College of American Pathologists

  Clinical Abstracts






September 2007

Michael Bissell, MD, PhD

Using HPLC-tandem mass spectrometry to monitor mycophenolic acid levels
Circulating osteoblasts
A case-control study of chromosome analysis and miscarriages
SARS colonization in health care workers
Vascular endothelial growth factor and calcium signaling in smooth muscle
CD1d expression on antigen-presenting cells
MMP-13 and nitric oxide activation of endothelial cells
Infected blood units in CAP Surveys of blood banks
Genetic susceptibility to Kawasaki disease

bullet Using HPLC-tandem mass spectrometry to monitor mycophenolic acid levels

Mycophenolic acid is the pharmacologically active form of the immunosuppressant prodrug mycophenolate mofetil. It is also available as the enteric-coated sodium salt. The drug is a reversible noncompetitive inhibitor of inosine monophosphate dehydrogenase type 2, an enzyme that is important in the de novo synthesis of guanosine nucleotides within lymphocytes. Because the mode of action of mycophenolic acid (MPA) differs from that of cyclosporine, tacrolimus, and sirolimus, it can be co-prescribed as part of a multiple-drug regimen. Therefore, it may be important to analyze total MPA and the inactive metabolite MPA glucuronide (MPAG) in serum and, in some circumstances, to determine free MPA. To address this, the authors developed the following method: After adding carboxybutoxy ether of MPA (MPAC) as an internal standard, they isolated MPA and MPAG from serum by acidification followed by solid-phase extraction. They performed gradient chromatographic separation on a Waters Atlantis reversed-phase liquid chromatography (HPLC) column and quantified the compounds by electrospray ionization tandem mass spectrometry (MS/MS) in the multiple-reaction monitoring mode. They compared results obtained by HPLC-MS/MS with those from an HPLC assay using ultraviolet detection (HPLC-UV) performed at a reference laboratory. The authors found that MPAG, MPA, and MPAC were fully separated during a seven-minute run time. Precision at low and high concentrations of MPA and MPAG met the suggested method validation criteria from a consensus panel report on MPA. The extraction efficiencies were 99 percent for MPA and MPAG. The assay was linear to 16 mg/L for MPA and 200 mg/L for MPAG. Limits of quantification were 0.1 mg/L for MPA and 1 mg/L for MPAG. Regression analysis gave the following results: HPLC-MS/MS= 1.03 (HPLC-UV)–0.03 mg/L (R2= 0.982) for MPA; and HPLC-MS/MS= 0.93 (HPLC- UV)+0.89 mg/L (R2= 0.967) for MPAG. The authors concluded that this HPLC-MS/MS assay can be used to reproducibly quantify MPA and MPAG across a large analytical range in serum from organ transplant patients.

Annesley TM, Clayton LT. Quantification of mycophenolic acid and glucuronide metabolite in human serum by HPLC-tandem mass spectrometry. Clin Chem. 2005; 51:872–877.

Reprints: Thomas Annesley, University Hospital, Room 2G332, 1500 E. Medical Center Drive, Ann Arbor, MI 48109-0054;

