Understanding the Immunobiology of Weak D and D Variants

Namaste everyone I'm Dr M Chri senior consultant in New Delhi today we'll be talking about understanding the immunobiology of weekd and D variants this is transconnect April 2024 so let's begin with this new topic of weekd very important and uh also we'll be covering some part of the partial D the molecular aspect of it and understanding actually where to imply the VD and its variant so I'll be talking about the RH system the genetics and biochemistry RH antigens RH genotyping and some RH deficiency syndromes so we all know that RH is the most important blood group system after AO in transfusion medicine and it is very critical component of the pre-transfusion testing it is very immunogenic complex and polymorphic and so what is polymorphic polymorphic means many forms so it is got various forms various Al of the normal form are present so currently there are 55 antigens that have been characterized and they have five principal antigen that is D capital D Capital C capital E small C and small e and these are responsible for clinically significant antibodies finally note that there is no small D so these RH antibodies how are they produced and how are they different from the AO antibodies Is AO are naturally occurring and these are formed only after exposure to the foreign red blood cells and they are commonly associated with the hdn or the hemolytic disease of the newborn and also with the transfusion reaction the term D positive and D negative actually refers to the presence and absence of the RH antigen D so if you are D positive you are uh that is then you are RH positive if it is D negative you are RH negative so that is how it is uh termed as now the basic importance of why we need to know v d and variant D is that we do not want to transfuse an RH positive blood to an Rh negative individual and similarly in females uh pregnant females we do not want to immunize them by giving RH positive if they are RH negative and also where to give the rum or RH imunoglobulin to the mothers who are RH negative so so that needs to be taken care of and that needs to be understood so coming to the landmarks in the history there are 1939 Levine and Sten were the first who describ the HDR in Ops patient and postulated that the fetus and father possess the same common factor that the mother lack in 1940 laner and Wier used antisera from ginipic and they found that with red cells razes Mass to distinguish RS positive from the RS negative red cells very important genetics of R Gene please remember there are two genes which are located on chromosome one that is the rhb Gene and in the rhc gene so the gene rhd codes for the presence of rhd protein whereas RH c e the other Gene which I which we just discussed quotes for RH Capital C Small E RH small C capital E RH small C Small E or RH Capital C capital E so rhd and rhc both are codominant so both are expressed and there have been numerous mutations that I been described in the r Gene and that's why so many Al and so many forms that you can see greater than 250 Al have been determined in RSD genes and 50 Al have been found in the RX C now these many imitations good thing about them is that they are rare and they do not change the serology so rest assured everything is okay so this is how the gene structure looks like just a minute so you can see this is the LOI 1 Loi 2 so you can see that rhd gen there are 10 exons similarly rhc Gene also has 10 exons so in RH positive individuals you can see these 10 exons are there and this will give you a d antigen similarly rhce you have the 10 Exon hair as well so when you actually have uh you place them together they are like in a Hair Pen Arrangement so 10 will come here and one will come here and that's how it it is uh present in the body in the chromosome so because they are so close to each other there is very less chance that there is a crossing over and that is why there are other mechanism by which a new blood group may be formed one of them is single nucleotide polymorphism then there are other forms like Gene conversion Gene insertion deletion Etc so if you seen an RS positive individual you will have the D antigen if there the person is Rh negative you can see there is no D antigen because the absent of the gene here so if there is rele of that Gene there will be no and that is why no de antigen however they are independent of each other both these genes and that's why if you have an rce Gene you will still have the C antigens so you can see they are identical the rhd and the rhc and they are almost 97% identical to each other there's another Gene that which we need to care about and that is the RH AG or a CO expressor gene or a regulator Gene which controls the RH so this is known as RH AG RH antigen expression is what it's linked in and it resides on the chromosome 6 this peptide is similar in structure to the RH protein only difference being that it is glycosilated that is that it has a carbohydrate to it so within the RBC membrane it forms a complex with the RH protein and we'll be discussing about this RH complex which is Rh Gene and Rh AG Gene along with the various other cytoskeleton proteins so it this should be coexpress so once it is expressed it gives the successful expression of the other RH antigens by itself this glycoprotein does not express any RH antigens and mutation of this Gene may result in the alteration in the rhd and C rhce proteins so this is the RH membrane complex if you see this is Rh Gene along with this is Rh AG Gene now RH AG Gene is or linked to the protein 4.2 anarine spectrin Band 3 all of this is which is involved in the structure of the RBC and that is why sometimes when you have a pathology in these proteins you may also have a pathology related to the RBC membrane defect like stomatocytes elliptocytes spherocytes Etc so you can see that this is linked together with a protein or integr Associated protein known as cd47 and this whole thing is a complex or the RH membrane complex which is very important for various diseases so when do you call an individual R positive we have already discussed if you have one or two copies of RSD genes that is from one parent you have one RSD and from the other parent either you have or don't have so if you have e even