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Fisher Race nomenclature
- follows order of loci genes DCE
- DCE genes are inherited and linked
- each gene produces Ag
- absence of D = d
- little c and little e means gene are present present
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weiner nomenclature
- 1 RHo gene codes for agglutinogen which encodes for Rh antigens
- Rh',rh"
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weiner and fisher race conversion
- r = d
- ' = C
- " = E
- Rz = DCE
- ry = dCE
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ISBT (international society of blood transfusion)
- 6 digit number for each Ag specificity
- first 3 indicate 004 = Rh
- last 3 indicate Ag specifity = 004001 = D
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follow the ISBT numerical code
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genotype frequencies
- Ro = Dce = blacks
- R1 = DCe = whites
- R2 = DcE = 14% white, 9% black
- r = dce =38% white, 25% black
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why Molecular testing for phenotyping and genotyping is becoming more popular
- cannot use anti sera on recently transfused individuals
- anti sera not available for some antigens
- fetal genotyping for D can be done on fetal DNA present in maternal plasma
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Rh antigens
- nonglycosylated proteins
- transmembrane proteins with loops exposed to surface of RBC
- transport CO2 or ammonia
- across plasma membrane
- integral part of RBC membrane
- Rh null people have mild hemolytic anemia
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typing for phenotype and genotype
- only anti D required for routine testing
- other typing sera used for typing RBCs to resolve antibody problems or family studies use statistical probability to determine most probable genotype
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Rh phenotyping used for
- parental testing
- predicting hemolytic disease of newborn
- confirmation of Rh Ab specificity
- finding compatible blood for Rh Abs specificity.
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protocol for Rh phenotyping
- mix unknown RBCs with Rh antiserum (forward typing)
- take tubes through phases ( IS, 37C, AHG, CCC)
- use published frequencies
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location of RHCE and RHD and RHAG
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function of Rh and RhAG
- Rh and RhAG form a complex
- maintain cell stability
- regulate cell volume
- transports ammonia
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genetics of Rh
- Rh locus on chromosome 1
- 2 closely linked Rh genes RHD and RHCE carry Rh antigens
- alleles are autosomal codominant
- linked together as a haplotype (DCe/dce, R1r)
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Rh associated glycoprotein (RhAG
- ABH antigens on 1st loop, no Rh antigens
- on chromosome 6
- serves as a helper for expression of Rh antigens
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Weak expression of D protocol
- if RBC not immediately agglutinated by anti D must be tested for weak D
- 1. incubate cells with anti D at 37 C, will sensitize if present
- 2 wash x3 add AHG
- AHG will bind to anti D coating cells if present
- if negative, individual is D negative
- if positive individual is D positive
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3 mechanisms for weak D
- genetic - inheriting D genes resulting in lowered densities of D Ags on RBC membranes
- C-trans position -D gene is in trans arrangement big D is trans to C (Dce/dCe)
- D mosaic/partial D - parts of D Ag is missing, may result in production of anti D
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are people with weak D considered Rh+ or Rh-
- Rh+ and will receive Rh+
- except mosaic which will receive Rh-
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weak D gene will result in ___ densities of D antigens on RBC membranes
lowered
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Weak D in the lab
- Dw mothers do NOT receive RhoGAM
- D negative do not receive Dw
- Dw may receive D neg
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enhanced D antigen
cDe/cDe (C has limiting effect on expression)
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Allelic deletions of D
- very rare
- may be Ee or Cc
- must be homzygous for deletion to be detected
- no reaction when RBCs are tested with anti serum
- requires transfusion of other D deletion red cells, bc these individuals may produce antibodies with specificities
- written as D - - or -D-
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Rh null
- will have NO Rh antigens on RBCs
- genotype written as ---/---
- can only give an Rh null to an Rh null
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what effects of RBC will an Rh null have
- stomatocytosis
- hemolytic anemia
- why? lack of antigens will disrupt structural integrity
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two types of Rh null phenotypes
- regulator type - Rh gene inherited, but not expressed due to mutation in RHAG gene
- amorph type - RHD gene is absent or no exxpression of RHCE gene, when RHAG is normal
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what is Rhmod
weakened expression of all Rh Ag
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compound antigens occur when and would react only with
- when two Rh genes are on the same chromosome, cis position
- f positive cells will only react with other f positive cells in cis position, not trans
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types of compound antigens
- f antigens - c and e are in cis (dce/dce)
- rh1 or Ce antigen - when C and e are in cis (dCe/dce)
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G antigen
- encoded by the same gene for C or D
- present in cells with C or D
- anti G mimics anti C and anti D
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weak C (Cw)
- C & Cw usually seen together
- 2% whites, very rare in blacks
- anti Cw may be naturally occuring and shows dosage
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D testing ( anti D reagents )
- saline based: for low protein, long incubation times, cannot perform weak D testing
- protein based: faster, incs false positives, requires control. can be used for weak D
- chemically modified anti D: in low protein, few false positives, saline control performed. can be for weak D testing
- blends of mAbs: using hybridized myeloma cells. Specific for Ab cells can be used for weak D. standard technique used now
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protocol for D testing
- have two tubes with Anti D (labeled D) and Rh control (labeled C)
- centrifuge and read
- if D pos: Rh positive
- if D neg: continue testing for weak D
- add adjuvant and incubate
- centrifuge
- was 3 to 4 times with saline
- add AHG
- centrigue
- if D pos in any phase and control is neg, RBCs are Rh+
- if D and control are neg = RBC are neg
- if control is positive = indeterminate
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why must you wash anti D with saline 3 to 4 times?
to get rid of fibrin or protein, or else they would neutralize the coombs reagent giving a false negative
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Rh antibodies
- IgG
- react warm or with AHG
- does not bind to complement, RBC destruction by Rh Abs isĀ extravascular
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what is the order of immunogenicity for Rh antibodies
D > c > E > C > e
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