College of American Pathologists
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  Clinical Abstracts





October 2009

Michael Bissell, MD, PhD, MPH

The Dombrock blood group system null phenotype
Multimeric adiponectin and metabolic syndrome
Development of memory progenitor B cells
Search for genetic associations in lupus
Prognostic value of fetuin-A as a cardiac marker
Clinical laboratory assay for metallo-ß-lactamases
High-prevalence antigens in the Cromer blood group system

The Dombrock blood group system null phenotype The Dombrock blood group system null phenotype

The Dombrock blood group system consists of two antithetical antigens, Doa (ISBT 014001) and Dob (ISBT 014002), and three antigens of high prevalence, Gregory (Gya; ISBT 014003), Holley (Hy; ISBT 014004), and Joseph (Joa; ISBT 014005). These antigens are carried on the Dombrock (Do) glycoprotein, which is a glycosylphosphatidylinositol (GPI) linked protein on red blood cells. The Do glycoprotein CD297, previously called Gregory glycoprotein, has an apparent Mr of 47,000 to 58,000 by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE) under nonreducing conditions. It has several potential N-linked glycosylation sites and four cysteine residues in the membrane-bound protein. The susceptibility of Do antigens to sulfydryl compounds suggests the tertiary conformation of the glycoprotein is dependent on disulfide bonds. The Do glycoprotein is encoded by DO (GenBank accession numbers AF290204 and XM_017877), which is identical to ART4 (GenBank accession number X95826). DO consists of three exons distributed over 14 kb of DNA. The messenger RNA is predicted to encode a protein of 314 amino acids that has a signal sequence and a glycosylphosphatidylinositol-anchor motif. The Do glycoprotein is a member of the mono-ADP-ribosyltransferase family but has no demonstrable enzyme activity on red blood cells. DOA and DOB are associated with 793A or 793G, respectively, in exon 2 of DO. This missense mutation is predicted to encode asparagine for Doa and aspartate for Dob at amino acid 265. Based on the structure of the rat ART2.2 crystal structure, the Do glycoprotein structure consists of nine β strands and seven α helices. Most of the β strands are located in the membrane-distal region, or the part of the molecule farthest from the cell membrane, whereas the helices are primarily situated in the membrane-proximal region closest to the membrane. The Doa/Dob polymorphism is located in the ninth β strand, which is exposed on the outermost part of the molecule and would be accessible to antibody binding. Conversely, the Hy site, found in a loop between the third and fourth helices, and the Joa site, found in the fourth helix, are located in the region closest to the cell membrane. Four genetic back-grounds for Dombrock null-type (Gy[a-]) red blood cells have been identified: a mutation in the acceptor splice site of intron 1 (ISV1–2A>G) deletion of nucleotides 343 to 350 in exon 2, a mutation in the donor splice site on intron 1 (lVS1+2T>C), and introduction of a premature stop codon in exon 3 by a 442C>T mutation (Gln148Stop). The authors conducted a study in which they reported a Donull phenotype due to a single-nucleotide mutation (185T>C in exon 2), which replaces phenylalanine with serine at amino acid 62 and prevents the Do glycoprotein from reaching the cell surface of transfected human embryonic kidney (HEK) 293T cells. For the study, DOA and DOB were determined by polymerase chain reaction-restriction fragment length polymorphism, and DO (ART4) exons and flanking regions were sequenced from genomic DNA. Expression analysis was performed by transfection of wild-type and mutant cDNAs into HEK 293T cells followed by flow cytometry and immunoblotting. Homology modeling was used to map the mutation on the protein structure. The authors found that the patient’s sample carried nt 793G/G, indicating a Dob/Dob background. Exon 2 sequencing showed the sample carried a new mutation, nt 185T>C, causing a Phe62Ser substitution. This variant Do was not expressed on the surface of transfected HEK 293T cells. The mutation maps to a highly conserved FDDQY motif located between the β1 strand and α1 helix near the COOH terminus in the native molecule. The authors concluded that the Dombrock null reported is due to a single Phe62Ser mutation. Expression data confirmed that 62Ser is responsible for lack of cell surface Do, and protein modeling suggests the mutation disrupts important aromatic side chain interactions between Phe62 and His160. Production of an antibody to a high-prevalence Dombrock antigen (anti-Gya) in this patient was consistent with complete absence of Dombrock/ART4 protein.

