Rh lecture
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Transcript of Rh lecture
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Rh
specific red cell antigen complex blood group system
History of the Rh System
1939, Levine and Stetson Ab in serum of mother of stillborn child;
responsible for the death of fetus.
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1940, Landsteiner and Weiner
reported on an antibody by guinea pigs and rabbits RBCs reacts with 85% of human subjects name Rh was retained for the human produced
antibody. Anti-rhesus formed by the animals was renamed anti-
LW (Landsteiner and Wiener).
1941, Levine et al
Erythroblastosis fetalis (HDN) linked with Anti-Rh
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Nomenclature:The terminology used to describe the Rh system is
derivedfrom 4 sets of investigators.
Two of the terminologies based on the postulated genetic mechanisms of
the Rh system. 3rd terminology the presence or absence of a given antigen. 4th terminology International Society of Blood Transfusion (ISBT)
Working Party on Terminology for Red cell Surface antigens.
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Fisher-Race (DCE terminology)
Suggested 3 sets of closely linked alleles (D and d, C and c, E and e)
Each gene (except d, which is an amorph) causes production of an Ag
Inherited from parents in linked fashion as haplotypes
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D, d, C, c, E, and e.
(d ) -is considered an amorph (silent allele) or the absence of D antigen.
The phenotype (blood type observed during testing) of a given red cells is define by the presence or absence of D, C, c, E, and e.
C, c, E, e represent actual Ags recognized by specific antibodies.
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Wiener (The Rh-Hr terminology)
Multiple alleles at 1 complex locus 1 locus encodes for production of an
agglutinogen which has 3 factors (antigens or epitopes)
Abs can recognize single or multiple factors
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Fisher-Race nomenclature may be converted to Wiener nomenclature and vice versa.
Agglutinogen - presence of a single haplotype composed of three different antigens.
R= D antigen r = absence of the D antigen (d).
C= indicated by 1 or a single ( ′ ) c= when there is no 1 or (′)
E=is indicated by 2 or ( ″ ) e= there is no 2 or ( ″ )
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When referring to the Rh Ag (factor) in wiener nomenclature the single prime ( ' ) refers to either C or c.
The double prime ( ″ ) refers to either E or e.
If the r precedes the h ( rh′ or rh″ ) we are referring to C or E Ags respectively.
When the h precedes the r we are referring to either c ( hr′ ) or e ( hr″ ) Ags.
Rho = D
There is no designation for the absence of D Ag.
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Rosenfield (Alpha numeric terminology)
No genetic assumptions made Numerical system
▪ If listed alone, the Ag is present (Rh:1 = D Ag)▪ If listed with a “-”, the Ag is not present (Rh:1,
-2, 3 = DcE)▪ If not listed, the Ag status was not
determined Adapts well to computer entry
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If Ag has not been typed for, its number will not appear in the sequence .
D is assigned =Rh1 C=Rh2E=Rh3 c =Rh4 e = Rh5
For cell that typed D+ C+ E+ c- e-, the Rosenfield designation is Rh:1,2,3,-4,-5.
If the sample was not tested for e, the designation would be Rh:1,2,3,-4.
All Rh system antigens have been assigned a number.
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Numeric terminology (ISBT)
6 digit number for each Ag specificity First 3 indicate the blood group, eg., 004 = Rh Last 3 indicates the Ag specificity, eg., 004001
= D Ag of Rh system For recording of phenotypes, the system adopts
the Rosenfield approach
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Nonglycosylated proteins (A,B,H are CHOs)
Transmembrane molecules
D and CE are epitopes of proteins with 417 Amino acids as that traverse the membrane 12 X
DNA sequences of D and CE differ by only 44 base pairs; CE, Ce, cd and cE are even more similar to D
Density of Rh Ags on RBCs varies by phenotype
D– greatest number of D Ag
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Rh phenotypeRh phenotype # of D antigen sites# of D antigen sites
RR11rr 990,0-146,00990,0-146,00
RR00rr 120,00-200,00120,00-200,00
RR22rr 140,00-166,00140,00-166,00
RR11RR11 145,00-193,00145,00-193,00
RR11RR22 230,00-310,00230,00-310,00
R2R2R2R2 158,00-333,00158,00-333,00
D--D-- 110,000-202,000110,000-202,000
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Weak D overview
Some cells require addition of AHG (IDAT) to demonstrate agglutination with Anti-D
3 mechanisms causing weak D expression
▪ Genetic - inheritance of D genes which result in lowered densities of D Ags on RBC membranes
▪ C trans - position effect; the D gene is in trans to the C gene, eg., Dce/dCe (C and D Ag arrangement causes steric hindrance weakening D expression)
▪ D mosaic - 1 or more parts of the D Ag is missing; may result in production of Anti-D
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There are instance when an accurate Rh type can not be determined through routine testing
1- If the new-born’s cell are coated with maternal IgG anti-D in utero, very few D Ag sites will be available to react with reagent anti-D.
