LKE is a sialylated, globo-family glycosphingolipid (GSL)[1, 2] that is biosynthetically related to the Pk (Gb3) and P (Gb4) antigens. Like Pk and P antigens, LKE is a high-frequency antigen on human red blood cells (RBCs), with 98% to 99% of donors typing LKE-positive.[2-7] LKE varies in strength between individuals, with 80% to 90% typing LKE-strong (LKE-S) and 10% to 20% individuals as LKE-weak (LKE-W). LKE-negative (LKE-N) is reported in 1% to 2% of P+ donors, as well as rare p and Pk individuals who are deficient in complex globo-GSLs. Interestingly, there is an apparent inverse relationship between LKE and Pk, with many LKE-N donors showing elevated Pk expression reminiscent of a “Pk variant” phenotype.[4, 7, 8] LKE may also have interactions with other carbohydrate blood groups. In uroepithelial tissues, LKE can be weakened in the presence of secretor. Serologic studies with rare human anti-LKE also suggest an association between LKE-W, A1, and P2 phenotypes.[5, 6]
Although 1% to 2% of the population types as LKE-N, alloantibodies to LKE are actually quite rare, with only five cases mentioned in the literature.[5-7, 10] All acted as direct panagglutinins, with most capable of fixing complement in vitro (Table 1). The first example was found in an untransfused man with Hodgkin's disease. Two examples were identified in prenatal samples[6, 7] and an additional two were identified in routine testing. Based on available literature, anti-LKE appears to be clinically insignificant.
Table 1. Examples of anti-LKE reported in the literature
|Case||Age (years)/sex||Ethnicity||Diagnosis||Prior sensitization||Antibody titer||IATa||Significant||Reference|
|Unmodified RBCs||Enzyme RBCs|
|1||32/male||Black||Hodgkin's lymphoma||No||256||512||C3||NR||Tippett, 1965|
|2||NA/female||NA||Prenatal||Yes, G2P1b||2||4||C3 only||No||Bruce, 1988|
|3||32/female||Danish||Prenatal||Yes, G2P1c||4||16, IAT||IgGc||No||Moller, 1988|
|6||58/male||White||Large B-cell lymphoma||Unknownd||ND||256||C3||Yes||This study|
We now report a sixth example of an alloanti-LKE. Unlike prior cases, this patient had clinical evidence of shortened RBC survival and hemolysis after repeated transfusions with LKE+ blood that was compatible in prewarmed testing. In addition, we show evidence that the LKE-N phenotype arose from a “weak P” RBC phenotype, with weakened expression of all globo-GSLs. This is the second case of a weak P and the first associated with a LKE-N phenotype. Finally, we provide the first direct evidence of human alloanti-LKE binding to monosialogalactosylgloboside (MSGG), the putative LKE antigen.
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The patient was a 58-year-old white man with a history of gastroesophageal reflux disease, hypertension, Type II diabetes, atrial fibrillation, congestive heart failure with an ejection fraction of 25% to 30%, chronic microcytic anemia, and a 1-year history of a nonpainful right groin mass. He was initially seen in a local primary care facility, where he was transfused with 2 RBC units for a hemoglobin (Hb) of 9.7 g/dL. He was referred to a local hospital 1 week later for further evaluation of his anemia. At that time, his Hb was 7.0 g/dL with a weakly positive direct antiglobulin test (DAT; 1+, anti-C3 only). In addition, a cold agglutinin with “P-like” specificity was identified in his plasma. He received no additional transfusions due to difficulty finding compatible blood. He was subsequently transferred to a large regional hospital for management of his anemia and investigation of his left groin mass.
Upon admission to Hospital 2, the patient was afebrile, tachycardic (pulse 104) with a 5 × 3-cm firm, nontender, left groin mass. A complete blood count was significant for a hypochromic, microcytic anemia (Hb 7.1 g/dL, MCV 79, MCH 25.9), mild leukocytosis (17.6 × 109/L), and thrombocytosis (614 × 1011/L). Chemistry studies showed a normal serum iron, normal bilirubin, mildly depressed transferrin, and elevated ferritin (11,600 ng/mL; normal range, 12-300 ng/mL) and haptoglobin (375 mg/dL; normal range, 23-200 mg/dL). There was no evidence of gastrointestinal bleeding by endoscopy and stool guaiac tests. A CT scan revealed splenomegaly with periaortic, iliac, and inguinal lymphadenopathy: There was no evidence of retroperitoneal or intrabdominal hemorrhage.
