Test Your Knowledge – Rh Blood Group System

Now you know what I am going to quiz you about after ABO!

Test Your Knowledge – Rh Blood Group System

Being nostalgic is the right for many of us - the retired bunch. :-)

Circa mid 1970’s at Calgary Red Cross
I remember dipping a capillary tube into a vial of saline reactive anti-c to let capillary action aspirate about an inch of the antiserum into the tube, then wiping the outside of the tube with gauze or a piece of tissue with my bare fingers and then dipping into a sample of red cells in a 2-3% suspension to allow an equal volume to be taken up. Again, the tube is wiped and sealed at the other end by placing it on an Rh view box.

Just wonder who might share a picture of it.

I am sure that this was a quick and the cheapest way to identify little c or little e negative donors on a routine basis and for reagent use, as antibody screening cells for the single autoanalyzer in the Rh laboratory once every three weeks! :-(

Using the same format as for ABO, I hope the following True or False game will entertain/update you on the Rh Blood Group System.

My suggestion: Please do a pre test and then look up references to improve your knowledge on the subject.
 
Proceed with Caution: beware of some esoteric but interesting tidbits. :-)
 

101 true or false questions on the Rh Blood Group System

History
 

1. Macacus rhesus commonly known as rhesus monkeys were introduced to Florida in the 1930’s from Asia. Red cells from rhesus monkeys were used by Landsteiner and Wiener to search for more human blood groups.
2. After absorbing out the anti-species antibody, Landsteiner and Wiener discovered that the new antibody obtained from guinea pigs immunized by the old world monkey blood, reacted with 85% of human blood samples. They called it “anti-Rh”
3.  In 1937, while Landsteiner and Wiener were working on the animal experiments in order to discover more blood groups other than, M,N and P1, Levine and Stetson encountered an antibody from a post partum sample causing a severe transfusion reaction and a fatal hemolytic disease of the newborn that reacted to 80% of random donors. That sample was named by Landsteiner, Wiener and Peters, in a separate paper, a year later, as anti-Rh.
4. In 1944, J Murray first proposed the use of a numerical notation to assign antigens for the Rh blood group system (18 years before Rosenfield et el’s proposal) which is very useful in our computer age.
5. After the discoveries of the four common Rh antigens during the second world war, the British terminology of DCE from the three closely linked genes advanced by Fisher and Race became more popular than the single gene producing an agglutinogen that has three factors postulated by Wiener in the United States.
6. In 1963, Wiener was pleased when Levine suggested the animal anti-Rh be renamed as anti-LW to honour Landsteiner and Wiener.
7. Decades long dispute over the inheritance of Rh blood group was put to rest by molecular analysis of Rh genes to confirm Tippett’s model of two genes, one of the antigen D and the other CcEe antigens.
8. In 1938, a year before Levine and Stetson’s report of maternal antibody causing HDN, Ruth Darrow, a physician in Chicago, postulated that the maternal antibody can cross the placenta to destroy fetal red cells.
9. The three-day rule of post exposure Rh immunoglobulin (RhIg) prophylaxis was established in 1961 by the three main researchers, Gorman, a blood bank physician from Australia, Freda, an American obstetrician and Pollack, an English immunologist conducting research using voluntary inmates at the Sing Sing Penitentiary in Ossining, New York.
10. Kath Gorman, Dr. Gorman’s sister-in-law, was the first Rh negative female received Rh immunoglobulin in 1964, she remained unsensitised after five more Rh positive babies! 

Racial Distribution

11. V or ce^S (not Vel!) is 10  times more prevalent among blacks than whites.
12. It is at least 3 times easier to find e-negative donors of Asian descent.
13. Anti-E and -c are 100 times more frequently seen in Asia than anti-D.
14. Ro^Har (Rh33) and Crawford (Rh43)are low incidence antigens  among Whites and Blacks respectively, they may be mistyped as D positive by some reagent anti-D, thus at risk of producing anti-D
15.  D_el is a lot more frequent among Chinese than German Rh negative individuals.

