The Rh System
Unlike the situation with A&B ,persons whose red cells lack the D –antigen donot regularly have anti D in their serum . Formation of the antibody almost always from exposure - through either transfusion or pregnancy – to immunizing red cells possessing the D antigen .
The immunogenicity of D that is to say the likelihood of its provoking an antibody if introduced into a D negative recepient is greater than that of virtually all other red cell antigens studied .
Of D negative persons who receive asingle unit of D positive blood 50 –70 % can be expected to develop anti D .
New discoveries have brought the total of Rh –related antigens to over 40.
Du is not to be considered an antigen separate from D itself , but merely a weaker form of D . Du form of the D antigen seems to be substantially less immunogenic than normal D . Experimental transfusion of 68 units of Du blood into 45 D negative recepient ( 15 of whom were admittedly receiving immunosuppresant therapy ) failed to produce a single example of anti D , although one person in the series made anti E & a second made anti K.
Du cells may suffer accelerated destruction if introduced into the circulation of recepient whose serum already contains anti D . A severe hemolytic transfusion reaction has been reported in these circumstances , & a case of hemolytic disease of the newborn occurred in a Du infant whose D negative mother has been immunized by the cells of D positive fetus during an earlier pregnancy .
Rh Antibodies :
· most Rh antibodies result from immunization by pregnancy or transfusion except anti –E &anti Cw that occur without known stimulus .
· D is considered the most potent immunogen followed by c (small) &E .
· Most Rh antibodies react best in high protein medium .
· Immunization usually persists for many years even if levels of circulating antibody fall below detectable threshold ,subsequent exposure to the antigen often results in a rapid secondary immune response .
· Rh antibodies donot bind complement when they combine to their antigens , at least to the extent recognizable by techniques currently used .
· Anti D seldom shows any difference in reactivity between cells with homozygous or heterozygous expression of the D antigen.
· Dosage effect can sometimes be demonstrated with some antibodies directed at the E ,c ,e antigens & occasionally at the C antigen .
· Some Rh antibodies ( anti c ,anti E ) occur commonly together & the components are not invariably of equal strength , the anti c component may be substantially weaker & may not be detectable at the same time the anti E is found . this antibody combination may cause a hemolytic transfusion following transfusion of seemingly compatible E negative , c positive blood .since anti-c occurs so commonly with anti E in immunized people whose cells are E negative ,& c negative , it is prudent to select blood of the patient own R1R1 phenotype for transfusion even when the presence of anti c cannot be demonstrated by the test procedure routinely used . serum that contains clearly detectable anti c doesnot as consistently contain anti E as the patient can easily have been exposed to c without being exposed to E .
· There is less clinical significance ( importance ) in definetly demonstrating anti E in serum known to contain anti c because R1Rz (Cde /CDE ) phenotype is rare .
Rh Grouping tests :
Tests for the other Rh antigens are performed when ther are specific reasons for additional testing such as :
1- Identification of unexpected antibodies .
2- Obtaining compatible blood for pt with an antibody .
3- Investigation of disputed parentage or other family studies .
4- Selecting a panel of donors known to lack certain antigens or in an attempting to determine whether a person is probably homozygous or heterozygous for D antigens .
· In selecting blood for recepient whose serum contains a comparatively weak Rh antibody , testing with reagent antisera may allow more reliable detection of antigen–negative cells than relying merely on negative cross match .
· Testing the antibody –markers own cells may help to provide conrmation of specificity& will suggest which other Rh antibodies might develop .
· In most cases it may be assumed without actual testing that cells negative for the E antigen are e positive .
· High molecular weight medium may cause spontaneous agglutination of red cells coated with immunoglobulin as in autoimmune conditions & hemolytic disease of the newborn . so it is recommended to use reagent containing high molecular weight medium as negative control which is provided by the manufacturer .using 22% or 30% bovine albumin detect even fewer false positives .
· Too heavy suspension in the tube or too weak cell suspension in the slide test may result in poor agglutination .
· Either a diluent control test or direct antiglobulin test must accompany Du test procedure .
· Saline agglutinating Rh antiserum should be used when the control system for high –protein anti D gives a positive test that persist even when washed red cells are tested .
· Cells with positive direct antiglobulin test can usually be tested successfully with antiserum that contain saline agglutinating antibodies & no additives that promote spontaneous agglutination of immunoglobulin – coated cells. .
· Two kinds of saline –reactive Rh antisera .
1- Traditional saline reagents are made from raw material containing predominately IgM antibodies .
- Require the test to be incubated at 37 c usually for 15 min. or longer
- Cannot be used for the Du test because IgM antibodies generally perform poorly in the indirect antiglobulin test .
2- Chemically modified IgG antisera .
- The IgG antibodies in the raw material are converted to direct saline agglutinins by chemical reduction of their interchain disulfide bonds by using 0.01M DTT .
- Saline –reactive antisera made from reduced & alkylated IgG antibodies show stronger reactivity than those prepared from IgM antibodies &are usually suitable for tests performed on slide .
- Chemically modified IgG antisera may be confidently used when immunoglobulin coating precludes the use of high –protein anti D on a red cell sample .
- These chemically modified anti D sera are just as suitable as high –protein reagents for the detection of Du by the indirect antiglobulin.
D Grouping in hemolytic disease of the newborn :
In this case the direct coomb,s test will be positive ( this means that immunoglobulin coated the cells ) this will give false positive if tested with high –protein anti D reagent because of spontaneous agglutination so saline reactive reagent should be used .
In some cases the cells will be heavily coated with anti D & no D antigenic sites to react with the reagent .So the antibody should be eluted from the cells by incubation at 45C to give chance for the anti D reagents to react with D antigens .
You should think about this case if the cells give negative result with anti D but positive by Du test using indirect antiglobulin test . To solve it you should do direct coombs first if negative ---report Rh (Du) positive .
If positive you should do the elution technique at 45 C & retest with anti D reagent .
بقلم
عادل عبد الحليم السلايمه
اخصائي طب مخبري
مسؤول مختبر جودة المياه / بلدية الخليل
الخميس 30 أكتوبر - 8:37