Westhoff C, Vege S, Yazdanbakhsh K, et al. A DOB allele encoding an amino acid substitution (Phe62Ser) resulting in a Dombrock null phenotype. Transfusion. 2007;47:1356–1362.

Correspondence: Connie Westhoff at or

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Multimeric adiponectin and metabolic syndrome Multimeric adiponectin and metabolic syndrome

Adiponectin is an adipocyte-specific secretory protein that possesses antidiabetic and antiatherosclerotic properties. It circulates in serum in at least three forms, including a low molecular-weight, middle molecular-weight, and high molecular-weight multimer form including 12mer and 18mer. Serum adiponectin level is reported to correlate well with insulin sensitivity and lipid metabolism. Adiponectin has been linked to metabolic syndrome, type 2 diabetes mellitus, obesity, and arteriosclerosis. Recent studies have demonstrated that the high molecular-weight (HMW) multimer form is the active form of this protein. In patients with type 2 diabetes mellitus, HMW-total adiponectin ratio was reported to be more useful than total adiponectin for predicting insulin resistance and metabolic syndrome. The authors investigated the hypothesis that measuring only HMW adiponectin may be as effective as HMW-total ratio for predicting insulin resistance or metabolic syndrome, or both. They conducted a working community-based cross-sectional study involving 637 healthy Japanese males aged 30 to 65 years who were not tak-ing medication. Total and HMW adiponectin concentrations in serum were measured by enzyme-linked immunosorbent assay using commercially available kits. The authors found that serum HMW adiponectin level was inversely correlated with homeostasis model assessment of insulin resistance (HOMA-IR; r=–0.375, P< .0001), even after adjusting for age and body mass index (r=–0.245, P<.0001). When the authors divided the study subjects into quartile groups with equal numbers of subjects, HOMA-IR in the four groups based on serum HMW adiponectin levels differed significantly (P<.01). Metabolic syndrome scores in the four groups based on serum HMW adiponectin levels also differed significantly (P<.01). Area under the curve of receiver operating characteristic curves of HMW adiponectin (0.73) to evaluate the presence of insu-lin resistance (HOMA-IR >2.5) was larger than that of total adiponectin (0.68) or HMW-total ratio (0.70). Area under the curve of receiv-er operating characteristic curves of HMW adiponectin (0.70) to evaluate the presence of metabolic syndrome (body mass index-based modified criteria) was also larger than that of total adiponectin (0.65) but equal to that of HMW-total ratio (0.70). These results suggest that HMW adiponectin measurement may be as effective as HMW-total ratio for evaluating the presence of insulin resistance and metabolic syndrome, at least in nondiabetic subjects who are not receiving medication.

Seino Y, Hirose H, Saito I, et al. High molecular weight multimer form of adiponectin as a useful marker to evaluate insulin resistance and metabolic syndrome in Japanese men. Metabolism. 2007;56:1493–1499.

Correspondence: Hiroshi Hirose at

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Development of memory progenitor B cells Development of memory progenitor B cells