bullet Circulating osteoblasts

Bone marrow contains osteoblast and osteoclast precursors that can become the mature osteoblasts and osteoclasts that are believed to be needed for normal bone remodeling on trabecular surfaces contiguous to bone marrow. In addition to residing in bone marrow, substantial numbers of osteoclast precursors are detectable in the peripheral circulation, and these cells may be able to travel to sites of active bone remodeling distant from red marrow. However, it is unclear whether a parallel process involving circulating osteoblast lineage cells exists and, if so, what role these osteoblastic cells play in normal or pathologic bone remodeling. The authors reasoned that the use of flow cytometry with antibodies to bone-specific proteins (osteocalcin and alkaline phosphatase) would better identify circulating osteoblast-lineage cells. Therefore, they determined the concentration of these cells in the circulation of adult men and adolescent boys who had markedly increased bone-formation indexes since they were going through the pubertal growth spurt. Although evidence suggests that only a minuscule number of osteoblast-lineage cells are present in peripheral blood, the authors hypothesized that such cells circulate but that their concentration has been vastly underestimated due to the use of assays that required adherence to plastic. They further reasoned that the concentration of these cells is elevated during times of increased bone formation, such as during pubertal growth. The authors used flow cytometry with antibodies to bone-specific proteins to identify circulating osteoblast-lineage cells in 11 adolescent males and 11 adult males (mean [±SD] age, 14.5±0.7 years versus 37.7±7.6 years). Gene expression and in vitro and in vivo bone-forming assays were used to establish the osteoblastic lineage of sorted cells. Cells positive for osteocalcin and for bone-specific alkaline phosphatase were detected in the peripheral blood of adult subjects (1% to 2% of mononuclear cells). There were more than five times as many cells positive for osteocalcin in the circulation of adolescent boys (whose markers of bone formation were clearly increased as a result of pubertal growth) than in adult subjects (P<0.001). The percentage of cells positive for osteocalcin correlated with markers of bone formation. Sorted osteocalcin-positive cells expressed osteoblastic genes, formed mineralized nodules in vitro, and formed bone in an in vivo transplantation assay. Increased values were also found in three adults with recent fractures. The authors concluded that osteoblast-lineage cells circulate in physiologically significant numbers, correlate with markers of bone formation, and are markedly higher during pubertal growth; therefore, they may represent a previously unrecognized circulatory component of the process of bone formation.

Eghbali-Fatourechi GZ, Lamsam J, Fraser D, et al. Circulating osteoblast-lineage cells in humans. N Engl J Med. 2005;352:1959–1966.

Reprints: Dr. Sundeep Khosla, Endocrine Research Unit, Mayo Clinic College of Medicine, 200 First St., SW, 5-194 Joseph, Rochester, MN 55905;

bullet A case-control study of chromosome analysis and miscarriages

Couples who have had two or more miscarriages are at increased risk of either of the partners carrying a structural chromosome abnormality. The incidence of carrier status increases from approximately 0.7 percent in the general population to 2.2 percent after one miscarriage, 4.8 percent after two miscarriages, and 5.2 percent after three miscarriages. No consensus exists regarding whether chromosome analysis should be offered after two or three miscarriages. It is not known if the probability of carrier status is modified by maternal age, factors related to previous reproductive outcome, or family history. If it is, the possibility of withholding chromosome analysis from couples with a low probability of carrier status could be considered. The authors aimed to identify additional factors that influence the probability of carrier status in couples who had two or more miscarriages and to calculate the associated probability of carrier status for every combination of these factors. They conducted a nested case-control study in six clinical genetics centers in the Netherlands. Participants were couples referred for chromosome analysis after two or more miscarriages between Jan. 1, 1992 and Jan. 1, 2001. The study identified 279 carrier couples as cases and 428 noncarrier couples as controls. The main outcome measures were independent factors influencing the probability of carrier status and the corresponding probability of carrier status. Factors influencing the probability of carrier status were maternal age at second miscarriage, history of three or more miscarriages, history of two or more miscarriages in a brother or sister of either partner, and history of two or more miscarriages in the parents of either partner. The calculated probability of carrier status in couples referred for chromosome analysis after two or more miscarriages varied between 0.5 percent and 10.2 percent. The authors concluded that the probability of carrier status in couples who had two or more miscarriages is modified by additional factors. Selective chromosome analysis would result in a more appropriate referral policy, could decrease the annual number of chromosome analyses, and could therefore lower the associated costs.

Franssen MTM, Korevaar JC, Leschot NJ, et al. Selective chromosome analysis in couples with two or more miscarriages: case-control study. Brit Med J. 2005;331:137–141.

