College of American Pathologists
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  Clinical Abstracts





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January 2006

Michael Bissell, MD, PhD, MPH
Ronald Domen, MD

bullet Drug-associated ANCA in Graves’ disease

Anti-neutrophil cytoplasmic antibodies are highly specific and sensitive markers for primary systemic vasculitides, which include Wegener’s granulomatosis and microscopic polyangiitis. Evidence from human studies and animal models indicates that the antibodies play a role in disease pathogenesis. Anti-neutrophil cytoplasmic antibodies (ANCA)-associated vasculitis is associated with serious morbidity and increased mortality, as well as potentially life-threatening toxicity from immunosuppressive therapies. The etiology of this condition is unknown, although it has been suggested that propylthiouracil, a common treatment for Graves’ disease, may be one factor. A number of cases of myeloperoxidase (MPO)-ANCA-positive vasculitis have been reported in patients with Graves’ disease treated with propylthiouracil, and a smaller number of cases have been reported in those treated with carbimazole or methimazole. The authors found no studies comparing the prevalence of ANCA in patients treated with these agents. Consequently, the authors compared, in a large cross-sectional study, the prevalence of ANCA in those patients treated with carbimazole and propylthiouracil. Because a small study of patients with MPO-ANCA-positive vasculitis had suggested an association with hypothyroidism, the authors defined the prevalence of ANCA in a group of patients with Hashimoto’s thyroiditis and in a large group of normal controls without thyroid disease. The occurrence of ANCA and the relationship to thionamide treatment was investigated in a cross-sectional study in a consecutive series of 407 patients with Graves’ disease, 200 with Hashimoto’s thyroiditis, and 649 normal euthyroid subjects. ANCA was measured by indirect immunofluorescence and enzyme-linked immunosorbent assay for proteinase 3 and MPO-ANCA. The prevalence of ANCA, as measured by indirect immunofluorescence, was increased in the Graves’ disease cohort (19.9 percent) compared with euthyroid controls (4.6 percent; P<0.001). The prevalence of MPO-ANCA was more strongly associated with propylthiouracil treatment than carbimazole (P=0.0265), although risk of ANCA was also higher in Graves’ patients treated with carbimazole than in controls (relative risk, 2.2; P<0.0001). ANCA positivity was not increased in patients with Hashimoto’s thyroiditis. The study revealed a high prevalence of ANCA in treated patients with Graves’ disease but not in those with Hashimoto’s thyroiditis. Furthermore, ANCA development within the Graves’ disease population was associated with propylthiouracil usage to a greater extent than carbimazole. These findings suggest that the altered immune environment associated with autoimmune thyroid disease is not sufficient to develop ANCA, but treatment with thionamides is important in promoting ANCA development.

Harper L, Chin L, Daykin J, et al. Propylthiouracil and carbimazole-associated antineutrophil cytoplasmic antibodies (ANCA) in patients with Graves’ disease. Clin Endocrinol. 2004;60:671-675.

Reprints: L. Harper, Division of Medical Sciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom;

bullet Using viral load and CD4+ cell counts in HIV prognosis for women on HAART

The medical community has seen dramatic improvements in HIV-related morbidity and mortality since the introduction of highly active antiretroviral therapy (HAART). Several studies have addressed the decision to initiate HAART by demonstrating that adverse clinical outcomes are less likely in those who initiate HAART with CD4+ cell counts of greater than 0.200 x 109 cells/L, HIV-1 RNA levels of less than 50,000 copies/mL, and absence of a clinical AIDS-defining event. However, we do not know the extent to which the improvements in prognostic markers that occur during treatment with HAART can modify the prognosis conferred by the values of these markers before HAART was initiated. It is important to know the extent to which post-HAART laboratory markers compared with pre-HAART laboratory markers predict clinical manifestations of HIV-1 infection. Therefore, the authors investigated the patterns of change and the prognostic value of time-varying measurements of CD4+ cell counts and quantitative HIV-1 RNA level after HAART was initiated, with adjustment for clinical disease stage, CD4+ cell count, and quantitative HIV-1 RNA level at the initiation of HAART. Their analysis was conducted using data from the Women’s Interagency HIV Study (WIHS), an ongoing cohort study of HIV-1-infected women in the United States. WIHS was a prospective cohort study involving 1,132 participants in which HIV-1 RNA level, CD4+ cell counts, AIDS-defining illness, and death were measured. In multivariate analyses with a median follow-up of 3.9 years, women with CD4+ cell counts of less than 0.200 x 109 cells/L compared with women with CD4+ cell counts of greater than 0.350 x 109 cells/L after HAART initiation had a relative hazard of death from all causes of 2.66 (95 percent confidence interval [CI], 1.42-4.99) and a relative hazard of death from AIDS of 47.61 (CI, 5.69-398.40). The relative hazard of all-cause death was 3.44 (CI, 1.67-7.09) in women with RNA levels of more than 10,000 copies/mL compared with women with attained RNA levels of less than 80 copies/mL. The relative hazard of AIDS-related or all-cause death did not increase for women with post-HAART CD4+ cell counts of between 0.200 and 0.350 x 109 cell/L compared with women with CD4+ cell counts of greater than 0.350 x 109 cells/L. In addition, the relative hazard did not increase in women with post-HAART HIV-1 RNA levels between 90 copies/mL and 10,000 copies/mL compared with women with post-HAART HIV-1 RNA levels of less than 80 copies/mL. Of the laboratory markers, only post-HAART CD4+ cell count and HIV-1 RNA level were predictive of new AIDS-defining illness. The authors concluded that post-HAART laboratory markers predicted death and new AIDS-defining illness. Pre-HAART CD4+ cell count and HIV-1 RNA level were not predictive of clinical outcomes if adjusted for values attained after HAART was initiated, suggesting that even advanced immune suppression can be overcome with HAART that results in CD4+ cell counts of greater than 0.200 x 109 cells/L and RNA levels of less than 10,000 copies/mL.

