Alexander S. Wiener

A New Test (Blocking Test) for Rh Sensitization.

As was first shown by Wiener and Peters, 1 sensitization of Rh-negative individuals against the Rh factor can often be detected by in vitro tests for anti-Rh agglutinins in the individuals plasma. However, it was soon found 2 , 3 that there are many Rh-negative patients who are strongly sensitized to the Rh factor, as proved by the occurrence of an intragroup hemolytic transfusion reaction or a baby with erythroblastosis (hemolytic disease of the fetus and newborn), yet the plasma does not contain demonstrable anti-Rh agglutinins. The purpose of this paper is to describe a new in vitro test, the “blocking test,” with the aid of which Rh sensitization can be detected in many of these problem cases. The first blocking experiments were tried in 1941, when retests of some of the stored post-transfusion sera from the patients described by Wiener and Peters the year before showed these sera to be no longer active. It occurred to the writer that the antibodies might still be present and capable of combining with the test cells but incapable of agglutinating the cells. When the mixture of test cells and apparently inactive serum was first allowed to combine and subsequently active (capable of agglutinating Rh+ cells in control experiments), anti-Rh serum was added, it was found that the test cells were not agglutinated, apparently because the action of the active agglutinin had been blocked. However, the results obtained were irregular, and therefore experiments on this “blocking test” for Rh antibodies, which is a counterpart of the inhibition test for haptens and group-specific substances, were temporarily abandoned. Recently, when more satisfactory anti-Rh testing sera became available, the experiments were resumed.

Genetic Theory of the Rh Blood Types.

Summary Three varieties of anti-Rh agglutinins have been encountered, designated as anti-Rh (standard), anti-Rh1, and anti-Rh2, and agglutinating, respectively, approximately 85%, 70%, and 35% of bloods from white individuals in New York City. Human sera containing agglutinins anti-Rh and anti-Rh1 together are designated anti-Rh sera containing the two agglutinins anti-Rh and anti-Rh2 are designated anti-Rh”. By means of the 3 sorts of Rh agglutinins, 5 varieties of Rh agglutinogens are demonstrable, designated as Rh1, Rh2, Rh, Rh′, and Rh”. These in combination give rise to 8 Rh blood types (including the Rh-negative type), of which all but one, the rarest, have actually been encountered. A theory to explain the heredity of the Rh blood types is proposed in which the existence of 6 allelic genes is postulated. Evidence in support of the theory is presented and some of the implications of the theory are mentioned.

Diagnosis and treatment of anemia of the newborn caused by occult placental hemorrhage

Abstract 1. 1. Attention is called to a previously unrecognized entity, namely, severe anemia of the newborn caused by unapparent hemorrhage from the fetal surface of the placenta. 2. 2. Two cases are described of newborn infants exhibiting this picture of severe posthemorrhagic anemia, one of whom recovered following treatment by blood transfusion, while the other died despite transfusion. In both these cases the mothers were Rh positive as revealed by routine antenatal blood tests, and isosensitization was ruled out as playing any role by detailed serological tests and the absence of any icterus of the cord serum. 3. 3. In a third fatal case of severe anemia in the newborn baby of a sensitized Rh-negative woman, the anemia was apparently due primarily to hemorrhage rather than hemolysis. The significance of this finding in relation to the treatment of such cases is that if exchange transfusions are given to such erythroblastotic babies, a margin of 150 c.c. instead of only 50 c.c. should be maintained between the amount of blood injected and the amount of blood withdrawn, in order to correct the reduction in blood volume as well as the anemia.

New Data on the Distribution of the Rh Blood Types.

Summary New data have been compiled with the aid of improved testing sera, on the distribution of the 8 Rh blood types among white individuals in New York City. Statistical analysis of these data by the gene frequency method yielded results in satisfactory agreement with the expectations under the six-gene theory. The distribution of the Rh blood types among Negroes is strikingly different from the distribution among white individuals; the most pronounced differences involving the frequencies of type Rho and the so-called intermediate types. Striking differences between whites and Negroes also exist with respect to the distributions of agglutinogens P and Hr.

Treatment of erythroblastosis fetalis by exchange transfusion: Statistical analysis of results*

Summary Among thirty-six stillbirths causedby Rh sensitization, all but one of the patients had Rh antibody titers of 16 units or higher by the albumin-plasma conglutination method. (In the single exceptional case, where the albuminplasma titrations were negative, it was possible to show that antibodies were present, but of an unusual type, since the titer was high by the antiglobulin and enzyme techniques.) Therefore, when using antibody titer as a criterion of severity, it was decided to consider 8 units by the albumin-plasma titration method as the upper limit for cases classified as “mildly” affected. The results of treatment by exchange transfusion in a series of 222 erythroblastotic infants were compared with the results obtained in a series of sixty-seven erythroblastotic babies who were left untreated or given one or more simple transfusions. Among “mildly” affected babies, i.e., those whose mothers had antibody titers of 8 units or less, no significant difference in the mortality rate in the two series was demonstrable. On the other hand, among severely affected babies treated by exchange transfusion, the mortality rate was 15.5±2.0 per cent, while among the babies receiving simple transfusions or no treatment, the mortality rate was as high as 46.4±6.2 per cent. The difference in mortality rate, 28.9±6.5 per cent, is statistically significant. During the past few years changeshave been introduced into our method of carrying out exchange transfusions. In the method now used by us, part of the citrated plasma is removed from bank blood not more than three days old, which has been allowed to sediment in the refrigerator in order to reduce the volume to 400 ml., and the resulting concentrated blood (hematocrit, approximately 0.50) is used for the exchange transfusion, equal volumes of blood being withdrawn and injected. In more severely affected babies, a second such exchange transfusion is given twenty-four to forty-eight hours after birth. Among seventy-eight erythroblastotic babics treated in this way, of whom fifty-three had mothers with antibody titers above 8 units by the albumin-plasma method, the mortality rate was only 5.1±1.6 per cent. (In this discussion, mortality rate is used to include neurological sequelae as well as deaths.) Case reports are presented in orderto illustrate the course of Rh sensitization in women followed through several pregnancies and to compare the results of treatment of several erythroblastotic babies born to the same mother. A case is cited which demonstrates the importance of icterus per se as a criterion of severity, and therefore this sign is a strong indication for treatment by exchange transfusion even in the absence of anemia. In view of the innocuousness of exchange transfusion carried out by the radial artery-saphenous vein technique and the excellent results obtained by this treatment, our tendency has been to widen the indications for its use in treating erythroblastotic babies.

