THE Rh FACTOR AS APPLIED TO THE OPERATION OF
BLOOD BANKS*
LESTER J. UNGER, M.D., M. WEINBERG AND M. LEFKON
From the Blood Transfusion and Plasma Division cf the New York Post-Graduate
Medical School and Hospital, New York, N. Y.
The discovery of the Rh factor and the recognition of its clinical importance
as related to transfusion have raised many problems in connection with the
operation of a blood bank. Ideally not only the A-B-0 group but also the exact
Rh type of the blood of every donor and patient should be determined and only
compatible blood of the same A-B-0 group and Rh type should be transfused.
If in addition the Hr factor is taken into consideration, this would make it
necessary for a blood bank to stock bloods of 40 different varieties. Certainly
for the present, this procedure is impracticable for general hospital practice.
It is too time-consuming, and the necessary diagnostic serums are now available
only to specialists in this field. Some go to the other extreme and make use of an
Rh-negative donor as a universal donor with no preliminary Rh factor determinations on the patient's blood. Such procedure is analogous to using, in
emergencies, a group 0 donor as a universal A-B-0 donor. This practice should
be frowned upon because if the patient is Hr-negative, he may become sensitized
to the Hr antigen which is always present in Rh-negative blood. The practice
is also uneconomical and depletes the blood bank of its supply of Rh-negative
blood, so that when such blood is indicated and needed for an Rh-negative
patient it is possible that none may then be at hand.
Another practice is to examine, by the test tube method, the bloods of all
donors using only anti-Rh 0 serum. This acts as a screening test and divides all
donors into Rh 0-positive and Rh 0 -negative. In such tests, the Rh' and Rh"
factors are deliberately disregarded and donors belonging to types Rh', and Rh",
and Rh'Rh" are classified as Rh-negative donors. In table 1, series II shows
the results of Rh tests done in this fashion on 4504 donors. In this particular
series 17.1 per cent were Rh 0 -negative and 82.9 per cent were Rh0-positive.
It will also be seen from this table that when the bloods of these Rh0-negative
donors were examined by the test tube method using three varieties of anti-Rh
diagnostic serum (anti-Rh 0 , anti-Rh' and anti-Rh"), 15.4 per cent were Rhnegative, about 1 per cent were Rh', and 0.7 per cent were Rh".
As far as the patient is concerned, with this routine the Rh test is performed
only when indicated. Such tests are definitely indicated and should not be
omitted in females from birth to the end of the child-bearing period. If Rhnegative females, even during infancy, are exposed to the Rh antigen by the
transfusion of Rh-positive blood, one in twenty-five will become sensitized.
Such a sensitized woman may be unable to have a normal child if later she
marries an Rh-positive man. Rh tests should not be omitted in women after
the climacteric if they have borne children since they may have become sensi*Received for publication, May 4, 1946.
498
R h IN OPERATION OF BLOOD BANKS
499
tized by repeated pregnancies even though there is no history of any of the
children having had erythroblastosis.2 Certainly tests must be performed if the
mother has given birth to a child with erythroblastosis, or gives a history of one or
more stillbirths. Tests must be performed on the blood of either male or female
if repeated trnasfusions have been or are to be given. When transfusions of
Rh-positive blood are given to an Rh-negative patient, here too, one in twentyfive may become sensitized. It must also be borne in mind that, in certain
instances, even an Rh-positive patient may become sensitized. Such sensitization may in rare instances be caused by the Rh' or Rh" or Hr factors if these
factors are lacking in the patient. As the list of indications for the test grows
and the subject becomes more complex, it becomes evident that the simplest,
safest and best procedure is at least to perform the test for the Rh 0 factor on
every patient and every donor prior to transfusion.
To carry out this procedure and to solve this practical and important problem
it is necessary that the routine of the test be so simple that it can be performed
by the average technician, and so rapid that it can be performed prior to every
transfusion, simultaneously with the ordinary test of the A-B-0 group. In
addition there must be a plentiful supply of the necessary diagnostic serums.
A satisfactory and practical routine has been developed at our blood bank.
During the year 1945 the Blood Bank of the New York Post-Graduate Medical
School and Hospital obtained blood from 25,515 donors. This very large volume
made it possible to test and evaluate various procedures and to determine their
practicability as applied to everyday hospital requirements. Donors in such
numbers were needed because our blood bank renders service not only to patients
within our own institution, but also to those of approximately 150 other hospitals,
and the number of tests is increasing daily. The blood of these donors was
used for 21,093 blood transfusions and 2,794 plasma transfusions, or for a total
of 23,887 transfusions during one year.
