A review: the Duffy group system

K.M. BEATTIE

In 1950, two reports described an that had neuraminidase. On the other hand, Fy3, Fy4, and Fy5 been found during the investigation of a hemolytic are not affected. Reactionswith purified trypsin show transfusion reaction in the serum of a 43-year-old man that the Fysup(a) antigen is unaffected and Fy sup(b) is only slightly suffering from hemophilia. 1,2 After the unidentified reduced in strength; Fy3 and Fy6 are slightly enhanced. antibody had been separated from the anti-D, anti-A, The trypsin most commonly used by blood bankers and anti-Bin his serum, it was tested against the blood is a crude preparation that is contaminated with chymo- of 205 unrelated English adults; 64.9 percent were ,agglu- trypsin; therefore, results obtained with that product tinated. With the permission of the patient, the new would approximate those resulting from chymotrypsin blood group system was named Duffy The antigen was treatment. designated as Fysup(a), the responsible for it, Fy sup(a), and Inactivation of Fy determinants is such that they are its hypothetical allele, Fysup(b). Anti-Fysup(b) was reported the no longer capable of adsorbing their antithetical anti- following year in the serum of a German woman who bodies. This is not due to the removal of sialic acid but had been pregnant three times but not transfused.sup(3) probably represents proteolytic action on cell membrane Four years later, Fy(a-b-) was reported to be the . Fy(a-b-) red cells are not sialic acid deficient most common phenotype in American blacks.sup(4) and their electrophoretic mobility is normal. 13 Either In 1965, a new allele, Fysup(x), was described,5 but new formaldehyde treatmentor heating the red cells to 56°C in the system-to Fy3, Fy4, and Fy5—were for 10 minutes denatures the Fy antigens, indicating not reported until 1971 and 1973.sup(6-8) Another 14 years that the receptors are proteins. elapsed before the most recently described antibody- Treatment with 2-aminoethylisothiouronim bromide anti-Fy6, a monoclonal murine antibody, became part affects neither Fysup(a) nor Fysup(b) antigens,sup(14) but Fy sup(b) may be of the Duffy system.sup(9) weakened by 2-hour treatment with chloroquine diphos- This chronology of events must include the 1963 phate. 15 report showing a probable close linkage between the In 'an assay using quantitative immunoferritin micro- Duffy locus and the locus for congenital zonular scopy,sup(16) red cells having a double dose of Fysup(a) antigens cataractsup(10) and five years later the assignment of the were found to have 13,300±3500 sites per cell and Duffy locus to human 1. 11 Another pro- 6,900 ± 610 sites per cell when only a single dose was foundly significant finding came with report of an present. The latter number was the same for single doses association between the Fysup(a) and Fysup(b) antigens and the of Fy sup(b)antigen, while double doses measured 13,700± invasion of red cells by certain species of malarial 4000 sites per cell. parasites. 12 Duffy antigens appear to be limited to red cells, Fysup(a), Fy sup(b), and Fy3 being absent from leukocytessup(13) and Fysup(a) Biochemical and Serological Characteristics and Fy sup(b) not found on . 17 While the Duffy anti- of the Antigens and Antibodies gens are not natural constituents of body fluids, anti- Duffy antigens genic substances can be shed from red cells into the The Duffy antigens fall into two groups in regard to supernatant when they are stored in a low-pH, low- their sensitivity to enzymes. Fy,sup(a) Fy sup(b), and Fy6 deter- ionic-strength medium. These substances specifically minants are destroyed by proteolytic enzymes such as inhibit their antithetical antibodies—anti-Fysup(a), anti-Fysup(b), ficin, papain, bromelin, and chymotrypsin, but not by or anti-Fy3.sup(18) The investigators reported that these Fysup(a)