Biochemistry and Genetics

16. Both RhD and RhCE are hydrophobic polypeptides traversing the red cell lipid bilayer 12 times with  both carboxylic and amino terminus are embedded in cytoplasm.
17. The RhD and RhCE polypeptide differ by 31-35 amino acids thus explains D is much more immunogenic than C/c (4 amino acids) and E/e with just a single amino acid substitution.
18. While FY, KN and CROM genes are found on the short arm (p) of chromosome 1, the syntenic RH and SC are found on the long q arm.
19. The gene for Rh associated glycoprotein, RHAG, is also located in chromosome 1.
20. Both RhAG and LW antigens are glycoproteins and less hydrophobic than Rh proteins.
21. The majority of D- among Caucasians are due to complete gene deletion while 2/3 of D- black Africans have inherited a RHD pseudogene which has an insertion of a 37 base pair nucleotides causing the generation of a stop codon.
22. Homozygous RHD gene deletion among East Asians are rare (0.1-0.3%), between 62-77% of these D-individuals are due to a hybrid gene of RHD-CE-D or DEL gene.
23. Although still less than perfect, weak D’s ( mostly due to membrane or intracellular amino acid substitution) and partial D’s (mostly due to hybrid genes) are better defined by molecular than serological methods.
24. There are over 30 D epitopes have been defined by monoclonal antibodies.
25. RhD and RhCE proteins are encoded by 10 exons each.

Structure and Function

26. Rh proteins are integral to the red cell cytoskeleton as inferred by abnormal red cell morphology in the Rh_null phenotype.
27. Along with the RhAG, Rh proteins are part of a macrocomplex with band 3, the anion exchanger where the Diego blood group system is located. Rh proteins have the typical membrane transport protein topology, they are responsible for transporting carbon dioxide, oxygen and ammonia.
28. The tetrameric band 3, RhD,RhCE and RhAG are linked to cytoskeletal proteins Ankyrin and protein 4.2. Other blood group containing components are LW, glycophorins A and B. CD47,the ‘self’ marker, having immunoglobulin superfamily (IgSF) structure, is also linked.
29. The dimeric band 3, RhD and RhCE but not with RhAG are complexed with Xk, Kell, Duffy protein are attached to the cytoskeleton through glycophorin C.
30. It is interesting to note that there is an association in the absence of Rh protein and the weakened (S,s,U)or absent(LW, RhAg, Fy:5) expression of certain blood group antigens.

Serology
Rh antigens

31. DcE/DcE red cells have the most number of D sites per cell whereas DcE/dce have the least between 10-15,000 per red cell.
32. Certain weak D types may have fewer than 1000 D sites per red cell.
33. D--/D—suspended in saline can be clumped by IgG anti-D by immediate spin.
34. Low frequency antigens in the Rh blood group system are sometimes referred to as “signature antigens” to most partial D’s. For example, DAK(DIIIa); Go^a(DIVa); Evans (DIVb); Dw(DV);BARC(DVI); Tar (VII); FPTT(DFR) etc.
35. D is very immunogenic, alloimmunization following D+ to D- transfusions exceeds 50% in hospitalized patient.
36. DVI is the most significant partial D as anti-D is most frequently made by partial D+ individuals. Severe HDFN have been reported associated with DVI.
37. DVI has both qualitative (missing epitopes) and quantitative (fewer sites) defects.
38. Reagent IgM monoclonal anti-D must be selected to detect DVI by immediate spin.
39. Similar to A_el, D_el has the weakest D expression (<22-36 sites per cell) can only be demonstrable by adsorption and elution.
40. Over 90% of weak D individuals are of types 1,2 and 3 and only in rare cases where type 1 and 2 have produced anti-D.
41. D^HAR  and Crawford (ceCF) may cause typing discrepancies with different antisera, that is very strong with some antisera but negative with others.
42.  C/c and E/e antigen frequencies are similar in different races.
43. Partial C and e are more frequently found in blacks than whites.
44. MAR or RH51, a high frequency antigen is antithetical to the low incidence C^w and C^x. Amongst Caucasians, expression of C and e are weakened in the presence of C^w or C^x.
45. Weakened C expression is seen in DCE (R^z) haplotype whereas e is weakened when the low frequency VS+( RH20 or e^s) is present. 
46. Four E variants have been classified (EI-IV), all with Pro226 but there is an additional amino acid substitution in the RHCE in each category.
47. e+ individuals producing alloanti-e (DAT-) are of African descent, these antibodies are anti-hr^s (RH19) and hr^B(RH31); 1/3 to 2/3 of these individuals are positive of STEM (RH49), a low frequent antigen.
48. Transfusing e- (R2R2) blood to hr^s- or hr^B- individuals with sickle cell disease could be at risk of producing anti-E and anti-D as these patient are often partial DIII typed as D+.
49. ce or f is antithetical to G.
50. Most anti-C made by D+ individuals also contain anti-Ce or -rhi while most samples of anti-C and anti-D+C also have an anti-Ce component.
51. With the advent of monoclonal anti-C, the use of rare C+,Ce- ( R^zr or r^Yr) as a positive control is now obsolete.
52. Almost all D+, C+ are G+, rare D- that G+ is also JAHK (a low frequency RH53) positive.
53. D- -homozygotes (C-c-,E-e-) produce only three Rh antigens: Rh29, G and many more D sites (5X-10x)
54. D.. homozygotes(C-c-,E-e-) have 2-3x as many D sites, positive for Rh29, G and the high frequency Dav (RH47) as well as the signature low frequency Evans (RH37)
55. Rh_mod red cells have depressed Rh antigens due to missense mutation of RHAG.
56. Presence of few Rh antigens can be demonstrated by IAT on enzyme treated Rh_mod
57. Rh _null can be a result of a deletion of RHD and inactivating mutation of RHCE (the amorph type) or inactivating mutations of RHAG (the regulator type).
58. RhAG is absent on amorphic Rh_null whereas regulator Rh null have reduced amount of RhAG. 
59. Rh_null cells lack the high frequency LW^a and LW ^ab.
60. S,s and U are depressed on Rh_null cells as there is a 60-70% reduction of glycophorin B on Rh_null cells.

