variant D

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Variants of D Antigen : 

Variants of D Antigen By Dr Ravi C Dara

Organization of Rh gene : 

Organization of Rh gene

Immunogenicity of D antigen : 

D antigen is the most immunogenic of Rh antigens and is most important clinically. Why D antigen is most important? If a unit of D-positive blood is transfused to a D-negative recipient, the recipient forms anti-D in some 90% of cases. In Rh incompatible pregnancy 1 in 6 cases shows primary immunization to D antigen. D antigen is also involved in specificity of some of the warm autoantibodies of AIHA. D+/D- are often referred as Rh+ and Rh-. Immunogenicity of D antigen

Frequency in different ethinic groups : 

Frequency in different ethinic groups 82% Europeans 88% white americans D positive 95% black africans Nearly 100% far east

D variants : 

Why there are variants of D antigen? D and CE antigens are encoded by two separate but highly homologous genes adjacent on the same chromosome. exchange of DNA between the two genes hybrid genes encoding variant D polypeptides (part of the normal D sequence replaced by CE polypeptide sequence) D variants

Mechanisms for Antigenic diversity : 

Two general mechanisms for generating antigenic diversity: Nucleotide substitutions Gene conversion Nucleotide substitution: single amino acid change in the protein sequence is the commonest mechanism for generating antigenic change in all systems other than Rh and MNS system. Gene conversion:occurrence of two adjacent, highly homologous genes predisposes to misalignment between the genes when chromosomes pair at meiosis. For eg: D with CE rather than D with D Results in insertion or deletion of stretches of DNA sequence in the misaligned genes Novel DNA sequences Novel protein Novel antigens Mechanisms for Antigenic diversity

Gene conversion : 

Predominant mechanism. Formation of RHD–CE–D and RHCE–D–CE hybrids. Macroconversion events give rise to an Rh gene in which a substantial segment is replaced by the equivalent segment from its homologue, Microconversion events can result in exchange of one or more small regions, often leading to single amino acid changes Effects of gene conversion are often associated with untemplated mutations, which change nucleotides to those not derived from either gene. Gene conversion

Weak D or Du : 

Stratton first coined the term Du for a D antigen detected by only some anti-D. Du Weak D or Du

Inheritance of Du : 

Du shown to be inherited as Du dominant over d, but Du recessive over Normal D. Definition of Du: D of those red cells that are not agglutinated by IgM anti-D, but which react with IgG anti-D in an antiglobulin test. Inheritance of Du

Weakened D : 

Defines as any D phenotype where expression of D antigen is quantitatively weaker than normal. Inherited weak D More common in African Americans These individuals may inherit the RHD gene that encodes for a weaker expression. Quantitative, meaning less is expressed Detected best at AHG phase Weakened D

Weak D : 

Weak D

Partial D : 

Defines as D phenotype which is qualitatively different from Normal D. Missing one or more parts of the D antigen Since the antisera is specific for the whole D antigen, a weak reaction may result if patient has a partial antigen. Partial D Missing portion

Position Effect of weak D : 

Gene interaction effect C allele is in trans position to D allele Does not occur when C is in cis position Steric hindrance causes the anti-D reagent to weakly attach (C antigen crowds the D antigen) Position Effect of weak D

Weak D and Partial D : 

Assumption that Weak D cannot make alloanti-D Partial D can make alloanti-D if transfused with D positive blood. This assumption was challenged by Fegel et al by demonstration of nucleotide substitution in RHD encoding amino acid changes in different weak D samples And by evidence that weak D phenotype can produce alloanti-D Weak D and Partial D

Variation in amino acid sequence : 

Variation in amino acid sequence

Variants of D : 

Variants of D

Clinical relevance of D variant. : 

For a Donor with D variant red cells- Whether these red cells will be immunogenic if transfused to a D-negative patient (or a patient with a different D variant) or not. For a patient with D variant phenotype. Whether they will make anti-D if transfused with red cells of normal D phenotype. Clinical relevance of D variant.

DIII variant : 

Only D category that cannot be defined by monoclonal anti-D, because category III red cells react with all monoclonal anti-D. 4 subcategories (DIIIa, DIIIb, DIIIc, DIII type IV.) DIIIa: DAK antigen is present. RHD shows 3 amino acid substitutions encoded by RHCE at exons 3-5. DIIIb: Cells are G- ( mostly D+ are G+) RHD sequence shows exon 2 is replaced by exon 2 from a c allele of RHCE There is Ser103Pro substitution in the second extracellular loop. DIIIb has Pro103 thus expresses no G. DIII variant

DIII variant : 

DIIIc: These are present in whites. RHD sequence shows exon 3 is replaced by exon 3 from RHCE. DIII type IV: Added in 2000 React with all monoconal anti D but not with anti D from a DIIIc individual. 3 amino acid substitution encoded by RHD gene. DIII variant

DIV variant : 