On Hospital Day 2, the patient became hypotensive and was transfused with 2 units of group O RBCs. Both units were strongly incompatible (3+) by immediate-spin (IS) cross-match and weakly (±) reactive in polyethylene glycol (PEG) indirect antiglobulin tests (IATs), but were compatible using a prewarmed technique.[12, 13] Both units were transfused without incident but failed to elicit the expected increase in Hb, with a posttransfusion Hb of only 7.7 g/dL (Fig. 1).
Figure 1. Patient clinical course following transfusion of P+ RBC units compatible by a prewarmed cross-match technique over the course of hospitalization at Hospital 2. (A) Patient's Hb, (B) LDH, and (C) haptoglobin (−) and total bilirubin (- -). Arrows indicate transfusion of group O+, leukoreduced irradiated RBC units.
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On Day 5, the patient received an additional 2 RBC units before a scheduled lymph node biopsy. As before, units were cross-matched by a prewarmed technique. The patient was medicated before transfusion with acetaminophen, diphenhydramine, and dexamethasone. The patient tolerated transfusion with an appropriate 2 g/dL increase in Hb; however, his Hb quickly decreased to pretransfusion levels within 24 hours. Over the next 6 days, the patient was transfused an additional 5 units with no sustained increase in Hb (Fig. 1A). Transfusions were accompanied by increased lactate dehydrogenase (LDH), total bilirubin, and decreasing haptoglobin (Fig. 1B). By Day 11, the patient was noted to be jaundiced with a total bilirubin of 17.9 mg/dL (64% direct bilirubin), elevated reticulocytes (4.9%), and low haptoglobin. In addition, his DAT was 2+ (anti-C3) with spherocytes and rouleaux noted on a peripheral blood smear.
Given laboratory evidence of a delayed hemolytic transfusion reaction, it was decided to forego any further transfusions unless the patient became symptomatic. A histologic examination of his left inguinal lymph node revealed a large B-cell lymphoma. On Day 15, the patient was treated with one course of rituximab, cyclophosphamide, and vincristine. He was discharged on Day 17 to the care of a local oncologist. The patient died 3 months after discharge of unknown causes.
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The first example of alloanti-LKE was identified in “Mr. Luke P.,” a 32-year-old, group B, P+, untransfused, African American man with diffuse Hodgkin's lymphoma. Serologic studies showed that this “anti-Luke” recognized a high-incidence RBC antigen that was expressed by P1 and P2 donors, but not on rare p and Pk RBCs, suggesting that the Luke antigen was related to the globo-GSL family. This was strengthened by family and population studies showing increased Pk expression on LKE-N RBCs.[4, 7] Over the next decade, four additional anti-Luke were identified in P+ individuals.[6, 7, 10] Unlike the original anti-Luke, these later examples were low-titer panagglutinins (Table 1). Despite a 1% incidence of LKE-N in the general population, anti-LKE remains a rarely encountered antibody. The latter may reflect the fact that LKE is also expressed on many nonerythroid tissues.[4, 39-43]
The formal identification of “Luke” as the globo-ganglioside MSGG occurred in 1988 with the demonstration that MoAb MC813-70, a MoAb developed against murine embryonic stem cells, had “Luke-like” activity in serologic testing.[1, 2] This was also consistent with the known RBC serology since MSGG would be absent on p and Pk RBCs due to the absence of required upstream GSL precursors necessary for MSGG synthesis (Fig. 6). With the presumed identification of MSGG as “Luke,” the antigen was officially renamed LKE for “Luke antigen on erythrocytes.” Direct evidence of human anti-LKE binding to MSGG, or any other RBC GSL, has been lacking due to the scarcity of anti-LKE antisera.
