 #3 a n t i 4 #4 a n t i 4 #5 anti-M #6 anti4 anti-Lea #1 anti-Lea #2 anti-Lea#3 anti-PI #1 anti-P, #2 anti-PI #3 anti-P, #4
Normal Saline
LISS
23
28
73
73
73 4
76 20 54 13 26
53
10 12 8 7
9 36 27 44 25
identified in the LISS system. It contained anti-Fyb reactive against all Fy(b+) cells of a test panel suspended in LISS, but reactive against only three of seven Fy(b+) cells suspended in each of the other media. No false positive reactions were encountered.
33
0
Enhancement of hemagglutination in polymerizedalbumin (37 C)
Transfusion January-February 1979
12
7 14 36 28 43 26
Sera from Patients Sera from 33 patients referred to our Reference Laboratory were also tested by the four methods under investigations (Table 1). Antibody identification procedures were performed with each reactive serum using cells suspended in either the low ionic strength salt solution or the polymerized albumin and the results were compared with previously recorded specificitiesobtained by conventional techniques. After 37 C incubation, test cells suspended in polymerized albumin were agglutinated by two sera containing anti-c, by one containing antiLea and by one containing anti-H. Test cells suspended in the other media were not agglutinated at that point, although these antibody specificities were apparent in the other media either at RT or by the antiglobulin test. A patient’s serum containing anti-M identified by conventional techniques was found to have anti-c specificity also in the antiglobulin test after incubation with cells in LISS; other methods failed to demonstrate the anti-c antibody, Three patients’ sera contained anti-Jka. Two of these had been thought to have specificity which could be determined only by enzyme premodification of the test cells. All three had markedly enhanced agglutination in the antiglobulin test after incubation with cells suspended in the low ionic strength salt solution compared with cells suspended in other media. In addition, two anti-Bp antibodies were detected by the low ionic strength salt solution methods and were not demonstrated by the other procedures. Sera Containing Known Antibodies Hemagglutination scores derived from titrating 51 examples of sera containing antibodies of known specificity against test cells suspended in each of the media under study are depicted in Tables 2-4. One of the six anti-M sera and the anti-S serum shown in Table 2 produced markedly enhanced agglutination at RT when the cells were suspended in LISS. No enhancement was noted in three of three examples of anti-Lea nor in four of our anti-P, antibodies. Hemagglutination scores were compared after incubation at 37 C (Table 3). Two examples of anti-D, one of anti-c and two of anti-Kell
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showed marked enhancement of direct agglutination when the test cells were suspended in the polymerized albumin. These antibodies were also detected by antiglobulin test, however, when cells were suspended in other media. Hemagglutination scores derived by the antiglobulin test indicated that for most of these antisera, 30 minutes incubation at 37 C provided adequate antibody uptake for detection no matter which medium was used to suspend the test cells. However, all antisera appeared to give at least slightly increased sensitivity when the cells were suspended in the LISS (Table 4). However, two of three anti-S antibodies reactive only by the antiglobulin test were markedly enhanced in sensitivity when the test cells were suspended in LISS, and the three antiJk' sera were also enhanced by this method. Six anti-#ell antibodies, three anti-k's, eight anti-Fy' sera and one anti-Fyb (not shown on Table 4) were also tested and equally reactive by all methods. Degree of Agglutination
Table 5 depicts the strength of agglutination exhibited by 36 of the known antisera. Most of the antibodies exhibited the greatest degree of agglutination in tests with cells suspended in the low ionic strength salt solution. Weaker agglutinations were produced when the cells were suspended in any of the other media. Since these results were obtained by antiglobulin test, they appeared to be indicative of the increased sensitization which occurred when the test cells were suspended in the low ionic strength salt solution.
Table 3. Hemagglutination Scores After 37 C Albumin
anti-Lea X1 anti-Lea X2 anti-CD anti-D X1 anti-D #2 anti-c anti-e anti-K X1 anti-K X2
LISS
Polymerized
22%
18 31 39 0 5 0 31 0 0
21 26 42 5 12 0 54 0 0
29 31 56 26 48 46 56 39 23
25 28 55 5 24 0 55 0 0
tected by normal saline, 22% albumin and polymerized albumin, and were detected by LISS.