VOLUME 5, NUMBER 2. 1989 45 K.M. BEATTIE

and Fysup(b) structures have a molecular weight of 35-55 of testing, and the anti-Fysup(b) used. kD.sup(19) Other workerssup(20) have reported that the Fyy sup(a) struc- Quantitative hemagglutination assays for Fsup(b) and Fy3 rure has a mobility similar to PAS 2, a band associated antigens on red cells from seven Fysub(x)Fy sub(x) two Fysup(b)Fy sup(x), with MN sialoglycoprotein. This would indicate that and four sup(a)Fy sup(x) persons showed that moderate depres- it is located on or near A in the extracellular sion of Fy3 occurs in sup(a)F sup(x) and F Fy sub(b)F sup(x) persons and domain of an integral membrane .sup(20,21) marked depression of Fy3 occurs in Fysub(x)Fy sub(x) people.sup(26) Weak reactions with anti-Fy5sup(13) and anti-Fy6sup(9) were also Anti-Fy sup(a) and -Fysup(b) found on testing the cells of Fy sup(x)Fysup(x) individuals. Like the first example of anti-Fysup(a), most Duffy anti- bodies require the antiglobulin test for detection. Saline- Fy3 and anti-Fy3 reactive Duffy antibodies are rarely found. Their reac- An antibody found in the serum of a Fy(a-b-) Cau- tivity is enhanced in a low-ionic-strength medium. Many casian Australian woman reacted with Fy(a+) and Fy(b +) examples of anti-Fysup(a) can be detected in low-ionic hexa- but not Fy(a-b-) red cells. The antibody was shown dimethrine bromide (Polybrene®).sup(22) The antibodies are not to be a mixture of anti-Fysup(a) and -Fysup(b) and also dif- stable at 70°C and are not denatured by 0.1M 2-mer- fered by agglutinating enzyme-treated Fy(a+) or Fy(b+) captoethanol. red cells. The authors of the report6 named the anti- These antibodies react optimally at 37°C and many body anti-Fy3 and speculated that Fy3 might be the activate the complement cascade to bind C3b to antigen- precursor for Fyy sup(a) and Fy sup(b). positive red cells. During a second pregnancy, anti-Fysup(a) and the second Most examples of these antibodies are 7S gamma example of anti-Fy3 were found in the serum of a globulins having a molecular weight of 160,000.IgG1 16-year-old American black woman whose red cells has been found as the only immunoglobulin subclass in typed as Fy(a-b-) Fy: - 3.sup(27) She had received one unit 22 of 25 examples of anti-Fysup(a) and FFy sup(b).sup(23) of Fy(a+ b +) Fy:3 red cells during her first pregnancy. Some examples of anti-Fysup(a) and anti-Fysup(b), when titrated The red cells of her second child typed as Fy(a- b +) against red cells of known phenotype, can distinguish Fy:3, but the direct antiglobulin test was negative at between cells having a single dose of the antigen and birth. those with a double dose. Dosage can be determined A third example was identified in the serum of a Cana- more clearly through antibody-binding techniques such dian Cree Indian woman who had been transfused. She as the enzyme-linked immunosorbent assaysup(24) and produced anti-Fy3 and not anti-Fysup(a), although five of fluorescence-activated flow cytometry.sup(25) her children were Fy(a+).sup(28) Neither her mother nor her maternal aunt, who also had Fy(a-b-) red cells FY sup(x) and had a total of 15 children, produced Duffy anti- The Fysub(x) antigen was detected because of what ap- bodies, suggesting that the risk of immunizationthrough peared to be a discrepancy in inheritance in 20 of 537 pregnancy may be minimal. families tested.sup(5) One or more Fy(a+b-) children were born in nine families where one of the parents tested Fy4 and anti-Fy4 as Fy(a- b+). The silent allele Fy did not account for The suggestion that the Fy(a-b-) phenotype com- the anomaly because in most of these cases the apparent monly observed in blacks represented a null phenotype Fy(a+ b-) person's red cells reacted weakly by anti- was challenged by the report of an antibody found in globulin test with some examples of anti-Fysup(b). a black child who reacted with the red cells of blacks The original investigators felt that Fy sub(x), as they but not whites.' At least in blacks, Fy(a-b-) could not designated the gene, was a new allele to ysup(a), F ysup(b), and be the resultant product of two silent Fy . The Fy. However, antibody eluted from Fysub(x) cells had anti- child’s red cells typed as Fy(a+b+),and her serum agglu- Fsup(b) specificity, and efforts to separate an antibody tinated Fy(a-b-) and some Fy(a+b-) or Fy(a-b+) red specific for Fysub(x) from anti-Fysup(b) failed. The difference cells from blacks but did not agglutinate the red cells between Fsup(b) and Fysub(x) may simply be quantitative. The from blacks with the Fy(a+b+)phenotype using anti- distinction between the products of Fy and sup(x) in globulin or enzyme-antiglobulin tests. The reactions were forensic applications depends on the methods, extent weak and some variations were observed by the three

46 I M M U N O H EM AT O L O G Y Duffy system review

laboratories studying the serum. Nonetheless, it was The authors of the reportsup(9) determined that red cells concluded that the antibody was part of the Duffy from persons presumably homozygous for Fysup(6) had a system. It was named anti-Fy4; its antigen, Fy4, was mean number of antigenic determinant sites of 12,200± said to be the product of a Fysup(4) gene. Fysup(4)Fy sup(4) rather than 1260 per cell, closely approximating the 13,300 Fysup(a) FyFy would represent the genotype of blacks having sites on cells having Fy sup(a) in double dose. No attempt the common Fy(a - b -) phenotype. Since the Fy(a - b - ) was made to measure sites on cells having a single dose phenotype is extremely rare in Caucasians having no of Fy6. admixture of black genes, their genotype would be FyFy. Anti-Fs Fy5 and anti-Fy5 A third antibody was found in a mixture of anti-D When an antibody was found in the serum of an and anti-V in the serum of a multiparous, multitransfused 11-year-oldblack boy whose red cells typed as Fy(a-b-), Brazilian black woman whose red cells typed as it was thought to be another example of anti-Fy3.sup(8) His Fy(a + b + ) Fy: 3,5. Testing of the unidentified antibody, serum reacted with enzyme-treated or untreated Fy(a+) after separation from the others, showed that it reacted and Fy(b+) red cells but not with Fy(a-b-) cells from by antiglobulin test with 79 percent of Fy(a - b - ) red random blacks. The antibody differed from anti-Fy3 cell samples and about 14 percent of Fy(a+b-), by not reacting with Rh sub(null) red cells in spite of their Fy(a-b+), and Fy(a+b+)samples from either black being either Fy(a+) or Fy(b+). Only weak reactions were or white donors. The reactions were so weak that a obtained with -D- cells. In further contrast, the serum cutoff score bad to be established to differentiate agglutinated the Fy(a-b-) cells of the Caucasian woman positives from negatives; agglutination ranged from who had made anti-Fy3. microscopic to barely recognizable macroscopically. The antibody was designated as anti-Fy5 and the anti- Nonetheless, Fy(a- b-) red cells were clearly targeted gen as Fy5. The investigators noted that since the makers by the antibody; the significantly higher frequency of of anti-Fy5 had red cells lacking Fy5 but had normal reactions for that phenotype, as opposed to other Duffy Rh antigens, Fy5 could not be a common precursor for types, indicated that the antibody was not directed both Rh and Fy. They suggested that Fy5 might originate against an unrelated high-incidence antigen in blacks. by interaction of Rh and Fy gene products. A second The authorsup(31) named the antibody anti-Fs and example of anti-Fy5, reported 3 years later, failed to pointed out that it differed from anti-Fy4 by reacting react with either the amorphic or regulator type of with Fy(a+b+)red cells. Anti-Fs did not bind comple- Rh sub(null) red cells, leading the authors to conclude that ment, and its reactivity was enhanced by papain treat- “it must be the Rh gene itself and not the X sup(1)r modifier ment of Fs-positive red cells. The Fs antigen was found that is inv01ved.”sup(29) Lending credence to this is a recent on the red cells of either black or white newborn infants. report showing that anti-Fy5 also does not react with Although family studies were not done because of the e variants in the Rh system.sup(30) weakness of the antibody, limited tests showed that Fs- positive children may be born to Fs-negative parents. Fy6 and anti-Fy6 “If Fs is inherited as a simple Mendelian charcteristic, The observation that Fy(a- b-) red cells resist penetra- the controlling gene segregates independently of the tion by certain malarial parasites led to the search for Duffy genes”sup(31) (Tables 1, and 2). an alloantibody that might detect the specific Duffy receptor responsible for invasion.sup(9) A monoclonal anti- Nomenclature body raised in mice reacted with all human red cells The known alleles in the Duffy system are Fy sup(a) and except those of the Fy(a- b-) phenotype. What set this Fybsup(); in the numerical system, they are Fy sup(1) and Fysup(2). The antibody apart from anti-Fy3 was its failure to react with products of these genes are Fysup(a) and Fysup(b) or Fy1 and Fy2; enzyme-treated cells. It differed from anti-Fy5 by giving the antibodies to these antigens are anti-Fysup(a) and anti- positive reactions with rh sub(null) cells of both the amorphic Fysup(b), respectively. The product of the Fysup(x) gene is an anti- and regulator types. Fy6, as the antigen was named, gen called Fysup(x), but as far as can be determined with behaved like Fysup(a) and Fysup(b) in being destroyed by ficin, antiserums (specific for anti-Fysup(b)), the Fysup(x) antigen is papain, and chymotrypsin. simply a weak variant of Fysup(b).

