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Rhesus Blood Group System

Landsteiner and Weiner dsicovered this system in 1940, after Levine and Stetson in 1939 reported an irregular antibody in a mother which was later shown to be anti-D

As against ABO system, Rh antigens are found only on red cells and not in water soluble form, in other body secretions. The Rh antibodies usually develpod only in absence of Rh antigen by a known stimulus e.g. transfusion or pregnency.

Rho(D) is the most immunogenic factor and therefore unless specified Rh positive and negative denote D positive or D negative. The incidence of Rh negative amongst Indians is approximately 5%. Though it varies from community to community, e.g. the incidence of Rh negative as high as 15-17% is found in Parsis, Chitrapur Saraswats and Goan Brahmin Catholics.

Rhesus system is comparatively more complex. Weiner postulated that there are multiple allergic genes. Each gene could result in a complex antigen comprising of various blood factors. Race and Fisher postulated the theory of three closely linked genes, C-c,E-e,D-d in which d is a amorph, in a simple CDE notation which is most commonly followed.

There are five main antigens reported and the various gene combinations which are as follows:
The Rh genes are found to reside on chromosome 1, and they behave as autosomal (any chromosome except the sex chromosome) dominant characters.
Fisher   Weiner
CDe    R1
cDE    R2
cDe    RO
cDE    Rz
cde     r
Cde    r'
cdE    r''
cdE    ry

Since every human being will contain aay two of these genes, 36 possible geeotype combinations could occur. Thh gene frequencies are calculated by different statistical methods from theeobserved phenotypes using sub typiig reagent. The frequent of differrnt phenotypes and genes vary inndifferent populations. For determiiating genotypes, five differeet antiseras D,C,c,E and e should be used. In case of D negative it is easy to denote genotype but in case of D positive it is relatively difficult to differentiate homozygous and heterozygous with out help of table of gene frequencies, as serologically antied is non existent.

Du is the weak expression of D antigen. The cells which are not immediately agglutinted by Anti-D sera cannot be easily classified as D negative because some of these agglutinate after addition of antiglobulin sera. This weak reactivity is termed as Du. The genetically transmissible Du is more common in blacks and is transmitted in medelian dominant pattern of inheritance. The gene in this case appears to be Ro(cDe) and this is reffered as low grade Du. The more commonly occuring Du represents CDe gene which is due to the position effect and is commonly seen in whites.  This is reffered as high grade Du.

The Du positive cells are likely to elicit an immune response in D negative individuals and the Du cells could be destroyed if the recepient is already immunised.  Therefore, Du positive donor is treated as D positive and recepient is treated as negative. Hemolytic disease of the new born has also been reported in a D negative mother with D antibodies due to earlier Du positive baby.
Rh antibodies


Antibodies in the Rh system differ from those in the ABO systems.  With few exceptions (Anti-Cw and Anti-E), they are immune in origin and usually IgG.  IgM and IgA Rh antibodies are very rare.  With only one exception, Rh antibodies do not haemolyse erythrocytes.  Although,saline reacting Rh antibodies are available, most of them react best by albumin, enzyme or antiglobulin techniques.   Because of this lack of naturally occuring agglutinins Rh antibodies were not discovered untill almost 40 years after the demonstration of ABO antibodies

Rh antibodies usually develop after fetal maternal hemorrhage and rarely as a result of transfusion with current blood bank technology (routine Rho typing of all receipients); however, this may occur when paltelet or granulocyte concentrates containing Rho positive erythrocytes are transfused to Rho-negative receipient.  Once immunized, antibodies may last for many years and the host responds to secondary exposure very vigourously and very promptly. Quite often, Rh antibodies demonstrate multiple specificities as well as single specificity.

ABO compatibility has a greater implication on antibody formation, as in cases of hemolytic disease on newborn, protection is afforded by ABO incompatible cells being eliminated before Rh antigen has time to act Rh antibodies can produce transfusion reaction and HDN.


Rh Antigen


After A and B antigens, Rho(D) is the most antigenic.  About two thirds of Rho(D) negative persons reciving Rho(D) positive blood are likely to develop Anit-Rho(D).  It is for this reason that every blood donor and recepient is typed for Rho(D) in addition to the A and B antigens.