Discussion
Although the way in which the agglutinating efficiency of albumin preparations is enhanced by an increase in their content of albumin polymers is not clear, a degree of correlation between the dimer plus polymer content of various albumin solutions and their efficiency in agglutinating sensitized cells has been reported.*.' Our data support the enhancement of the second Table 4. Antiglobulin Test Scores after Incubation at 37 C for 30 Minutes Albumin
Incubation Times
Thirty-six of the above antisera were diluted in AB serum at a dilution selected to produce 2+ agglutination by the indirect antiglobulin test after a 30-minute incubation at 37 C. The diluted antisera were incubated with the appropriate cell suspension at vaned incubation times as indicated in Table 6. After five minutes' incubation, approximately 50 per cent of the antibodies were detected against cells suspended in normal saline or in either of the albumin solutions. With the same incubation time 81 per cent of these antibodies were detected if the test cells were suspended in LISS. This advantage with LISS was demonstrated in each of the other time periods as well. At the end of the 15-minute period, the only antibody not detected by any method was an anti-k; but an anti-D and an anti-Fys were not de-
Normal Saline
anti-Lea R1 anti-Lea X2 anti-D X1 anti-D C2 anti-D X3 anti-D X4 anti-D X5 anti-CD anti-E anti-c X1 anti-c #2 anti-c #3 anti4 X1 anti4 X2 anti4 X3 anti-Jka X1 anti-Jka #2 anti-Jka X3
Normal Saline
LISS
Polymerized
22%
18 39 69 99 72 86 45 53 44 24 15 41 8 36 0 28 59 24
21 42 71 103 77 100 51 75 55 30 26 46 25 50 16 40 65 30
18 44 68 96 72 76 43 57 36 27 14 38 9 41 0 14 52 14
20 40 69 97 72 89 45 59 44 26 19 43 12 43 0 26 53 19
FITZSIMMONS AND MOREL
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Table 5. # Antisera Vs. Degree of Agglutination (Antiglobulin Test) Albumin
1 to 2+ 3 to 4+
Normal Saline
LlSS
Polymerized
22%
26 10
11 25
28 8
23 13
.stage of hemagglutination in polymerized albumin systems when the antibodies in our study failed to agglutinate even the cells suspended in 22% albumin. A reagent with this quality could be most helpful in investigation of serum containing multiple antibodies. Increased sensitivity of the antiglobulin test when cells and serum were incubated in the presence of albumin led one group of workers to conclude that albumin enhanced the uptake of antibody.8 A later report suggested that this effect may have been related to the ionic strength of the albumin solution.* Increased sensitivity in albumin systems as demonstrated by the antiglobulin test was not substantiated in our work and we found that normal saline as a cell suspending medium was equally as effective as either 22% albumin or polymerized albumin, if the incubation period at 37 C were as long as 30 minutes. It has been postulated that at low ionic strength, ionized groups on antibody and antigen become exposed, leading to an inTable 6. Percentage of Antibodies Detected by Antiglobulin Test at Different Incubation Times Albumin ~
Minutes
Normal Saline ~~
5 10 15 20 30
~
LlSS
Polymerized
81 92 97 100
42 78 92 100
22%
~
50 78 92 97 100
Not detected: anti-k.
t Not detected: anti-k, anti-D. anti-Fya.