Reprints: M.T.M. Franssen, Centre for Reproductive Medicine, Dept. of Obstetrics and Gynaecology, Academic Medical Centre, University of Amsterdam, P.O. Box 22660, 1100 DD Amsterdam, Netherlands;

bullet SARS colonization in health care workers

It was reported that as many as 21 percent of 8,098 patients worldwide confirmed to have probable severe acute respiratory syndrome from November 2002 to July 2003 were health care workers. The nosocomial spread of the virus in large hospitals was the major epidemic feature of early severe acute respiratory syndrome (SARS) outbreaks, causing high morbidity and mortality among health care workers. The transmission route for this emerging disease is yet undetermined, but it is believed that the illness is primarily transmitted by close contact with contaminated droplets. The authors conducted a prospective observational study to report the efficacy and findings of a large-scale preventive screening program for SARS-associated coronavirus (SARS-CoV) using amplification of the virus from nasopharyngeal swabs. The swabs were obtained from 230 health care workers at a medical center in Taiwan during an outbreak of SARS. SARS-CoV was examined by two nested reverse transcriptase-polymerase chain reactions (RT-PCR) and one quantitative RT-PCR. Serum-specific antibodies were assessed by enzyme immunoassay and indirect immunofluorescence. The study monitored 217 first-line health care workers and 13 non-first-line workers. Negative results in both nested RT-PCR assays were found for 190 first-line health care workers and 13 non-first-line workers. Two first-line health care workers who were positive on both nested RT-PCR assays had SARS. They had 16,900±7,920 copies (mean±standard deviation) of RNA per milliliter in the nasopharyngeal swab and detectable anti-SARS antibodies. The remaining 25 first-line health care workers were negative for the first nested RT-PCR but positive for the second. Their corresponding titers were 338±227 copies of RNA per milliliter; antibodies did not develop in any of these 25 health care workers. The expression and function of angiotensin-converting enzyme-2 did not differ among these health care workers. This study showed that colonization of SARS-CoV occurred in 25 of 217 well-protected first-line health care workers on a SARS-associated service but that the workers remained seronegative. With the second RT-PCR assay being more sensitive than the first, the authors were able to show that approximately 11.5 percent of well-protected health care workers exposed to SARS patients or specimens may have colonization without seroconversion. Only those with significant clinical symptoms or disease would have active immunity. The authors concluded that regular nasopharyngeal swab screening for nested RT-PCR assays in conjunction with a daily recording of body temperature in all first-line health care workers may provide an effective method of early detection.

Ho H-T, Chang M-S, Wei T-Y, et al. Colonization of severe acute respiratory syndrome-associated coronavirus among health-care workers screened by nasopharyngeal swab. Chest. 2006;129:95–101.

Reprints: Dr. Yen-Ta Lu, Division of Chest Medicine, Dept. of Internal Medicine, Mackay Memorial Hospital, 92, Sec 2, Chung-Shan North Rd., Taipei, 10449, Taiwan;

bullet Vascular endothelial growth factor and calcium signaling in smooth muscle

Neointimal hyperplasia is a pathologic process that continues to be a major cause of graft restenosis and failure despite continued advances in the surgical treatment of peripheral vascular disease. Several studies have focused on mitogens in the milieu of the injured artery, particularly platelet-derived growth factor-BB (PDGF-BB), vascular endothelial growth factor (VEGF), and basic fibroblast growth factor, and their effects on vessel remodeling. The authors hypothesized that the selective ability of VEGF to promote vascular smooth muscle cell migration stems from a unique mitogen-stimulated calcium-signaling pathway. With immunoprecipitation, the authors documented the presence of VEGFR-1, VEGFR-2, and PDGFR-β in primary cultured vascular smooth muscle cells. They conducted a study in which primary cultured vascular smooth muscle cells were grown to subconfluency and assigned to the following experimental groups: no stimulation, stimulation with PDGF-BB (20 ng/mL) as the positive control, and stimulation with VEGF165 (40 ng/mL). Total increase in [Ca2+]cyt and intracellular calcium release was quantified using a fura-2 fluorescence assay. Assays for VEGFR-1 (Flt-1), VEGFR-2 (KDR/Flk-1), and PDGFR-β were performed by immunoprecipitation, while PLCγ1, Akt 1/2, and phospholamban B phosphorylation were assessed with Western immunoblotting. Vascular smooth muscle cells stimulated with VEGF165 exhibited no intracellular Ca2+ release, compared with a 75±30 nmol/L intracellular calcium release after PDGF-BB stimulation (P<0.02). VEGF165-stimulated vascular smooth muscle cells in CA2+-containing media exhibited 192±26 nmol/L increase in [CA2+] compared with 354±54 nmol/L increase after PDGF-BB stimulation (P<0.02). VEGF165 did not phosphorylate PLCγ1 after one, five, or 10 minutes of treatment. VEGF165 treatment did not result in PI3-K/ Akt activation at one-, five-, or 10-minute time points. Calmodulin-dependent kinase II (CaMKII) was activated by VEGF165 and PDGF-BB after one and five minutes of stimulation. The presence of VEGFR-1, VEGFR-2, and PDGFR-β was confirmed in all experimental groups. The authors concluded that VEGF induces extracellular calcium influx but not intracellular calcium release in vascular smooth muscle cells. This lack of intracellular Ca2+ release stems from the inability of VEGF165 to activate the PLCγ1 cascade and IP3 receptor-mediated Ca2+ release. The lack of PI3-K/Akt activation at these time points indicates a novel extracellular Ca2+ influx pathway sufficient to activate CaMKII. A paradigm relating extracellular Ca2+ influx to CaMKII activation and migration is suggested and may account for the selective effects of VEGF on vascular smooth muscle cell migration.

