Vol. 16, No.

11
Printed in U.S.A.

APPLIED MICROBIOLOGY, Nov. 1968, p. 1761-1763

Copyright 1968 American Society for Microbiology

Effect of Type of Red Blood Cells on

Antistreptolysin 0 Titer
GEORGE C. KLEIN, BETTY V. ADDISON, JAMES S. BOONE, AND MAX D. MOODY
Streptococcus Unit, National Communicable Disease Center, Atlanta, Georgia 30333

Received for publication 29 July 1968

The antistreptolysin 0 (ASO) test is a widely

used serological test for the detection of streptococcal antibodies. It is particularly useful as a
diagnostic aid in rheumatic fever and glomerulonephritis. The test requires the use of red blood
ceUs (RBC) as an indicator system to determine
whether the streptolysin 0 has been neutralized
by antibody (ASO). Different types of RBC
are used in the ASO tube test. Todd's (9) original
technique employed a 5% suspension of rabbit
RBC. Sheep (5) or horse (2) RBC have also been
used. The use of human RBC in the ASO test
has been described in the literature (8) and has
been suggested as an alternative for rabbit RBC
by some of the commercial suppliers of ASO
reagents.
The purpose of this study was to determine
whether the type of RBC used in the test had an
effect on the ASO titer.
MATERIALS AND METHODS
A buffered saline solution was prepared by rehy-

drating Streptolysin 0 Buffer (Difco) with distilled

water. The Difco buffer is composed of 7.4 g of NaCl,
3.17 g of KH2PO4, and 1.81 g of Na2HPO4 per liter
of distilled water. The pH is 6.5 to 6.7.
Type 0 human blood in ACD solution, rabbit
blood in Alsever's solution, and sheep blood in citrate
solution were used to prepare the RBC suspensions.
The RBC were packed by centrifugation in a graduated conical centrifuge tube and were washed in
buffered saline until the supernatant fluid was clear.
A 5% suspension of each type (human, rabbit, sheep)
of RBC was prepared in buffered saline; a 2.5% suspension of sheep RBC was also prepared.
A total of 117 human serum specimens were tested
for ASO titer with the four RBC suspensions as indicator systems. The sera were diluted according to the

modified Rantz-Randall tube technique (7). The final

dilutions of serum in the test were: 1: 100, 1:125,
1:166, 1:250, 1:333, 1:500, 1:625, 1:833, 1:1,250,
and 1:2,500. Each tube contained 1 ml of the final
dilution of serum.
Streptolysin 0 (Difco) was rehydrated just prior to
use with cold distilled water and was kept in an ice
bath during use. The same lot of Streptolysin 0 was
used for all cells. A sufficient number of vials of
streptolysin 0 from the same lot were rehydrated and
pooled to eliminate any variation from different vials.
All tubes except the RBC control received 0.5 ml of
streptolysin 0. The tubes were shaken to mix the
contents and were incubated in a water bath at 37 C
for 15 min. An 0.5-ml amount of RBC was added to
each tube and mixed by shaking. The tubes were incubated in the water bath at 37 C for 45 min (shaken
one time at the end of the first 15 min of incubation
and reincubated for 30 min). At the end of the incubation period, they were centrifuged at 300 to 400 X g
for 5 min and read. The ASO titer was recorded as the
reciprocal of the highest dilution of serum which
showed no hemolysis.
RESULTS

The distribution of ASO titers among the 117

sera is shown in Table 1. If a titer of 250 is
considered the upper limit of normal, then the
number of sera with titers of 333 or above for
each type of RBC is as follows: 72 with 5%
rabbit; 82 with 5% human; 85 with 5% sheep;
and 86 with 2.5% sheep.
The comparability of ASO titers for various
combinations of RBC is shown in Table 2.
Although the differences in titer were never more
than one dilution step, the difference is statistically significant by a chi-square test at the 5%
level. According to the hypothesis that ASO
titers are not affected by the type of cells, any

1761

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Antistreptolysin 0 (ASO) titers were determined on 117 human sera with rabbit,
human, and sheep red blood cells (RBC) as the indicator system in the ASO tube
test. The titers of 65% of the sera were one dilution step higher when a 5% suspension of sheep RBC was used instead of a 5% suspension of rabbit RBC. Titers
were one dilution step higher in 42.7% of the sera when a 5 % suspension of human
RBC was used instead of a 5% suspension of rabbit RBC. The best comparability
of titers was between a 5% suspension of human RBC and a 2.5% suspension of
sheep RBC.

