.PREPARATION AND ASSAY OF A R E D CELL FRACTION OF
T H E Rh FACTOR*
BETTINA B. CARTER, M.S.
From the Inslilute of Pathology, Western Pennsylvania Hospital, Pittsburgh, Pennsylvania
In 1947 a preliminary report 1 was made concerning a substance which inhibits anti-Rh serum. In 1949 this substance was described2 under the name of
"Rh hapten" and, in 1950 its occurrence was confirmed by Goldsmith 4, 6 and
by Schubert and Grunberg. 8
Wolf and co-workers9 have stated, "A number of our observations suggest
that the term Rh hapten may correctly be applied to the extract described by
Carter." Others have questioned whether its action is that of a hapten or whether
some other mechanism, possibly hormonal in nature, may be involved. Speculation in this regard has gone so far as to contemplate the possibility that the current theories of the pathogenesis of erythroblastosis may be subject to challenge.
In this paper, then, I shall refer to the active substance simply as the red cell
fraction, in the hope that further work may prove conclusively whether it is a
hapten or of some other nature. It is my purpose to detail the methods in use at
the present time in the production of this fraction and in the study of its nature
and properties.
PREPARATION
The first step in production of the red cell fraction is to obtain outdated blood
from one or more blood banks. This blood may be of any blood group and should
be Rh-positive. It may be of any age, as long as there is no bacterial contamination and the cells remain superficially intact. The cells are grossly freed from
plasma and are pooled to give one or more volumes Of one liter each. Then the
liter of cells is partially laked by the addition of 500 ml. distilled water. This
volume of 1500 ml. containing partially laked cells is placed in an Erlenmeyer
flask having a capacity of 6 liters and to it is added very sloioly, with constant
shaking, 4 liters of 95 per cent ethyl alcohol. A dark red precipitate forms. The
flask is shaken thoroughly and allowed to stand overnight at 4 C. Then the material is filtered with suction, leaving a dark red powdery residue. To this residue
is added an aliquot of 4 liters of 50 per cent alcohol and then the mixture is shaken
for ten minutes and filtered again. The powder is suspended in 25 per cent alcohol,
shaken for 10 minutes and again filtered. Finally, the precipitate, dried by suction until it is friable, is divided roughly in halves and to each aliquot is added
2 liters of ether. This represents 4 liters of ether for each original liter of cells.
This procedure differs only in volumes of reagents used, but not in kind from that
described previously.2 Goldsmith4 has observed that the use of U.S.P. ethyl
* This work was supported in part by a grant from Eli Lilly and Company, Indianapolis,
Indiana.
Received for publication, November 6, 1950.
561
562
CARTER
ether is satisfactory for production and we also find it so. The use of anesthesiagrade ether, as described in earlier papers, 1 ' 2 was specified in order to avoid as
much water as possible during the drying procedure which follows. As Howe
and Rustigian 6 have noted, no other value is inherent in its use.
The red cell precipitate in' ether is shaken vigorously for ten minutes and then
placed in the cold room at 4 C. for five days. Each day of the five-day extraction
period, the flasks containing the red-cell powder in ether should be shaken for
ten minutes. At the end of five days, which has proved to be the optimum period
of extraction, the powder-in-ether is filtered with suction and this time the
ethereal filtrate is retained. While it is possible to obtain active material with a
shorter extraction period, the best yield and activity are obtained in five days
A longer period, on the other hand, tends to bring out nonspecific inhibitors,
which possibly may be related to the development of peroxides in the ether.
The ethereal filtrate containing the active fraction may be evaporated by repeated fillings of a crystallization dish placed before an electric fan in a hood or,
more economically, the ether may be distilled off. The residue may be dried under
vacuum over phosphorus pentoxide or, in some localities, it may be dried in air.
The yellow-white residue should have the consistency of vaseline. This is weighed
and dissolved in absolute alcohol, at present, usually in a concentration of 200
mg. per milliliter. Whereas in previous reports 1 • 2 warming of the alcohol to hasten
evaporation was recommended, this has been found undesirable, since occasional
lots show a sensitivity to heat and even a slight increase above room temperature,
seems to impair activity.
Aerobic and anaerobic cultures of our material have shown no growth.
However, Goldsmith 5 uses ultra-violet irradiation to destroy viruses and
Chown3 dissolves his fraction in ethyl oleate and subjects it to filtration to
remove possible bacterial contamination. The final product appears to be
stable at room temperature and is stored thus.
The procedure may be summarized as follows:
T o one liter Rh-positive red cells add 500 ml. distilled water.
Add 4 liters 95 per cent alcohol.
Keep overnight a t 4 C. Filter. Retain residue.
Add 4 liters 50 per cent alcohol.
Shake for 10 minutes. Filter. Retain residue.
Add 4 liters 25 per cent alcohol.
