R H - H R FACTORS AND THEIR SPECIFIC ANTIBODIES, AS
APPLIED TO BLOOD TRANSFUSION
LESTER J. UNGER, M.D.
Blood and Plasma Bank, New York University-Bellevue Medical Center, Nexo York
As recently as 1921 it was claimed by some that the crossmatching test preliminary to transfusion was superfluous. These workers believed that if the ABO
blood-grouping tests were carried out correctly on blood specimens of patient
and donor and that if the results showed that they belonged to the same blood
group, no further tests were necessary, and the donor's blood could be transfused
without danger of a hemolytic reaction. The author found this to be incorrect33
and was the first to report that occasionally serum of a patient would clump cells
of a donor even though they both belonged to the same ABO blood group, and
that transfusion of such blood was followed by chills and fever which he attributed to the observed incompatibility. He called the antibodies responsible for the
intragroup'incompatibility "minor" agglutinins to distinguish them from the
anti-A and anti-B agglutinins. Crossmatching tests carried out by him employed
a 2 per cent suspension of cells in the individual's own plasma, whereas at that
time the custom was to use saline-suspended cells. Unwittingly, he carried out
the "conglutination" test and detected "univalent" antibodies and although
their specificity was not established, they were probably Rh antibodies. Subsequently, Culbertson and Ratcliffe, Pondman, Zacho, Neter, Mosonyi, and Levine and Stetson18 described similar intragroup incompatibilities.
With the discovery in 1937 by Landsteiiier and Wiener11"13 of the first anti-Rh
serum as a result of injecting blood of rhesus monkeys into rabbits, the solution
of most hemolytic transfusion reactions was at hand. Later, with the development of new tests for the detection of immune isohemagglutinins, methods for
the detection of hitherto unknown antibodies were described and their specific
blood factors recognized. Along with the acquisition of knowledge, and with the
development of blood banks, there was a great increase in the number of patients
transfused and the number of transfusions given to the same patient. The repeated administration of blood exposes the recipient to a great variety of antigens, and because of this he may become isosensitized. It is practically impossible
to transfuse a patient with blood that is identical with his own with regard to all
of the known combinations of blood factors. Related blood factors are grouped
into blood-group systems, and those now known to exist indicate the occurrence
of more than 50,000 varieties of blood. Some day a complete blood grouping may
become as distinctive as a thumb print, in conformity with the prediction made
long ago by Karl Landsteiiier.
Received for publication August 10, 1953.
Presented as part of the Symposium on Blood Bank Methodology at the joint meeting
of the College of American Pathologists and the American Society of Clinical Pathologists
in Chicago, October 13, 1953.
Dr. Ungcr is Director of the Blood and Plasma Bank.
275
276
UNGER
VOL. 2 4
Isosensitization may result not only from repeated blood transfusions, but
also from intramuscular injections of blood or repeated pregnancies or a combination of these. 16 ' "•19'36' 66 Such isosensitization may in turn be responsible for
posttransfusion hemolytic reactions. The development of isoantibodies requires
at least 2 exposures to the antigen, with an interval of several months between
each exposure.49, 69 The first exposure primes the individual, while subsequent
exposures may stimulate the production of isoantibodies. Yet experience with a
recent case shows that a long interval is not always necessary and isosensitization
may result from continuous stimulation over a short period of time, provided
huge quantities of the antigen are given. The patient, Rh-negative, was a man
who was losing huge quantities of blood from the rectum. The first 11 transfusions, totaling 5500 ml., were of Rh-negative blood. Because no more Rh-negative
blood was available and the patient was almost moribund, he was given an additional 54,500 ml. of Rh-positive blood within a period of 7 days. Then 10 ml. of
10 per cent calcium gluconate was given after each 1000 ml. of blood, and the
bleeding finally ceased. Further transfusions were required prior to a contemplated operation. Our routine pretransfusion laboratory tests for Rh sensitization
were carried out before each transfusion but were negative until the 108th pint
was to be administered, when it was found that the patient had become sensitized by the Rh 0 factor and had developed univalent antibodies. The titers by
the various tests, at that time, were as follows: saline agglutination method, zero;
albumin-plasma conglutination method, 32 units; indirect blocking method, 8
units; antiglobulin method, 16 units; trypsin-treated cell method, 96 units;
trypsin-treated cell antiglobulin method, 96 units. After Rh sensitization developed, of course, no further transfusions of Rh-positive blood were given; 2500
ml. of blood from type rh donors was then transfused without reaction. The
patient recovered after receiving a total of 62,500 ml. of blood and, as far as we
could determine, he had been given the largest amount of Rh-positive blood
ever deliberately administered to an Rh-negative patient within such a short
period of time. This case illustrates that continuous exposure to large amounts
of incompatible Rh0-positive blood over a relatively short period of time can
stimulate the development of isoantibodies. Although the patient must have
been exposed to many blood factors absent from his own blood, he developed no
antibodies other than those for the Rh 0 factor, obviously because this is the most
antigenic factor.
We must be careful to distinguish between agglutinogens and blood factors.
Wiener73 has repeatedly pointed out the difference between these. An agglutinogen is a definite hereditable substance of unknown chemical composition on
the red cell envelope, and the red cells of each individual have a number of such
agglutinogens. Blood factors, on the other hand, are attributes or properties of
the structures called agglutinogens. Each agglutinogen generally has multiple
blood factors, which are the serologic attributes by which the agglutinogen can
be identified. This may be compared to the identification of glucose in urine by
its abihty to reduce copper solution or to its identification as a chemical with
multiple physical properties. Agglutinogens are antigenic and are capable of in-
MAR. 1954
R H - H R FACTORS
277"
ducing in susceptible persons the formation of antibodies specific for one or more
of its various blood factors, and such serums are used for diagnostic purposes.