if you have a one RSD Gene copy you are terms as RSD positive if there is a mutation in the rhd antigen and then you can have a Rh negative phenotype when you don't have any copy of RTG so it is more common in European and as Asian it is less common but in europian if you see the mechan mechanism is the deletion of the RSD Gene that is no RSD Gene but you have two RSC gen so if there is no RSD Gene you are rs negative whereas if you have in see in the Asian there is a mutation in the RH Gene there is no deletion so in European there is generally a duration of the rhd gene in Asian ethnicity there is mutation in the rhd gene in African ethnicity there is an unusual form of rhd Gene which is known as rhd pseudo Gene now this is because of some missense mutation in Exon 5 and six or a nonsense mutation in a Exxon 6 nonsense mutation basically means that you have a stop codon which is produced and this causes further uh translation of that protein to be inhibited coming to a small uh pictorial representation of the biochemistry of the rht gene you can see that both the genes are here rht protein is here rhc is protein is here and you have the RH Associated glycoprotein you can see the both of them rhd and Rh see there is a uh there is this proteins are traversing through the membrane this is a transmembrane protein and it goes through the cell membrane 12 times so 12 times there is a loop so this total amino acids are the same in both 417 amino acids are present and they are both non- glycosilated whereas RH AG does not have any RH protein these have Rh proteins so you can see that these are the proteins that are there here also you can see now this is very important because this is the part that is exposed and these are the epitopes against which an antibody may be produced and this happens in a partial D which we'll be discussing later but if you have a mutation in the inner area or the inner cytoplasm inner inner part of the cell membrane along with the inside of the cell membrane you can see this is because of the uh this leads to the formation of v d so for partial D basically you have a mutation in the iops are the outer part and for BD you have a mutation in the inner part of the cell membrane so RH antigens are highly immunogenic we are all aware of that they are present on the red blood cell as transmembrane proteins they do not exist in soluble form and they are very immunogenetic in the sense that even 1. 0.1 mL of RH positive RBC can stimulate an antibody production in then Rh negative individual so immunogenicity of Rh antigens you can see the capital is the most immunogenetic then we have the cap small C capital e capital C and G coming to the main topic that is the weak D variation of D antigen expression now we already know that there is a d antigen there is there are two genes that is rhd and rhc that are involved so this when we started doing the serology many of times a patient would come to your lab and would say that I was knowing my blood group as rhd positive or rhd negative and now you have suddenly given me an R positive so basically what you're doing is you are using different anticas so somebody was typed with a different anti gave the result as RS negative but when you type with a better S or S which contains a blend of IGM and IGG you would be able to detect the we D generally the reaction strength is also very important in the sense that you tend to have a reaction strength of four plus in a normal individual but if you see a weak D the reaction strength is one or two plus in those cases the RBC carrying weer D antigens have historically been referred to as du type we used to do du typing for them later it was known that it was the same thing and these individual did not produce antiu they produced only the anti-d so we D is the right terminology and there are various mechanisms by which we D antigen expression happens important among these are the C in the trans to RSD weak D partial d d that is DL and D epitopes on RC protein coming to the first mechanism C in trans to RSD very important this is a position effect and Gene interaction effect uh basically there is a steric endurance so you can see that whenever there the D and the C capital D and the capital c is trans to each other that means they are on the different uh the eles are on the different chromosomes that is capital D is here you can see and capital c is on the other chromos other part of the chromosome so you can see that that causes a steric hindrance and that is why it decreases the activity of the VD so now if they are on the same side that is capital D and capital c are there in this case there is no interference and it is a normal rhd now these individuals cannot be distinguished from the genetic we de which we'll be discussing later only the reaction strength is reduced and they are actually dep positive individuals with only the strength of the D reduced the second and very important mechanism is the weak D is the genetic weak D this is basically because of your RSD Gene with a weakened expression of the D antigens so what happens is because of this there is a reduced density of the D antigen itself less than amount to less than 500 antigen on the rbcs and they are basically the epitopes are all normal but the density is reduced that is it is a quantitative defect so when that happens obviously the strength decreases and you have a weaker reaction now there are various subtypes of weekd right from 1 to 20 in fact there are 150 weekd types that have been deciphered but important amongst them are type 1 2 3 which we'll be discussing later why it is very important these type 1 2 3 have to be determined and they form approximately 90% of the VD so that is very important and we need to know which type of BD it is in the genetic uh framework so just to represent this we D you can see this is the relative density of the the antigen is reduced the D antigen is reduced in a partial D if you see there is a missing epitope so when of the epitopes you can see the mutation is generally happening on the outer surface whereas this is intracellular a DL is a super super weak form of a week D in which nothing can be seen and you require special techniques to actually go through and detect this the L form now this is another representation of the