APPL. MICROBIOL.

KLEIN ET AL.

1762

TABLE 1. Distribution of ASO titers by RBC type

of 333
Titers
or above

Titer
RBC type

5% Rabbit.......
5% Human ......
5% Sheep........

2.5% Sheep..

100

125

166

250

333

500

625

833

1,250

2,500

No.

Per cent

8
1
0
0

4
8
7
7

19
15
12
13

14
11
13
11

31
31
25
29

15
16
20
20

14
19
22
17

4
8
10
12

7
7
7
7

1
1
1
1

72
82
85
86

61.5
70.1
72.6
73.5

Type of RBC

5% Sheep vs. 5% rabbit

Sheep > rabbit ...........
Sheep = rabbit ...........
Sheep < rabbit ...........
2.5% Sheep vs. 5 % rabbit
Sheep > rabbit ...........
Sheep = rabbit ...........
Sheep < rabbit ...........
5% Human vs. 5% rabbit
Human > rabbit .........
Human = rabbit .........
Human < rabbit .........
5% Sheep vs. 5% human
Sheep > human ..........
Sheep human ..........
Sheep < human ..........
2.5% Sheep vs. 5% human
Sheep > human ..........
Sheep = human ..........
Sheep < human ..........
2.5% Sheep vs. 5% sheep
2.5% > 5% ...............
=

2.5% = 5% ...............
2.5% < 5% ...............

specimens

Per cent

76
41
0

65.0
35.0

71
45
1

60.7
38.5
0.9

50
67
0

42.7
57.3

27
89
1

23.1
76.1
0.9

24
92
1

20.5
78.6
0.9

5
104
8

4.3
88.9
6.8

differences in titer for a particular comparison

should be evenly divided between the two types
of cells being compared. This is obviously not the
case with the above comparisons except for
2.5% sheep RBC versus 5 % sheep RBC. All
other differences are highly significant (P <
0.01) by the chi-square test. In no instance were
titers with 5% rabbit RBC higher than those
with 5 % sheep or 5% human RBC. Only one
of 117 specimens had a higher titer with 5%
rabbit RBC than with 2.5% sheep RBC. The
two types of RBC which differed most frequently
in titer were 5% rabbit and 5% sheep. The titers
of 65% of the 117 sera tested were one dilution
step higher with 5 % sheep RBC as compared to
5% rabbit RBC. The comparison of 5% human
with 5 % rabbit RBC revealed that 42.7 % of

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TABLE 2. Agreement of ASO titers with different

types of RBC

the specimens had a titer one dilution step higher

with human RBC. The two types of RBC which
were most comparable in titer were 5% human
and 2.5% sheep, and the sheep RBC resulted
in higher titers in 20.5% of the sera tested. Both
human and sheep RBC resulted in higher ASO
titers when compared with rabbit RBC; sheep
RBC also gave higher titers when compared
to human RBC.
DISCUSSION

Many laboratories performing ASO tests

use human or sheep RBC instead of rabbit RBC
as a matter of convenience or economics. Most
hospital laboratories have human RBC available
at no additional cost in the form of an outdated
bank blood, whereas rabbit blood usually must
be purchased. Some laboratories have sheep
RBC on hand for use in complement fixation
tests. They prefer to use them in the ASO test
rather than go to the trouble and expense of
obtaining rabbit blood. It is evident from the
results presented above that the use of different
types of RBC in the ASO test does have an effect
on the titer.
At first glance, the difference in size between
rabbit and sheep RBC would appear to be a
possible explanation for the difference in titers.
Sheep RBC have an average diameter of 4.8
,um (10) as compared to 7.5 ,um for rabbit RBC
(1). Thus, a 5% (v/v) suspension of sheep RBC
contains more cells, with more surface area
available for binding streptolysin, than an equal
volume of a 5% suspension of rabbit RBC.
To correct for this discrepancy in total surface
area, a more dilute suspension of sheep RBC
can be used. However, other data in this study
indicate that the differences in titers are not due
to differences in cell size. There is almost as much
disparity between 2.5% sheep and 5% rabbit
RBC as there is between 5 % sheep and 5 %
rabbit RBC. Also, human RBC have an average
diameter of 7.5 ,um (6), which is the same size
as rabbit RBC, yet the titers are significantly
different.

