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Review: conditions causing weak expression of Kell system

R. ØYEN, G.R. HALVERSON, AND M.E. REID

Key Words: Kell system, KEL gene, weakened Kell Kpa Cis-Modifying Phenotype antigens Kell system antigens (other than Kpa) on Kp(a+b–) RBCs may be weaker than on RBCs of common Kell type Background a 1 due to the cis-modifying effect of the Kp allele. This phe- Since the discovery of anti-K in 1946, the Kell 9 2 nomenon was first noted decades ago by Allen et al., group system has grown to include 22 antigens. The sys- who found that K+k+Kp(a+b+) RBCs were weakly reac- tem has five sets of alleles that produce antithetical anti- tive with some examples of anti-k. We have seen numer- gens, one or two of low-incidence and the others of ous examples of anti-k that react 2+ with RBCs of high-incidence. In addition, eight antigens of high-inci- common Kell phenotype but are nonreactive (by direct dence and three of low-incidence have been recognized testing) with a K+k+Kp(a+b+) RBC sample that is rou- (Table 1). The system also includes a null phenotype, Ko, tinely included on our in-house panel. Kell system anti- in which the red blood cells (RBCs) are devoid of all Kell gens, including the Kpa , are expressed weakly on system antigens, and a Kellmod phenotype, in which all RBCs from Kpa/Ko individuals.9–11 In fact, the cis-modi- Kell antigens are expressed weakly. fying effect of Kpa is most evident in Kpa/Ko individu- The occurrence of low-incidence Kell system antigens 10 a als but is also evident for the k antigen if the partner differs considerably in different ethnic groups: K, Kp , and carries K. Since the RBCs of most Kp(a+) K17 are found mainly in Caucasians; Jsa in blacks; and Ula 3 people also type as Kp(b+), the antigens produced by the in Finns and Japanese. K23, K24, and K25 have been iden- Kpb allele mask the cis-modifying effect of the Kpa allele. tified in one family each, all Caucasian. A single Kell gene The cis-modifying effect of Kpa varies from one individu- (KEL) has not encoded more than one low-incidence anti- al to another and it is difficult to demonstrate on RBCs gen. For example, a gene that produces K has never pro- a a a a a 3 from Kp /Kp people. None of the genes encoding other duced Kp , Js , Ul , K17, K23, K24, or K25. Since now it Kell system antigens has shown a similar cis-modifying is known that polymorphism in the Kell blood group sys- 4–6 effect. Unlike other RBCs that have a weak expression of tem is the result of point , the presence of Kell blood group system antigens, the Kx antigen is not more than one low-incidence Kell system antigen on a sin- enhanced on Kp(a+b–) RBCs. gle chromosome is statistically an unlikely event. In fact, The cis-modifying effect has not been described with this status is similar to the situation with low-incidence anti- Kpc, the other low-incidence antithetical antigen to Kpb. gens in Lutheran and Cromer blood group systems. c c This may be because Kp is rare and K+Kp(c+) or Kp /Ko Kell blood group antigens are inactivated by treatment RBCs have not been found. of RBCs with disulfide reagents such as dithiothreitol (DTT) and aminoethylisothiouronium bromide (AET), due to disruption of the multiple disulfide bonds.7 The Table 1. Antigens in the Kell blood group system remarkable susceptibility of Jsa and Jsb antigens to such Allelic High-incidence Low-incidence Obsolete treatment, as compared to other Kell antigens,8 is proba- K(K1)/k(K2) Ku(K5) Ula(K10) Kw(K8) Kpa(K3)/Kpb(K4)/Kpc(K21) K12 K23 K9* bly due to the location of the substitution asso- Jsa(K6)/Jsb(K7) K13 K25(VLAN) K15* ciated with this polymorphism, which is within a cluster K11/K17 K16 5 K14/K24 K18 of cysteine residues. K19 This paper will review causes of weak expression of K20 antigens in the Kell blood group system, compare their K22 *K9(KL) has been replaced by K15(Kx) and K20(Km). Since K15 is not part relative strengths, and discuss how this information can of the Kell blood group system, it has been given system status by the ISBT be used in identification studies. working party: 019(XK).

IMMUNOHEMATOLOGY, VOLUME 13, NUMBER 3, 1997 75 R. ØYENETAL.