same so you can see that the first picture this is normal D so let's assume there are two epitopes on this antigen one and two and this is the actual Den it which should be there in a normal D when you compare it that in the weak D you can see the epitopes are normal you have both the epitopes here but the problem here is that the density of the D is reduced so in Weak D the density is reduced and that is why the strength is reduced they are actual D positive in partial D if you see there is a missing of epitope 2 so the density is same but you have a missing epitope so it is a qualitative defect so partial D is a qualitative effect so if you have one missing epitope obviously the antibodies are formed against the epitope so once you have a missing epitope you can form an antibody against that episode so that is why a d positive individual who has a missing epitope who has a missing epitope will still form an antibody against the D antigen so this is the molecular basis of we D you can see this is a diagram that has been taken from a journal this descript surprise that if you see on the epitope part or the or the outer part on the rroy surface that is where the epitopes are located and this is against which the antibodies will be produced so if you have a missing epitope here you may have a partial D now if you have some mutation inside that may reduce the density of the antigen and this may lead to a weak deformation so all these red marks are the potential side for weak D mutation and all these blue marks are the site for partial D mutation so before we do anything we should know how to do it and that is why molecular typing needs a genetic background for all blood group antigen since the formation of Human Genome Project we have every blood group antigen that can be typed to its molecular marker or a corresponding DNA market so all the blood groups have been deifer and how do you do that how do you differen shate now we have a mechanism known as single nucleotide polymorphism that is you have nucleotide and in one nucleotide there is a change in the uh uh nucleotide at that position and this can lead to formation of a new blood group so usually the genotyping methods are based on the PCR that is the polymerized Chain Reaction or the molecular photocopy with the use of gen genomic DNA and in the PCR we have a PCR SSV or PCR sequence specific primer which is the most common and most cost effective method for determining so from DNA to blood group this is the Watson and cric model of DNA that is double helical model which was discovered by Watson and cck and you can see that we have purines and pyramid in that is atgc these are the Base PS nucleotide base and this is actually the unwinding of that DNA you can see that there is a sugar phosphate background backbone and you have the nucleotide base which form the it is like a ladder and this forms the rungs of the ladder so you have these nucleotide bases which have three hydrogen bond between G and c and two hydrogen bond between a and t so once you have this DNA that can be Amplified by a technique known as polymerase Chain Reaction or PCR coming to polymorphism like I said it is many form or set of two or more alternate form of a normal phenotype so if any or two or more ales there is an occurrence or frequency is more more than 1% It is known as polymorphism if it is less than 1% It is called as a rare variant example for polymorphism is MN group and a rare variant like o Bombay blood group so polymorphism is everywhere we have seen it in normal differences occur between people such as eye color hair color blood type nail Etc all of this are a part of polymorphism although they have no negative effect but they can also these variation May influence the risk of developing a certain disorder so coming to single nucleotide polymorphism you can see at this base pair this purple gentleman has a a blue gentleman has g and an orange gentleman has a t base pair so you can see that a pairs with T so obviously there's going to be a difference in there G pairs with C and ta pairs with a so all of this at one base pair you can see there is some polymorphism in single nucleotide this leads to the formation of a new blood group or a new variant of a blood group so most blood group polymorphism result from one or more snps encoding amino acid substitution in either the glyos cell transferase or extracellular domain of membrane protein a very good example is a Duffy system in which an SNP occurs in a rroy transcription Factor binding site in the promoter region of theine which is responsible for the theal phenotype in Africans why VD is important there are it's the this is explained by a very good algorithm or a flowchart that is depicted here now what happens is if there is a negative somebody's Rh negative there is no problem that person if that female is pregnant obviously she is a candidate for rum or rhig and that person is considered as rhd negative for transfusion so if you are transfused with RH positive obviously they're going to make some allo antibodies however if your RH positive and everything is concordant with the history you are not a candidate for rhig you don't require a rum if you're a pregnant female and you are an Rd positive individuals now the problem happens when there is a discrepant or inconclusive result or the strength of reaction is weak that is where you go for a molecular typing nowadays especially in the international scenario their RSD genotyping is done for VD types I told you there are more than 50 or 100 uh VD types but only thing is in those cases we need to determine what weekd types we want to detect basically because weekd 1 12 3 if you have that person behaves like an RSD positive individuals they not they are not at risk of forming anti-d they are not a candidate for rum or rhig and they are considered as rhd positive for transfusion so yes you need to determine type one two 3 if type 1 two 3 is there this is a better method in the sense that molecular genotyping is done and the financial modeling study is also suggesting that this is a better method to detect rather than going for a serological typing however if you are not any of these types that is not we d123 you may be at risk of forming entity you are a candidate for rum and rhd negative