These reagents are obtained from persons accidentally sensitized by pregnancy
or transfusion or by deliberate injection of the appropriate antigen.
At the blood bank of New York University-Bellevue Medical Center, approximately 125,000 prospective donors present themselves annually. Many of these
are rejected prior to the completion of all our routine blood examinations, yet a
great number of bloods are subjected to all routine tests. It is axiomatic that adequate methods must be established in order to determine Rh factors and accurately to identify Rh antibodies or, for that matter, other atypical antibodies.
Our routine examinations 37 ' 38 ' 48 ' 61 carried out for the Rh-Hr blood group system
in connection with blood transfusions* are divided into 4 steps:
1. Rh testing
2. Rh-Hr typing
3. Tests for sensitivity for Rh-Hr antibodies
4. Identification of the specificity of and titration of Rh-Hr antibodies.
Rn
TESTING
The most antigenic of the Rh-Hr factors and the one to which human beings
respond most readily is the Rh 0 factor. Clinically, therefore, this is the most important factor of this blood group system. It is, by far, the most common cause
of intragroup posttransfusion hemolytic reactions, as well as of erythroblastosis
fetalis. No patient whose blood lacks the Rh 0 factor, except in dire emergencies,
should be transfused with blood that contains it. This is of particular importance
for females, from birth to climacteric. Therefore, our routine procedure starts
with Rh 0 testing. This test separates persons into 2 groups, Rh-positive and Rhnegative or, to be more explicit, Rh0-positive and Rh 0 -negative. The test employed by us is a screening procedure, and is called the rapid slide test. 9
METHOD
For this test (rapid slide test) oxalated whole blood is preferable, as it is approximately a
40 to 50 per cent suspension of cells in the individual's own plasma. If the patient is anemic,
the whole oxalated blood should be centrifuged, and enough of the supernatant plasma
removed to make the volume of the remaining plasma equal that of the cells. The tube is
then shaken and a 40 to 50 per cent cell suspension results. Clotted blood may be used. By
breaking up the clots, cells are freed, and then a 40 to 50 per cent suspension of these is
made in the individual's own serum. On a microscope slide, place 2 drops of the 40 to 50 per
cent cell suspension and add 1 drop of anti-Rh0 slide test serum which meets the standards
of the National Institutes of Health. Then mix it with the blood and spread the mixture on
the slide so that it covers an area about lJ/£" X 1". The slide is placed on a viewing box that
is illuminated from below and slightly warmed (40-50 C.) and rocked back and forth for 2
to 3 minutes. If the cells are Rho-positive, macroscopic clumping appears in about 30
seconds and large clumps in 2 or 3 minutes. If no clumping results, the cells are Rh0-negativo. Do not examine the mixture with a microscope.
For the rapid slide test, serums are available also of specificities other than anti-Rho.
The technic for using them is identical, irrespective of the specificity of the univalent anti* Since this symposium was limited to blood transfusion, no consideration is given in
this paper to Rh problems related to pregnancy.
278
VOL. 24
UNGER
serum, but the interpretation of the results, of course, depends upon the specificity of the
diagnostic serum.
This rapid test can be done in a test tube (rapid tube test), taking 1 drop of a 2 per cent
suspension of the individual's cells in his own plasma, adding 1 drop of slide test serum
followed by incubation for 15 minutes, centrifuging at low speed for 1 minute, gently shaking to dislodge the sediment, and then reading the result.
At the same time that the unknown blood is examined, known Rh0-positive and known
Rho-negative bloods are similarly tested, as controls. In addition, the patient's cells are
tested with his own serum instead of the anti-Rh0 serum. In some instances there may be a
slightly granular appearance, particularly around the edges of the specimen, giving a doubtful positive appearance. This is rouleaux formation and occurs in persons having a rapid
sedimentation rate. The addition of 2 drops of normal saline solution, followed by rocking
the slide back and forth will almost always cause this to disappear, and the result becomes
clearly negative. False-positive results owing to rouleaux, as well as those caused by cells
coated with antibodies, may be clarified by carrying out the slide test, employing 20 per
cent bovine albumin instead of anti-Rh 0 serum, because under such circumstances the results will be equally positive. In such cases, the test must be repeated by the test tube
agglutination method employing a saline suspension of cells.
R H - H R TYPING
In contrast to Rh testing, which determines the presence or absence of the
Rh 0 factor only, Rh-Hr typing identifies various Rh-Hr factors. 14 ' 16 - 22 - B6- 60
For this purpose various specific diagnostic antiserums of known antibody content are utilized to test cells of unknown Rh-Hr antigenic composition. The
antiserums commonly employed are anti-Rh 0 , which react with blood of approximately 85 per cent of Caucasoids; anti-rh', giving 70 per cent positive reactions;
anti-rh", giving 30 per cent positives; anti-hr', giving 80 per cent positives; and
anti-hr", giving 97 per cent positives (Table 1). If clumping results, the factor for
which the serum is specific is present, and each factor is named for the serum that
produces clumping. There is also a less common factor, rhw, to determine which
specific anti-rh w serum (giving 4 to 8 per cent positives) is required. 3, 4 ' 63, 72
Recently an antiserum giving approximately 65 per cent positive reactions was
found. It identifies still another blood factor that has been named "hr." In addiTABLE l
INCIDENCE OF REACTIONS TO R H - H R ANTISERUMS (CAUCASOIDS)
Anti-Rho
Anti-rh'
Anti-rh"
Anti-hr'
Anti-hr"
Anti-rh"
Anti-hr
Anti-Hro*
APPROXIMATE POSITIVE
APPROXIMATE NEGATIVE
per cent
per cent
85
70
30
80
97
5
65
63
15
30
70
20
3
95
35
37
* Although this hypothetic antiserum is as yet not available, it is included for the
sake of completeness.