VOLUME 5, NUMBER 2, 1989 47 K.M. BEATTIE

Table 1 Characteristics of Duffy system antibodies

Typical Serological Reactivity Saline LlSS or Albumin Enzyme Ability to lmplicated in Specificity 22° C 37" C AGT* 22°C 37" C AGT 37" C AGT Bind Complemcnt HTR† HDN‡

'Antihuman globulin test †Hemolytic transfusion reactions ‡Hemalylic of the newborn

Table 2 Reactions of Duffy System antibodies with red cells of various phenotypes

Race of Anti- Donor Red Cell Phenotypes Fysup(a)* Fysup(b)* Fy3 FY4 Fy5 Fy6* Fs

Caucasian Fy(a+ b-) + 0 + 0 + + 14%+ Caucasian Fy(a - b + ) 0 + + 0 + + 14%+ Caucasian Fy(a +b + ) + + + 0 + + 14%+

Caucasian Fy(a+b-).D- - + 0 + 0 +sup(w) NT Caucasian Fy(a+b+).Rhsub(null)(cegulator type) + + + 0 0 + NT Caucasian Fy(a-h+),Rh (amorphic type) 0 + + NT 0 + NT Caucasian Fy(a- h + genotype 0 + sup(w) + sup(w) NT + w + sup(w) NT Caucasian Fy(a-b-) producer of anti-Fy3 0 0 0 +sup(w) + 0 + Cree Indian Fy(a-b-) producer of anti-Fy3 0 0 0 + † NT NT Black Fy(a- b -) 0 0 0 most+ 0 0 79%+ Black Fy(a+ b+ ) + + + 0 + + 15%+ Black Fy(a+ b -) + n + most+ + + 15%+ Black Fy(a- b+) 0 + + most + + + 15%+ Black Fy(a+) or Fy(b+) card blood + + +sup(w) NT + + ‡ +§

NT–not tested 'negative reactions are obtained with these antiscra when antigen-positive red cells are enzyme treated †test could not he properly controlledsup(28) ‡same strength as adult red cells (Nichols ME, personal communication) §same strength as adult red cells

There is no known product for the Fy gene when a black donor with the same reagents would be written: it occurs in Caucasians or Asiatics. While Fy can also Fy(a - b -) Fy : - 3,4, - 5,- 6. act as a silent gene in blacks, the common Fy(a-b-) phenotype is usually accompanied by the Fy4 antigen. Clinical Significance of the Other Duffy system notations follow this pattern: the Antigens and Antibodies genes Fysup(3) and Fysup(4) each produce their antigens Fy3 and Relationship of and the Duffy antigens Fy4, which are recognized by anti-Fy3 and anti-Fy4, Two important observations were made in 1955: I) respectively. Phenotypes express the actual results of that the red cell phenotype of the majority of blacks testing, not presumptions. For example, typical reactions was Fy(a-b-)sup(4) and 2) that blacks were resistant to for red cells from a Caucasian donor tested with anti- infection with Plasmodium vivax. 32 Almost two Fy sup(a), anti-Fysup(b), anti-Fy3, anti-Fy4, anti-Fy5, and anti-Fy6 decades elapsed before the association between those could result in a phenotype written as Fy(a-b+) two facts emerged. sup(12,33) Fy3, -4,5,6. The common phenotype found by testing Using the merozoites of P. knowlesi, a simian malarial