for transfusion this is the technology that I was talking about PCR SSP this is a gel based technology you can see and this is the uh internal control on the above this is the DNL adder and when you compare this position third position this come positive because there are two band internal control and a positive band and this is the method for determining the weak D typing molecular we DP types I've already told you there are various various subtypes you have 1 two 3 which is more common in fact 1 12 4 represent 94% of the weekd types in the Caucasian population especially in the whites and 6% of all weekd samples are 5 to 11 the rest 5 to1 now you can also see that the four VD type four is split into 4.1 and 4.2 and that is why because 4.1 behaves like we D type 123 and 4.2 behaves like weekd type 5 to 11 or further so we have another weekd 4.2 which is also known as Dar this is very common in Portuguese population so you need to know where uh weekd types and which weekd types occur where we do not have R data which represent the molecular VD types but yes we should have it very very soon this is another diagram which represent the various weekd phenotypes and their prevalence in this is an article from Germany so this is their prevalence and you may notice that they are considered as D posited up to D VT type 4.1 and they are D negative after from 4.2 to other subtypes very important to know which weekd type it is and of course there are other mechanism apart from single nucleotide polymorphism which can cause the V the expression or formation of other VD type and these are some splicing mutation deletion or duplication of RSD the exons so the Crux of the matter is in donors v d blood is regarded as v d positive in recipient VD are only given RS negative blood coming to partial D we had already discussed a part of it partial D is basically when you have a missing epitope or altered epitope and that's why an Alo antibody may be formed to the missing epitope if exposed to RVC that possess the complete de antigen so there was earlier there were seven categories now these categories have become obsolete they were denoted through a Roman numerals that is 1 to7 category now is actually Obsolete and category six is what we talk about D6 which is seen that it is relatively more commoner in the Caucasians we don't have our data which suggest which category is more common and none the less D6 has is the one which we all have seen it more often than Norm so partial D antigens also need to be detected on a molecular level so there can be attributed to hybrid genes can also occur and this can result from a RSD Gene being replaced by portion of rhc e Gene and this happens due to Gene conversion because they are so closely there there is no crossing over but one of the portion of the rhd is replaced by a portion of rhc this results in certain portion of rhd and rhc in various combinations so they will make an antibody to the portion of rhd protein that is missing and can result in hdfn so for a pregnant woman with partial d she's always given RH imunoglobulin because she can make an antibody to the missing part and as a donor you are a d positive and a recipient you are a d negative this is another representation of VD you can see the most of the mutation that are happening are happening intracellularly and you can see this type one type two and type three which reflects the amino acid change predicted intracellular transmembrane region of rhd whereas a partial D you can see that so many mutations that are occuring are mostly extracellular and they cause the partial D pype same thing in this diagram again to reiterate normal D good density all the epitopes are present weak D all the epitopes are present but the density is less partial D the density is okay and only an part of the epitope or an epitope is missing another subtype DL this is why it is known as L is because it is been detected through absorption illusion studies now this is a super uh low density form of the VD in which the the the numbers are so undetectable levels that you can do it or you can locate it through only a special techniques like flow cytometry or Illusion studies you can also do a molecular study to detect the RSD Gene that alters the expression of the RSD protein this is relative L common in the individuals of Asia you can also have an RH deficiency trome like an RH null which can be because of both regulator Gene that is the RH AG Gene which is not expressed sometimes and because of which rhd or rhc is subsequently altered or you can have an amorphic Gene in which mutations happen in the rhce genes inherited from each parent and the rhd gene is deleted so you have a mutation in rce and a RSD Gene which is deleted and this is found in D negative individuals the RH AG Gene is normal in this case so RH Nal individuals how are they special they are negative for high prevalence antigen like LW and duffy5 also s and U antigens may be depressed they have compensated hemolytic anemia and rap finding point to a muscular the skeletal defect in the RVC membrane like you may have stomat cyes decrease in the hemoglobin because of the compensatory htic anemia decrease in serum apoglobin features of hemolytic anemia like elevated verid L transfusion you can only transfuse the RS null blood to these individuals you can also have an R modified basically you have mutation in an R AG Gene whereas the quantity of that rhg Gene may be altered that rhg AG protein is altered therefore normal RH antigens are also altered this for it causes the weakened expression of normal RH antigen rarely clinically remarkable so not that important but you should know that it exist these are some other unusual phenotypes that are there we'll not be discussing it in this lecture like CWF rs1 C rs17 rs23 rs30 rs40 Crawford evariant and LW antigens so you can go through them in your list and uh some of them may come as a short note for you so thank you so much I hope this lecture helps to understand what week how to do molecular genotyping for them what are partial how to deifer them and what implications we have in the htic disease of the newon and hemolytic transfusion reaction thank you so much