At least three pathways contribute to Ab formation in T cell-dependent immune responses, including a short-term Ab-forming cells (AFC) response in extrafollicular regions of secondary lymphoid tissue, long-lived AFC response in bone marrow by germinal center emigrants, and memory B-cell response that provides recall immunity for the life of the animal. T cell-dependent immune responses generate long-lived plasma cells and memory B cells, both of which express hypermutated Ab genes. The relationship between these cell types is not entirely understood. Both appear to emanate from the germinal center reaction, but it is unclear whether memory cells evolve while obligatorily generating plasma cells by siblings under all circumstances. The authors reported on experiments in which plasma cell development was functionally segregated from memory cell development by a series of closely spaced injections of Ag delivered during germinal center development. The injection series elevated serum Ab of low affinity, supporting the idea that a strong Ag signal drives plasma cell development. At the same time, the injection series produced a distinct population of affinity/specificity matured memory B cells that were functionally silent, as manifested by an absence of corresponding serum Ab. These cells could be driven by a final booster injection to develop into Ab-forming cells. This recall response required that a rest period precede the final booster injection, but a pause of only four days was sufficient. The authors’ results support a model of memory B-cell development in which extensive affinity/specificity maturation can take place within a B-cell clone under some circumstances in which a concomitant generation of Ab-forming cells by siblings does not occur.

Aviszus K, Zhang X, Wysocki LJ. Silent development of memory progenitor B cells. J Immunol. 2007;179:5181–5190.

Correspondence: Dr. Lawrence J. Wysocki at

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xxxxxxxxxxxxxxxxxxxx Search for genetic associations in lupus

Recent reports indicate that a genomic segmental duplication, which included the murine Toll-like receptor 7 gene, and the translocation of this segment to the Y-linked autoimmune accelerator locus were associated with autoreactive B-cell responses to RNA-related antigens. Similar findings of a duplication of the Y-linked autoimmune accelerator (Yaa) locus have been independently reported. The Yaa locus has been shown to increase the severity of lupus-like disease in males of the BXSB mouse strain and to change their autoantibody specificity. This has led to the hypothesis that increased expression of Toll-like receptor 7 (TLR7) due to this increase in genomic DNA may affect the autoimmune phenotype of these mice. Mouse models can provide important insights into human immune function and disease; however, they require careful validation since many immunologic differences exist between the two species. A role of TLR7 in humans with systemic lupus erythematosus (SLE) is consistent with its ability to induce the release of interferon-α (IFN-α), a cytokine found to be increased in the serum of patients with SLE. Because TLR7 is located in a syntenic region of the X chromosome in humans and mice and because an increased prevalence of SLE in women suggests an X-linked genetic component, the authors sought to determine whether, similar to the findings in the Yaa mouse, there were increased gene copy numbers of TLR7 in humans with SLE. The authors used a modified real-time quantitative polymerase chain reaction protocol to calculate the relative TLR7 gene copy number according to the comparative 2-ΔΔCt method in 99 SLE patients and 91 healthy control subjects matched for gender and ethnicity. Autoantibody profiles were determined by standard methods. The relative number of TLR7 gene copies in SLE patients and healthy controls varied; however, the authors found no significant concordance between the number of relative gene copies and the SLE phenotype and no difference in variation by ethnic group. Comparison of the relative gene copy numbers according to the presence or absence of antinuclear antibodies (ANAs), ANA staining patterns, and presence or absence of anti-RNA-associated antigen antibody showed no statistically significant difference in the SLE patients. The authors concluded that although the relative gene copy number of TLR7 varied in SLE patients and healthy controls, it was not significantly increased among the SLE patients compared with the controls. The authors found no detectable trend for an association between the relative gene copy number and autoantibody profile of SLE patients.

Kelley J, Johnson MR, Alarcon GS, et al. Variation in the relative copy number of the TLR7 gene in patients with systemic lupus erythematosus and healthy control subjects. Arthritis Rheum. 2007;56:3375–3378.

Correspondence: Dr. Jeffrey C. Edberg at jedberg

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Prognostic value of fetuin-A as a cardiac marker Prognostic value of fetuin-A as a cardiac marker