48 IMMUNOHEMATOLOGY Duffy system review

parasite able to invade human red cells, it was shown of immunization involved the production of anti-Fysup(b) by in vitro tests that Fy(a+) or Fy(b+) cells were invaded, by a woman who received an intrauterine transfusion while Fy(a- b-) cells or those whose Fysup(a) or Fysup(b) anti- for her fetus.sup(37) She and her husband typed as Fy(a+ b-) gens had been removed by enzyme treatment were resis- and the blood donor as Fy(a+b+). tant to invasion. l2 Although the merozoites attach Autoantibodies in this system are uncommon. One themselves to all human red cells regardless of Duffy report of an autoantibody mimicking anti-Fysup(b) was status, they detach from Fy(a- b-) cells, whereas in the found in the serum of a woman having Fy(a + b - ) red case of Fy(a+) or Fy(a+) cells, attachment proceeds cells.sup(38) That it was not an alloantibody was shown by to invasion. Fy(a+b-) as well as Fy(a-b+) red cells adsorbing it; In West Africa, there is high resistance to infection Fy(a-b-) cells did not remove the autoantibody. Anti- by E! knowlesi and P. vivax as well. Although E! vivax Fy3 and anti-Fy5 were ruled out because the antibody could not be cultured in vitro, in vivo studies showed produced only weak reactions with enzyme-treated that of 16 volunteers who had been bitten by E! vivax- Fy(a+) red cells. infected mosquitoes, only the five Fy(a- b -) blacks failed An ailtoantibody mimicking anti-Fysup(a) has also been to be infected. The other 11 Fy(a+) and/or Fy(b+) blacks reported. 39 and whites developed malaria.sup(34) Only 32 percent of American blacks are positive for Fy sup(a) and Fysup(b), but a Frequency and Immunogenicity study of 13 black soldiers who had contracted E! vivax Usually, Duffy system antibodies are found in mixtures malaria in Vietnam showed all to be Fy(a+) and/or of other antibodies, but they can occur as single specif- Fy(a+).sup(35) The p value for this to occur by chance cities. Anti-Fysup(a) is not rare in Caucasians, but it is found would be < 0.001. 20 times more often than anti-Fysup(b). Since 98 percent In spite of some relationship between the Duffy type of blacks lack Fysup(a) and/or Fysup(b), one might expect to find and P. vivaxmalaria, no association could be made with twice as many Duffy antibodies in blacks as are found P.fulciparum, the major malarial parasite in humans. in the 51 percent of Caucasians at risk. However, reports A study of 420 persons living in Honduras involved from five reference laboratories indicate that Duffy testing for Duffy blood groups and markers of infection system antibodies occur much less frequently in blacks with E! vivax and E! fulciparum.sup(36) While the results than might be expected from their phenotypes, com- confirmed previous findingsbetween P. vivax and Fysup(a) pared with Caucasians.sup(40-44) Of 110 Duffy antibodies and/or Fysup(b), infection with P. fulciparum was found found by one laboratory, only 18 were formed by blacks equally among those persons negative and positive for in a patient population having a ratio of 1 black to 1.7 the Duffy factors. whites.** Based on the occurrence of other antibodies, The hypothesis that Fysup(a) and Fy sup(b) serve as receptors this racial difference cannot be attributed to an unusual for invasion of erythrocytes by E! vivax merozoites has distribution of immune response genes. Rather, it may been questioned by the finding that human Duffy- reflect a protective effect afforded by Fy4. negative red cells after trypsin treatment, and chim- Alloimmunization to Fysup(a) is not high even in Cauca- panzee Duffy-positive cells after chymotrypsin treatment, sians, indicating that the antigen has low immuno- are invaded by E! knowlesi even though these cells have genicity. In three series of patients immunized to Fy serologically undetectable Duffy antigens. and K antigens, the frequency of Duffy system antibodies The foregoing points to different receptors for the was 11.5 percent (507 antibodies in 4401 patients), various Plasmodium species and suggests that Fy3 may compared to 28.5 percent for anti-K. This great dif- be necessary for invasion by E! knowlesi, Fy6 for P. ference in immunogenicity is best appreciated when vivax, and a yet-to-be-discovered epitope for E! one considers that a K+unit would occur only once fulciparum. (statistically) among 11 donor units whereastwo of three would be Fy(a+). The relative immunization potential Allo- and Autoimmunization of various blood group antigens has been calculatedsup(45 Exposure to red cells through or by comparing the frequency of the combination of a transplacental passage is the most common cause of K+ donor and K- recipient (0.09x0.91=0.08)with alloimmunization to Duffy antigens. An unusual route the frequency of a Fy(a+) donor and a Fyta-) recipient

VOLUME 5, NUMBER 2, 1989 49 K.M. BEATTIE

(0.65x0.34=0.22).Thus, while the opportunity for Fy3 or -Fy5 suffered delayed transfusion reactions.sup(47) immunization to Fysup(a) is almost three times more fre- Anti-Fy5 was incriminated in another report, but anti-C quent than for K, anti-K is found almost three times and anti-E in the patient's serum may have been respon- more often than anti-Fysup(a). Therefore, K is about nine sible for the reaction.sup(55) times more immunogenic than Fysup(a). The part played by Fysup(a) in kidney transplantation was Several racial differences must be noted involving anti- examined when a kidney from a Fy(a+) sibling was Fy3. First, in spite of 68 percent of blacks having the transplanted into a patient who had an anti-Fysup(a) titer Fy(a-b-) phenotype and approximately 230,000 blacks of 16. The titer rose to 64 three days after the transplant annually receiving blood transfusions, most of which but decreased to 32 by the eighth day and continued would be Fy:3 donor units, anti-Fy3 is an uncommon at that level through the 44th day in spite of an acute antibody. It is unclear whether the scarcity of this anti- rejection episode that was successfully controlled. It body is due to the low immunogenicity of Fy3 or the was concluded that Fysup(a) is not important in transplan- presence of Fy4 impedingimmunization of these Fy: - 3 tation.sup(56) persons. Second, while the Fy(a-b-) phenotype is an In spite of Fysup(a) being well developed early in fetal extremely rare phenotype in other racial groups, the life and anti-Fysup(a) being an IgG antibody capable of cross- first and third examples of anti-Fy3 were found in a ing the placenta, anti-Fysup(a) is not a frequent cause of Caucasian sup(6) and a Cree Indian.sup(28) Third, five of five ex- hemolytic disease of the newborn (HDN). Although amples of anti-Fy3 produced by blacks were accom- some infants have required , most panied or preceded by anti-Fya.sup(27,46,47) Although the are not severely affected. It was pointed out in a report number of examples of anti-Fy3 in nonblack persons involving 11 cases of HDN due to anti-Fysup(a) that five of is small, neither contained a separable anti-Fysup(a) or the mothers had been immunized by transfusions of -Fysup(b)6,28 Fourth, it has been suggested that anti-Fy3 pro- Fy(a+) blood. Two of the infants died.sup(57) duced by blacks is different in being weakly reactive In the only reported case of HDN caused by anti- or nonreactive with cord red blood cells in indirect anti- Fyb, the infant received phototherapy and two red cell globulin tests.sup(48) Adsorption-elution may give positive transfusions.58 results.sup(27) Strong reactivity with cord cells was reported Anti-Fy3 has been responsible for mild HDN of the for the anti-Fy3 made by the non-blacks.sup(6,28) third child of the Australian Caucasian womansup(6) and The paucity of examples of anti-Fy4 and anti-Fy5 may the ninth child of the Canadian Cree Indian woman.sup(28) be attributable to some of the same factors in the afore- The infant of the American black woman, maker of mentioned discussion of anti-Fy3. As in the situation the second example of anti-Fy3, was unaffected.sup(27) with anti-Fy3, two patients' anti-Fy5 was preceded by So fw, anti-Fy4 and anti-Fy5 have not been implicated the appearance of anti-Fysup(a). Subsequent to the identifca- in HDN. tion of either anti-Fy3 or anti-Fy5, the anti-Fysup(a) was no longer demonstrable even by adsorption Distribution of the Duffy Antigens and Phenotypes Clinical Impact of the The racial distribution of the Duffy system antigens Duffy System Antibodies is widely divergent. The frequency of Fysup(a) varies from anti-Fysup(a) has frequently been cited as causing a very low percentage in African blackssup(59-60) to almost hemolytic transfusion reactions. However, only seven 100 percent in the people of Oceania.sup(61) About two- (four immediate, three delayed, none fatal) were detected thirds of North American and European Caucasians are among 268,000 transfusions given at the Mayo Clinic Fy(a+) and over 80 percent are FY(b+).sup(62) Fy sup(b) is much in the 10-year period S964-1973.sup(49) Fatal reactions have less frequent in blackssup(59-61) and Asians.sup(61,63,64) also been reported.sup(50,51) Anti-Fysup(b) has also caused de- The Fy(a-b-) phenotype is found in 68 percent of layed reactionssup(52) and deaths.sup(53) American blacks and approaches 100 percent in some Although anti-Fy4 has not been implicated, anti-Fy3 African blacks.sup(59,61) This phenotype does not occur in caused a severe hemolytic reaction following partial Australian aborigines sup(65) in spite of their skin color being red cell exchange for (SCD).sup(54) Four black. Instead, Fysup(a) ranges from 90 to 100 percent, indi- of five SCD patients in another study who made anti- cating Asian origins.