Namaste everyone I&#39;m Dr M Chri senior consultant in New Delhi today we&#39;ll be talking about understanding the immunobiology of weekd and D variants this is transconnect April 2024 so let&#39;s begin with this new topic of weekd very important and uh also we&#39;ll be covering some part of the partial D the molecular aspect of it and understanding actually where to imply the VD and its variant so I&#39;ll be talking about the RH system the genetics and biochemistry RH antigens RH genotyping and some RH deficiency syndromes so we all know that RH is the most important blood group system after AO in transfusion medicine and it is very critical component of the pre-transfusion testing it is very immunogenic complex and polymorphic and so what is polymorphic polymorphic means many forms so it is got various forms various Al of the normal form are present so currently there are 55 antigens that have been characterized and they have five principal antigen that is D capital D Capital C capital E small C and small e and these are responsible for clinically significant antibodies finally note that there is no small D so these RH antibodies how are they produced and how are they different from the AO antibodies Is AO are naturally occurring and these are formed only after exposure to the foreign red blood cells and they are commonly associated with the hdn or the hemolytic disease of the newborn and also with the transfusion reaction the term D positive and D negative actually refers to the presence and absence of the RH antigen D so if you are D positive you are uh that is then you are RH positive if it is D negative you are RH negative so that is how it is uh termed as now the basic importance of why we need to know v d and variant D is that we do not want to transfuse an RH positive blood to an Rh negative individual and similarly in females uh pregnant females we do not want to immunize them by giving RH positive if they are RH negative and also where to give the rum or RH imunoglobulin to the mothers who are RH negative so so that needs to be taken care of and that needs to be understood so coming to the landmarks in the history there are 1939 Levine and Sten were the first who describ the HDR in Ops patient and postulated that the fetus and father possess the same common factor that the mother lack in 1940 laner and Wier used antisera from ginipic and they found that with red cells razes Mass to distinguish RS positive from the RS negative red cells very important genetics of R Gene please remember there are two genes which are located on chromosome one that is the rhb Gene and in the rhc gene so the gene rhd codes for the presence of rhd protein whereas RH c e the other Gene which I which we just discussed quotes for RH Capital C Small E RH small C capital E RH small C Small E or RH Capital C capital E so rhd and rhc both are codominant so both are expressed and there have been numerous mutations that I been described in the r Gene and that&#39;s why so many Al and so many forms that you can see greater than 250 Al have been determined in RSD genes and 50 Al have been found in the RX C now these many imitations good thing about them is that they are rare and they do not change the serology so rest assured everything is okay so this is how the gene structure looks like just a minute so you can see this is the LOI 1 Loi 2 so you can see that rhd gen there are 10 exons similarly rhc Gene also has 10 exons so in RH positive individuals you can see these 10 exons are there and this will give you a d antigen similarly rhce you have the 10 Exon hair as well so when you actually have uh you place them together they are like in a Hair Pen Arrangement so 10 will come here and one will come here and that&#39;s how it it is uh present in the body in the chromosome so because they are so close to each other there is very less chance that there is a crossing over and that is why there are other mechanism by which a new blood group may be formed one of them is single nucleotide polymorphism then there are other forms like Gene conversion Gene insertion deletion Etc so if you seen an RS positive individual you will have the D antigen if there the person is Rh negative you can see there is no D antigen because the absent of the gene here so if there is rele of that Gene there will be no and that is why no de antigen however they are independent of each other both these genes and that&#39;s why if you have an rce Gene you will still have the C antigens so you can see they are identical the rhd and the rhc and they are almost 97% identical to each other there&#39;s another Gene that which we need to care about and that is the RH AG or a CO expressor gene or a regulator Gene which controls the RH so this is known as RH AG RH antigen expression is what it&#39;s linked in and it resides on the chromosome 6 this peptide is similar in structure to the RH protein only difference being that it is glycosilated that is that it has a carbohydrate to it so within the RBC membrane it forms a complex with the RH protein and we&#39;ll be discussing about this RH complex which is Rh Gene and Rh AG Gene along with the various other cytoskeleton proteins so it this should be coexpress so once it is expressed it gives the successful expression of the other RH antigens by itself this glycoprotein does not express any RH antigens and mutation of this Gene may result in the alteration in the rhd and C rhce proteins so this is the RH membrane complex if you see this is Rh Gene along with this is Rh AG Gene now RH AG Gene is or linked to the protein 4.2 anarine spectrin Band 3 all of this is which is involved in the structure of the RBC and that is why sometimes when you have a pathology in these proteins you may also have a pathology related to the RBC membrane defect like stomatocytes elliptocytes spherocytes Etc so you can see that this is linked together with a protein or integr Associated protein known as cd47 and this whole thing is a complex or the RH membrane complex which is very important for various diseases so when do you call an individual R positive we have already discussed if you have one or two copies of RSD genes that is from one parent you have one RSD and from the other parent either you have or don&#39;t have so if you have e even if you have a one RSD Gene copy you are terms as RSD positive if there is a mutation in the rhd antigen and then