53 81 92 97 100
Transfusion J p n ~ a r y - F ~ h1979 ~y
crease in attraction between oppositely charged groups on antigen and a n t i b ~ d y . ~ Several workers have investigated the effects of low ionic strength media in hemagglutination test systems, noting enhancement of red blood cell sensitization, reduction in reaction time and also the undesirable aspects of false-positive r e s ~ l t s . ' ~ ~ ~ ~ ~ ~ ~ Our work confirms that of earlier workers who reported increased sensitivity and shorter incubation periods when test cells were suspended in low ionic strength salt solutions. In indirect antiglobulin tests with some of the anti-Rh and anti-Jka antibodies, uptake of antibody (as measured by the antiglobulin test) by the cells suspended in low ionic strength salt solution was markedly enhanced, a characteristic most often produced by enzyme modification of the test cells. With other antibodies, such as some examples of anti-M and an anti4 which are not reactive in enzyme systems, marked enhancement of agglutination occurred when the cells were suspended in the low ionic strength salt solution. With some other nonagglutinating antibodies which are also not reactive against enzyme-treated cells, such as several with anti4 specificity ' and an anti-Fyb, sensitization as demonstrated by the antiglobulin test was enhanced in the low ionic strength salt solution system. It was important that equal volumes of serum and cell suspension be mixed for the test. Our preliminary investigation indicated that sensitivity was not increased if two volumes of serum were mixed with one volume of the cell suspension. An excess of serum appeared to raise the ionic strength of the system and thereby diminish sensitivity. In addition, we noted the production of more clearcut reactions by the LISS method. We would recommend an incubation period of at least 10 minutes. This is the point at which all but one of the antibodies in our study were detected. Earlier workers who suggested a five-minute incubation
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period were frequently using the low ionic strength solution itself as a diluent when preparing dilutions of their test sera, which would of course affect the ionic strength of the medium with which they began their testing. Our diluent was serum, which we believe contributed to a more valid test system, since the serum concentration remained the same from the beginning to the end of each titration. In our work, we had no problems with false-positive results. Moore and Mollison reported there was no risk of false-positive reactions as long as no more than three volumes of cells in low ionic strength salt solution were added to one volume of serum.6 They did report that weak falsepositive reactions with anticomplement were observed when testing cells which had been stored in the low ionic strength salt solution. It was noted that it is important to prepare fresh cell suspensions each day, and the red blood cells must be washed in normal saline before suspending them in the low ionic strength salt solution to prevent false-positive results. Unless care is taken to remove completely the last saline wash, we suggest that the final wash solution should be LISS. The addition of 0.1% sodium azide to the suspending medium does not affect the osmolality of the solution significantly, but appears to produce a more stable reagent than other workers have described. Our study suggests considerable advantages in the use of a low ionic strength salt solution as a suspending medium for red blood cells in our antibody detection procedures. The solution is easy to prepare and the procedure is easily integrated into existing frameworks since the only necessary change is to prepare test cell suspensions in this solution instead of another medium such as saline. Our study indicates that incubation times then can be safely shortened to 10 to 15 minutes without
loss of sensitivity. It seems reasonable to conclude that this method is more timesaving and certainly as reliable as more conventional methods. Acknowledgment The authors would like to thank Dr. Herbert Perkins and Mr. George Garratty for their assistance in the preparation of this manuscript.
1.
References Elliot, M., E. Bossom, M. E. Dupuy, and S. P.
Masouredis: Effect of ionic strength on the serologic behavior of red cell isoantibodies. Vox Sang. 9:3%, 1964. 2. Goldsmith, K. L. G., and other members of a working party of the Joint International Committee of Standardizatiodhternational Society of Blood Transfusion on the Standardization of Blood Group Reagents: A study performed on batches of serum albumin used as diluents in Rh testing. Br. J. Haematol. 32215, 1976. 3. Hughes-Jones, N.C., M.J. Polley, R. Telford, B. Gardner, and G. Kleinschmidt: Optimal conditions for detecting blood group antibodies by the antiglobulin test. Vox Sang. 9 3 8 5 , 1964. 4. Low, B., and L. Messeter: Antiglobulin test in low-ionic strength salt solution for rapid antibody screening and cross-matching. Vox Sang. 2653, 1974. 5. Marsh, W. L.: Scoring of hemagglutination reactions. Transfusion 12:352, 1972. 6. Moore, H. C., and P. L. Mollison: Use of a low-
ionic-strength medium in manual tests for antibody detection. Transfusion 16:291, 1976. 7. Ortho Research Institute: Bovine albumin reagents. Immunohematology Seminar Report, 1975. 8. Stroup, M.,and M. MacIlroy: Evaluation of the
antiglobulin technic in antibody detection. Transfusion 5: 184, 1965. 9. Wicker, B., and C. H. Wallas: A comparison of low ionic strength saline medium with routine methods for antibody detection. Transfusion 16:469, 1976.
Joan Fitzsimmons, M.T.(ASCP)SBB, Supervisor of Special Studies, Sacramento Medical Foundation Blood Bank, Sacramento, California. Phyllis A. Morel, M.T.(ASCP)SBB, Technical Director, Reference Laboratory, (reprints) Los Angeles Red Cross Blood Program, 1130 South Verri~ontAvenue, Los Angeles, California 9OOO6.