MAR. 1954
R H - H R FACTORS
279
tion, variants 2 ' 26~28' 30' 50, 54' 63' " of the Rh-Hr factors exist, and the most important is the variant of the Rh 0 factor, which occurs far more frequently in
Negroids than in Caucasoids.71
When only the 3 basic anti-Rh serums are employed, 8 Rh types are determined (Table 2). 6 3 ' 6 1 , 6 2 ' 7 1 This is not as complicated as it seems, if one keeps in
mind the reactions obtained with anti-A and anti-B serums, which determine
the 4 ABO groups. Here too, if one employs only 2 serums, namely, anti-rh' and
anti-rh", 4 types are established and the reactions possible are the same as for the
4 ABO groups. But when the third serum, anti-Rho, is added, the 4 types established by the anti-rh' and anti-rh" serums are all either positive or negative to
this anti-Rho serum. As a result, there is a double set of 4, or 8 possible Rh types.
These 8 are likewise named for the Rh antiserums that produce the clumping.
Thus, type rh (triple Rh-negative) blood reacts with none of the Rh antiserums,
type rh' blood reacts only with anti-rh' serum, type rh" reacts only with antirh", type rh'rh" (rhy) reacts with both rh' and rh" antiserums, while type Rh 0
reacts only with anti-Rho. The remaining 3 types react with more than 1 antiserum and the names are shortened, viz., Rhi reacts with anti-Rh 0 and anti-rh',
Rh 2 reacts with anti-Rho and anti-rh", while RhiRh 2 (Rhz) reacts with anti-Rho,
anti-rh' and anti-rh". Wiener selected the names Rhi and Rh 2 rather than Rh 0 rh'
and Rh 0 rh" for the sake of simplicity, and also to indicate that the blood factors
in such bloods are usually attributes of a single unit agglutinogen inherited by a
corresponding gene.
If the fourth anti-Rh serum is utilized, namely, anti-rh w , types rh', Rhi and
RhiRh.2 are subdivided into those that give negative results and those that give
positive results, and the latter are named rh' w , Rh" and Rhi'Rh 2 .
The frequencies in the general population of the 8 Rh blood types are unequal; rh'rh" (0.02 per cent) is by far the rarest, and Rhi (51.2 per cent) is the
most common (Table 2). There are also racial differences (Table 3); for example,
type rh is most common in Caucasoids (13.5 per cent), less common in Negroids
(7.5 per cent), and least common in Chinese (1.5 per cent) and Japanese (0.6 per
cent).
For the table of reactions obtained with the 2 Hr antiserums, one must remember that the Hr factors identified by these antiserums are the reciprocals of the
corresponding Rh factors. This is indicated in the name that Levine devised, by
the reversal of the letters "Rh". If an Rh factor is absent, the corresponding Hr
factor is present, although recently exceptions to this rule have been observed.
These exceptions are, however, very rare. If an Rh factor is present, the corresponding Hr factor may or may not be present. As a result, when 3 anti-Rh serums
and 2 anti-ITr serums are simultaneously employed, 18 Rh-Hr types are determined. If the third Hr antiserum, namely, anti-Hi'o, should become available,
the number of Rh-Hr types would be increased to 27 (Table 2).
As stated previously, the Rho factor is clinically the most important of the
Rh-Hr blood-group system, since it accounts for the overwhelming majority
both by intragroup incompatibilities and of cases of hemolytic disease of the newborn. Yet the other Rh-Hr factors cannot be completely ignored even though
isoseiisitization by them occurs less frequently. During the past year I have
85.1
Rh-posi-
tive
14.9
(%)
Rh-negative
Designations
t
Approximate Fre- Reaction
quencies
with
in New
Anti-Rho
York City (or AntiWhites
rhesus)
2 R h PHENOTYPES
Rhi
Rh0
51.2
2.5
+
-
+
+
.02
+
-
0.4
rh"
rh'rh"
(rh y )
-
+
1.1
-
-
rh'
Antirh"
Antirh'
13.4
(%)
rh
Designations!
