Ragnhild Øyen

Chronic Granulomatous Disease and the Kell Blood Groups

Summary. Fifteen antigenic determinants are known to be related to the Kell blood group. Some boys with X‐linked chronic granulomatous disease have the very rare McLeod or K0 phenotype on their red cells. Serological studies of the McLeod type suggest that the weak Kell antigens that are present differ qualitatively and quantitatively from those on red cells of common Kell type. A new antigen, Kx, has been characterized and shown to be present on red cells and neutrophil leucocytes. Lack of red‐cell Kx is associated with the McLeod phenotype, lack of leucocyte Kx is associated with chronic granulomatous disease.

Kx antigen, the McLeod phenotype, and chronic granulomatous disease: further studies.

Abstract. Leukocytes of nine unrelated boys with X‐linked chronic granulomatous disease lack Kx antigen. In three of these cases, the red cells also lack Kx and have the McLeod phenotype and abnormal morphology. X‐linked chronic granulomatous disease CGD can thus be separated into two types. Type I cases have an antigenic deficiency that is restricted to the phagocytic leukocytes while in type II, the deficiency involves both leukocytes and red cells. Red cells of type II CGD patients have enhanced i antigen activity suggesting that they are under hemopoietic stress. Normal Kx synthesis is directed by an X‐linked gene named X1k. Three rare variants, X2k, X3k, and X4k order the different permutations of leukocyte and red cell Kx antigen production that have been recognized.

A DNA‐based immunization protocol to produce monoclonal antibodies to blood group antigens

A major challenge facing transfusion medicine is the establishment of immunological methods to produce specific and avid blood group typing reagents to the many polymorphic blood group antigens. This is especially true when sources of human antibody are limited. Based on the knowledge that inoculation with plasmid DNA can induce a humoral response in the host animal, we inoculated mice with plasmid DNA followed by a single boost injection with plasmid‐transfected cells that have a high level of expression of the same target protein. Using this method, several hybridoma clones that produced strongly reactive antibodies specific for the Kell polymorphic antigens (anti‐K, anti‐k, anti‐Kpa) were isolated. The monoclonal antibodies that were produced with this method have potential clinical utility for identifying a patients blood type and for screening for antigen‐negative donor blood.

First example of anti-Kx in a person with the McLeod phenotype and without chronic granulomatous disease.

BACKGROUND: Kx is lacking in the RBCs of patients with the McLeod syndrome. This condition is sometimes associated with chronic granulomatous disease (CGD). If given allogeneic RBCs, CGD patients with the McLeod phenotype may produce anti‐Kx and anti‐Km, and only phenotypically matched McLeod blood would be compatible. McLeod phenotype persons without CGD have made anti‐Km but not anti‐Kx (2 examples), and thus both McLeod and KO blood would be compatible.

Summary of the clinical significance of blood group alloantibodies.

The knowledge of the potential clinical significance of alloantibodies to blood group antigens is essential for selecting appropriate red blood cell (RBC) products for transfusion. This article provides a summary of clinical and technical aspects of many alloantibodies.

Anti-K13 and the K:-13 Phenotype

Abstract. A ‘new’ antibody that recognizes a high‐frequency red‐cell antigen in the Kell blood group system has been found in the serum of a K–k+, Kp(a–b+), Js(a–b+) person, who is heterozygous for the silent Ko gene. The antibody, named anti‐K13, reacted with all of 1,000 unselected blood samples, but does not react with the red cells of the propositus, one of his five siblings, nor with Ko cells. Red cells of a child with chronic granulomatous disease and the McLeod phenotype were weakly reactive.

Production and characterization of anti-Kell monoclonal antibodies using transfected cells as the immunogen

Monoclonal antibodies (Mabs) to blood group antigens are valuable as diagnostic reagents for typing red blood cells (RBCs) in the clinical setting, and for structure–function studies of proteins. Here, we report a powerful system that enabled us to produce Mabs to blood group antigens. A murine erythroleukaemia (MEL) cell line expressing Kell protein, a transmembrane glycoprotein that carries a number of clinically relevant antigens, was used as a novel immunogen. Mabs with different specificities to the Kell protein were produced from a single mouse fusion: an anti‐Jsb (MIMA‐8), and two antibodies (MIMA‐9 and MIMA‐10) with novel specificities, that reacted with RBCs with the common Kell phenotype but not with RBCs with K+k− or Kp(a+b−) or K0 phenotypes. The non‐reactivity with both K+k− or Kp(a+b−) RBCs implied that the epitope was influenced by the molecular changes associated with an absence of the k or Kpb antigens. MIMA‐8 is the first example of a Mab anti‐Jsb and was used in the clinical laboratory for screening donor RBCs for Js(b−) blood and for typing RBCs from patients even when the RBCs were coated with anti‐IgG as is the case in autoimmune haemolytic anaemia. Heavy and light chain variable regions of MIMA‐8 were cloned and the sequence is given. This study illustrates the potential of this novel immunization approach for making monoclonal antibodies to blood group antigens.