Chandra A, Angle N. Vascular endothelial growth factor stimulates a novel calcium-signaling pathway in vascular smooth muscle cells. Surgery. 2005;138:780–787.

Reprints: Dr. Niren Angle, UCSD Section of Vascular and Endovascular Surgery, 200 W. Arbor Drive, San Diego, CA 92103-8402;

bullet CD1d expression on antigen-presenting cells

Antigen-presenting molecules play a fundamental role in T-cell development, T-cell priming, and regulation of immunity. The cell surface expression of antigen-presenting molecules is particularly critical for host defense against intracellular pathogens because the presentation of microbial antigens (Ags) by class I or class II major histocompatibility complex (MHC) molecules is the principal way the immune system identifies infected cells. Although Ag-presenting molecules are constitutively expressed by many cells, they are also regulated by various stimuli, such as cytokines and microbial products. MHC class II is constitutively expressed by B cells, dendritic cells, and macrophages; it is also induced by cytokines, principally IFN-γ on a variety of cells, including macrophages and endothelial and epithelial cells. Like class I and class II MHC, the Ag-presenting molecule CD1d is constitutively expressed by many cell types. It is prominently expressed by splenic B cells, and CD1d is found on macrophages, dendritic cells, and T cells. Whether cell surface expression of CD1d is subject to additional regulation by cytokines or microbial products, as observed for MHC, is an important question. Although class I and class II MHC induction increases the efficiency of T cell activation, it is unknown whether increased surface expression of CD1d will do the same. The observation that mice lacking CD1d-restricted natural killer T (NKT) cells have diminished host resistance to certain pathogens, impaired tumor immunity, and alterations in their predisposition to autoimmune disease indicates that NKT cells participate in these immunological responses. The authors conducted a study to show that like MHC class I and class II molecules, cell surface CD1d expression on Ag-presenting cells is regulated and affects T cell activation under physiological conditions. Although IFN-g alone is sufficient for optimum expression of MHC, CD1d requires two signals, one provided by IFN-g and a second mediated by microbial products or by the proinflammatory cytokine tumor necrosis factor. IFN-g-dependent CD1d up-regulation occurs on macrophages following infection with live bacteria or exposure to microbial products in vitro and in vivo. Ag-presenting cells expressing higher CD1d levels more efficiently activate NKT cell hybridomas and primary NKT cells independent of whether the CD1d-restricted T-cell receptor recognizes foreign or self-lipid Ags. The authors’ findings support a model in which CD1d induction regulates NKT cell activation.

Sköld M, Xiong X, Illarionov PA, et al. Interplay of cytokines and microbial signals in regulation of CD1d expression and NKT cell activation. J Immunol. 2005;175:3584– 3593.