18.0
33.2
Rhirh
2.5
.02
.0003
.0001
.000001
.003
0.4
.02
1.1
13.4
(%)
RhiRhi
Rho
rhyrh
rhyrh'
rhj.rh"
rhyrhy
rh"rh"
rh"rh
rh'rh'
rh'rh
rh
Designations
Approximate Frequencies!
in New
York City
Whites
+
*
*
*
*
+
+
+
+
rV
r'r
*
*
R,Ri
Rtr'
RiRo
Rir
RoRo
Ror
ryr
ryr'
ryr"
ryry
r"r
rr
*
+
*
*
+
*
Antihr'
16.9
1.1
2.6
30.6
0.1
2.4
.02
.0003
.0001
.000001
.003
0.4
.02
1.1
13.4
+
+
+
*
*
*
*
*
*
*
*
*
Reaction
with
AntiHro§
27 R h - H r PHENOTYPES
Calculated
Frequencies
Designain New
tions
York City
AntiWhites
hr*
(%)
Reaction
with
18 R h - H r PHENOTYPES
( I n t e r n a t i o n a l Classification)**
Reaction with
8 R h PHENOTYPES
Approximate Frequenciesj
in New
York City
Whites
TABLE 2
T H E R H - H R P H E N O T Y P E S AND G E N O T Y P E S
RlRl
Rlr'
RlR°
R'r a n d
ROR"
R«r
ryr'
RV
r"r a n d r'r"
r"r"
r"r
r'r'
r'r
rr
GENOTYPES
<
o
H
a
o
+
+
14.9
RhiRh2
(Rh,)
+
—
—
+
.09
.03
.0001
Rh 2 Rhi
RhzRh2
Rhjlh,
+
+
*
-
+
13.6
Rh 2 rh
*
R h 2 R h 0 14.8
2.9
Rh 2 Rh 2
2.7
0.2
1.1
12.5
RzRo 13.9
R,r
0.9
RJti
.08
Rzr'
.001
.03
RzR 2
R,r"
.0004
.0001
R,R,
R*r y
.00002
R 2 r"
R 2 Ro
R2r
XV2XV2
** After Wiener. Reprinted with his permission from the May, 1949, issue of the Laboratory Digest,
f In this table Rhi is used to indicate Rh 0 ; Rh 2 , Rh 0 ; rh y , rh' "; and Rh z to indicate Rh 0 .
I Based on more than 1000 tests.
§ Hypothetic anti-Hr 0 serum is not available, but anti-hr is.
* The asterisks indicate tests which need not be made since they are invariably positive.
+
-
16.5
Rh 2
+
+
+
+
_
+
+
R'R" and R'R'
R'r, R'r", R'r', R°r»
R'R1
R'r' and R'r"
R'R'
R'r" and R'r"
R'R'
R'r"
R'R'
R'r"
R*R<>
R'r and R'r"
»
O
o
CO
282
UNGER
VOL. 2 4
TABLE 3
R A C I A L D I S T R I B U T I O N OF R H T Y P E S *
R h BLOOD TYPE (PER CENT)
RACE
Caucusoids (New York City)
Negroids (New York City)
Chinese
Japanese
rh
rh'
rh'
13.5
7.5
1.5
0.6
1.0
1.5
0
0
0.5
0
0
0
* Modified after Wiener, An Rh-Hr Syllabus.
rh'rh»
.02
0
0
0
Rho
Rhi
Rhi
RhiRlu
2.5
45.0
0.9
0
53.0
25.0
60.6
51.7
15.
15.5
3.0
8.3
14.5
5.5
34.4
39.4
Grune & S t r a t t o n , I n c . , 1954, p. 39.
encountered 4 instances of hr' sensitization alone. Sensitization by the hr' and
hr" factors as well as rh' and rh" can cause clinical complications indistinguishable from those caused by the Rh 0 factor.
Ideally, a complete typing for all known blood factors should be done on every
patient and donor and only type-specific blood transfused. If this were done,
sensitization resulting from blood transfusion would be eliminated. Since such
an ideal procedure is impossible, our present routine is to do Rh testing alone
and to add complete Rh-Hr typing only for the following:
1. Rh typing, including the special test for the Rh 0 variant, but omitting Hr
typing, is carried out on the blood of each donor found to be negative for the Rh 0
factor by the rapid slide method. This is necessary to detect 9ih0 (Rh0 variant)
and the rh' and rh" factors. Donors positive for any of these factors are classified
as Rh-positive, even in the absence of the standard Rh 0 factor, in order to avoid
sensitization of susceptible recipients.
For clinical purposes, a patient whose blood is negative for the Rh 0 factor and
its variant (9ih0 or D" of the English nomenclature) is to be considered Rhnegative even though the rh' or rh" factor, or both factors may be present. This
is necessary to avoid possible sensitization by the Rho factor. A patient whose
blood is positive for the 9ih0 factor falls into 1 of 3 categories, namely, type 9ih0
or 9?hi or 9{h2. A patient belonging to the first of these 3 may be considered Rhpositive and may be given Rh0-positive blood. A patient whose blood belongs to
either of the other 2 types, for the sake of safety, should be considered Rhnegative and only given Rh-negative blood. This is necessary because a positive
serologic result for the Rh 0 variant, when associated with either the rh' or
rh" factor, is difficult of interpretation. Such positive results may or may not
mean that the Rho variant factor is actually present, because to test for the Rh 0
variant, Rh 0 conglutinating serum is used. Such serums almost always react with
the rh' factor and more rarely with the rh" factor, and such reactions may erroneously lead to the conclusion that the Rh 0 variant is present, whereas in reality,
the positive reaction of the serum might be due to the rh' or rh" factor. For example, recently a case was published28 in which anti-Rho was found in the serum
of a patient reported to be type Rhi with an Rh 0 variant (9?hi). It is possible that
actually this blood was type rh', for the reason just given, and then, of course,
the finding of Rh 0 antibodies would not be unexpected.