DIV are initially subdivided by reactions with anti Goa. DIVa cells- Goa positive. DIVb cells- Goa negative. DIVa individuals are mostly black. Three amino acid changes distinguish DIVa from normal D two, encoded by exons 3 and 7, probably represent microconversion events (exon 2) represents an untemplated mutation. DIVb is associated with an RHD–CE–D gene in which the 3end of exon 7, exon 9, and probably exon 8 are derived from a RHCE gene. DIVb are seen in mostly in whites. four DIVb individuals were found among Japanese blood donors DIVb (J) results from an RHD–CE–D gene in which the whole of exons 7 and 9 are RHCE derived DIV type III and type IV were defined primarily on a molecular basis. Both have an RHD–CE–D gene: Type III exons 6–9 have the RHCE sequence, Type IV only part of exon 7 is exchanged DIV variant

DV variant : 

DV are subdivided due to reaction with antibody against low frequency antigen anti Dw. DVa are Dw + Molecular background shows always involves replacement of all or part of exon 5 of RHD by the equivalent region of RHCE DV variant

DVI variant : 

DVI shows very few epitopes And most monoconal anti D do not react with category DVI cells. Mostly seen in whites or Japanese DVI travels with Ce in most families and less commonly with cE . Anti-BARC, an antibody to a low frequency antigen, is a marker for the DVICe haplotype. DVI variant

DVI variant : 

The most common partial D associated with anti-D is DVI. Most DVI cells have low density of D and behave like weak D, many monoclonal anti-D agglutinate weak D, but not DVI cells. For D typing of patients, anti-D reagents that do not react with DVI cells should be selected, as it is preferable that DVI cells be typed as D–, so that DVI patients receive D– blood. Anti-D immunoglobulin should be given to partial D mainly DVI type women during and after pregnancy . DVI variant

DVI variant : 

Molecular genetic analysis has revealed three types of RHD–CE–D encoding DVI DVI type I- shows exons 4 and 5 derived from a E allele of RHCE. DVI type II shows exons 4–6 DVI type III shows exons3–6 DVI variant derived from an e allele of RHCE

Exon rearrangements associated with some other partial D types : 

Exon rearrangements associated with some other partial D types

DHAR variant : 

The Ro Har ‘haplotype’ differs from all others described in that it comprises only one gene: there is no RHD or RHCE, but an RHCE–D–CE hybrid with only exon 5 representing RHD. DHAR variant

D variants : 

Common D variants in White people DVI appears to be most abundant. DVII and DNB Common D variants in black people DIVa, DAU Common D variants in Asians DEL D variants

DEL (Del ) : 

A very weak form of D found in the Far East and asians is called DEL. Can only be detected reliably by adsorption–elution tests. The DEL gene is almost exclusively in cis with a Ce allele of RHCE. DEL was associated with a 1013-bp deletion of RHD extending from intron 8 to intron 9 and encompassing the whole of exon 9. DEL (Del )

Rh null phenotype : 

First described by Vos et.al Here no Rh antigens can be detected on RBC’s Two types: Amorph type Regulator type Rh null phenotype

Rh Antigens/Rh null Pathways : 

Rh Antigens/Rh null Pathways

Rh null phenotype : 

Amorph type: Shows homozygosity for a silent or amorph gene at the RH locus, resulting from inactivating mutations in RHCE and a deletion of RHD. Only 4 cases are reported japanese/ german/ norwegian/ spanish. Symbol – – –/– – – was used for the genotype. Regulator type: Rh genes are normal. There is homozygosity for inactivating mutations in RHAG gene. Rh null phenotype

Rh null phenotype : 

Red cells shows Spherocytosis Somatocytosis with diminished life span and mild hemolytic state. Red cells: increased content of HbF react more strongly with anti-i; increased osmotic fragility and increased Na+–K+pump activity. Anti Rh:29(anti total Rh) antibody may be produced. Rh null phenotype

Rh mod phenotype : 

Some mutations in RHAG give rise to low level expression of Rh antigens, a phenotype called Rh mod. Rh mod shows greatly weakened Rh antigens. Easily mistaken for Rh null if only limited testing carried out. This phenotype is due to homozygosity for a modifier gene (called XQ), at a locus separate from the Rh complex locus. Rh mod phenotype

D Testing : 

Routine D antigen testing involves testing the washed patient RBCs with anti-D commercial antisera If the D antigen is present, it should agglutinate strongly with anti-D at Immediate Spin (IS) If you’re Rh+, you have the D antigen If you’re Rh-, you do not have the D antigen D Testing

Weak D testing : 

If negative at IS, patient cells and anti-D reagent are incubated at 37° for 20 minutes, then centrifuge If still negative, wash x3 with NS and form a cell button. add AHG If negative, add CC and report as Rh negative (if CC agglutinate) If positive, report as Weak D Positive. Weak D testing

Donor D typing : 

Adopt procedures to maximise detection of weak D and partial D as RhD positive Determined on each donation New donors tested with at least two anti-D (different clones) capable of detecting DIV, DV and DVI between them Unequivocal + with both sera, donor RhD+, unequivocal –with both sera, donor RhD- Donor D typing

Donor D typing : 