Reprints: Dr. Samuel M. Behar, Division of Rheumatology, Immunology, and Allergy, Brigham and Women’s Hospital, Smith Building, Room 516B, 1 Jimmy Fund Way, Boston, MA 02115;

bullet MMP-13 and nitric oxide activation of endothelial cells

Wound healing is an orchestrated cascade of enzymatic activities that converge toward damage repair. Wound healing involves inflammation and angiogenesis and is tightly regulated by cytokines. Vascular endothelial growth factor (VEGF) is a critical cytokine involved in angiogenesis, and nitric oxide is a downstream effector. VEGF increases nitric oxide levels in endothelial cells by activating endothelial nitric oxide synthase (eNOS/NOS3). The role of eNOS in endothelial cell migration recently has been demonstrated in vivo and in vitro, but the precise mechanism by which nitric oxide regulates migration is unknown. Endothelial cells migrate as a result of an injury and during angiogenesis from pre-existing vessels. The process is tightly regulated by matrix turnover, in which matrix metalloproteinases (MMPs) play a pivotal role. Nitric oxide induces MMP-13 expression and activity in bovine aortic endothelial cells (BAECs). MMPs are extracellular matrix-degrading endopeptidases. MMP expression and activity can be found in physiological and pathological situations, such as tissue development, atherosclerosis, ovarian function, arthritis, osteoarthritis, cancer, angiogenesis, and wound healing. MMP-13 was initially discovered in mammalian cell carcinomas and is also expressed by several cell types, including endothelium. The authors developed a wound-healing model in BAECs and aortic cells from eNOS WT and eNOS-deficient mice. They showed that aortic endothelial cells lacking MMP-13 experience delayed migration, and aortic endothelial cells from eNOS null mice present delayed cell migration and a significant decrease in MMP-13 expression. The authors also presented data showing that MMP-13 exists in association with caveolin-1 in resting cells, and that this complex is disrupted in the presence of nitric oxide, leading to secretion of MMP-13 to the extracellular media. The authors postulated that nitric oxide induces endothelial cell movement in part via the disruption of the MMP-13/caveolin-1 complex, which in turn releases the secretion of active MMP-13 to the extracellular matrix. The authors concluded that MMP-13 is an important effector of nitric oxide-activated endothelial migration.

Lopez-Rivera E, Lizarbe TR, Martinez-Moreno M, et al. Matrix metalloproteinase 13 mediates nitric oxide activation of endothelial cell migration. Proc Nat Acad Sci (US). 2005;102:3685–3690.

Reprints: Santiago Lama, Fundacion Centro Nacional de Investigaciones Cardiovasculares, Ronda de Poniente S Tres Cantos, 28760 Madrid, Spain; or

bullet Infected blood units in CAP Surveys of blood banks

The preoperative collection of autologous blood or components offers patients an alternative to allogeneic blood transfusion during elective surgical or invasive medical procedures, such as total joint replacement. Patients often bank their own blood out of fear of acquiring HIV, hepatitis B virus (HBV), or hepatitis C virus (HCV) from an allogeneic transfusion. In reality, however, the risk of acquiring HIV, HBV, or HCV from transfusion is remote because of improved screening and testing schemes. It is estimated that in the United States the frequency of a repeat blood donor transmitting HIV, HBV, or HCV via transfusion is approximately 1 in 2.1 X 106 U for HIV, 1 in 2.0 X 105 U for HBV, and 1 in 1.9 X 106 U for HCV. To determine if hospital transfusion services store or transfuse, or both, autologous blood or components infected with HIV, HBV, and/or HCV, an educational enhancement subsection of a College of American Pathologists Proficiency Testing Survey (J-C 2003) assessed transfusion service practices for storing or transfusing, or both, HIV-, HBV-, and HCV-infected autologous blood and components. For the survey, 4,251 people were asked whether they stored or transfused, or both, autologous blood or components and whether these stored blood products included those infected with HIV, HBV, or HCV. A total of 3,561 survey respondents provided data regarding their autologous blood and component storage or transfusion practices, or both. Of these, 2,988 reported that they store or transfuse, or both, autologous blood or components. A total of 2,390 respondents reported that they do not test autologous donations collected in their own institution for evidence of infection with HIV, HBV, or HCV. Most survey participants reported that even if an autologous donation were tested and found to be infected they would still be willing to store and transfuse the blood component according to which agent was causing the infection: HIV (n=1,867), HBV (n=2,158), or HCV (n=2,233). The authors concluded that most North American hospitals do not test autologous blood donations that they collect in their own institution for evidence of infection with HIV, HBV, or HCV, leading to the conclusion that infected autologous blood components are being stored and transfused. Even when autologous donations are tested and found to be infected with HIV, HBV, or HCV, most North American hospitals would be willing to store or transfuse, or both, the infected autologous blood components.