2. Rh-Hr typing is carried out on persons who have experienced posttrans-
MAR. 1954
283
R H - H R FACTORS
fusion reactions, or when difficulties in crossmatching present themselves. In
addition, tests for M and N and other factors, such as the Kell and Duffy factors,
are made if indicated, and if the antiserums are available. 6 - 70 Such patients are
transfused only with blood that lacks the factor or factors to which they have
been sensitized.
3. Although not pertinent to the subject of this symposium, Rh-Hr typing is
carried out in cases of disputed paternity,'• 20 ' "• 43 - b8- 68 to aid in the exclusion
of a falsely accused man, and for obstetric patients to determine whether or
not isosensitization is possible and to determine homozygosity or heterozygosity.
METHOD
Rh typing. Into each of 4 narrow test tubes (7-S mm. inside diameter) is placed 1 drop of
a 2 per cent suspension in normal saline of once-washed fresh cells. Into each of the first 3
tubes 1 drop of a different saline agglutinating antiserum is placed in the following order:
anti-rh', anti-rh", anti-Rho. Into the fourth tube 1 drop of conglutinating anti-Rh 0 serum
is placed. All tubes are then incubated in a water bath at 37 C. for 1 hour and the cell sediments read under the microscope, using the scanning lens. If the third tube containing
anti-Rho saline agglutinating serum shows no clumping, an antiglobulin (Coombs) test is
then carried out on the cell sediment of the fourth tube. Otherwise, this fourth tube is discarded without further testing, because its purpose is to determine the presence of the
3fh0 (Rho variant) factor, and will invariably be positive if the third tube is positive.
Clumping of cells in any tube establishes the presence of a factor that is named for the
serum producing the clumping. As previously mentioned, S different phenotypes are established (Table 2) by these tests.
Hr typing. Into each of 2 tubes is placed 1 drop of a 2 per cent saline suspension of oncewashed fresh cells. To the first tube is added 1 drop of saline agglutinating anti-hr' serum
and to the second tube 1 drop of saline agglutinating anti-hr" serum, then shaken and
incubated for 1 hour at 37 C. and read under the microscope with the scanning lens. Clumping indicates the presence of the corresponding factor. When both Rh and Hr typing is
carried out, persons are divided into 18 phenotypes, as already pointed out (c/. Table 2).
Anti-hr' and anti-hr", besides occurring as bivalent antibodies, are found in serum as
univalent antibodies. When such antiserums are used, the test is carried out in the same
fashion as that described for Rh testing with univalent anti-Rho serum (rapid slide test).
TEST FOR SENSITIZATION BY R H - H R
ANTIBODIES
Besides determining an individual's Rh-Hr type, our routine is to determine
the presence or absence of atypical antibodies in the serum of both patient and
51
donor.33'35'48•49'
It is true that if the crossmatching technic is adequate, if the
tests are accurately performed and correctly interpreted, and if the results show
that the bloods of donor and patient are compatible, routine tests for sensitization, from one point of view, are unnecessary. However, antibodies in the patient's blood and in very low titer might be unobserved in the crossmatching test
and be responsible for posttransfusion reaction. This has occurred particularly
when the hr' factor is involved. To such Rh-positive patients, only blood of the
same Rh type as that of the patient is routinely transfused, omitting examinations for rare blood factors. The Rh-Hr factors capable of sensitizing such Rh0positive patients are of relatively low antigenicity (rh', rh", rhw, hr', hr"), and
preliminary sensitization tests might avoid such errors and their consequent
untoward clinical results.
284
UNGER
VOL. 24
It also seems desirable to obtain this information even when the crossmatching
test is compatible, if only to be aware of the existence of atypical antibodies in
either the patient or donor prior to transfusion. If posttransfusion reaction or
death should be a medicolegal problem, it may be difficult to avoid responsibility
merely by stating that crossmatching was compatible, if subsequent examinations
showed the existence of isosensitization for which no examinations had been
made prior to transfusion. Further if an Rh-negative donor's blood containing
atypical antibodies is transfused, we are passively sensitizing the patient41 and
it is conceivable that such passive sensitization might affect the fetus of a pregnant Rh-negative woman.
In emergencies, occasionally group 0, Rh-negative persons are used as universal donors without preliminary ABO grouping or Rh testing of the recipient.
If such a patient is in reality Rh-positive, it is also conceivable that transfused
Rh antibodies may attach themselves to the cells of the patient and produce a
condition not unlike acquired hemolytic anemia. Further, especially if the recipient is very anemic or has had a hemorrhage, the antibodies may even produce
a hemolytic reaction. For these reasons every Rh-negative donor whose blood we
send for transfusion at another institution and every donor and patient, whether
Rh-negative or Rh-positive, preliminary to transfusion at our own institution, is
examined for isosensitization. These tests are merely the reverse of Rh-Hr typing,
since here the antigenic composition of the diagnostic cells is known but the
antibody content of the serum under test is unknown.
Since Rh-Hr antibodies may differ in specificity, and since antibodies of identical specificities may be of different varieties, the test must be planned so that
antibodies of all Rh-Hr specificities and of all varieties are detected. While
the sensitization test can be arranged so as to identify the exact specificity of the
Rh-Hr antibody involved by employing a panel of cells of different phenotypes
(Table 5), in our own tests we make no attempt to do this at this time but rather
postpone it until the serum is titrated. For the sensitization test, blood of a single
phenotype, namely group 0 , type Rh z Rh 0 , is chosen because such blood cells
contain all 5 Rh-Hr factors (cf. Table 2). This acts, therefore, as a screening test
and detects Rh-Hr antibodies irrespective of their specificity. As an alternative,
however, 3 sets of cells may be used; group 0, type Rhi; group 0, type Rh 2 and
group 0, type rh. Using 3 sets of cells rather than 1 set increases the chance of
the presence of antigens of other blood-group systems. The sensitization test is
qualitative and not quantitative and does not determine the concentration of
antibodies.