In a bank of this size, the demand for Rh-negative blood is necessarily great.
During 1945 Rh-negative blood was supplied for 2,882 transfusions. To render
the service demanded of us, not only Rh-negative blood, but also blood of the
various Rh types must be on hand. This is necessary in problem cases where
incompatibilities are detected, and it seems desirable to follow the ideal procedure
and, have blood of a donor of the same Rh type as the patient. It is also necessary to have available blood cells of various Rh types for use in the detection
of anti-Rh agglutinins produced in a patient as a result of pregnancy or of repeated blood transfusions, blood of such patients being frequently sent to us
for examination. The blood of every donor on our roster is examined for the
Rh 0 factor, and in about 20 per cent of the total a complete Rh typing
is performed.
It is evident from this volume of work that any "procedure adopted must be
rapid, efficient and accurate. We have finally adopted the following procedure.
At the time the phlebotomy is performed, besides taking specimens of blood
for serologic examination and for cross-matching with the prospective patient's
blood, a specimen is also taken for determination of the Rh factor. The test
500
L. J. TJNGER, M. WEINBERG AND M. LEFKON
tube used contains dried oxalate, which is dissolved by inverting the tube several
times immediately after the blood is added (0.2 cc. of a stock solution, containing 3 per cent ammonium oxalate and 2 per cent of a solution of potassium
oxalate in distilled water, is placed into a test tube and evaporated to dryness
in a hot air oven; approximately 4.8 cc. of blood obtained by venipuncture is
then added to the tube.) When such oxalated whole blood is used the cells are
usually present in a 50- per cent suspension. For anemic individuals, the blood
should be centrifuged and enough supernatant plasma removed to make the
volume of plasma approximately equal to that of the erythrocytes. This same
oxalated specimen is also used to check the original A-B-0 grouping of the blood
of the donor which has been taken from his ringer.
DONOR'S NUMBER
A SERUM
B SERUM
R h SERUM
F I G . 1. R U L I N G AND L E G E N D ON G L A S S S L I D E U S E D FOB SIMULTANEOUS
G B O U P I N G AND R h
A-B-0
TYPING
T h e glass slide measures 7 x 5 inches
The slide used for the test (fig. 1) is a piece of glass 7 by 5 inches, marked off
into 20 squares (four, horizontal and five vertical). Each set of horizontal
squares is used for the examination of a single donor's blood. In this way the
blood of as many as five individuals can be examined simultaneously. In the
first column of squares under the heading "number" are written the number
or name of each individual whose blood is to be tested. Each donor's fresh
oxalated blood is transferred to the slide as follows, from left to right: a small
drop in the square under the heading "A", another small drop in the square
under the heading " B " , and two full-sized drops in the square under the heading
"Rh". In order to avoid the labor necessary to wash glass pipettes, an ordinary
drinking straw is used for transferring the drops of blood. These straws, as
R h IN OPERATION OF BLOOD BANKS
501
purchased, are of such length that they can be cut in two for the sake of further
economy. Each half is used once and is then discarded. A drop of saline is
added to each drop of blood except those under the column headed "Rh". To
each of the squares under the heading "A", starting at the top, a drop of A
(anti-B) serum is added, on each of the squares under the heading " B " a drop of
B (anti-A) serum is added, and on each of the squares under the heading "Rh"
a drop of serum containing pure anti-Rh 0 blocking antibodies is added.6 The
drops within each square are thoroughly mixed with the end of an applicator
stick, using a separate stick for each square. The glass plate is then placed on
a "light box" similar to the one described by Diamond and Abelson,1 but modified
slightly to meet the needs of large-scale examination. The box is so constructed
that it can accommodate two slides such as just described. In this way, ten
bloods can be examined at one time. The box is rocked back and forth and
the electric light underneath furnishes not only light but also enough heat to
facilitate the reaction with the anti-Rh serum. The A-B-0 grouping and the
Rh factor are simultaneously determined in from two to five minutes. Agglutination or lack of agglutination by either the anti-A, anti-B, or anti-Rh serum
(Rh 0 blocking) is obvious to the naked eye. The A-B-0 grouping is diagnosed
according to the well-known criteria. If clumping (conglutination)6 results
with the Rh 0 blocking serum, the cells contain the Rh 0 factor. If no clumping occurs, the cells do not contain the Rho factor. This routine quickly
and easily divides the donors into two groups, those who are Rh 0 positive and those who are Rh 0 -negative. On the latter group a complete Rh
typing is then performed employing the standard test tube method, using
Rh 0 , Rh' and Rh" serums.4 Only individuals who are negative to all three
serums are classified as Rh-negative. While this is the routine technic followed
at our blood bank, for the average hospital where fewer tests are performed
certain modifications may be made. The tests can, of course, be carried out
on three ordinary glass microscope slides, and these can be held over a microscope
lamp instead of the special box to which reference has been made. The last
step which tests all Rh 0 -negative bloods for the Rh' and Rh" factors must
necessarily be omitted by the average hospital until such time that these rarer
serums are available for general hospital practice.