K:–13 Phenotype linked XK gene was responsible for the observed weak- The original propositus and his sister are the only indi- ening of the Kell system antigens.3 There is no known viduals identified with RBCs of the K:–13 phenotype. family in which the McLeod phenotype has been passed Their RBCs have a decreased expression of common anti- from father to son. In one family, a normal K+k+ man and gens in the Kell system. It is not known if this decrease is a K–k+ McLeod carrier female have two McLeod pheno- due to a cis-modifying effect as is seen with the Kp(a+b–) type sons, one K+wk+w and the other K–k+w.19 These phenotype, since the low-incidence allele to K13 has not data indicate that the Kell genes are normal in the McLeod been discovered. It became apparent during family stud- phenotype but expression of the antigens is modified. ies that the two K:–13 individuals may have inherited the If immunized to allogenic RBCs, non-CGD McLeod indi- Ko gene as judged by tests with anti-Kx. Titration studies viduals produce anti-Km and CGD McLeod individuals pro- using RBCs from presumed K:13/Ko family members did duce anti-Kx+Km. Anti-Km reacts with all RBCs of common 12 not exhibit weakening of Kell system antigens. Kell type but not with those of either McLeod or Ko phe- Individuals with RBCs of common Kell/Ko type cannot be notype. Anti-Kx+Km reacts with all RBCs except those with identified by quantitative studies if the Kpb gene is pres- McLeod phenotype (of CGD and non-CGD types).15,20 13–15 ent. Therefore, it is unlikely that the weak expression Thus, crossmatch-compatible McLeod or Ko RBCs may be of Kell antigens observed in the K:–13 phenotype is a con- used to transfuse immunized non-CGD McLeod individuals, sequence of the Ko allele. while only McLeod RBCs will be compatible with from immunized CGD McLeod individuals. Kmod Phenotypes Anti-Kx+Km is required to identify conclusively Kmod is an umbrella term used to describe RBCs that McLeod phenotype RBCs. This antibody, which is only have a permanent decreased expression of Kell system made by individuals with the CGD-type of McLeod, is in antigens.16 The degree of Kell system antigen depression short supply and thus detection of McLeod phenotype of Kmod RBCs varies from individual to individual (rang- RBCs mainly relies on observed weakening of Kell anti- ing from nonreactive to 1+ on direct testing) but appears gens on the individual’s RBCs by . Since to be a permanent feature of an individual’s own RBCs. different Kell system vary in their ability to RBCs from Kmod individuals from the same family show a detect weakened expression of the corresponding anti- similar degree of depression.17 The antigen depression gen of RBCs with the McLeod phenotype, we recom- may be so severe that adsorption and elution using potent mend that suitable sera be preselected for this purpose. Kell system antibodies may be needed to differentiate In our experience, anti-K14 (polyclonal and monoclonal) Kmod RBCs from Ko phenotype RBCs. Indeed, some is the most effective specificity. Flow cytometry using examples originally thought to be of the Ko phenotype anti-K14 demonstrates the weakening of this antigen on have been reclassified as Kmod. The Kx antigen is RBCs from McLeod phenotype males and clearly shows 21,22 enhanced on Kmod phenotype RBCs, a feature that dis- the two RBC populations in McLeod carrier females. tinguishes the phenotype from the McLeod phenotype. McLeod carrier females have two populations of RBCs: Individuals with the Kmod phenotype, if immunized, one is of the McLeod type and the other is of common Kell produce an antibody similar to anti-Ku (called anti- type. The proportion of McLeod RBCs to common Kell RBCs Ku–like), an antibody that is nonreactive against Ko RBCs varies in different females but is consistent within a female 21 and autologous RBCs (Table 2). The antibodies from Kmod over time and in different females in the same family. individuals are not mutually compatible when crosstest- 18 a ed against other Kmod RBCs. Kp cis-modifying in conjunction with McLeod Severe depression of Kell antigens has been reported McLeod Phenotype in one family in which the propositus appeared to have 23 RBCs of the McLeod phenotype (both chronic granu- RBCs of the Ko phenotype. However, his RBCs had a lomatous disease [CGD] and non-CGD types) show very weak expression of the Kx antigen. His son’s RBCs marked weakening of all Kell system antigens and lack the typed Kp(a+b+)Kx+ and his two daughters’ RBCs typed Kx antigen (Table 2). The phenotype was initially con- Kp(a+wb+w)Kxw. Molecular studies showed he had the sidered to belong to the Kell blood group system; how- Kpa/Kpa genotype and that his XK gene had a single base ever, the inheritance of the McLeod phenotype is change within the donor splice consensus sequence of X-linked. Later it became apparent that an interaction intron 2. These findings explain the RBC typings of the between products of the autosomal KEL gene and the X- propositus and of his children.