50 IMM U N O H EM AT O LOGY Duffy system review

Because of African admixture, the Fy(a-b-) pheno- Development of Fy Antigens on Fetal Red Cells type is found in other ethnic groups such as native white Fysup(a) has been found on the red cells of a fetus at 6 Sicilians,sup(66) Moslem, Christian, and Druze Arabs, and weeks' gestation and Fy sup(b) at 6½ weeks.sup(68) At birth, both Jewish immigrants from Yemen and of these antigens are well developed.sup(2) Fy3 and Fy5 are The Fy gene occurs in Caucasians but with a fre- developed at birth but may give weaker reactions than quency < 0.001, Inasmuch as no Fy(a-b-) blood adult red cells with some anti-Fy3 or -Fy5 re- samples were found in testing almost 10,000 whites, agent~.~,~,~~,~~,~The Fy3 antigen increases in strength the frequency of persons homozygous for the Fy gene shortly after birth.sup(68) There is no information on Fy4 (FyFy) must be much less than 0.001.sup(5,6,62) In at birth, but Fs has been detected on the red cells of contradistinction, Fy reaches 100 percent in some newborn infants.sup(31) African tribes59 (Tables 3 and 4).

Table 3 Racial distribution of Fy phenotypcs (in percent)

Fy(a+ b- ) Fy(a - b +)† Fy(a+b + ) Fy(a- b-) Fy(a+b+ sup(w))‡ Fy(a- b + w)§

Asian Chinese' 90.8 0.3 8.9 0 Japanesesup(63) 81.5 0.9 17.5 0 Thaisup(64) 69 3 28 0

93 0.1 7 0

Black S Africansup(59) 6 6 0.2 88 American sup(4) 9 22 2 68

Caucasiansup(62) in 33 48 0 1.3 0.02

*frequencies observed by M. Lin-Chu, MD, personal communication †Fy(a-b+) includes the products of Fysup(b) Fysup(b) Fysup(b)Fy sup(x) and Fysup(b)Fy ‡Fy(b+ ") is the product of the Fy sup(x) gene §includes the products of Fy sup(x)Fy sup(x) and Fy sup(x)Fy

Asian Chinese' 0.945 0.047 0.8930 0.0088 0.0022 Japanesesup(63) 0.903 0.097 0.8150 0.1755 0.0094 Thaisup(64) 0.8289 0.1711 0.6871 0.2836 0.0293 0.000

Australian Aboriginsup(65) 0.964 0.0160 0.000 0.9291 0.0694 0.0013 0.000

Black S.Africmsup(59) 0.0314 0.0302 0.9384 0.00098 0.0019 0.00091 0.0589 0.0567 0.8806 American sup(4) 0.0534 0.1220 0.8246 0.0029 0.0130 0.0149 0.0081 0.2012 0.6799

Caucasiansup(62) 0.424 0.560 0.015 0.001 0.1798 0.4749 0.3136 0.0001 0.0127 0.0168 0.0002 0.0001

*M.Lin Chu, MD, personal communication

VOLUME 5, NUMBER 2, 1989 51 K.M. BEATTIE

Duffy Antigens in Other Duffy system may consist of two closely linked genes Eleven of 12 were typed as Fy(a- b+) at adjacent loci with Fysup(a), Fy sup(b), Fy sup(x), or Fy at one locus Fy:3 and the 12th as Fy(a-b-) Fy: -3, giving approx- and Fysup(3), or its allele Fysup(4) at a nearby site.’ Fysup(4) may be imate gene frequencies of Fy sup(a) 0.00, Fysup(b) 0.70, and Fy an allele to Fy3 since most blacks who have the 0.30.sup(9,13,69) In descending the evolutionary scale, it was Fy(a-b-) Fy: - 3 phenotype are positive for Fy4. Fur- found that the only tested typed as Fy:3, weak thermore, no Fy(a+b+) Fy:3 red cells are Fy:4. In Cauca- Fy sup(b), and Fy:6, but had no detectable Fysup(a). None of the sians, Fysup(a) and/or Fy sup(b) would be at one locus and Fy3 other primates had Fysup(a) either. Two Macacus at the other locus (Fig. la).The most common haplotype facicularis monkeys had Fy3, but their Fy sup(b) was detect- in blacks would contain Fy and Fysup(4) (Figure 1c). able only by adsorptionelution. Two M. rhesus monkeys and two marmosets typed as Fy(a-b-) Fy:3, but two tree shrews were only weakly positive for Fy3. Lower HUMAN CHROMOSOME PAIRS primates had no detectable Fy sup(b) or Fy3 antigens.sup(13,69) Two of the new world monkeys and none of the old world monkeys had Fy6, and the presence of this epi- tope corresponded with susceptibility of the animal’s (a) red cells to penetration by P. vivax merozoites.sup(9) In the chain of evolutionary events, Fy3 seems to have RBC Phenotype appeared before Fy sup(b) and later during human develop- ment, Fysup(a) arose.