For this rapid slide test, serum of a high titer as tested by the conglutination
method must be used. Although in our experience serum containing Rh 0
blocking antibodies is preferable to that containing anti-Rh 0 agglutinins, the
latter type serum yields satisfactory results providing the titer is high. Serum
containing Rh 0 blocking antibodies in a low titer, when tested by the test tube
technic,* may show the presence of antibodies in a relatively high titer when
*A drop of each serum dilution to be tested is added to a series of tubes; to one set a drop
of a 2 per cent suspension in saline of type Rhi cells is added, to a second set type Rh2 cells
is added. The mixtures are allowed to react in a water bath at 38°C. for 45 to 60 minutes,
then a drop of anti-Rh 0 testing serum is added to each tube. After a second period of
incubation the tubes are gently shaken and the degree of agglutination read under the low
power of a microscope.
502
L. J . U N G E R , M. W E I N B E R G AND M. L E F K O N
tested by the conglutination method.* In our experience, as well as that of
Wiener, the titers by the conglutination method are on the average about ten
times as high as by the blocking technic. In order to increase the supply, such
serum can be diluted with AB serum. It must, however, never be diluted with
saline. In fact prior to the completion of the reaction no watery solution of
any sort should be mixed with either the cells or the serum. All glassware or
pipettes must be dry. The anti-coagulant used for the specimen of blood must
be dried. The phenomenon responsible for this test depends upon the presence
of univalent antibodies, and also upon the presence of X protein in a definite
concentration. The addition of watery solutions of sodium chloride, sodium
citrate or potassium oxalate so dilutes the X protein that the phenomenon fails
to develop. Occasionally rouleau occurs, expecially at the periphery of the
mixture which might erroneously be interpreted as a positive reaction. After
four or five minutes have elapsed and sufficient time for the completion of the
reaction has passed, a drop of saline may be added to the mixture, and the slide
is again rocked back and forth for one or two minutes. False positive readings
due to rouleaux will then disappear and the mixture will become homogeneous.
If, however, the result is truly positive, the clumps persist, or become even more
apparent.
It is very important to use blood with the correct concentration of cellsto
avoid occasional false negative readings caused by weak suspensions of cells.
Because of the tendency toward drying of the specimen, it is advisable not to
examine more than 10 bloods at one time. In the earliest stages of the reaction,
if the slide is held at an angle so that most of the mixture accumulates at the
lower end of the slide and a thin layer remains at the upper half, reading will
be facilitated. Examination of the specimen should be macroscopic because
the microscopic appearance is often confusing. For the inexperienced it is
helpful to test the unknown blood simultaneously with bloods known to be
Rh-positive and Rh-negative.
The slide test as originally described by Diamond and Abelson was meant to
diagnose the existence of sensitization of an Rh-negative patient during pregnancy, or as a result of transfusion with blood of an Rh-positive donor. They
used cells known to be Rh-positive (types Rhi and Rhi) and Rh-negative to
determine the presence or absence of anti-Rh agglutinins or blocking antibodies
in the serum of the patient under investigation. The procedure described in
this paper uses serum known to contain Rh 0 blocking antibodies as an anti-Rh
testing serum in order to determine the presence or absence of the Rho factor
in the cells of the individual under investigation.
During the course of examining many hundreds of bloods to determine the
existence of sensitization to the Rh factor, on three occasions tests performed by
us by the slide technic were positive, yet tests with the serums of these three
patients made by the agglutination, blocking and conglutination technic in
test tubes were entirely negative. On the other hand, at no time had serum
*The technic of this test is the same as that for anti-Rh agglutinins except that the
serum dilutions to be tested are made with AB serum and the test cells are diluted with AB
serum. No watery solutions are used. Pipettes must be dry or rinsed with AB serum.