A Novel Common Kell Antigen, TOU, and Its Spatial Relationship to Other Kell Antigens

A 47‐year‐old native American (TOU) was admitted to hospital for hip surgery. His serum agglutinated all red blood cells (RBCs) tested except Ko and DTT‐treated RBCs and was weakly reactive with RBCs known to have a weak expression of Kell antigens, namely Kmod, McLeod, Kp(a+b‐) (KEL:3,‐4) and K:‐13 (KEL:‐13) phenotypes. RBCs from three siblings, a son and a daughter were incompatible with TOUs antibody. TOUs RBCs had the common Kell phenotype: K‐ k+ Kp(a‐b+c‐) Ku+ Js(a‐b+) Ul(a‐) K:11, ‐17 K:14,‐24 K:12,13,18,19,22,‐23 (KEL:‐1,2,‐3,4,5,‐6,7,‐10,11,12,13,14,‐17,18,19,‐21,22,‐23,‐24). Since TOUs RBCs were not agglutinated by an unidentified Kell‐related antibody (IAN), tests were performed to show that TOU and IAN were mutually compatible. IAN is a Latino female hospitalised for a hysterectomy. The TOU antigen was shown to be located on the Kell glycoprotein by a monoclonal antibody immobilisation of erythrocyte antigen (MAIEA) assay. The unique pattern of reactivity obtained with TOU and IAN antibodies using this assay indicated the TOU epitope to be in an area remote from other Kell antigens, namely K, k, Kpa, Kpb, Kpc, Ku, Jsa, Jsb, Ula, K11, K12, K13, K14, Wka, K18, K19, K22 and K24 (KEL1, KEL2, KEL3, KEL4, KEL5, KEL6, KEL7, KEL11, KEL12, KEL13, KEL14, KEL17, KEL18, KEL19, KEL21, KEL22 and KEL24) but close to the low‐incidence antigen K23 (KEL23). Investigation of antibodies to previously identified antigens on the Kell glycoprotein by MAIEA using the mouse monoclonal antibodies BRIC18, BRIC68, BRIC107 and BRIC203 has identified six patterns of reactivity and has provided evidence for Kpc being located in the same region as Kpa and Kpb.

A New Low-Incidence Antigen in the Kell Blood Group System: VLAN (KEL25)

A multilaboratory investigation has identified a new low‐incidence antigen ‘VLAN’ on the red cells of a blood donor. The VLAN antigen is destroyed by 2‐aminoethylisothiouronium bromide treatment of the donors red cells suggesting an association with the Kell system. Monoclonal antibody‐specific immobilization of erythrocyte antigen analysis with anti‐VLAN and with several mouse monoclonal antibodies directed at epitopes on the Kell glycoprotein gave positive results, indicating that the VLAN antigen is located on the Kell glycoprotein. The VLAN red blood cells have the common Kell phenotype: KEL:–1,2,–3,4,5,–6,7,–10,11,12,13,14,–17,18,19,–21,22,–23,–24. Additional serologic data indicate that the VLAN antigen is not part of any other ISBT blood group system, collection or series. A family study showed that the VLAN antigen is inherited since the red cells of two sisters and one niece of the propositus are also VLAN+. The ISBT Working Party on Terminology for Red Cell Surface Antigens has assigned VLAN to the Kell blood group system as KEL25 (number for computer listings 006025).

First Example of Hemolytic Disease of the Newborn Caused by Anti-Or and Confirmation of the Molecular Basis of Or

Background and Objectives: The rare MNS antigen Or (MNS31) is sensitive to ficin, papain and sialidase, but partially resistant to trypsin (0.05%); the effect of α-chymotrypsin is not known. A point mutation, 204C → T in exon 3 of GYPA, is associated with the Or+ phenotype. We report here the first case of hemolytic disease of the newborn (HDN) caused by anti-Or, and expand the information on the nature of the Or determinant. Materials and Methods: A woman, gravida 4, para 0, delivered a baby whose red blood cells (RBCs) were positive (2+) on the direct antiglobulin test (DAT). The mother’s serum, an eluate made from the baby’s RBCs and the RBCs of the baby’s father were investigated. Exon 3 of GYPA, extracted from the father’s genomic DNA, was amplified and sequenced. Results: The mother’s serum reacted at room temperature, 37°C and on the indirect antiglobulin test with RBCs from the baby’s father. The father’s RBCs were M+N+S–s+Or+. The antibody in the mother’s serum and in the baby’s eluate was identified as anti-Or. The serum did not react with the father’s RBCs treated with trypsin (180,000 U/ml), but did react with his α-chymotrypsin-treated RBCs. Amplification and sequencing of DNA from the father revealed a single point mutation, 204C → T, in GYPA exon 3. At birth, the baby had clinical symptoms of HDN and was transfused with 36 ml of packed RBCs and received phototherapy for eight days. At week 11, the baby’s M+N+S+s+Or+ RBCs were negative on the DAT. Conclusion: This is the first case of HDN caused by anti-Or. The observed point mutation, 204C → T, confirms that of a previous report and predicts a change of Arg (Or–) to Trp (Or+) at amino acid 31.