Figure 6. Schematic of GSL synthesis in patient and weak P individuals. Unlike normal individuals, there is an apparent decrease in α4GalT1 activity, the first committed enzyme for globo-GSL synthesis. As a result, there is a global decrease in all globo-GSLs including Pk, P, and LKE and a compensatory increase in neolacto-series GSLs. There is also an increase in CDH and GM3 in these individuals.
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We now present a sixth case of alloanti-LKE. Like the original Mr. Luke P., the anti-LKE in our patient was a high-titer, complement-fixing, direct agglutinin capable of in vitro hemolysis in a two-stage IAT with enzyme-treated RBCs. Unlike previous anti-LKE described,[5-7, 10] our patient's antibody was also clinically significant with evidence of shortened RBC survival, a positive DAT, and laboratory evidence of hemolysis in vivo. It is interesting that the only two known high-titer anti-LKE both occurred in male patients with newly diagnosed lymphoma, raising the possibility that anti-LKE arose in response to malignancy. Carbohydrate antibodies are not uncommon in patients with advanced lymphoma, which commonly express Gb3, Gb4, and GM1 (Galβ1-3GalNAc, Table 2).[44-47] Globo-specific antibodies have been reported in other globo-rich cancers.[48-52]
We were also able to demonstrate, for the first time, direct binding of a human anti-LKE to MSGG, thus confirming MSGG as the LKE antigen. In addition to MSGG, the patient's antibody showed weak reactivity to Gb5, the biosynthetic precursor to MSGG. Gb5 is also recognized by MoAb MC631 and many alloanti-P. Furthermore, alloanti-P demonstrating strong Gb5 binding also recognized MSGG, suggesting that the Galβ13GalNAcβ1-R may be a common epitope among alloanti-P (Fig. 5D). Our findings complement an earlier study by Hansson and colleagues, who demonstrated broad reactivity by a high-titer alloanti-P, which recognized Gb4, Gb5, Forssman, and a placental ganglioside running near snLc4. The demonstration of alloanti-P with anti-LKE activity suggests that some examples of alloanti-P could behave as an anti-P/LKE with stronger reactivity against LKE-S RBCs due to higher expression of both P and LKE antigens (Table 3).
Most examples of LKE-N described to date share features of the Pk variant phenotype, with elevated Gb3/Pk expression.[4, 8] Our patient is the first known example of LKE-N arising from a weak P phenotype and is only the second example of weak P characterized in the literature. The first weak P was identified serendipitously in a normal donor after routine phenotyping showed weaker agglutination with some anti-PP1Pk. Analysis of the donor's RBC GSLs showed a 30% decrease in globo-GSL expression, accompanied by a two- to fourfold increase in CDH, nLc4, GM3, and total RBC ganglioside. As we have shown, our patient also displayed significant decreases in Gb3 and Gb4 with compensatory increases in CDH, nLc4, and GM3. In fact, a comparison of relative neutral RBC GSL expression between our patient and the original weak P are virtually identical (Table 4). Unlike our patient, the original weak P donor was of Chinese ancestry and did not possess any atypical RBC antibodies in his plasma.
The etiology of the weak P phenotype is unknown. Kundu and coworkers hypothesized a partial block in α4GalT1, the α1,4-galactosyltransferase responsible for Gb3 synthesis. As a result, weak P RBCs have a decrease in all globo-GSLs with a compensatory increase in CDH, GM3, and neolacto-GSLs (nLc4, snLc4) similar to that observed in the Pk and p phenotype (Fig. 6).[11, 25] The basis for decreased α4GALT1 activity is unknown although one obvious possibility is heterozygosity for a null A4GALT1 allele. Family studies in p kindreds, however, have shown normal RBC GSL expression in heterozygous family members.[11, 25] Alternatively, Kundu and colleagues proposed inheritance of a mutant A4GALT1 allele with functionally decreased enzyme activity. Unfortunately, we were unable to sequence the patient's A4GALT1 gene.
In summary, we report the first case of a clinically significant anti-LKE, capable of binding MSGG, in a male patient with lymphoma. This is the second case of weak P described and is the first associated with an anti-LKE.