Genetics of the System (b) Chromosomal assignment and gene linkage The and alpha- genes located Rh,sup(70) Fy,sup(11) are RBC on chromosome 1. Although Fy and alpha-spectrin Phenotype genes are linked,sup(71) Rh and Fy are only syntenic. The Rh gene is located near the tip of the short arm. Fy is near the centromere, but there is controversywhether it is on the short (p) or long (q) arm somewhere in the (c) region of the chromosome. Three studies place Fy on the long One study presents RBC 4 evidence for linkage of Fy and the gene for antithrombin Phenotype IIIsup(74) while the third study concludes that Fy and the FIGURE1. (a) Common Duffyy system haplotypes; (b) rare haplotypes gene for type 1 hereditary motor and sensory in Caucasians; (c)common Duffy haplotypes in American blacks. neuropathy are linked.sup(73) The rare person of races other than blacks who has Genetic background of the system and synthesis the Fy(a-b-) phenotype is also Fy:-3 but may not of the antigens have the Fy4 antigen, in contrast to blacks who usually Presently, it is believed that there are four alleles at have Fy4 in double dose (Fig. 1b,c).The inference has locus–Fy the Duffy locus–Fy Fysup(b), Fysup(x), and Fy. The products been drawn that there may be a different genetic basissup(a), of these genes are the antigens Fy sup(a) and Fysup(b). Fysup(x) gives for the inheritance of Fy in other races.sup(75) The Fy(a- b-) rise to an antigen that is a weak variant of Fysup(b). Fy is phenotype does not arise through homozygous inheri- a silent gene that has no known product. The other tance of a suppressor gene independent of Fy; this is antigens, Fy3, Fy4, etc., are not products of genes at ruled out by failure to find Fy(a+b+) children from the same locus as the aforementioned four alleles, but matings of Fy(a-b-) Fy:-3 persons with Fy(a+) or the Fy3 antigen might be an interaction product of them. Fy(b+) spouses. It cannot be an allele to them because the red cells of The dampening effect on a Fysup(b) gene and an asso- some people type as Fy(a+b+)and Fy3 positive. The ciated Fysup(3) gene to produce the Fysup(x) phenotype can be