Variety. There are 2 main varieties of Rh-Hr antibodies (Table 4) of any given
specificity, and each reacts better under different test conditions. The first variety
clumps specific cells when suspended either in normal saline solution or in high
colloid medium or the same cells enzyme-treated,21' 23,29.44,45, es aric i SUS p eri ded
in normal saline. Such antibodies are variously called agglutinins or bivalent
antibodies, or complete or early immune antibodies. The second variety does not
produce visible clumps of specific cells suspended in saline but simply combines
with the factor for which it is specific and "coats" or "blocks" it.'26' 5S This coat-
MAR. 1954
RH-HR
FACTORS
TABLE 4
VARIETIES OF R H - H R ANTIBODIES
METHOD OF TESTING
SALINE AGGLUTININ*
GLUTININ**
Saline agglutination
Albumin-plasma conglutination
Enzyme-treated cell
Antiglobulin
+
+
+
_
+
+
+
* This antibody is also known by the terms bivalent, complete, early immune.
** This antibody is also known by the terms univalent, incomplete, hyperimmune,
blocking.
TABLE 5
I D E N T I F I C A T I O N OF SPECIFICITY O F R H - H R A N T I B O D I E S
REACTION OF AN UNKNOWN SERUM WITH GROUP 0 CELLS OF
VARIOUS Rh-Hr PHENOTYPES
Anti-rh'
Anti-rh"
Anti-Rho
Anti-Rho
Anti-Rho
Anti-hr'
Anti-hr"
Anti-hr
RhiRhi
Rhirh
RhiRhi
Rhirh
rh'rh'
rh'rh
+
+
—
_
+
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
—
—
rh'rh*
rh'rh
+
—
—
-
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
—
-
-
—
-
rh
—
-
+
+
+
Rh.Rho
+
+
+
+
+
+
+
—
-+• indicates agglutination; —, no agglutination.
ing of red cells by antibodies can be detected by the antiglobulin method.6"8,64
This kind of antibody does, however, clump specific cells by its combined action with conglutinin present in serum or by such colloid substitutes as acacia
or gelatin (conglutination),67 and also when the specific cells are enzyme-treated
and suspended in normal saline. Antibodies of this variety are variously called
glutinins or blocking, or incomplete, or hyperimmune antibodies.
•The existence of these 2 major forms of antibodies of each specificity is firmly
established and generally accepted. There is some evidence that there is an additional third form of antibody that is said to "coat" red cells but lacks the capacity
to clump them when suspended either in normal saline or high colloid medium.10
However, here too, this coating can be detected by the antiglobulin method. To
take into consideration antibodies of all specificities and varieties, the serum
under investigation is tested against one or more sets of cells containing all the
Rh-Hr factors and prepared and examined by the various methods necessary for
detection of the different varieties of antibodies.
METHODS
Fresh group O, t y p e Rh z Rho cells are washed thrice in normal saline. Some of these are
retained untreated and employed for making a 2 per cent cell suspension in normal saline
286
TJNGER
VOL. 24
solution. Some are treated with trypsin (the technic for this procedure is described later
in this paper), and then employed to make a 2 per cent saline suspension of enzyme-treated
cells. Optionally, a third portion is taken to make a 2 per cent cell suspension in albuminplasma mixture, consisting of 3 parts of oxalated plasma of the same individual whose cells
are being used, plus 1 part of 30 per cent bovine albumin. Into separate test tubes is placed
1 drop of each variety of cell suspension prepared. To each tube is then added 1 drop of the
inactivated serum under test. The tubes are shaken and placed in a water bath at 37 C. for
1 hour. The cell sediment is then examined witli the naked eye after which the tubes are
gently shaken and again examined for clumping, using the scanning lens of the microscope.
After reading the results of these tests, antiglobulin tests are carried out on both the untreated and trypsin-treated saline-suspended cells. If no clumping results from any of these
tests, isosensitization by the Rh-Hr factor is believed to be absent. If any of the tests
result in clumping, antibodies specific for one or more of the factors in the test cells are
present in the serum and the variety of the antibodies, but not their specificity, can usually
be deduced from the results obtained (Table 3).
IDENTIFICATION OF SPECIFICITY AND TITRATION OF R H - H R ANTIBODIES
In the event that the screening test for sensitization is positive, the specificity
and the titer of the antibody remain to be established. The specificity of the
antibody is determined by the specificity of the antigen to which the individual
responds. The titer is dependent upon the constitutional susceptibility of the
individual, the antigenicity of the agglutinogen, the number of exposures and,
to some degree, on the dose of antigen. The specificity is determined by examining the serum against a panel of test cells the antigenic content of which, in each
instance, is known. Depending upon the blood factors present in the diagnostic
cells clumped by the serum, the specificity of the antibody is deduced (Table 5).