you can have a Rh negative phenotype when you don&#39;t have any copy of RTG so it is more common in European and as Asian it is less common but in europian if you see the mechan mechanism is the deletion of the RSD Gene that is no RSD Gene but you have two RSC gen so if there is no RSD Gene you are rs negative whereas if you have in see in the Asian there is a mutation in the RH Gene there is no deletion so in European there is generally a duration of the rhd gene in Asian ethnicity there is mutation in the rhd gene in African ethnicity there is an unusual form of rhd Gene which is known as rhd pseudo Gene now this is because of some missense mutation in Exon 5 and six or a nonsense mutation in a Exxon 6 nonsense mutation basically means that you have a stop codon which is produced and this causes further uh translation of that protein to be inhibited coming to a small uh pictorial representation of the biochemistry of the rht gene you can see that both the genes are here rht protein is here rhc is protein is here and you have the RH Associated glycoprotein you can see the both of them rhd and Rh see there is a uh there is this proteins are traversing through the membrane this is a transmembrane protein and it goes through the cell membrane 12 times so 12 times there is a loop so this total amino acids are the same in both 417 amino acids are present and they are both non- glycosilated whereas RH AG does not have any RH protein these have Rh proteins so you can see that these are the proteins that are there here also you can see now this is very important because this is the part that is exposed and these are the epitopes against which an antibody may be produced and this happens in a partial D which we&#39;ll be discussing later but if you have a mutation in the inner area or the inner cytoplasm inner inner part of the cell membrane along with the inside of the cell membrane you can see this is because of the uh this leads to the formation of v d so for partial D basically you have a mutation in the iops are the outer part and for BD you have a mutation in the inner part of the cell membrane so RH antigens are highly immunogenic we are all aware of that they are present on the red blood cell as transmembrane proteins they do not exist in soluble form and they are very immunogenetic in the sense that even 1. 0.1 mL of RH positive RBC can stimulate an antibody production in then Rh negative individual so immunogenicity of Rh antigens you can see the capital is the most immunogenetic then we have the cap small C capital e capital C and G coming to the main topic that is the weak D variation of D antigen expression now we already know that there is a d antigen there is there are two genes that is rhd and rhc that are involved so this when we started doing the serology many of times a patient would come to your lab and would say that I was knowing my blood group as rhd positive or rhd negative and now you have suddenly given me an R positive so basically what you&#39;re doing is you are using different anticas so somebody was typed with a different anti gave the result as RS negative but when you type with a better S or S which contains a blend of IGM and IGG you would be able to detect the we D generally the reaction strength is also very important in the sense that you tend to have a reaction strength of four plus in a normal individual but if you see a weak D the reaction strength is one or two plus in those cases the RBC carrying weer D antigens have historically been referred to as du type we used to do du typing for them later it was known that it was the same thing and these individual did not produce antiu they produced only the anti-d so we D is the right terminology and there are various mechanisms by which we D antigen expression happens important among these are the C in the trans to RSD weak D partial d d that is DL and D epitopes on RC protein coming to the first mechanism C in trans to RSD very important this is a position effect and Gene interaction effect uh basically there is a steric endurance so you can see that whenever there the D and the C capital D and the capital c is trans to each other that means they are on the different uh the eles are on the different chromosomes that is capital D is here you can see and capital c is on the other chromos other part of the chromosome so you can see that that causes a steric hindrance and that is why it decreases the activity of the VD so now if they are on the same side that is capital D and capital c are there in this case there is no interference and it is a normal rhd now these individuals cannot be distinguished from the genetic we de which we&#39;ll be discussing later only the reaction strength is reduced and they are actually dep positive individuals with only the strength of the D reduced the second and very important mechanism is the weak D is the genetic weak D this is basically because of your RSD Gene with a weakened expression of the D antigens so what happens is because of this there is a reduced density of the D antigen itself less than amount to less than 500 antigen on the rbcs and they are basically the epitopes are all normal but the density is reduced that is it is a quantitative defect so when that happens obviously the strength decreases and you have a weaker reaction now there are various subtypes of weekd right from 1 to 20 in fact there are 150 weekd types that have been deciphered but important amongst them are type 1 2 3 which we&#39;ll be discussing later why it is very important these type 1 2 3 have to be determined and they form approximately 90% of the VD so that is very important and we need to know which type of BD it is in the genetic uh framework so just to represent this we D you can see this is the relative density of the the antigen is reduced the D antigen is reduced in a partial D if you see there is a missing epitope so when of the epitopes you can see the mutation is generally happening on the outer surface whereas this is intracellular a DL is a super super weak form of a week D in which nothing can be seen and you require special techniques to actually go through and detect this the L form now this is another representation of the same so you can see that the first picture this is normal D so let&#39;s assume there are