Simple Method of Preparing Potent Blood Grouping Sera.

Conclusions The intramuscular injection of small amounts of autoclaved saliva (0.2 cc) from secretors of groups A and B often stimulates a considerable increase in the isoagglutinin titer. This method simplifies the largescale production of potent blood grouping sera.

Demonstration of the Properties A, B, M, N and Rh in Red-Cell Stromata.

Summary From 250 cc of packed erythrocytes approximately 10 cc of red-cell stromata can be obtained. The titers of the group substances, A, B, and Rh, in the stromata are almost proportionately higher than the corresponding titers of the original erythrocytes. However, the calculated rise in titer was not obtained for properties M and N, suggesting that these may have been damaged by the processing of the cells. The possible use of red-cell stromata for treating sensitized Rh-negative individuals is discussed.

Treatment of erythroblastosis fetalis, with special reference to sensitization to Rh-Hr factors other than Rh0

Summary To date, 166 babies with erythroblastosis fetalis caused by Rh 0 sensitization have been treated by our latest modification of exchange transfusion by the radial artery-saphenous vein technique. In this latest modification, partially concentrated blood (hematocrit 0.5), preferably not more than three days old, is used. This is prepared by removing part of the citrated plasma after sedimentation has occurred. Depending on the severity of the disease and the size of the baby, from 450 to 900 ml. of such concentrated blood is used for the transfusion. In severe cases the exchange transfusion is repeated. In no instance are more than two exchange transfusions deemed necessary. Among the 166 babies treated this way, only five babies died or had neurological sequelae, a crude mortality rate of only 3.0±0.9 per cent. On the other hand, among babies treated by our earlier modifications, or by only simple transfusion, or receiving no treatment at all, the mortality rate ranged from 17 to 21 per cent. The difference in results is even more striking when the severity of the disease is taken into account. Thus, among severely affected babies who received no treatment the mortality rate was 65.5±10.6 per cent; among those receiving simple transfusion therapy the mortality rate was 42.1±7.8 per cent; among those treated by our previous methods of exchange transfusion the mortality rate was 19.8±2.6; while among those treated by our latest modification of exchange transfusion, the mortality rate was only 3.8±1.0 per cent. The cases of erythroblastosis fetalis caused by sensitization to Rh-Hr factors other than Rh 0 pose an important problem, since the routine antenatal screening test for Rh 0 sensitization does not take them into account. Such cases are rare, but when they occur they are just as serious as instances of Rh 0 sensitization. Thus, among nine cases of erythroblastosis fetalis caused by hr′ sensitization, one baby died before treatment could be instituted. Of the remainder, four were treated by exchange transfusion and recovered uneventfully, while among the four treated by simple transfusion one died and one of the three who survived had severe neurological sequelae. A simple screening test for Rh 0 antibodies consists in testing the serum of the patient against ficin-treated cells of types Rh 1 Rh 1 , Rh 2 , and rh, respectively. This test should be carried out on all patients in labor admitted to the obstetrical service, and on patients admitted to the surgical or other services, who might require blood transfusion therapy. Case histories are presented, which illustrate the value of this screening test. It is pointed out that in cases of hr′ sensitization the antibodies, even when present in high titer, may react only in tests against enzyme-treated cells and may fail to react in the conglutination and antiglobulin tests. This accounts for puzzling, severe cases of erythroblastosis fetalis in which the direct antiglobulin test for coating of the babys red cells by Rh-Hr antibodies is negative. It is emphasized that certain antibodies, such as anti- F and anti- J are not detected by the enzyme test, so that the screening test against ficin-treated cells should supplement and not replace the other tests for isosensitization.

Heredity of Rh Blood Types. V. Improved Nomenclature; Additional Family Studies with Special Reference to Hr

Summary New additions to and further simplifications of the designations of Rh blood types, subtypes and genes are proposed in order to encourage the more general use of the Rh and Hr tests by medical men and geneticists. Investigations on the hereditary transmission of the 8 Rh blood types in 300 families with 620 children yielded results in conformity with the theory of 6 major allelic genes. Studies on the Hr factor; in 81 families with 126 children and on a random series of 645 Caucasians in New York City fully support the theory that factors Rh′ and Hr′ are related to each other genetically and serologically like the agglutinogens M and N.

Vitamin A and infection.

Summary Three groups of children whose ages varied from seven to seventeen years were studied. One group received supplementary halibut liver oil in the diet; the second, viosterol; and the third group served as a control. No statistically significant difference in the incidence of respiratory infections was found.