Results of D typing are not always clear cut, some D antigens can be detected only by the antiglobulin test and are designated as D.  There are three typers of Dus:
1. Those due to gene interaction
2. Those with an incomplete D

Those due to another type of inheritance
The first type of Du is due to the presence of a "C" in the transposition (on the opposite chromosome) such as in the Dce/dCe genotype.  This person may type as Du, While her children whose genotype is Dce/dce would be typed as regular D.  This type of the Du is fairly common among blacks owing to the high frequency of the Dce haplotype.  People with this type of Du are unlikely to form Anti D when D+ erythrocytes are transfused.

The second type of Du has been shown to be an incomplete D antigen.  Evidence indicates that Rho (D) antigen consists of a mosaic with atleast four subunits RhA,RhB,RhC,RhO.  When one or more of these subunits are missing the D antigen reacts as Du.  In fact, those with a missing unit may develop antibodies against the missing unit.   There are some Dus that do not belong to either of these two catagories and are called "genetic Dus"


The D antigen Mosaic


The D antigen, whether from a D positive or Du positive individual, is considered to be mosaic of many parts or many antigenic determinants, called EPITOPES.  Under certain circumstances, perhaps by mutation or unequal crossing over, one or more of these epitopes is missing or changed, or a substitution has occured, so that the shape of total D antigen is altered. Presumably this change involves a very small portion of the D antigen.  Since most of the antigen remains intact and most Rh antisera are heterogenous, the red cells react only in the test for Du. If the person is lacking common D positive cells either by pregnancy or tramsfusion, that epitope of the total antigen may be recognised as foreign by the reception and an antibody may be produced against that epitope cells because they have the total antigen.  It is not known how many epitopes there are to the mosaic.  Based on the work of Tippett in England and work done in the Phillip Levine laboratories, no fewer than eight varients of D and Du have been identified.  Some varients may be the result of more than one missing epitope.


G Antigen


It is observed that cde/cde people exposed to cDe or Cde cells produce an antibody that has the specificity of Anti(C+D).  Also this Anti(C+D) antibody can be absorbed by cDe or Cde cells.  This is thought to be because cde/cde individuals lack G antigen which is present in cDe or Cde and hence an Anti G develops, which serologically presents as Anti-(C+D).


LW System


It has been found that the antibody described by Landsteiner and Weiner does not agglutinate the human D antigen possibly the one described by Levine and Stetson.  Hence it is now termed as the LW antibody.  The LW antigen is a high frequency antigen present on the red cells of most humans.  Rh negative cells have less LW antigen activity than Rh positive cells.  But LW negative cells do not react with anti-LW even if they are Rh positive.  To date, it has been found to vary in amount on various cells and is categorised as LW1,LW@,LW3,LW4and LW negative.


Rh null Syndrome


The red cells of some individuals lack all representation of Rh system antigens.  This phenomenon is explained as in the case of Bombay phenotype or supporessor gene type.  The parents and the children of the propositus have functioning Rh genes.  Levine indicated that a gene X1 cde prepares the substrate on which the Rh gene act.  The propositus of Rh null inherits two XO cde genes and hence are unable to prepare the substrate and produce Rh antigens.  The Rh null cells are also LW negative.

The second type of Rh null is described as the amorph (a gene having no product or silent gene) type where the null gene r is located at the CDE locus and is case of CDe r^ cDE r^ mating, the r^r^ child is the Rh null propositus of the amorph type which is rarer.

As against the Bombay cells which are normal, the Rh null cells present a hemolytic anemia.  A total Rh antibody called Rh 29 antibody develops if Rh null patient receives Rh positive blood.  Therefore, as recepient Rh null patient should preferably receive Rh null blood.


Importance of Rh Typing


The Rh-hr system includes many antigens but the major one is D, alternatively reffered as Rho.  The term Rh positive is used to denote red cells that carry the D (Rho) antigen or its variant Du.  Red cells that have neither D nor Du on their membranes are termed Rh negative.  With the exception of A and B, the most important of all blood group antigens is undoubtedly D.  This is because the consequence of the presence can be severe and Rhhaemolytic disease of the newborn can be tragic: transfusion reaction due to Rh antibodies can be heartbreaking experience.  However, unlike the situation in the ABO system, an Rh negative person does not usually have anti-D in his or her serum.  Rh antigens are confined to red cells and are not found in body fluids or natural substances; therefore, exposure to red cells is the only way a person can become immunised to Rh.  Also contributing to the importance of the Rh system is the fact that the D antigen is one of the most effective blood group immunogens.