R h IN OPERATION OF BLOOD BANKS
503
that reacted positively, as tested by the test tube methods for Rh 0 blocking
antibodies or anti-Rh agglutinins, failed to yield positive results when tested
on the slide. These facts show that occasionally the slide technic may yield
results at variance with those obtained in the test tube method. At the moment
we are not attempting to explain these differences. As far as the procedure
described in this paper is concerned, these few differences are of no consequence
except insofar as they concern the selection of serums to be used as diagnostic
reagents. All serums selected for that purpose must contain anti-Rh agglutinins
or Rh 0 blocking antibodies as determined by the test tube method. Such serums,
on the slide, will invariably give results identical with those obtained by the
longer standard test tube method. More than 2000 of the bloods, the findings
of which are shown in table 1, were tested both by the slide and the tube method,
and the results were always the same. One advantage of the slide method for
testing for the Rho factor is that, as Diamond and Abelson pointed out, it uses
serum formerly considered of no value for Rh testing (Rh 0 blocking serum).
In addition, by the dilution of such serums with AB serum as suggested in this
paper, the available supply of our test reagent is greatly increased. Even more
important, however, is the fact that it is a rapid and simple procedure and with
it every patient and every donor can be examined in a few minutes prior to
transfusion. Furthermore, it is human serum. Experience has shown that
animal serum is not as reliable as that obtained from human beings. The bloods
of all newborn infants, Rh-negative as well as Rh-posilive, read positively with
anti-Rh serum of animal origin.
The bloods of 10,810 donors, (table 1, series III) were examined by the screening method using Rh 0 blocking serum on the slide. All bloods belonging to
types Rh 0 , Rhi, Rh 2 or RhiRh 2 react positively and are called Rh-positive.
This group constituted 84.1 per cent of the total, whereas 15.9 per cent were
negative. On this latter group complete Rh typings were performed using the
standard test tube method with anti-Rh 0 , anti-Rh' and anti-Rh" serums. Of
the 15.9 per cent, 14.4 per cent were found to be completely negative, 1.0 per
cent belonged to type Rh', 0.5 per cent to type Rh", and none to Rh'Rh".
These results compare almost exactly with those obtained when complete Rh
typings (table 1, series I) were performed by the tube method on the bloods of
another group of donors without any preliminary screening out of Rho-positives.
A total of 7,317 bloods were examined in this fashion. For this series, the
percentage of types Rho, Rhi, Rh2 and RhiRh 2 are given, and when these are
combined, as when the Rh 0 screening method is used, they constitute 83.6 per
cent of the total. In this series, moreover, 14.7 per cent were completely
negative, 1.1 per cent were type Rh', 0.6 per cent were type Rh", and none were
type Rh'Rh". The two series of donors examined consisted of entirely different
individuals and yet the results are practically identical. Accordingly, one is
justified in concluding that the slide method using anti-Rh 0 blocking serum
yields results identical with those obtained by the more difficult and more timeconsuming test tube method and is, therefore, satisfactory for routine hospital
work.
Table 1 also shows the results of tests of the three series combined, blood
504
L. J . TJNGER, M. W E I N B E R G AND M.
LEFKON
from a total of 22,631 individuals having been examined. This large series
establishes the percentage of Rh-negative donors taken at our blood bank at
14.7 per cent, R h ' 1.0 per cent, Rh" 0.6 per cent, and Rh'Rh" 0.004 per cent,
whereas the remaining Rh types constitute 83.7 per cent of the total. As
indicated by these results among the total of 22,631 donors, there was one
individual belonging to the rare type Rh'Rh". Although in this series this
type of blood occurred once in approximately 20,000 cases, this does not differ
significantly from the theoretical frequency of 1 to 10,000.3 The rarity of
this type of blood prompted us to make a complete study of other members of
this donor's family. The donor himself was a male belonging to group O,
type N, type Rh'Rh". His mother's classification was ON Rha, his father's 0
MN Rhi. This family study was made in the hope that other individuals of
TABLE 1
D I S T R I B U T I O N O F R h FACTORS ACCORDING TO V A R I O U S M E T H O D S OF T E S T I N G
METHOD USED
SERIES
NUMBER
OF INDIVIDUALS
TESTED
Rho
PERCENTAGE OP R h TYPES
SCREENING
Pos.
Neg.
Neg.
Rh'
Rh" Rh'Rh" Rho
83.6 16.4 14.7 1.1 0.6 0.01
Rhi Rh2 RhiRh.