To determine the specificity of Rh-Hr antibodies, the panel of test cells must
have each Rh-Hr factor represented in at least 1 set and, as a control, absent in
at least 1 set. A panel consisting of cells all belonging to group 0 and of types
RhiRhi, Rh 2 Rh 2 , rh'rh', rh"rh" andrh will accomplish this. Such an ideal panel
is, or course, extremely difficult to have available, so that deviations from this
become necessary, particularly for the very rare varieties of cells. Usually types
RhtRhi, Rh 2 rh and rh may have to be substituted. This short-cut may not
permit, in all instances, the exact identification of the specificity of the antibody
or antibodies present, in which case further studies must be made.
The titer or concentration of the antibodies is determined by making a series
of tests employing progressively doubled dilutions of the serum and determining
the highest dilution in which the antibody produces a distinct (1-plus) clumping.
There are 6 different methods for titration, namely: (1) saline agglutination
method; (2) antiglobulin method; (3) trypsin-treated cell method; (4) albuminplasma conglutination method; (5) trypsin-treated cell antiglobulin method;'12 (6)
indirect blocking method. Although I use all 6 simultaneously for routine hospital work all 6 are not necessary; 3 methods are generally sufficient, and we
would select the saline agglutination, the antiglobulin and trypsin-treated cell
methods. Each of the other 3 methods detects blocking or "incomplete" antibodies, and this is well taken care of by the enzyme-treated cell and antiglobulin
methods, which, in addition, eliminate any complicating prozone effect. Furthermore, the differences in their results are largely a question of titer. The indirect
MAR. 1954
R H - H R FACTORS
287
blocking method yields the lowest titer and the trypsin-treated cell antiglobulin
method described by the author 42 ' 46 yields the highest titer. The real value of
this last method lies in the fact that it will detect antibodies in such low titer
that they cannot be detected by other methods, and also that it would aid in
detecting new antibodies. This latter prediction materialized when this test aided
in clearly identifying a new antibody, the anti-Jkb.24
When Rh-negative persons are sensitized by an Rh factor, the specificity of
the antibody that is almost always involved is anti-Rh 0 . In many such instances
anti-rh' is also present, and far less frequently anti-rh". Sensitization of Rhopositive persons35' 59' M by an Rh-Hr factor occurs only rarely and in such
instances the specificity of the antibody may be anti-rh', anti-rh", anti-rh", antihr', or anti-hr", or a combination of these. Not only may the serum have antibodies of more than one Rh-Hr specificity, but also of more than one variety of
the same specificity. Multiple Rh-Hr sensitization may be further complicated
by the presence of antibodies specific for other blood-group systems,5 so that
this may also have to be taken into account when choosing a panel of test cells.
METHODS
/ . Saline agglutination method. The ideal panel of cells all belonging t,o group 0 consists
of types K h i R h i , Rh 2 Rh 2 , rh'rh', rh"i-h" and rh. These may not all be available and then
substitutes must bo utilized such as 2 examples each of type R h i R l n or R h i r h , Rh 2 rh and
rh. If possible, rh'rh and r h ' r h should be included. T h e cells should bo fresh. T h e y are
washed once in normal saline solution and, of each variety of test cells, a 2 per cent suspension in terms of cell sediment is made in normal saline. Rows of 10 small test tubes (7-S mm.
inside diameter) arc placed in a rack. The number of rows equals the number of varieties of
test cells. A series of 10 progressively doubled saline dilutions of the serum is m a d e ; i.e.,
undiluted, 1:2, 1:4,- 1:8, etc., up to 1:512. In making these dilutions, great care must be
taken to avoid "carry o v e r " of serum of a higher concentration to a lower one. A different
pipet may be used for each dilution, or thorough rinsing of the pipet in fresh saline after
each dilution is equally satisfactory. Into the first test tube of each row is placed 1 drop of
the patient's undiluted serum. I n t o the second tube of each row is placed 1 drop of the 1:2
saline dilution. This is repeated for each successive tube employing the next higher dilution, so t h a t 1 drop of the 1:512 dilution is placed in the tenth tube.
To each of the 10 tubes in the first row is added 1 drop of one of the cell suspensions of
the panel of cells. Similarly Jx> each successive row of 10 tubes is added a different variety
of the panel of cells. The tubes arc shaken, placed in a water bath at 37 C. for 1 hour and
the reactions read in the usual fashion under the microscope, using the scanning lens. T h e
last tube showing a definite 1-plus reaction is the end point and the titer is expressed in
units equal to the reciprocal of the serum dilution in this tube. If the titer is greater than
512 units additional progressively doubled saline dilutions of the scrum must be made and
tested as just described until an end point is reached.
2. Antihuman globulin method. This method is merely a continuation of the saline agglutination method. T h e cell sediment, in each tube in which no clumping is observed, is
washed 4 times with large volumes of normal saline solution and then packed. This is to
remove all plasma because it contains globulin and would react with antihuman globulin
and produce a false-negative result. T h e saline is removed as completely as possible. To the
packed cells, 2 drops of antihuman globulin serum (rabbit) is added, mixed, and then centrifuged lightly (500-1000 r.p.m.) for 1 minute. T h e sediment is gently resuspended and examined for clumping under the scanning lens of the microscope. T h e tube with the highest
dilution showing cell clumping is regarded as the end point, and the titer in units is the
reciprocal of the dilution of serum present in t h a t t u b e .
288
UNGER
VOL. 24
S. Trypsin-treated cell method. This procedure is identical with that of the saline agglutination method, except that each variety of test cells, prior to use, is trypsin treated in the
following manner: The cells are wTashed 3 times in large volumes of normal saline and
packed. This is to remove all serum, since it contains trypsin inhibitor. One part of 1 per
cent trypsin (Difco) in standard buffer solution is added to 9 parts of these washed packed
red cells, the mixture placed in a water bath at 37 C. for 1 hour and shaken every 15 minutes.