Elution of the sensitizing Ab (removing the Ab) and identifying it as anti-D will verify that the infant’s red cellare D positive
2- Warm autoimmune hemolytic anemia, Abs are directed against the patient’s own red cell and react as though they are Rh specific.
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Is essential when test donor blood sample.
Blood considered Rh positive if either the D or Du
test is positive
If any donor blood sample that types Rho(D)
negative by either slide or rapid method must be
tested further by indirect anti-globulin test (IDAT).
If both test results are negative, the donor sample
is considered Rh negative.
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Immune IgG Abs (IgG1 and IgG3 most important)
React optimally at 37oC or with AHG
Order of immunogenicity:D > c > E > C > e
Do not bind complement (RBC destruction by Rh Abs is extravascular)
Exposure to less than 1 ml of Rh positive red cells can stimulate Ab production in an Rh negative person
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Anti-D reagents Saline-based - Low protein (fewer false positives);
long incubation times; cannot convert to weak D testing
Protein-based - Faster, increased frequency of false positives; requires use of Rh control tube, converts to weak D testing
Chemically modified - “Relaxed” form of Anti-D in low protein medium; few false positives; saline control performed; converts to weak D testing
Blends of mAbs
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Protocol Add Anti-D to “D” tube; Rh control to “C” tube Spin, read and record
▪ If “D” is positive, cells are Rh positive▪ If “D” is negative, continue testing
Add 22% albumin and incubate for 20” at 37oC Spin, read, and record Wash 3 X in saline Add AHG, spin, read, and record If “D” is positive after heat/albumin or AHG
cells are weak D positive; if negative, cells are Rh negative; “C” should always be negative
Add check cells to neg. tubes; spin, read & record
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Coomb’s cells (C.C)Control cellular used when AHG test is negative.To confirm that washing has been adequate and the anti-globulin reagent is reactive
Control Cell
A group O Rh positive donor’s sample is mixed with 1:10 dilution of reagent anti-D and allowed to incubate.After sensitization of RBC, they are washed with saline and suspended to a 50% RBC suspensionThese sensitized RBCs then used to confirm the anti-IgG activity of the AHG.
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Coomb’s cells (C.C)
They can be used to ensure that AHG test with neg results are not false neg because of inactivation of the AHG reagent.When AHG test is neg , they should be free AHG reagent in the test tubeWhen the CC are added, the free AHG in the test should cause agglutination of the sensitized RBCs.
This positive reaction is a mixed field in nature because;half of RBCs in the mixture lack IgG on their surface and are free cells
2nd half of RBCs have IgG (CC) and are agglutinated
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Weak C (Cw) Not allelic to C and c (C and Cw usually seen
together) 2% of whites; very rare in blacks Anti-Cw may be naturally occurring and
shows dosage
f (ce) When c & e are in cis, eg., dce/DCe Combination Ag Anti-f may be helpful in phenotyping
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Ce/rhi When C and e in cis Compound Ag Ab helpful in phenotyping
G Always found with C-positive RBCs; usually with
D-positive cells Anti G appears to bind to D, C, and G
Many others
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No Cc and/or Ee epitopes DC-, Dc-, D-E, D-- Enhanced or exalted D Ag expression
Rhnull (no Rh Ag expression at all) ---/--- (double bar rr) Or, because of independently inherited
suppressor genes If exposed to any Rh Ags, make Abs to those
and to Rh 29 (“pan” or “total” Rh) Causes a mild hemolytic anemia
Rhmod - weakened expression of all Rh Ags
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Severe HDN Rh Ags are well developed in fetal cells Rh Abs can cross placenta Rh Immune Globulin
IgG and Anti D Only for D Ag
Severe transfusion reactions Highly immunogenic 1st- 120 days; 2nd -2-7 days Extravascular destruction of RBCs
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Symptoms:
Fever, mild bilirubin elevation, decrease in Hgb and haptoglobin
AHG positive