Complete R h t y p ings
7,317
II
Anti-Rho in test tube
followed by complete typing of all
negatives
4,504
82.9 17.1 15.4 1.0 0.7 0.0
82.9
III
Rh 0 blocking serum
on slide, followed
by complete t y p ing of all negatives
10,810
84.1 15.9 14.4 1.0 0.5 0.0
84.1
22,631
83.7 16.3 14.7 1.0 0.6 0.004
83.7
I
Total
2.2 53.5 15.0 12.9
83.6
type Rh'Rh" might be found. We were disappointed, but at any rate the
results conformed with the genetic theory.
When using either anti-Rh 0 agglutinating serum or Rh 0 blocking serum' to
screen out all Rh 0 -positive donors, approximately 1.6 per cent of bloods belonging
to types Rh', Rh" or Rh'Rh" will be included as negative bloods. For ordinary
hospital work this is of very little consequence, and the method described is
entirely satisfactory for practical purposes. It is very rare for sensitization
following transfusion to be caused by the R h ' or Rh" factors. If, however,
one is somewhat more exacting and wishes to eliminate all R h ' donors the
technic can be carried out as described (using Rh 0 blocking serum) when examining the bloods of all patients, but when examining the bloods of donors use
anti-Rho serum, or serum containing Rh 0 blockers and R h ' agglutinins. This
M l IN OPERATION OF BLOOD BANKS
505
will quickly screen out all donors whose bloods contain either or both of the
factors, Rh 0 and Kh'. The ideal procedure, of course, is the routine followed
in our blood bank. We consider it absolutely essential that the donor be negative
to all three factors, and the more complicated and time-consuming test tube
method is used on the blood of all those who react negatively to the slide test.
SUMMARY
A total of 10,810 bloods was examined by a rapid slide method. Rh 0 blocking
serum was used as the diagnostic reagent. This screened out Rh0~positive
bloods. As a control, 4,504 other bloods were tested using the standard test
tube method and anti-Rh 0 agglutinating serum as the diagnostic reagent for
screening with identical results. In still another series, 7,317 complete Rh
typings were performed using anti-Rh 0 , anti-Rh' and anti-Rh" serums in order
to determine the frequencies of each of the eight Rh types. Out of a total of
22,631 persons tested 14.7 per cent were Rh-negative, 1.0 per cent type Rh',
0.6 per cent type Rh", and 0.004 per cent type Rh'Rh".
Sensitization of an Rh-negative patient by the Rh factor causes approximately
90 per cent of post-transfusion hemolytic reactions. Such sensitization is almost
always caused by the Rh 0 factor. Therefore, an Rh-negative patient should
not be exposed to this factor by blood transfusion. To avoid such exposure,
the blood of every patient and every donor must be examined prior to transfusion.
Complicated and tedious tests, together with a scarcity of testing serum, have
prevented the adoption of such a routine. Our results establish the slide technic
using Rh 0 blocking serum as a reliable substitute for the lengthy test tube
method using anti-Rh serum. This method has the advantage of speed which
is desirable in preparing for transfusions in hospitals. It is a simple procedure,
using human serum as the test reagent, the supply of which is plentiful. Such
human serum yields reliable results in all cases, in contradistinction to animal
anti-Rh serums to which the bloods of all newborn infants, Rh-positive as well
as Rh-negative, react positively. The blood of every patient and every donor
can now be examined rapidly prior to transfusion, simultaneously with the test
for the ordinary A-B-0 blood grouping. Such a routine procedure will make it
possible effectively to avoid post-transfusion hemolytic reactions due to sensitization by the Rh 0 factor from repeated transfusions.
REFERENCES
1. DIAMOND, L. K., AND ABELSON, N . M . : The demonstration of anti-Rh agglutinins. A n
accurate and rapid slide test. J . L a b . and Clin. Med., 30: 204-212, 1945.
2. U N G E H , L E S T E R J . , AND W I E N E R , ALEXANDER S.: Observations o n sensitization t o t h e
R h factor by blood transfusion. Am. J . Clin. P a t h . , 15: 280-285, 1945.
3. W I E N E R , A. S.: Genetic theory of the Rh blood types. Proc. Soc. Exper. Biol, and Med.,
54: 316-319, 1943.
4. W I E N E R , A. S.: Nomenclature of the R h blood t y p e s . Science, 99: 532, 1944.
5. W I E N E R , A. S.: A new test (blocking test) for Rh sensitization. Proc. Soc. Exper. Biol.
and Med., 66: 173-176, 1944.
6. W I E N E R , A. S.: Conglutination test for R h sensitization. J . Lab. and Clin. Med., 30:
662-667, 1945.