52 1 M M U N O HEM AT O L O G Y explained by the action of an operator gene at another blood group determinants. Science 1975;189:561-3. locus. 13. Marsh WL. Present status of the Duffy blood group system. CRC Crit Rev Clin Lab Sci 1975;5:387-412. In spite of there being antibodies to Fy3, Fy4, Fy5, 14. AddH, Zamor J, Judd WJ, Johnson CL, Marsh WL. Inactivation Fy6, and Fs, it is not necessary to invoke separate genes of Kell blood group antigens by 2-aminoethylisothiouronium for each of these epitopes. Genes interacting with other bromide. Br J Haematol 1982;51:107-15. 15. Edwards JM, Moulds JJ, Judd WJ. Chloroquine diphosphate genes may give rise to multiple products, a common dissociation of antigen-antibody complexes: a new technique example being the Le gene acting in concert with H for phenotyping red cells with a positive direct antiglobulin and Se results in both Le sup(a) and Le sup(b) antigens. Based on test. Transfusion 1982;22:59-61. the similarities in serological behavior of Fy3, Fy5, and 16. Masouredis SP, Sudora E, Mahan L, Victoria EJ. Quantitative immununoferritin microscopy of Fy sup(a), Fysup(b), Jk sup(a), U, and Di sup(b) antigen Fy6, one gene, Fy3, could be responsible for each of site numbers on human red cells. Blood 1980;56:969-77. these epitopes. It can he speculated that Fysup(3) could be 17. Dunstan RA, Simpson MB, Rosse WF. Erythrocyte antigens on the Duffy gene necessary to interact with a normal Rb human platelets. Absence of Rh,Duffy Kell, Kidd and Lutheran antigens. Transfusion 1984;24:243-6. gene to produce the expression of Fy5. In this scheme, 18. Williams D, Johnson CL, Marsh WL. Duffy antigen changes on the Fy3 protein may consist of a protease-resistant area red blood cells stored at low temperature Transfusion and a protease-sensitive portion-the later serving as 1981;21:357-9. 19, Davies DM, Hall SJ, Graham HA, Chachowski R. The isolation the Fy6 product. and partial charactcerizationof Duffy antigens from human red Speculation on genetic pathways for the biosynthesis cells (abstract). Transfusion 1979;19:638. of Duffy system antigens and hypotheses to account 20. Moore S, Woodrow CF, McClelland DBL. Isolation of membrane for the various phenotypes may be found else- components associated with Rh(D), (c), (E) and Fysup(a). Nature 1982;295:529-31. where.sup(76,77) 21. Hadley TJ, David PH, McGinniss MH, Miller LH. Identification of an erythrocyte component carrying the Duffy blood group References Fysup(a) antigen. Science 1984;223:597-9. 22. Letendre PL, William MA, Ferguson, DJ. Comparison ofa com- 1. Cuthush M, Mollison PL, Parkin DM. A new human blood group mercial hexadimethrine bromide method and low-ionic-strength Nature 1950;165:188-9. solution for antibody detection with special reference to anti-K. 2. Cutbush M, Mollison PL. The Duffy. blood group system. Heredity Transfusion 1987;27:138-41. 1950;4:383-9. 23. HardmanJT, Beck ML. Hemagglutination in capillaries: correla- 3. lkin EW, Mourant AE, Pettenkofer HJ, Blumenthal G. Discovery tion with blood group specificity and IgG subclass. Transfu- of the expected haemagglutinin, anti-Fyb. Nature sion 1981;21:343-6. 1951;168:1077-8. 24. Caren LD, Bellavance R, Grumet FC. Denionstration of gene 4. Sanger R, Race RR,Jack JA. The Duffy blood groups of New dosage effects on antigens in the Duffy, Ss, and Rh systems using York Negroes. The phenotype Fy(a-h-). Br J Haematol an enzyme-linked immunosorbent assay. Transfusion 1955;1:370-4. 1982;22:475-8. 5. Chown B, Lewis M, Kaita H. The Duffy blood group system 25. Oien L, Nance S. Garratty G.Zygosity determinations using flow in Caucasians: evidence for a new allele. Am J Hum Genet cytometry—a superior method (abstract). Transfusion 1965;17:384-9. 1985;25 474 6. Albrey JA. Vincent EER, Hutchinson J, et al. A new antihody. 26. Habibi B, Perrier P, Salmon C. HD50 assay evaluation of the anti-Fy3, in the Duffy blood group system. Vox Sang antigen Fy3 depression in Fysup(x) individuals. J lmmunogenet 1971;20:29-35. 1980;7:191-3. 7. Behzad O, Lee CL, Gavin J, Marsh WL. A new anti-erythrocyte 27. Oberdorfer CE, Kahn B, Moore V, Zelenski K, Oyen Marsh antibody in the Duffy system: anti-Fy4. Vox Sang R, WL. A second example of anti-Fy3 in the Duffy blood group 1973;24:337-42. system. Transfusion 1974;14:608-11. 8. Colledge KI, Pazzulich M, Marsh WL. Anti-Fy5, an antibody disclosing a probable association between the Rhesus and Duffy 28. Buchanan DI, Sinclair M, Sanger R, Gavin J, Teesdale P. An Alberta Cree Indian with a rare Duffy antibody, anti-Fy3. Vox Sang blood group genes. Vox Sang 1973;24:193-9. 1976;30:114-21. 9. Nichols ME, Rubinstein Barnwell J, Rodriguez de Cordoba S, Rosenfield RE. A new human Duffy blood group specificity 29. DiNapoli J, Garcia A, Marsh WL, Dreizin D. A second exam- defined by a murine monoclonal antibody, J Exp Med ple of anti-Fy5. Vox Sang 1976;30:308-11. 1987;166:776-85. 30. Meredith LC. Anti-Fy5 does not react with e variants (abstract). Transfusion 1985;25482. 10. Renwick JH, Lawler SD. Probable linkage between a congenital catract locus and the Duffy blood group locus. Ann Hun-Genet 31, Palatnik M, Junqueira.PC, Alves ZMS. Fs: an antigenic determinant 1963;27:67-84. possibly related to the Duffy blood gruup. Rev Fr Transfus Immu- 11. Donahue RP, Bias WB, Renwick JH, McKusick VA. Probable nohematol 1982;6:629-37. assignment of the Duffy blood group locus to chromosome 32. Young MD, Eyles DE, Burgess RW, Jeffrey GM. Experimental 1 in man. Proc Nat Acad Sci USA 1968;61:949-55. testing of the immunity of Negroes to Plasmodium vivax. J 12. Miller LH, Mason SJ, DvorakJA, McGinniss MH, Rothman IK. Parasitol 1955;41:315-8. Erythrucyte receptors for (Plasmodiumknowlesi) malaria: Duffy 33. McGinniss MH, Miller LH. Malaria erythrocyte receptors and