Rh antibodies

61. “naturally-occurring” or non red cell immune anti-D,-C, -E, -C^w and -C^x have been reported.
62. IgG Rh alloantibodies may persist for more than 30 years.
63. Rh antibodies do not activate complement because there are fewer than 50,000 antigen sites per red cell.
64. Although Rh antibodies could be IgG1 and IgG 4, most Rh antibodies are IgG 2 and 3.
65. Anti-E and anti-c are more frequently detected than anti-D in Asia.
66. An apparent “D+C” specificities may  be due to anti-D+anti-C; anti-D+anti-G; anti-C+anti-G; anti-D+anti-C+anti-G; anti-G only.
67. If the rare rr^G reagent red cell is not available, the combinations in the previous question can not be differentiated.
68. Although anti-D can be measured semi-quantitatively by titration, it can be quantified by the use of a standard anti-D with the antibody nitrogen content expressed as µg/ml.
69. Anti-D’s indirect antiglobulin titre at 1 has been measured as 0.01-0.1µg/ml.
70. IgG anti-D will agglutinate D--/D--, enzyme treated normal D+ and normal+ red cells suspended in 20-30% bovine albumin by immediate spin.
71. Anti-rhi will not react with R^zr even though it is C+e+.
72. Anti-Jarvis is reactive with red cells from R^zr but not R¹/R²
73. Anti-f can be developed from D- patients.
74. Hemolysis due to Rh antibodies can occur following Rh antigens (D,c,E and e) positive recipients received bone marrow or solid organ transplant from the corresponding Rh antigen negative donors. This is due to the fact that the immunocompetent donor’s “passenger lymphocytes” recognize the recipient’s tissue, this this case, blood group antigens, are being foreign.
75. Rare Anti-RH17 (Hro), anti-RH18 (Hr) and RH34 (Hr^B)are alloantibodies react with all normal Rh  but not D--/D—or Rh_null red cells, when a warm autoantibody having the same specificity, it is called anti-nl which stands for anti-non (Rh) deleted.
76. Anti-pdl is an autoantibody with the same specificity as the allo anti-RH29 (total Rh) developed by immunized Rh_null.
77. Anti-RH50 (serum Mol) detects a low frequency antigen of less than 0.01% in the general population but it reacted with 3 out of four propositi who worked for the French Post Telegraph and Telecommunication, hence named FPTT.
78. Anti-e is the most frequently detected warm reactive autoantibody.
79. With rare exceptions, weak Rh antibodies detected by enzyme IAT only are clinically insignificant.
80. Use of monoclonal reagent at refrigeration temperature anti-D may cause a false positive result as some IgM monoclonal anti-D’s has a variable region of the heavy chain (V4-34) is also present on cold agglutinins of Ii specificities.