VOL. 16,1968

EFFECT OF RED BLOOD CELLS ON ANTISTREPTOLYSIN 0 TITER

1763

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According to the results of Howard and Wal- RBC are used instead of rabbit RBC in the ASO
-lace (4), the disparity in titers cannot be explained test.
-on the basis of differences in resistance of the
ACKNOWLEDGMENT
RBC types to lysis by streptolysin 0. They found
that for 50% lysis, rabbit RBC required 1 hemoWe thank E. C. Hall, Chief, Statistical Activities,
lytic unit (HU) of streptolysin 0, sheep RBC Laboratory Program, National Communicable Disrequired 0.75 HU, and human RBC required ease Center, for his evaluation of the significance of
only 0.25 HU. If our results were due to these the comparative data.
differences in resistance of the RBC to lysis,
LITERATURE CITED
then rabbit RBC would be expected to result
E.
C., ed. 1952. Standard values in
in higher titers more frequently than human 1. Albritton,
blood, p. 42. W. B. Saunders Co., Philadelphia.
RBC. This was not the case; in fact, higher titers
W. D., J. Lampard, and M. T. Parker.
-were obtained with human RBC in 42.7% of 2. Brighton,
A
1967.
comparison of two methods of estimatthe specimens. In no instance were titers higher
ing antistreptolysin 0. Monthly Bull. Min.
with rabbit RBC. Our results are not in agreeHealth (London) 26:208-214.
ment with those of Howard and Wallace; our re- 3. Friedemann, U. 1917. Ueber heterophile normal.sults show that sheep RBC are the most resistant
amboceptoren. Biochem. Z. 80:333-356.
and rabbit RBC the least resistant to lysis by 4. Howard, J. G., and K. R. Wallace. 1953. The
comparative resistance of the red cells of various
streptolysin 0.
species to haemolysis by streptolysin 0 and by
Comparability of results among laboratories
saponin. Brit. J. Exptl. Pathol. 34:181-184.
performing ASO tests could be improved if all
S. 1944. Bacterial contamination of
laboratories used the same type of RBC in the 5. Lofgren,
blood samples as a source of error in antistreptest. The difficulty is in agreeing on which type
tolysin titration. Acta Pathol. Microbiol.
of RBC to use. We prefer sheep or human RBC
Scand. 21:768-774.
for the following reasons: (i) they are less sus- 6. Osgood, E. E. 1935. Normal hematologic standceptible to spontaneous lysis than are rabbit
ards. Arch. Internal Med. 56:849-863.
RBC; (ii) there is closer agreement in ASO titer 7. Rantz, L. A., and E. Randall. 1945. A modification of the technic for determination of the antibetween them than between rabbit and human
streptolysin titer. Proc. Soc. Exptl. Biol. Med.
RBC or between rabbit and sheep RBC; and (iii)
59:22-25.
they are more readily available than rabbit RBC.
R. 1952. Determination of antistreptoSheep RBC have the disadvantage of being sub- 8. Robinette,
lysin titers: technic and significance. Am. J.
ject to lysis by the natural hemolysins which are
Med. Technol. 18:205-213.
found in some human sera (3). This does not 9. Todd,
E. W. 1932. Antigenic streptococcal
prevent their use with these sera because the
hemolysin. J. Exptl. Med. 55:267-280.
hemolysin is diluted out at 1:85 or 1:100. The 10. Wintrobe, M. M. 1933. Variations in size and
slightly higher titers obtained with human or
hemoglobin content of erythrocytes in blood of
various vertebrates. Folia Haematol. 51:32-49.
sheep RBC should be kept in mind when these