Anastos K, Barrón Y, Cohen MH, et al. The prognostic importance of changes in CD4+ cell count and HIV-1 RNA level in women after initiating highly active antiretroviral therapy. Ann Intern Med. 2004;140:256-264.

Reprints: Dr. Kathryn Anastos, Women’s Interagency HIV Study, 3311 Bainbridge Ave., Second Floor, Bronx, NY 10467;

bullet Haptoglobin measurements in gestational diabetes

Haptoglobin is an acute phase protein synthesized in the liver and, to some extent, in fat tissue in response to interleukin 6-type cytokines. The haptoglobin (Hp) gene is located on the long arm of chromosome 16. The two major Hp alleles—Hp1 and Hp2—code for proteins with one (Hp1) or two (Hp2) SH groups used in multimerization. The Hp1 molecule forms only dimers with a molecular weight of 86 kDa, whereas the Hp2 molecule forms multimers with a molecular weight of between 170 kDa and 900 kDa. A mixture of Hp1 and Hp2, as present in heterozygous individuals, exhibits dimeric Hp as well as oligomeric Hp, but the latter with a lower degree of polymerization than in Hp2 homozygotes. The Hp phenotype is inherited in a Mendelian pattern. Hp forms complexes with free Hb that are rapidly cleared by the liver and by macrophages. Rapid complexation of free Hb, stemming from common, low-grade intravascular hemolysis, is important because free Hb catalyzes the generation of reactive oxygen species, like the extremely reactive hydroxyl radical, by the so-called Fenton reaction. Reactive oxygen species promote endothelial activation and inflammation and play a crucial role in the development of endothelial dysfunction, which is also observed in gestational diabetes mellitus (GDM). Thus Hp functions as an antioxidant and an essential vascular endothelial protector. However, the Hb-binding capacity and antioxidant capacity of Hp1 is higher compared with Hp2. The increased antioxidant function of Hp1 is thought to confer protection from angiopathies. This has been reported for coronary and peripheral atherosclerotic lesions, cardiac transplant vasculopathy, diabetic nephropathy, mortality in coronary heart disease, restenosis after peripheral and coronary angioplasty or stenting, and cardiovascular disease in diabetics. The overall picture appears to confirm a protective role of Hp1 in clinical settings associated with increased oxidative stress. GDM is a common complication of pregnancy and carries considerable health risks for the fetus and mother. Furthermore, diabetics, especially women, carry a significantly greater cardiovascular risk than nondiabetic individuals, and even subtle disturbances in glucose metabolism appear to be linked with premature atherosclerosis. Women with prior GDM feature endothelial dysfunction in relation to insulin resistance and obesity. Obesity is the most prominent risk factor for GDM and type 2 diabetes in prior GDM and is linked to inflammatory processes and angiopathy, increasing cardiovascular risk. Because the pathogenesis of insulin resistance in type 2 diabetes is considered to be associated with endothelial oxidative stress, the authors expected the different anti-oxidative capacity of Hp phenotypes to contribute to the development of hyperglycemia at times of increased endothelial oxidative stress, such as GDM. The Hp phenotype has not been reported to be a risk factor for the development of GDM. The authors conducted a study to investigate a possible association between the Hp phenotype and GDM. The Hp phenotype was determined from consecutive Caucasian pregnant women (n=250) referred for oral glucose tolerance testing. Significance of distribution and odds ratios associated with Hp phenotype were calculated for women with GDM (n=110) and women with normal glucose tolerance (n=140). Frequency of GDM in Hp phenotype classes increased with the number of Hp2 alleles (P<0.001). Odds ratios for GDM in women heterozygous and homozygous for Hp2 were 2.7 (95 percent confidence interval [CI], 1.06-6.84) and 4.2 (CI, 1.67-10.55), respectively. The authors concluded that the Hp phenotype is an apparent risk factor for developing GDM, possibly due to the low antioxidative potential of Hp2 compared with Hp1.