Atherosclerosis is a chronic inflammatory disease of the arterial wall characterized by a progressive accumulation of lipids, cells (macrophages, T lymphocytes, and smooth muscle cells), and extracellular matrices. Inflammation is involved throughout the various stages of atherosclerosis and is a strong determinant of plaque disruption and thrombosis. This process is naturally counteracted by antiinflammatory cytokines such as interleukin (IL5)-10, an inhibitor of pro-inflammatory transcription factor NF-αB nuclear localization, leading to suppressed cytokine production. Furthermore, accumulating evidence suggests that inflammatory balance, especially anti-inflammatory factors, are determinants of the prognosis of atherosclerotic disease. Fetuin-A/α2-Heremans-Schmid glycoprotein, an anti-inflammatory mediator that is produced by the liver, helps deactivate macrophages. Fetuin-A enhances the cellular uptake of cationic inhibitors of proinflammatory cytokine synthesis, thereby preventing the self-amplification of inflammatory response. Fetuin-A also inhibits insulin receptor autophosphorylation and tyrosine kinase activity. Studies have suggested that fetuin-A exerts a protective effect against ischemia in the cardiomyocyte. In patients under-going dialysis, low plasma fetuin-A is associated with cardiovascular death. However, the prognostic value of fetuin-A has not been evaluated in patients with atherothrombosis and without end-stage renal disease (ESRD). To address this issue, the authors measured the fetuin-A plasma concentration in 284 consecutive patients with ST-segment elevation myo-cardial infarction (STEMI) and correlated the data with the occur-rence of death at six months (n=25). They also measured fetuin-A in a control group and chose the 95th percentile as the cutoff to define abnor-mality. Patient mean (standard deviation) age was 60 (14) years, and creatinine clearance was 83 (31) mL/min.; 82 percent of patients were men. Mean plasma fetuin-A concentrations at admission (188 [69] mg/L, P=0.01) and at day three (163 [57] mg/L, P&0.0001) were lower in patients than in controls (219 [39] mg/L; 95th percentile, 140 mg/L). Fetuin-A of less than 140 mg/L was observed in 20 percent of patients at admission versus 40 percent at day three (P&0.001). Fetuin-A concentrations did not correlate with peak cardiac troponin values but did correlate inversely with C-reactive protein (CRP) and NT-probrain natriuretic peptide (NT-proBNP). Fetuin-A of less than 140 mg/L at admission (odds ratio, 3.3; P=0.03) and at day three (odds ratio, 6.3; P=0.002) was an independent correlate of death at six months, irrespective of NT-proBNP, CRP, or Controlled Abciximab and Device Investigation to Lower Late Angioplasty Complications (CADILLAC) risk score. Conversely, fetuin-A of 140 mg/L or greater was associated with an excellent survival rate (negative predictive value [NPV], 97 percent overall), even in high-risk populations with a CADILLAC risk score of six or greater (NPV, 90 percent in patients). The authors concluded that fetuin-A is an important predictor of death at six months in STEMI patients, independent of NT-proBNP, CRP, and CADILLAC risk score.

Lim P, Collet J-P, Moutreau S, et al. Fetuin-A is an independent predictor of death after ST-elevation myocardial infarction. Clin Chem. 2007;53:1835–1840.

Correspondence: Pascal Lim at

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Clinical laboratory assay for metallo-ß-lactamases Clinical laboratory assay for metallo-ß-lactamases

Carbapenems are the drugs of choice for treating infections caused by multiresistant gram-negative bacilli. The carbapenemases involved in acquired resistance are of Ambler molecular classes A, B, and D. The class B enzymes, metallo-ß-lactamases (MBLs), are the most clinically threatening carbapenemases because they can hydrolyze all ß-lactams except aztreonam. Although various MBL-detection techniques have been investigated, no perfect phenotypic methods for detecting all transferable MBLs have been found. The authors conducted a study to evalu-ate the utility of double-disk tests (DDTs) by using disks containing a combination of the chelators 2-mercaptopropionic acid (MPA) and Tris-EDTA (TE) to detect MBLs. They tested 16 isolates—four Acinetobacter baumannii, six Pseudomonas aeruginosa, one Serratia marcescens, one Aeromonas hydrophila, one Aeromonas veronii, two Chryseobacterium meningosepticum, and one Stenotrophomonas maltophilia—that produced IMP-1, IMP-1-like, IMP-18, GIM-1, SPM-1, VIM-2, VIM-2-like, and chromosomal MBLs. They also tested 20 isolates—seven Klebsiella pneumoniae, three Escherichia coli, five Enterobacter cloacae, two S. marcescens, one Proteus mirabilis, and two A. baumannii—that produced non-MBL carba-penemases, AmpC ß-lactamases, and extended-spectrum ß-lactamases. The DDT method was evaluated using various concentrations of four types of chelator disks—TE, high-strength TE, MPA, and TE plus 20 µL of MPA—and the ß-lactams imipenem (IPM), meropenem (MEM), ertapenem (ERT), and ceftazidime (CAZ). DDTs with IPM and a TE disk supplemented with 1:320 MPA detected all MBLs and yielded no false-positive results. Some MBL producers were detected in IPM-based tests involving TE or MPA alone or by ERT- or CAZ-based tests. IPM-based tests with MPA concentrations other than 1:320 and all MEM-based tests had suboptimal sensitivities or specificities. The authors concluded that DDT with IPM and a TE disk supplemented with 20 µL of 1:320 MPA appears to be convenient for detecting MBLs in the clinical laboratory.