Shake for 10 minutes. Filter. Retain residue.
Add 4 liters U.S.P. ethyl ether.
Shake for 10 minutes, daily for five days. Filter. R e t a i n filtrate.
E v a p o r a t e or distill off ether.
METHODS
Assay of the activity of the red cell fraction has been accomplished chiefly by
means of complement fixation. As previously reported, 2 the work was based on an
anti-Rh serum of 32 units, carrying either saline agglutinins or antibodies demonstrable only in the presence of albumin. However, unpublished observations in
collaboration with Chown would seem to indicate that there is wide variation
563
RED CELL FRACTION OF Rh FACTOR
from one laboratory to another in results of titration of a given blood. Thus a
modification of the complement-fixation test as originally proposed has been
developed. This modification employs, as did the original procedure, guinea pig
complement at a dilution previously determined for use in a Kolmer7 complementfixation test against a Kolmer antigen. This provides a base, which, while entirely
unrelated to the system in use, nevertheless gives a good working structure and
avoids the titration of one unknown reagent against another. Two full units of
complement, as determined against the Kolmer antigen, are used in the test.
This is a quantitative procedure as set up, but actually provides only qualitative
information since anti-Rh serums are not standardized from laboratory to laboratory.
TABLE 1
ASSAY OF R E D C E L L F R A C T I O N BY C O M P L E M E N T - F I X A T I O N
1 0.2 ml.
2 0.1 ml.
3
0.05 ml.
4 . 0.025 ml.
5 0.005 ml.
6 0.2 ml. (control)
7
Antigen control: 0.5
ml. saline
Hemolytic control:
8
1.0 ml. saline
Sheep cell control:
9
2.5 ml. saline
(2 FULL UNITS
AGAINST KOLMER
ANTIGEN)
FRACTION,
ML.
0.5
0.5
0.5
0.5
0.5
None
0.5
None
+2
o3
g 0)
.S ta
1.0
1.0
1.0
1.0
1.0
1.0
1.0
1.0
*3
None
c
None
d.s
incub ation a
d b y l 0 minu
C. wa ter bat
A N T I - R h SERUM IN 0.5 ML.
rval of 1
oom tern
TUBE
~w P l -
?,£
J3S
METHODS
ML. (2
UNITS)
CELLS, ML.
(2 PER
CENT)
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
None
0.5
O
CO
a
•~J2
o «
s
f=4
The following respective amounts of saline are added to each of six tubes:
0.9, 0.5, 0.5, 0.5, 2 and 0.5 ml. A volume of 0.6 ml. of an anti-Rh serum with a
titer suitable for Rh-testing is added to the first tube, mixed and 0.5 ml. of this
mixture is transferred to tube No. 2 and 0.5 ml. to tube No. 6. After mixing, 0.5
ml. from tube No. 2 is transferred to tube No. 3. Similarly 0.5 ml. is transferred
from tube No. 3 to tube No. 4 and from tube No. 4 to tube No. 5. The contents
of tube No. 5 are mixed and 2 ml. are discarded. This series of tubes may be set
up for each dilution of red cell fraction used as antigen, with the omission of the
control tube (tube No. 6) in all but one set.
The red cell fraction in alcohol may be diluted 1:1000, 1:2000, 1:4000 and
1:6000 and one dilution used in 0.5 ml. volume in each tube of the appropriate
series. Dilutions of the fraction are made by blowing the alcoholic antigen from
a pipet into the appropriate volume of saline. For instance, to make a 1:1000
dilution of the red cell fraction which has a concentration of 200 mg. in 1 ml.
alcohol, one would pipet 0.1 ml. of the fraction into a tube containing 20 ml. of
0.85 per cent saline. Further dilutions can be made easily from this initial 1:1000
564
CARTER
dilution. After adding 0.5 ml. of antigen (red cell fraction) to each tube, one may
allow the tests to stand at room temperature for ten minutes, when two full
units of complement contained in 1 ml. are added. The tubes are then placed,
after shaking, in the cold at 4 C. for four hours, along with antigen, hemolytic
and cell controls. Then they are put in the water bath at 37 C. for ten minutes
and 0.5 ml. hemolysin, carrying two units, plus 0.5 ml. 2 per cent washed sheep
cells are added. The titration of hemolysin against sheep cells is performed
exactly as in Ivolmer's method for the serologic test for syphilis. The tests are
shaken and allowed to stand in the water bath at 37 C. for thirty minutes, at the
end of which time readings are made. The accompanying chart shows the procedure for the complement-fixation technic.