Clinical Significance

81. Since Rh antibodies do not activate complement, intravascular hemolysis leading to hemoglobinuria is not possible.
82.  If anti-c can be adsorbed completely by c- red cells from a c- patient, the apparent alloanti-c is a mimicking warm reactive autoantibody.
83. One must consider the possibility of development of a warm reactive autoantibody in addition to a delayed hemolytic transfusion reaction due to anti-c and anti-E when patient’s hemoglobin drops below the pre transfusion level in the absence of bleeding.
84. Along with anti-Jk^a and anti-K, anti-c and anti-E are frequently associated with delayed hemolytic transfusion reactions hemolytic disease of the fetus/newborn HDFN.
85. In the 1920’s, before the elucidation of HDFN’s etiology, exchange transfusions were performed successfully in Canada to treat HDN.
86. Fetal maternal hemorrhage, was first quantified by Marion Lewis in Toronto using capillary tube method and saline reactive anti-D.
87. Dr. William Pollack, an English immunologist, prepared the first Rh immunoglobulin (RhIg) for use in 1961. He also advanced the Zeta Potential Theory.
88. IgG level in cord serum is higher than that in the maternal circulation, this is likely explained by a possible active transport mechanism of maternal IgG in the placenta.
89. Maternal HLA antibody to the Fc receptor on the fetal macrophages may reduce the severity of FDFN . It has been illustrated by much milder outcomes on women who had previous history of severe HDFN.
90. Non invasive procedures such as Doppler ultrasonography and fetal D genotyping on maternal plasma established in recent years have improved clinical management of HDFN.
91. Over 20% of neonates whose cord total bilirubin level between 325-410 µmol/L(19-24mg/dl) developed kernicterus.
92. Routine antenatal injection of RhIg and postpartum screening of large fetomaternal bleed with appropriate dosing have completely iradicated HDFN due to anti-D
93. Volume of fetomaternal bleed is routinely measured by micro D^u test, column agglutination (gel), enzume-linked IAT, rosette test and acid elution techniques although flow cytometry, fluorecence in situ hybridization FISH and DNA technology are also available for special indications and research use.
94. Titration of maternal antibodies is no longer relevant when ultrasonography or amniocentesis takes over prenatal management.
95. Anti-D can be seen in primigravidas with no history of abortion can be explained by the “grandmother effect”. That is, primary alloimmunization by maternal-fetal bleed occurred when the mother was born.  
96. Although Keihauer-Betke Test did not correct only 92% of fetal cells are stained by eosin and they are 22% large than adult red cells that were included in Mollison’s calculation, the results compared favourably with Mollison’s calculation and it is easier to calculate.
97. Compatible blood (washed and irradiated) used for in HDFN due an antibody to a high frequency Rh antigen can be obtained from the mother, her siblings or from rare donor file.
98. RhIg is now prepared from blends of monoclonal anti-D by harvesting T cells from D immunized donors and human myeloma cells, thus eliminating plasmapheresis from D alloimmunized volunteers.
99. Intrauterine transfusion has significantly reduced the incidence of exchange transfusion as a result of Rh HDFN.
100.  Injection of RhIg still offer partial protection if it has not been administered within 72 hours after birth of a D+ infant.
101. For those who are Rh negative singles, there is a “date by type” web site to meet your future Rh negative partner/husband  

 
                                                          Acknowledgement
 
This entry is dedicated to our Canadian blood bankers and research scientists, especially those distinguished pioneers in Winnipeg, who contributed to the conquest of Rh-HDFN.
 
Best regards,

Eric

References

• Daniels G. Human Blood Group. 3^rd ed. Oxford:Wiley-Blackwell.2013
• Garratty G ed. Immunobiology of Transfusion Medicine. New York:Marcel Dekker, Inc. 1993
• Issitt PD Anstee JD. Applied Blood Group Serology. 4^th ed. Durham NC Mongomery. 1998
• Mollison PL. Blood Transfusion in Clinical Medicine. 5^th ed. London: Blackwell. 1972
• Race RR, Sanger R. Blood Groups in Man. 6^th ed. Oxford:Blackwell.1975 
• Reid ME, Lomas-Francis C. Blood Group  Antigens FactsBook. 2nd ed. San Diego: Academic Press. 2003