Kim S-Y, Hong SG, Moland ES, et al. Convenient test using a combination of chelating agents for detection of metallo-ß-lactamases in the clinical laboratory. J Clin Microbiol. 2007;45:2798–2801.

Correspondence: Kenneth S. Thompson at

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High-prevalence antigens in the Cromer blood group system High-prevalence antigens in the Cromer blood group system

The antigens of the Cromer blood group system are carried on decay-accelerating factor (DAF; CD55), which is a member of the regulators of complement activation family of proteins. DAF has four homologous short-consensus-repeat (SCR) domains, which are more commonly referred to as complement control protein (CCP) domains, followed by an O-glycosylated serine- and threonine-rich region attached to a glyco-sylphosphatidylinositol membrane anchor. The Cromer blood group system consists of nine high-prevalence antigens and three low-prevalence antigens, each of which, with the exception of IFC, is associated with a single-amino-acid change in DAF. The locations of the various Cromer antigens on DAF were identified by testing the corresponding antibodies against stable transfectants expressing full-length and deletion mutants of DAF. Tca/Tcb/Tcc, Esa, and WESa/WESb are located in CCP1; Dra and SERF are in CCP3; and Cra, UMC, and GUTI are in CCP4. The Dr(a-) phenotype is characterized by an amino acid substitution of Ser199Leu of DAF. The authors conducted a study in which they described three new high-prevalence antigens—ZENA, CROV, and CRAM—in the Cromer blood group system that are identified by specific correspond-ing alloantibodies, which are anti-ZENA, anti-CROV, and anti-CRAM, respectively. For the study, sequence analyses were performed on DNA from three probands whose serum samples contained an alloantibody to a high-prevalence antigen in the Cromer blood group system. Poly-merase chain reaction-restriction fragment length polymorphism analysis to detect the mutation encoding the CROV– phenotype was performed on 100 Croatian donors. To map the respective epitopes, DAF deletion mutants were tested by immunoblotting with eluates containing the antibodies. The authors found that in each proband, sequence analysis revealed a single-nucleotide substitution in DAF: ZENA, 726T>G mutation, predicted change His242Gln; CROV, 466G>A mutation, predicted change Glu156Lys; and CRAM, 740A>G mutation, predicted change Gln247Arg. By analysis of DAF deletion mutants, the CROV antigenic determinant mapped to the CCP domain 2, which is encoded by exon 3, whereas ZENA and CRAM mapped to CCP4, which is encoded by exon 6. The authors concluded that the antigens ZENA, CROV, and CRAM are associated with a single nucleotide and therefore predict single amino-acid substitution in DAF.

Hue-Roye K, Lomas-Francis C, Belaygorod L, et al. Three new high-prevalence antigens in the Cromer blood group system. Transfusion. 2007;47:1621–1629.

Correspondence: Dr. Marion E. Reid at

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Dr. Bissell is professor, Department of Pathology, Ohio State University, Columbus.