DISCUSSION
In this laboratory we have not seen nonspecific reactions in relation to syphilitic
serums. Normal serums also have not given nonspecific reactions. Some nonspecific reactions have been seen during the study of breakdown products of the
TABLE 2
INHIBITION
TITER
i
25G0
1280
A
640
320
160
80
40
20
10
5
• o
2
3
4
OF
ANTI-RH
5
6
7
SERUM
8
9
A
BY
10
RED
u
CELL
12
13
FRACTION
14
15
17
18
A
A
A
A
A
A
A
A
A
A
A
O
16
A
A
A
O
A
O
O
O
O
O
o
o
O
O
O
O
O
O
O
O
A indicates titer of u n t r e a t e d a n t i - R h serum; O , t i t e r of a n t i - R h serum exposed to red
cell fraction.
E a c h number represents a different serum. Each was treated with a different lot of
red cell fraction.
fraction with complement fixation. The fraction itself has shown as much specificity as a Kolmer antigen in a well conducted complement-fixation test for syphilis.
The use by some workers of fractions prepared from Rh-negative cells as a "control" seems to me to be not valid, since what we call Rh-negative is in all probability closely related biochemically to the various Rh-positive subtypes and
might easily show common serologic reactions under some conditions.
The failure of Howe and Rustigian 6 to demonstrate complement fixation with
a similarly prepared lipid extract from red cells may derive from several sources.
Since the authors do not state the time period for the ether extraction and since
they made use of such small volumes of red cells, it is hard to tell whether their
RED CELL FRACTION OF Rh FACTOR
565
material may have been active or not. But, granting that it was, in their studies
the lipid extract was used as antigen iii a proportion of one part in five in the
complement-fixation test. We use the red cell fraction as antigen in a proportion
no greater than one part in six since this seems to be the largest amount compatible with clear-cut results. Also, in Howe and Rustigian's tests, the complement was titrated against the lipid extract rather than against a Kolmer antigen
which, in our experience, means that less complement was used in the tests.
Activity of the red cell fraction may be shown also through inhibition of a
saline agglutinating anti-Rh 0 (anti-D) serum". Fifty mg. of the fraction is weighed
and incorporated in 1 ml. of an anti-D serum. Another tube containing the same
serum alone is set up as a control. Two other tubes, one carrying 1 ml. anti-A
serum with-50 mg. of the red cell fraction and a second with 1 mi. of the anti-A
serum alone, are placed in the same rack. The four tubes are kept in a water
bath at 37 C. for 18 hours. The tubes containing the serums plus red cell fraction
are centrifuged and the precipitate, which rises to the surface, is skimmed off.
Both the controls and the treated serums are then titrated, using serial dilutions
of the serum against appropriate cells-(with the anti-Rh 0 serum Group 0 , Rh 0 positive cells; with the anti-A serum, Group A cells) 0.1 ml. serum dilution plus
0.1 ml. of 2 per cent cell suspension. The tests are incubated at 37 C. in the water
bath for one hour, then centrifuged for one minute at 1500 R.P.M. and the results are read. Table 2 shows the decreases in titer after exposure of anti-Rh
serum to the red cell fraction. Alterations in titers with the anti-A serums after
exposure to the red cell fraction have never been observed.
Experiments are in progress now utilizing exact determinations of quantitative
antibody nitrogen adsorbed on the lipid particle. These should prove of some
value in determining whether the reaction involved with the red cell fraction
has immunologic specificity.
SUMMARY
The preparation of the red cell fraction obtained from Rh-positive pooled
bloods has been discussed. Details of the methods for assay, as well as the current modification of the production method, have been given.
REFERENCES
1. CAKTEB, B . B . : Preliminary report on a substance which inhibits a n t i - R h serum. Am.
J . Clin. P a t h . , 17:640-649, 1947.
2. CAKTEB, B . B . : R h h a p t e n : its preparation, assay and n a t u r e . J . Immunol., 6 1 : 79-SS,
1949.
3. C H O W S , B . : Personal communication t o the author.
4. GOLDSAIITH, J . W.: Experiences with R h hapten. Am. J . Obst. and Gynec., 69: 172-177,
1950.
5. GOLDSMITH, J . W.: Experiences with R h hapten. Wisconsin M . J . , 49: 381, 1950.
6. H O W E , C , AND RUSTIGIAN, R . : Studies with t h e R h 0 (D) Antigen. J . Immunol., 64:
505-513, 1950.
7. K O L M E R , J . A., AND B O E R N E R , F . : Approved L a b o r a t o r y Technic. E d . 4. New Y o r k :
D . Appleton-Century Co., 1945, pp. 674-698.
8. SCHUBERT, J . , AND GRUNBERG, A.: Predbezna p r a v a O haptenove Iecbe foetalni e r y t h r o blastosy. Pediatricke' listy, 5 : 1-11, 1950.
9. W O L F , A. M . , SCHLUTZ, C , F R E U N D L I C H , M . , AND L E V I N S O N , S. O . : Clinical s t u d y of
prevention of erythroblastosis with R h hapten. J . A. M . A., 144: SS-92, 1950.