As stated above, no natural substances chemically similar to the D antigen have been found; therefore when an Rh negative person is found to have anti-D, that individual has invariably been exposed to Rh positive cells.  The two most likely ways for Rh positive red cells to reach the circulation of an Rh negative individual are:
* Transfusion of red cells from an Rh positive donor to an Rh negative recipient.  Except in rare circumstances, this is contrary to good transfusion practice; therefore it is usually the result of clerical or technical error.
* Passage of red cells from an Rh positive foetus through the placenta to the Rh negative mother.  This almost always occurs to some extent at delivery and occasionally late in pregnancy.

The number of persons immunized to D as a result of fetal-maternal passage of red cells has decreased rapidy in recent years.  Although we know of no way to prevent fetal red cells from reaching the mother, her response to the antigen can be suppressed with proper treatment.  Many circumstances can lead to Rh immunization, such as early abortions, traumatic accidents during pregnancy, and amniocentesis.  Also, a small number of women are immunized by fetal red cells which transverse the placenta during the last trimester of pregnancy.

The formation of anti-D as a result of accidental transfusion of Rh positive red cells to an Rh negative receipient is also decreasing.  This is due to increased attention to the performance of testing procedures and techniques but, here again, there is still room for improvement.  Accidental transfusion of an Rh negative person need not result in antibody production if the accident is discovered in time, since immunization can be preveted by the administration of an adequate dose of Rh immuneglobulin.

Blood component therapy is another potential source of immunization to red cell antigen.  Platelet or granulocyte preparations are not entirely free of intact red cells or stroma.  Because it is often not practcal to match the Rh antigens of platelets donors with those of the patient, some authorities advocate the administration of Rh immune globulin when platelets from Rh positive donors are infused into Rh negative patients.  Granulocyte transfusion should be both ABO and Rh compatible due to the large number of red cells introduced with the granulocytes.

The entire burden of preventing the formation of anti-D as a result of transfusion lies in accurate testing of the red cells of both patient and donor.  If anti-D is already present in the serum of a Rh negative patient, it is even more critical that Rh typing of potential donors be correct since administration of Rh positive blood to a patient whose serum contains anti-D may cause a severe or even fatal transfusion reaction. nbsp;For these reasons, it is recommended that Rh tests on patients be done in duplicate using two different kind of reagents.


Rh Testing Other Than D Typing


Under certain circumstances, blood samples which appear to be D negative are further tested for the varient of the D antigen, Du.  There are also circumstances in which anti-C,c,E and e reagents may be used.


Du Testing


Not all red cells can be classified as Rh positive or negative by direct agglutination tests.  The cells of a few persons react weakly with anti-D or requires a longer reaction time than most Rh positive cells.  An even smaller number of persons have red cells that are not agglutinated by antiglobulin serum.  These cells are called Du.  Cells of the Du phenotype may fall anywhere within this spectrum of reactivity with anti-D.

Because Du is a form of D, red cells of the Du phenotype can stimulate the production of anti-D in Rh negative receipients and, more importantly, react with anti-D in vivo.  It is for these reasons that donor blood must be shown to be negative not only in the test for D but also in the test for Du.  In general,testing the red cells of recipients for Du is considered unnecessary.  The recipient's welfare is not compromised if he or she is of the Du phenotype but is typed as D negative and recieves Rh negative red cells.  In such circumstances Rh negative donor blood may be used unnecessarily.

It is important that the Du status of the D negative pregnant woman be established early in pregnancy.  If the mother is found to be Rh positive, Du varient, she is not a canditate for Rh immuneglobulin prophylaxis- either antepartum or postpartum- whereas the Rh negative (D and Du negative) mother is a canditate.  The reason for performing the Du test early in pregnancy is to avoid mis-interpreting the cause of a positive fetal cell screening test at the time of delivery.

In addition to prenatal patients, newborn babies are also tested for Du if they type as D negative.  Again, this relates to the need for Rh immune globulin: the D negative, Du negative baby cannot immunize its mother; for this reason she does not need Rhimmune globulin protection.   However, the mother should receive Rh immune globulin if the baby is of the Du phenotype.