Existing donors-one anti-D (or blended reagent) that detects weak D, DIV, DV, DVI (results must match with historical record) Repeat all discordant or equivocal results If doubt-classify as D+ Donor D typing

D typing patients : 

Test with IgM monoclonal anti-D which should not detect DVI. The IAT should NOT be used for Rh D grouping. Anti-CDE not recommended for routine typing of patients Weak D considered RhD +, partial D as RhD – If doubt-classify as RhD negative D typing patients

Reagent antisera : 

Polyconal : Polyclonal antibodies is a mixture of immunoglobulins against a specific antigen but recognising different areas (epitopes) of that antigen. Monoconal : These are antibodies produced by the same clone of a lymphocyte directed against a specific epitope of the antigen. Reagent antisera

Polyconal vs monoclonal : 

Polyconal vs monoclonal

Disadvantages of monoclonal antisera : 

Monoclonal antibodies are specific for a single epitope (overspecificity). They are susceptible to pH changes. This has been overcome by blending, dilution and standardization of the final product. Disadvantages of monoclonal antisera

Monoconal antibodies : 

Monoconal antibodies

Antisera for Rh typing : 

There are multiple antigens in the Rh system (D,c,C,E,e) Most prevalent and most important to identify is D. These antisera identify the presence or absence of the D antigen on patient RBC. Agglutination: D present, D (+) No agglutination: D absent, D(-) Anti D- IgG Anti D- IgM Anti D -IgG/IgM blend Antisera for Rh typing

Acceptable titre and avidity of anti D : 

Acceptable titre and avidity of anti D Weak D’ testing is done using IgG + IgM monoclonal antibody reagent

Hemolytic disease of new born : 

(HDFN) results from the transfer of IgG1 and IgG3 antibodies across the placental barrier from the mother to the fetus. Despite the immunoglobulin prophylaxis, anti-D remains the most common cause of HDFN. Thus a its blood group antibody that is capable of causing HDFN, it is beneficial to determine the corresponding blood group phenotype of fetus. If the fetus is antigen (negative)-no risk of HDFN and no invasive procedures If the fetus is antigen(positive) -then the appropriate management can be carried out. Hemolytic disease of new born

Prenatal diagnosis : 

Blood group phenotype can be predicted by with a high degree of accuracy by testing fetal DNA. In the past Highly invasive 1% risk of miscarriage Not possible before 11 weeks 20 percent risk of causing transplacental hemorrhage. Prenatal diagnosis

Non invasive prenatal diagnosis(NIPD) : 

Lo. et al reported Cell free fetal DNA in the maternal circulation Detectable from 5 weeks and throughout pregnancy. Cleared from circulation within 30 minutes of delivery 3 – 6% of total circulating cell free DNA Originates from trophoblast. This cell free fetal DNA acts as a valuable source for molecular diagnostics by minimally invasive methods using PCR based tests. Non invasive prenatal diagnosis(NIPD)

Current use for NIPD : 

Fetal sex determination Fetal RhD status in high risk pregnancies Some single gene disorders Current use for NIPD

Prenatal diagnosis and RhD : 

D+ / D- phenotype is usually due to the presence or absence of the RHD gene, respectively The RHD Type is the prime target If RHD gene sequences are detected in the plasma of a D- woman, the fetus is predicted to be D+ If no RHD is detected, the fetus is predicted to be D- Prenatal diagnosis and RhD

Potential in routine antenatal care : 

Potential in routine antenatal care Anti D to be given and proper follow up RhD +ve RhD -ve Anti D No anti D Reduction in AntiD Prevent exposure to blood products Repeat NIPD testing RhD- women - NIPD for fetal RhD status RhD -ve RhD +ve Check RhD on all cord bloods

Slide 52: 

STUDY DESIGN AND METHODS: A prospective study was conducted between November 2002 and December 2006. DNA extraction was performed in an automated closed tube system. Fetal RHD/SRY genotypes were detected in the plasma of 563 pregnant mothers by real-time polymerase chain reaction (PCR) targeting multiple exons 4, 5, and 10 of the RHD gene and targeting an SRY gene sequence.These were compared to the D phenotypes determined in the 581 babies they delivered. RESULTS: By combining amplification of three exons,the concordance rate of fetal RHD genotypes in maternal plasma and newborn D phenotypes at delivery was 100 percent. TRANSFUSION 2008;48:373-381

DNA EXTRACTION : 

Major problem in DNA extraction Low copy number of cf fetal DNA Background ‘contamination’ with cf maternal DNA (94 – 97%) In this study DNA extraction was performed with an automated sample preparation system (Cobas Ampliprep, Roche Diagnostics, Vilvoorde, Belgium) with the total nucleic acid isolation kit (Roche Diagnostics) DNA EXTRACTION

References : 

Jean-Marc Minon, Christiane Gerard, Jean-Marc Senterre, Jean-Pierre Schaaps, and Jean-Michel Foidart Routine fetal RHD genotyping with maternal plasma: a four-year experience in BelgiumTRANSFUSION 2008;48:373-381. Human Blood Groups by Goeff Daniels. Mollison’s blood transfusion in clinical medicine. 11edn. References

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