two epitopes on this antigen one and two and this is the actual Den it which should be there in a normal D when you compare it that in the weak D you can see the epitopes are normal you have both the epitopes here but the problem here is that the density of the D is reduced so in Weak D the density is reduced and that is why the strength is reduced they are actual D positive in partial D if you see there is a missing of epitope 2 so the density is same but you have a missing epitope so it is a qualitative defect so partial D is a qualitative effect so if you have one missing epitope obviously the antibodies are formed against the epitope so once you have a missing epitope you can form an antibody against that episode so that is why a d positive individual who has a missing epitope who has a missing epitope will still form an antibody against the D antigen so this is the molecular basis of we D you can see this is a diagram that has been taken from a journal this descript surprise that if you see on the epitope part or the or the outer part on the rroy surface that is where the epitopes are located and this is against which the antibodies will be produced so if you have a missing epitope here you may have a partial D now if you have some mutation inside that may reduce the density of the antigen and this may lead to a weak deformation so all these red marks are the potential side for weak D mutation and all these blue marks are the site for partial D mutation so before we do anything we should know how to do it and that is why molecular typing needs a genetic background for all blood group antigen since the formation of Human Genome Project we have every blood group antigen that can be typed to its molecular marker or a corresponding DNA market so all the blood groups have been deifer and how do you do that how do you differen shate now we have a mechanism known as single nucleotide polymorphism that is you have nucleotide and in one nucleotide there is a change in the uh uh nucleotide at that position and this can lead to formation of a new blood group so usually the genotyping methods are based on the PCR that is the polymerized Chain Reaction or the molecular photocopy with the use of gen genomic DNA and in the PCR we have a PCR SSV or PCR sequence specific primer which is the most common and most cost effective method for determining so from DNA to blood group this is the Watson and cric model of DNA that is double helical model which was discovered by Watson and cck and you can see that we have purines and pyramid in that is atgc these are the Base PS nucleotide base and this is actually the unwinding of that DNA you can see that there is a sugar phosphate background backbone and you have the nucleotide base which form the it is like a ladder and this forms the rungs of the ladder so you have these nucleotide bases which have three hydrogen bond between G and c and two hydrogen bond between a and t so once you have this DNA that can be Amplified by a technique known as polymerase Chain Reaction or PCR coming to polymorphism like I said it is many form or set of two or more alternate form of a normal phenotype so if any or two or more ales there is an occurrence or frequency is more more than 1% It is known as polymorphism if it is less than 1% It is called as a rare variant example for polymorphism is  MN group and a rare variant like o Bombay blood group so polymorphism is everywhere we have seen it in normal differences occur between people such as eye color hair color blood type nail Etc all of this are a part of polymorphism although they have no negative effect but they can also these variation May influence the risk of developing a certain disorder so coming to single nucleotide polymorphism you can see at this base pair this purple gentleman has a a blue gentleman has g and an orange gentleman has a t base pair so you can see that a pairs with T so obviously there&#39;s going to be a difference in there G pairs with C and ta pairs with a so all of this at one base pair you can see there is some polymorphism in single nucleotide this leads to the formation of a new blood group or a new variant of a blood group so most blood group polymorphism result from one or more snps encoding amino acid substitution in either the glyos cell transferase or extracellular domain of membrane protein a very good example is a Duffy system in which an SNP occurs in a rroy transcription Factor binding site in the promoter region of theine which is responsible for the theal phenotype in Africans why VD is important there are it&#39;s the this is explained by a very good algorithm or a flowchart that is depicted here now what happens is if there is a negative somebody&#39;s Rh negative there is no problem that person if that female is pregnant obviously she is a candidate for rum or rhig and that person is considered as rhd negative for transfusion so if you are transfused with RH positive obviously they&#39;re going to make some allo antibodies however if your RH positive and everything is concordant with the history you are not a candidate for rhig you don&#39;t require a rum if you&#39;re a pregnant female and you are an Rd positive individuals now the problem happens when there is a discrepant or inconclusive result or the strength of reaction is weak that is where you go for a molecular typing nowadays especially in the international scenario their RSD genotyping is done for VD types I told you there are more than 50 or 100 uh VD types but only thing is in those cases we need to determine what weekd types we want to detect basically because weekd 1 12 3 if you have that person behaves like an RSD positive individuals they not they are not at risk of forming anti-d they are not a candidate for rum or rhig and they are considered as rhd positive for transfusion so yes you need to determine type one two 3 if type 1 two 3 is there this is a better method in the sense that molecular genotyping is done and the financial modeling study is also suggesting that this is a better method to detect rather than going for a serological typing however if you are not any of these types that is not we d123 you may be at risk of forming entity you are a candidate for rum and rhd negative for transfusion this is the technology that I was talking about