After 1 washing with normal saline solution to remove trypsin, a 2 per cent cell suspension
is made in normal saline. These cells are then utilized exactly as described for the saline
agglutination method and the titer is also similarly determined. Other enzymes, such as
papain and ficin maybe employed in similar fashion. Enzymes damage blood factors M and
N and Duffy.44'46 Therefore, antibodies specific for the factors of these last 2 blood-group
systems cannot be identified or titrated with trypsin-treated cells.
Jf. Albumin-plasma conglutination method. This method of titration also consists of 2
stages. The steps of the first stage are identical with those of the saline agglutination
method. If the results are negative, at the end of the period of incubation the tubes are
centrifuged and with a fine Wright pipet as much as possible of the supernatant is removed
from each tube. Then to each tube is added 1 large drop of albumin-plasma mixture (1 part
of 30 per cent bovine albumin plus 3 parts of pooled, fresh, human oxalatcd plasma). The
tubes are shaken, returned to the water bath at 37 C. for 1 hour. The cells are rcsuspended
and examined for clumping as for the saline agglutination method. Again the titer is the
reciprocal of the last dilution giving a distinct (1-plus) reaction.
5. Trypsin-treated cell antiglobulin method. This method is a continuation of the trypsintreated cell method. After the readings have been made on the tubes used for the titration
of trypsin-treated cells, an antihuman globulin test is carried out on the cell sediment in
each tube in which no clumping of cells was observed. The method for this is the same as
that for the antihuman globulin titration and the end point is similarly determined. Hightiter antihuman globulin must be employed for this test. Low-titer diagnostic serum is of
little or no value. This method was described by the present author and is valuable when
the titer is extremely low, and in one instance it aided in the clear identification of a previously unidentified blood factor.
6. Indirect blocking method. This method of titration also consists of 2 stages. The steps
of the first stage are identical with those of the saline agglutination method. If the results
are negative, to each tube 1 drop of saline agglutinating anti-Rho serum is added. The tubes
are shaken and placed in the water bath at 37 C. for 1 hour and then read in the usual fashion. The tube containing the highest dilution of the serum under investigation in which no
visible clumping is seen with the scanning lens of the microscope is the end point. The
titer is expressed in units equal to the reciprocal of that dilution.
SUMMARY
<
It is essential to carry out a routine that will adequately detect Rh factors
present in the donor's blood and absent in the patient's blood so that sensitization by transfusion of such blood is avoided. The most antigenic and the one to
which persons respond most readily is the Rh0 factor. Clinically, this is the most
important factor of the Rh-Hr blood-group system. It is responsible for approximately 90 per cent of hemolytic posttransfusion reactions and of cases of erythroblastosis fetalis. Therefore, all specimens of blood are first examined for this
. by the rapid slide method. This screening test, or as it is called, Rh testing, permits
classification of persons into those who are Rh-positive (Rh0-positive) and those
who are Rh-negative (Rh 0 -negative). However, 2 to 3 per cent of specimens of
blood so classified as Rh-negative are positive for the 9ih0 (Rh0 variant or D") or
rh' or rh" factors or a combination of them. If such blood is transfused to patients
MAR. 1 9 5 4
R H - H R FACTORS
289
lacking one or more of these factors, sensitization may result. Therefore it is
necessary to examine the blood of all donors for these factors and if they are
found to be present to classify the person as Rh-positive. Only blood of donors
who are negative for the Rh 0 factor and its variant, as well as the rh' and rh",
is to be classified as Rh-negative (type rh) and to be used as such in blood transfusion. Patients, however, who are negative for the Rho factor or its variant,
clinically are to be regarded as Rh-negative whether or not the rh', rh", or both,
are present.
Rh typing is accomplished by testing erythrocytes with 3 diagnostic Rh antiserums. The various combinations of clumping reactions obtained separate all
bloods into 8 different Rh types. For Hr typing, 2 additional diagnostic serums
are employed. Thus, when tests are made with all 5 serums simultaneously (RhHr typing), 18 Rh-Hr types are identified. There are also rarer types owing to
variants of the different factors and the rh"' factor. When an untoward posttransfusion reaction results, or when difficulties in crossmatching are encountered,
it is necessary to identify as many of the patient's blood factors as possible and to
obtain a donor whose blood does not contain the factor or factors absent in that
of the patient, and responsible for the hemolytic reaction. When an individual
whose blood is negative for any Rh-Hr factor is exposed, by transfusion, intramuscular injections or pregnancy, to blood containing that factor, isosensitization
may result. If blood containing that factor is then transfused, a hemolytic reaction may follow.
The antibodies responsible vary both in varieties and specificities. Tests to
determine their presence (sensitization tests) should be carried out in order (1) to
avoid passive sensitization of the patient, (2) to be aware of the fact that sensitization exists and (3) to obtain material for research purposes.
If atypical antibodies are found, their specificity is then identified, testing the
serum against a panel of cells of known Rh-Hr type. The concentration of these
antibodies is then determined by titration and the result expressed in units.
There are 6 different methods to accomplish this, although at least 3 different
methods of titration are necessary because antibodies of the same specificity may
be of different varieties, each of which has different requirements for detection.
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