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Table 2. Kell system antigen expression associated with various phenotypes, in autoimmune hemolytic (AIHA), and with thiol-treated red blood cells (RBCs) Expression of antigen Associated Phenotypes Kell system Kx Km antibody in serum RBC shape Common* Normal Normal (weak) Normal Possible alloantibody Discoid Kp(a+b–) Decreased () Normal (weak) Weak () Possible anti-Kpb Discoid a b Kp /Ko Decreased () Slight increase () Weak () Possible anti-Kp Discoid K:–13† Decreased () Slight increase () Weak () Anti-K13 Discoid

Kmod Markedly weak or Moderate Markedly weak or Anti-Ku–like (not Discoid absent (/0)‡ increase () absent (/0)‡ mutually compatible)

Ko heterozygote Normal Moderate Normal None Discoid increase ()

Ko Absent§ Marked Absent Anti-Ku Discoid increase () McLeod CGD** Markedly weak () Absent Absent Anti-KL (anti-Kx+Km) McLeod non-CGD Markedly weak () Absent Absent Anti-Km Acanthocytes McLeod carriers Bimodal normal to Not known Not known None Discoid+acanthocytes markedly reduced Gerbich and Leach Slight decrease Normal (weak) Normal Not Kell-related Discoid+ in phenotypes*** Leach type AIHA Normal to markedly Slight increase Not known “Kell-related” antibodies Discoid or spherocytic reduced (N/)(/) or non-specific (due to ) Thiol-treated RBCs Not detectable Slight increase () Absent Not applicable Discoid * Inherited Kell system phenotypes ** Inherited Kx system phenotypes *** Other adverse factors † Only one pair of sibs described. May be a heterozygote KEL:–13/Ko ‡ Adsorb and elute § Do not adsorb and elute

Gerbich System Phenotypes— autoimmunity resolves, the Kell antigen strength returns Leach, Gerbich, and Yus to normal. This phenomenon was first reported by RBCs with the Leach and Gerbich phenotype may have Seyfried31 in a patient with autoimmune hemolytic ane- a depressed expression of Kell system antigens, while mia with autoanti-Kpb in the serum and severe depression those with the Yus phenotype do not. Although weak- of Kell system antigens. The weakening of Kell system ened Kell antigens on Ge-negative cells are not uncom- antigens is acquired but the responsible mechanism is not mon, they are not a consistent feature of all examples of known. However, in one case the absence of Kell anti- Ge-negative RBCs. The weakened expression is most gens was shown to be due to a loss of the Kell .34 notable with K11,24–28 which suggests that the presence In the early 1980s, the eluate from all patients with of amino acids encoded by exon 3 of GYPC are critical for warm autoantibodies referred to our laboratory were test- 29,30 normal Kell antigen expression. ed with Ko RBCs. More than 50 cases of Kell-related autoimmunity were recognized in a 5-year period, which Acquired Weak Expression of corresponds to 1 in 170 patients with warm autoanti- Kell System Antigens bodies. Of these cases, approximately half showed Cases of autoimmunity with Kell-related autoantibod- depression of Kell system antigens on the patient’s RBCs ies have been reported. The patient’s RBCs often show a and 75 percent had anti-K (IgM, IgG, or both) in their transient weakening of all Kell system antigens. The serum. Patients presenting with warm autoantibodies, depression may be so severe that the RBCs appear as Ko. weakly positive DATs, and anti-K in their serum should be The direct antiglobulin test (DAT) is usually weakly posi- investigated for possible Kell-related autoimmunity. In the tive due to the depression of the Kell antigens, and the 3 years that followed the initial 5-year investigation, and eluate from these RBCs may show broad Kell specificity, without changing our laboratory protocol, we only found 16 i.e., nonreactive with Ko phenotype RBCs only or the elu- three cases of Kell autoimmunity. ate may show specificity within the Kell system. Examples of autoanti-Kpb, -K13 and -K have been report- Antibody Identification ed.31–33 When Kell antigen depression is so pronounced Knowledge of weakened expression of Kell antigens is that the RBCs appear to be Ko, the DAT can be negative. important in investigation and identification of Kell sys- The individual’s serum usually contains the same autoan- tem antibodies directed against high-incidence antigens. tibody as is eluted from the autologous RBCs. As the Tests with enzyme-treated or chemically modified RBCs

IMMUNOHEMATOLOGY, VOLUME 13, NUMBER 3, 1997 77 R. ØYENETAL.