PCR SSP this is a gel based technology you can see and this is the uh internal control on the above this is the DNL adder and when you compare this position third position this come positive because there are two band internal control and a positive band and this is the method for determining the weak D typing molecular we DP types I&#39;ve already told you there are various various subtypes you have 1 two 3 which is more common in fact 1 12 4 represent 94% of the weekd types in the Caucasian population especially in the whites and 6% of all weekd samples are 5 to 11 the rest 5 to1 now you can also see that the four VD type four is split into 4.1 and 4.2 and that is why because 4.1 behaves like we D type 123 and 4.2 behaves like weekd type 5 to 11 or further so we have another weekd 4.2 which is also known as Dar this is very common in Portuguese population so you need to know where uh weekd types and which weekd types occur where we do not have R data which represent the molecular VD types but yes we should have it very very soon this is another diagram which represent the various weekd phenotypes and their prevalence in this is an article from Germany so this is their prevalence and you may notice that they are considered as D posited up to D VT type 4.1 and they are D negative after from 4.2 to other subtypes very important to know which weekd type it is and of course there are other mechanism apart from single nucleotide polymorphism which can cause the V the expression or formation of other VD type and these are some splicing mutation deletion or duplication of RSD the exons so the Crux of the matter is in donors v d blood is regarded as v d positive in recipient VD are only given RS negative blood coming to partial D we had already discussed a part of it partial D is basically when you have a missing epitope or altered epitope and that&#39;s why an Alo antibody may be formed to the missing epitope if exposed to RVC that possess the complete de antigen so there was earlier there were seven categories now these categories have become obsolete they were denoted through a Roman numerals that is 1 to7 category now is actually Obsolete and category six is what we talk about D6 which is seen that it is relatively more commoner in the Caucasians we don&#39;t have our data which suggest which category is more common and none the less D6 has is the one which we all have seen it more often than Norm so partial D antigens also need to be detected on a molecular level so there can be attributed to hybrid genes can also occur and this can result from a RSD Gene being replaced by portion of rhc e Gene and this happens due to Gene conversion because they are so closely there there is no crossing over but one of the portion of the rhd is replaced by a portion of rhc this results in certain portion of rhd and rhc in various combinations so they will make an antibody to the portion of rhd protein that is missing and can result in hdfn so for a pregnant woman with partial d she&#39;s always given RH imunoglobulin because she can make an antibody to the missing part and as a donor you are a d positive and a recipient you are a d negative this is another representation of VD you can see the most of the mutation that are happening are happening intracellularly and you can see this type one type two and type three which reflects the amino acid change predicted intracellular transmembrane region of rhd whereas a partial D you can see that so many mutations that are occuring are mostly extracellular and they cause the partial D pype same thing in this diagram again to reiterate normal D good density all the epitopes are present weak D all the epitopes are present but the density is less partial D the density is okay and only an part of the epitope or an epitope is missing another subtype DL this is why it is known as L is because it is been detected through absorption illusion studies now this is a super uh low density form of the VD in which the the the numbers are so undetectable levels that you can do it or you can locate it through only a special techniques like flow cytometry or Illusion studies you can also do a molecular study to detect the RSD Gene that alters the expression of the RSD protein this is relative L common in the individuals of Asia you can also have an RH deficiency trome like an RH null which can be because of both regulator Gene that is the RH AG Gene which is not expressed sometimes and because of which rhd or rhc is subsequently altered or you can have an amorphic Gene in which mutations happen in the rhce genes inherited from each parent and the rhd gene is deleted so you have a mutation in rce and a RSD Gene which is deleted and this is found in D negative individuals the RH AG Gene is normal in this case so RH Nal individuals how are they special they are negative for high prevalence antigen like LW and duffy5 also s and U antigens may be depressed they have compensated hemolytic anemia and rap finding point to a muscular the skeletal defect in the RVC membrane like you may have stomat cyes decrease in the hemoglobin because of the compensatory htic anemia decrease in serum apoglobin features of hemolytic anemia like elevated verid L transfusion you can only transfuse the RS null blood to these individuals you can also have an R modified basically you have mutation in an R AG Gene whereas the quantity of that rhg Gene may be altered that rhg AG protein is altered therefore normal RH antigens are also altered this for it causes the weakened expression of normal RH antigen rarely clinically remarkable so not that important but you should know that it exist these are some other unusual phenotypes that are there we&#39;ll not be discussing it in this lecture like CWF rs1 C rs17 rs23 rs30 rs40 Crawford evariant and LW antigens so you can go through them in your list and uh some of them may come as a short note for you so thank you so much I hope this lecture helps to understand what week how to do molecular genotyping for them what are partial how to deifer them and what implications we have in the htic disease of the newon and hemolytic transfusion reaction thank you so much

Transcript for:Understanding the Immunobiology of Weak D and D Variants

Transcript for:
Understanding the Immunobiology of Weak D and D Variants