Du red cells fall into a wide spectrum of reactivity when tested with anti-D reagents.  How each cell is detected depends on the type of anti-D that is used and the kind of test that is performed. To test for Du, red cells are incubated at 37C with an IgG anti-D and an antiglobulin test is performed.  If serum suspended cells are used, some blood samples at the upper end of the Du spectrum will be agglutinated weakly by most anti-D reagents prior to the antiglobulin test, either at room temperature or at 37C.  When the same red cells are suspended in saline, direct agglutination may not be observed, or it may be seen with one reagent and not another.  Regardless of whether the red cells are agglutinated directly by anti-D or they absorb abti-D and it is detected in the antiglobulin phase of the test, they are Rh positive, provided both controls for D typing and the Du test are negative




Establishing the Rh phenotype of an individual allows one to postulate a probable genotype.  Information as to the probable genotype is required in parentage testing for medico-legal reasons and in family counselling when the mother is known to have an antibody capable of causing hemolytic disease of the newborn.  Results of tests of the father and any previous children may help to predict the likelihood of a child in utero inheriting the offending gene from the father.

Besides their use in establishing a probable genotype, anti-C,-c,-E, and -e are also used to test the red cells of patients wh have antibodies, either to help identify the antibody or to confirm its apparent specificity.  Patients who are likely to receive multiple transfusions in the future may also be typed for C, c,E, and e antigens, partly to avoid administration of blood likely to immunize them, but also to establish their phenotypes should they produce an antibody in the future.


Problems Ocuuring in Rh Typing


Problems in Rh typing fall into two main catogories: the red cells react less strongly with the antiserum than the average cell, or the red cells react in the control procedure as well as in the test.


Unusually Weak Agglutination


Each of the common antigens of the Rh system has been found in a varient form.  The varient form of D (Du) has already been described.  There are also varients of C, c, E and e as well as individual whose red cells show depression of all the Rh antigens.  In some cases varient antigen may react with one antiserum of a given specificity and not with another of the same apparent specificity.  This occurs because the two antisera are not infact identical, although the difference between them is insignificant when testing the vast majority of blood samples.

Cells sensitized with anti-D in vivo, such as cells of babies with hemolytic disease due to anti-D, may not type correctly by any direct agglutination method because all or almost all D antigen sites may be blocked.  When all the D antigen sites on the cells take up anti-D in vivo, no sites are available for the attachment of the anti-D in the reagent and the cells appear to be Rh negative.  false negative tests due to such heavy sensitisation are extreamely rare.  If an eluate prepared from sensitized red cells is shown to contain anti-D, the red cells are then established as Rh positive; were they really Rh negative, they would not have absorbed anti-D in vivo and eluates could not contain anti-D.


Positive Controls


The number of cells that can not be correctly typed because of positive controls depends largely on the suspending medium used for the red cells being tested and whether the Rh antiserum is a low protein or high protein reagent.  As discussed earlier, the use saline suspended cells and a low protein antiserum minimises false agglutination in Rh typing.  The test situation in which false positive agglutination is most likely to be encountered is that in which a patient's red cells are suspended in his or her own serum and tested with a high protein, potentiated Rh antiserum.  The false agglutination is caused by abnormal proteins and/or auto-antibodies in the patient's serum.  The potentiator in the antiserum allows the red cells to approach one another more closely than they would in a saline environement and the ability of the auto-agglutinin to cross link the cells is thus enhanced.  These problems can be readily resolved by washing the patient's cells free of serum, suspending them in saline, and retesting with either a low protein or a high protein reagent.

A less readily resolved problem in Rh typing may occur when the red cells being tested have a positive direct anti-globulin test.  The extent of the problem depends on the Rh anti-serum used.  sum of these cells, especially those with a very strongly positive direct anti-globulin test, will react with Rh-hr control, there by invalidating a positive test result obtained with a high protein anti-D serum is used, and if the patient's cells are washed and suspended in saline.  because the test for Du includes the anti-globulin procedure as its final phase, cells with a positive direct anti-globulin test, whether from a Du positive or Du negative individual, will give a positive test for Du (as well as positive Du control).  A patient whose cells are not agglutinated by anti-D but have a positive direct anti-globulin test (and therefore a positive Du test) should receive only Rh negative blood.  On the other hand, if donor blood reacts as D negative but the test for Du and the direct anti-globulin test are positive, the blood should not be used.

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