may pinpoint to which blood group system the antibody RBCs with decreased Kell antigen expression have belongs. Kell system antigens are destroyed by thiol increased Kx expression (including DTT-treated RBCs). reagents but not by papain (similar to antigens from LW Exceptions are Kp(a+) RBCs and, of course, McLeod phe- and Scianna blood group systems). In order to establish notype RBCs, which lack Kx. Knowledge of weakened Kell system specificity, the serum must be nonreactive expression of Kell system antigens is a valuable aid in with Ko RBCs. The next step is to test the serum with drawing the right conclusions when identifying antibod- RBCs known to lack high-incidence antigens in the Kell ies directed against antigens in the Kell system. system, starting with the most common: k–, Kp(b–), and Js(b–). If specificity is established, the individual’s RBCs Acknowledgment should be tested and found negative for the appropriate The authors thank Jill Storry for reading the antigen. The patient’s RBCs should also be tested with an manuscript and Robert Ratner for assistance in preparing antibody directed against the corresponding low-inci- the manuscript. dence antigen, if available. Depending on the strength of the antibody under inves- References tigation, tests using RBCs with decreased Kell system anti- 1. Coombs RRA, Mourant AE, Race RR. In-vivo isosensi- gen expression may appear to be negative. For example, tisation of red cells in babies with haemolytic disease. a serum may be nonreactive with Ko and K:–13 RBCs on Lancet 1946;i:264–6. direct testing, but it does not follow that the antibody is 2. Daniels GL, Anstee DJ, Cartron J-P, et al. Blood group anti-K13. The individual’s RBCs must also type K:–13 terminology 1995. From the ISBT working party on before a conclusion can be drawn. Adsorption and elution terminology for red surface antigens. Vox Sang using the patient’s serum and the apparently nonreactive 1995;69:265–79. RBCs may be necessary for final interpretation of results. 3. Marsh WL, Redman CM. The Kell blood group sys- In cases with Kell system autoantibodies and transient tem: a review. Transfusion 1990;30:158–67. depression of all Kell system antigens, interpretation is 4. Lee S, Wu X, Reid ME, Zelinski T, Redman C. even more difficult. One of us (RØ) reported a case of Molecular basis of the Kell (K1) phenotype. Blood autoimmune hemolytic anemia caused by anti-K13 17 1995;85:912–6. years ago.35 However, upon recent review, the specifici- 5. Lee S, Wu X, Reid ME, Redman C. Molecular basis of ty is suspect, for although K:–13 RBCs did not adsorb and the K:6,–7 [Js(a+b–)] phenotype in the Kell blood elute the antibody, only direct testing was performed group system. Transfusion 1995;35:822–5. using McLeod phenotype RBCs, and Kmod RBCs were not 6. Lee S, Wu X, Son S, et al. Point mutations characterize included. By today’s criteria, this antibody may have been KEL10, the KEL3, KEL4, and KEL21 alleles, and the interpreted as Kell-related autoimmunity. In another case KEL17 and KEL11 alleles. Transfusion 1996;36:490–4. of autoimmunity, autoanti-Kpb was found in a Kp(a+b–) 7. Advani H, Zamor J, Judd WJ, Johnson CL, Marsh WL. patient.36 Inactivation of Kell blood group antigens by 2- When investigating samples from individuals with the aminoethylisothiouronium bromide. Br J Haematol apparent Ko phenotype, adsorption and elution using the 1982;51:107–15. patient’s RBCs and potent Kell system antibodies should 8. Branch DR, Petz LD. Disulphide bonds are a require- a be performed to differentiate Ko from Kmod RBCs. At the ment for Kell and Cartwright (Yt ) blood group anti- same time, an eluate from the patient’s RBCs must be gen integrity (abstract). Transfusion 1982;22:420. tested to ensure that this is not a case of autoimmunity. As 9. Allen FH Jr, Lewis SJ, Fudenberg H. Studies of anti- mentioned earlier, some cases with Kell-related autoanti- Kpb, a new antibody in the Kell blood group system. bodies show such severe depression of Kell antigens that Vox Sang 1958;3:1–13. the DAT is virtually negative. 10. Tippett P. Expansion of the Lutheran and Kell blood Over the last few years, individuals with apparent Ko group systems. In: Mohn JF, Plunkett RW, phenotype RBCs have been reclassified as Kmod. Some of Cunningham RK, Lambert RM, eds. Human blood these people are blood donors; however, retrospective groups. Basel, Karger, 1977:401–9. review did not reveal any reports of adverse transfusion 11. Ford DS, Knight AE, Smith F. A further example of reactions in the patients with antibodies to Kell system Kpa/Ko exhibiting depression of some Kell group antigens who received the Kmod units. antigens. Vox Sang 1977;32:220–3.

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