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the Duffy, blood group system. In: Steane EA, ed. Cellular anti- transplantation (letter). N Engl J Med 1978;299(14):775 gens and disease. Washington, DC: American Association of 57. Greenwalt TJ, Sasaki T, Gajewski M. Further examples of Blood Banks, 1977:67-77. hemolytic disease of the newborn due to anti-Duffy (anti-Fy sup(a)). 34. Miller LH, Mason SJ, Clyde DF, McGinniss MH. The resistance Vox Sang 1959;4:138-43. factor to Plasmodium vivax in Blacks. N Engl J Med 58. Carreras-Vescio LA, Farina D, Rogido M, Sola A. Hemolytic disease 1976;295:302-4. of the newborn caused by anti-Fysup(b) (letter). Transfusion 35. Miller LH, McGinniss MH, Holland PV, Sigmon P. The Duffy 1987;27:366. blood group phenotype in American Blacks infected with 59. Hitzeroth Hw,Bender K, Burckhardt K. South African Negroes: Plasmodium vivax in Vietnam. Am J Trop Med serogenetic polymorphisms (ABO, Rhesus, MNS, Duffy and Kell) 1978;27:1069-72. and- inter-ethnic genetic 'distances. Acta Anthropogenent 36. Spencer HC, Miller LH, Collins WE, et al. The Duffy blood group 1982;6(3):171-93. and resistance to Plasmodium vivax in Honduras. Am J Trop 60. Paul B. Duffy blood group distribution in Malawi. Trans R Soc Med 1978;27:664-70. Trop Med Hyg 1983;77(6):877. 37. Contreras M, Gordon H, Tidmarsh E. A proven case of maternal 61. Mourant AE, Kopec AC, Domaniewska-SobczakK. The distribu- alloimmunization due to Duffy antigens in donor blood used tion of the human blond groups and other polmorphisms. Lon- for intrauterine transfusion. Br J Haematol 1983;53:355-6. don: Oxford Press, 1976. 38. van't Veer MB, van Leeuwen I, Haas FJLM, Smelt M, Overbeeke 62. lewis M, Kaita H, Chown B. The Duffy blood group system MAM, Engelfriet CP. Red cell autoantibodies mimicking anti- in Caucasians. A further population sample. Vox Sang Fysup(b) specificity. Vox Sang 1984;47:88-91. 1972;23:523-7. 39. Mannessier L, Habibi B, Salmon C. Un nouvel example anti- 63. Okubo U, YamaguchiII, Seno T, Yoshimura K, TanakaM, Yokota Fy3 comportant une reactivite pseudo-anti-Fy sup(a) Rev Fr Transfus T. Some rare blood group phenotypes inJapanese. Proceedings Imunohematol 1979;22:195 (in French) cited in Issitt PD. Ap- of the XV Congress. Paris: International Society for Blood Transfu- plied blood group serology. 3rd ed. Miami: Montgomery Scien- sion, 1978. tific, 1985. 64. Chandanayingyong D, Sasaki TT, Greenwalt TJ.Blood groups 40. KosankeJ. Production of Anti-Fysup(a) in Black Fy(a-b-) individuals. of the Thais. Transfusion 1967;7:269-76. Red Cell Free Press 1983;8(3):4. 65. Simmons RT, GraydonJJ. Population genetic studies in Australian 41. Beattie KM Production of Anti-Fysup(a) in Black Fy(a-b-) individuals Aborigines of the northern territory Hum Biol Oceania (letter). Red Cell Free Press 1983:8(3):13. 1971;1:23-53. 42. Vengelen-Tyler V Production of Anti-Fysup(a) in Black Fy(a-b-) indi- 66. Sandler SG, Schiliro G, Russo A, Musumeci S, Rachmilewich viduals (letter). Red Cell Free Press 1983;8(3):14. FA. Blood group phenotypes and the origin of sickle cell hemo- 43. Issitt PD. Production of Anti-Fy sup(a) in Black Fy(a-b-) individuals globin in Sicilians. Acta Haematol 1978;60:350-7. (letter). lmmunohematology 1984;1:11-13. 67. Sandler SG, Kravitz C, Sharon R,Hermoni D, Ezekiel E, Cohen 44. Baldwin M, Shirey RS, Coyle K, Kickler TS, Ness PM. The inci- T. The Duffy blood group system in Israeli Jews and Arabs. Vox dence of anti-Fysup(a) and anti-Fysup(b) antibodies in Black and White Sang 1979;37:41-6: patients (abstract). Transfusion 1986;26:546. 68. Toivanen P. Hirvonen T. Antigens Duffy, Kell. Kidd. Lutheran 45. Giblett ER. A critique of the theoretical hazard of inter- vs. intra- and Xgsup(a) on fetal red cells. Vox Sang 1973;24:372-6. racial transfusion. Transfusion 1961;1:233-8. 69. Palatnik M, Rowe AW. Duffy and Duffy-related human antigens 46. Kosinski KS, Molthan L, White L. Three examples of Anti-Fy3 in primates. J Hum Evol 1984;13:173-5. produced in Negroes. Rev Fr Transfus Immunohematol 1984: 70. Ruddle F, Riccuti E McMorrisFA, et al. Somatic cell genetic assign- 27:619-24. ment of peptidase C and the Rh linkage goup to chromosome 47. Vengelen-TylerV. Anti-Fysup(a) preceding anti-Fy3 or -Fy5: a study A-1 in man. Science 1972;176:1429-31. of five cases (abstract). Transfusion 1985;25:482. 71. Huebner K, Palumbo AP, Isobe M, et al. The alpha-spearin gene 48. Oakes J, Taylor D, Johnson C, Marsh WL. Fy3 antigenicity of is on chromosome 1 in mouse and man. Proc Natl Acad Sci blood of newborns (letter). Transfusion 1978;18:127. USA 1985;82:3790-4. 49. Pineda AA,Brzica SM Jr. Taswell HE Hemolytic transfusion reac- 72. Cook PJL, Page BM, Johnston AW, Stanford WK, GavinJ. Four tion: recent experience in a large Mood bank. Mayo Clinic Proc further families informative for 1qand the Duffy blood group. 1978;53:378-90. Cytogenet Cell Genet 1978;22:378-80. 50. Freiesleben E. Fatal hemolytic transfusion reaction due to anti- 73. Guiloff RJ,Thomas PK, Contreras M, Armitage S, SchwartzG, Fysup(a) (Duffy). Acta Pathol Microbiol Scand 1951;29:283-6. Sedgwick EM. Linkage of autosomal dominant type 1 hereditary 51. Badakere SS, Bhatia HM. A fatal transfusion reaction due to anti- motor and sensory neuropathy to the Duffy locus on Duffy (Fysup(a)). Indiana J Med Sci 1970;24:562-4. chromosome 1. J Neurol Neurosurg Psychiatry 1982;45:669-74. 52. Boyland IP, Mufti GJ, Hamblin TJ. Delayed hemolytic transfusion 74. WinterJH, Bennett B, Watt JL, et al. Confirmation of linkage reaction caused by Anti-Fy sup(b) in a splenectomized patient (letter). between antithrombin III and Duffy blood group and assignment Transfusion 1982;22:402. of AT3 to lq22-q25. Ann Hum Genet 1982;46:29-34. 53. Badekere SS, Bhatia HM, Sharma RS, Bharucha Z. anti-Fysup(b) (Duf- 75. Race RR, Sanger R. Blood groups in man. 6th ed. Oxford: fy) as a cause of transfusion reaction. Indiana J Med Sci Blackwell Scientific, 1975. 1970;24:565-7. 76. Issitt PD, Issitt CH. Applied blood group serology 2nd ed. Ox- 54. Jensen N, CrossonJ, Grotte D, Anderson D. Severe hemolytic nard: Spectra Biologicals, 1975. reaction due to anti-Fy3 following partial red cell exchange for 77. Salmon C, CaruonJ-P, Rouger P. The human blood groups. New sickle cell disease (SCD). Transfusion 1988;28 (abstract suppI):8S. York: Masson Publishing, 1984. 55. Chan-Shu SA. The second example of anti-Duffy 5. Transfu- sion 1980;20:358-60. Kathryn M. Beattie, MT(ASCP)SBB, 20 Yacht Club 56. GinshurgJC, Singer MA. Role of the Duffy A antigen in kidney Drive, #107 North Palm Beach, FL 33408.

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