Answers
1.T
2.F Rabbits were used first, they switched to guinea pigs later because the guinea pig antibody was stronger.
3.T
4.T
5.T
6.F Wiener and Levine had a very public debate as to who discovered anti-Rh first, until the day of  Wiener’s death in 1976; although incorrect,  he still maintained that the animal anti-Rh was the same as human anti-Rh.
7.T
8.T
9.F It was the warden who made the decision based on his fear that a return visit two days in a row would post a higher risk of escape!
10.T
11.F Blacks- 30%; Whites 1% Asian ?
12.T
13.T
14.T
15.T
16.T
17.T
18.F RHD and RHCE are located at 1p36.13 and 1p34.3 respectively
19.F RHAG is located at 6p11-p21.1
20.T
21.T
22.F  2/3 of the D- East Asians due to D deletion whereas the remaining 1/3 are due to hybrid or DEL gene. The incidence of D- among east Asians is 1 in 350-1000. With a population 1.3 billion in China alone, there are millions of Rh- females that are at risk of Rh alloimmunization,  Rh prophylaxis is still a challenge.
23.T
24.T
25.T
26.T
27.F Rh protein are almost certain not a membrane transporter and their functions not clear.
28.T
29.T
30.T
31.F DCe/dce red cells have the least around 10,000 copies, compare with DcE/DcE cells which have around 30,000 copies per cell
32.T
33.T
34.T
35.F Healthy volunteers-80%; patients-20-30%
36.T
37.T
38.F DVI must be typed by immediate spin as Rh negative to avoid alloimmuniztion following transfusion of Rh positive blood
39.T
40.T
41.T
42.F            Caucasian      Black       Asian         Ref. Blood Group  Antigens Facts Book.
          C           68%            27%          93%                
           c           80                96             47
           E          29                22             39
           e           98                98             96
43.T
44.T
45.T
46.T
47.T
48.T
49.F Pure anti-ce or -f is rare and it is usually accompanied by anti-c or anti-e, G is usually with D or C although rare rr^G red cells have been identified.
50.T
51.T Some human reagent anti-C’s contained predominant anti-Ce than anti-C which may give false negative results.
52.T
53.T
54.T
55.T
56.F adsorption and elution is required
57.T
58.F RhAG is present on amorph Rh_null cells whereas regulator Rhnull has no RhAG.
59.T
60.T
61.T
62.T
63.F Two adjacent IgG molecules referred to as a “doublet” a distance of 200-400A^o (A^o=0.1nM) in order to accommodate C1q,  even distribution of the Rh antigens on the red cell membrane make the sensitizing Rh antibodies to be too far apart to bind C1q. IgG anti-Jk^a, on the other hand,  can activate complement with fewer antigen sites than D at 8-14,000 copies per cell. It may be explained by the clustering effect of the Kidd system antigens. Same explanation can be offered to the Kell and Duffy blood group system as well.
64.F Most Rh antibodies are IgG 1 and 3, few are 2 and 4.
65.T
66.T
67.F Use of rr’ and Ror red cells in adsorption and elution study can differentiate different combinations by identifying antibody in eluate and adsorbed serum.
68.T
69.T
70.T
71.T
72.T
73.T Although not reported (to the author’s knowledge), anti-f can theoretically be produced from the rare dCe/dcE ie. f- individuals.
74.T
75.F Anti-nl is an autoantibody developed by patients with normal Rh phenotypes. Immunized CE deleted or Rh null individuals are able to make allo anti-RH17, RH18 and RH34.
76.T
77.T
78.F Although auto anti-e is the most commonly seen warm reactive autoantibody against a single Rh specificity (e,c,E,D and C in descending order of occurrence), most of  these patients  are usually diagnosed in the early stage of warm auto immune hemolytic anemia. As the disease progresses, the autoantibody tend to “broaden” its specificity to be reactive with normal Rh phenotypes but not with partially deleted Rh and Rh null phenotypes (anti-nl). Sera from these patient may eventually also react with partially deleted cell (anti-pdl) and finally with Rhnull cells (anti-dl). Most warm reactive autoantibies that I had experienced were pan reactive with panel cells rather than seeing a simple Rh specific antigen.
79.T
80.T
81. F Hemoglobinuria was seen in the very first reported case of immediate hemolytic transfusion reaction due to anti-D. The best plausible explanation would the intravascular hemolysis was due to antibody dependent cell-mediated cytotoxicity ADCC by the circulating NK cells.
82.T
83.T
84.T
85.T
86.F Dr. Marion Lewis was working with Drs. Chown and Bowman in Winnipeg.
87.T
88.T
89.T
90.T
91.F less than 10% (1/13 in Mollison’s series).
92.F HDFN still occur at 0.2-0.4 %.
93.F micro D^u test is too insensitive that only 15% of technologists could see a positive result when a control of 30 ml FMH is set up!)
94.T
95.T
96.T
97.T
98.F Monoclonal antibodies are less effective than polyclonal anti-D in clearing D+ cells, clinical trials had not shown similar efficacy.
99.T
100.T
101.T