Shulman IA, Osby M. CAP laboratory improvement programs: storage and transfusion of infected autologous blood or components: a survey of North American laboratories. Arch Pathol Lab Med. 2005;129:981–983.

Reprints: Dr. Ira A. Shulman, Dept. of Pathology, Los Angeles County-USC Medical Center, 1200 N. State St., Room 2900 GH, Los Angeles, CA 90033;

bullet Genetic susceptibility to Kawasaki disease

The medical community recently has focused on determining CCR5-CCL3L1 gene-gene interactions and their role in susceptibility to infectious diseases. This interest stems from CCR5 serving as a high-affinity receptor for the potent chemokines CCL3 and CCL3L1, which are thought to play important roles in immunity and host defense, and the major coreceptor with CD4 for cell entry of HIV-1. Homozygosity for a 32-bp deletion in the coding region of CCR5 (CCR5-32) results in the loss of CCR5 surface expression, and this genotype, in turn, affords significant protection against HIV-1 infection. The distribution of the CCR5-32 allele in human populations is thought to follow the migration patterns of descendents of the Vikings, such that the frequency of the CCR5-32 allele is highest in individuals of northern European decent. Thus, Icelanders and Swedes have among the highest CCR5-32 allele frequencies (14.7% and 14.2%, respectively), and the allele is virtually absent in Asians and Africans. It is speculated that the CCR5 signaling pathway is important for productive infection with pox viruses and that natural selection driven by epidemics of infectious diseases, such as smallpox, fixed the CCR5-32 allele in the population of northern Europeans. Given the significant interest in elucidating the relationship between infectious disease susceptibility and the differential distribution of alleles among populations, the authors examined the relationship between the distribution of the CCR5-32 allele and Kawasaki disease, which is suspected to have an infectious trigger. Among the compelling reasons to focus on Kawasaki disease were: evidence of a striking racial-susceptibility pattern, with overrepresentation of Kawasaki disease in children of Asian ancestry; the clinical, seasonal, and epidemiologic features of Kawasaki disease, which suggest an infectious etiology; and studies that support a genetic predisposition to Kawasaki disease, including the growing recognition of Kawasaki disease pedigrees in Japan and the United States. The authors demonstrated a striking, inverse relationship between the worldwide distribution of the CCR5-32 allele and the incidence of Kawasaki disease. In 164 patient-parent trios, four CCR5 haplotypes, including the CCR5-32 allele, were differentially transmitted from heterozygous parents to affected children. However, the magnitude of the reduced risk of Kawasaki disease associated with the CCR5-32 allele and certain CCR5 haplotypes was significantly greater in people who also possessed a high copy number of the gene encoding CCL3L1, the most potent CCR5 ligand. These findings, derived from the largest genetic study of any systemic vasculitis, suggest a central role of CCR5-CCL3L1 gene-gene interactions in Kawasaki disease susceptibility and the importance of gene modifiers in infectious diseases.

Burns JC, Shimizu C, Gonzalez E, et al. Genetic variations in the receptor-ligand pair CCR5 and CCL3L1 are important determinants of susceptibility to Kawasaki disease. J Infect Dis. 2005;192:344–349.

Reprints: Dr. Jane C. Burns, Dept. of Pediatrics, UCSD School of Medicine, 9500 Gilman Drive, La Jolla, CA 92093-0830; or Dr. Sunil K. Ahuja, VA HIV/AIDS Center, 7703 Floyd Curl Drive, San Antonio, TX 78229;

Dr. Bissell is Professor and Director of Clinical Services and Vice Chair, Department of Pathology, Ohio State University Medical Center, Columbus.