Mustafa S, Vukovich T, Prikoszovich T, et al. Haptoglobin phenotype and gestational diabetes. Diabetes Care. 2004;27:2103-2107.

Reprints: Dr. Thomas Vukovich, AKH-Wien, KIMCL, Leitstelle 5H, Währingergürtel 18-20, A-1090 Wien, Austria; thomas.

bullet Heritability of Alzheimer’s disease late in life

With advances in molecular genetics and genetic epidemiology, expectations about the cause of Alzheimer’s disease have changed, as has the nomenclature for the disease. Before the first linkages to chromosome 21, scientists referred to familial Alzheimer’s disease (AD) and sporadic AD. In general, sporadic AD had a later age of onset than familial AD. The notion that early-onset AD has a stronger genetic component than late-onset AD was confirmed by family studies in which relatives of early-onset (before 72 years) cases had a higher risk of developing AD than relatives of late-onset cases. Furthermore, most of the positive genetic linkages with AD were for relatively early-onset disease—that is, before age 65—although the members of some families with mutations in the presenilin 1 gene may have been slightly older at onset. The association with APOE4 provided evidence that genes also contributed to susceptibility to late-onset AD, although the effect appeared to decrease at very old ages, particularly in nonfamilial samples. Despite these findings, there has been no quantification of the relative importance of genetic effects that contrasts onset before and after 80 years of age. Twin studies allow quantification of the relative importance of genetic factors for liability to AD, giving a sense of the total population burden due to the sum of all disease-relevant genes. Until recently, most research on risk factors for AD, both with non-twin and twin samples, has been based on prevalent cases. More recent studies of incident cases in nontwin samples have led to somewhat different conclusions about the risk associated with various environmental exposures compared with findings from prevalence studies. All of the twin studies reported have evaluated prevalent rather than incident cases. The authors of this study investigated a cohort of 662 pairs of Swedish twins who were 52 to 98 years of age and who were initially identified as nondemented. These twins were followed up for an average of five years to evaluate the relative importance of genetic and environmental factors for developing Alzheimer’s disease late in life. The authors tested whether heritability is greater in older (80 years or older at first screening) than in younger twin pairs. Incident dementia cases were detected through followup at two- to three-year intervals using cognitive testing or telephone screening followed by dementia workups. A physician, psychologist, and nurse gave consensus diagnoses. During the followup period, 5.8 percent of the sample population was diagnosed with Alzheimer’s disease. Average age of onset was 83.9 years (standard deviation, 6.3). Of the 26 monozygotic pairs in which at least one twin developed Alzheimer’s disease, five were concordant (probandwise concordance, 32.2 percent). The concordance rate for dizygotic pairs was 8.7 percent (two of 44 pairs). Structural model fitting indicated that 48 percent of the variation in liability to Alzheimer’s disease could be attributed to genetic variation. Estimates did not differ significantly between twins younger than 80 years and those older than 80 years at baseline. The authors concluded that although these genetic estimates for incident disease are lower than those for prevalent disease, the importance of genetic factors for liability to Alzheimer’s disease is considerable, even late in life.

Pederson NL, Gatz M, Berg S, et al. How heritable is Alzheimer’s disease late in life? Findings from Swedish twins. Ann Neurol. 2004;55:180-185.

Reprints: Dr. Nancy L. Pederson, Dept. of Medical Epidemiology, Karolinska Institutet, Box 281, SE 171 77 Stockholm, Sweden;

bullet Stability of glycemic parameters in nondiabetics

Direct comparison between the glycohemoglobin results obtained using a boronate affinity, high-performance liquid chromatography method that measures all glycosylated hemoglobin molecules and a cation-exchange HPLC method used to quantify the hemoglobin A1c fraction of hemoglobin has shown that the determination of percent total glycohemoglobin is likely to be a better method for estimating the long-term integrated glucose concentration in people with diabetes than is the determination of percent HbA1c. It also may be more useful in surveying populations for diabetes. Current diagnostic criteria for diabetes do not include glycohemoglobin (GHb) values due to a lack of uniformity in methods used to determine percent GHb and to a reported lack of sensitivity and specificity. In part, the latter may be due to the nonspecificity of the methods used. For example, hemoglobinopathies and the derivatization of hemoglobin by adducts other than glucose affect the interpretation of HbA1c values when determined by the method used in the Diabetes Control and Complication Trial (DCCT) and by other methods that depend on a change in charge on the hemoglobin molecule. None of these methods have been reported to significantly affect total glycohemoglobin (tGHb) values. Since the reproducibility and specificity of the HPLC affinity method for determining tGHb are excellent, the major variables in interpreting tGHb results are likely to be due to biological variations in circulating glucose concentration and in red blood cell survival in the same person over time and between individuals at a single time. The authors decided to investigate these variables. They determined the correlation of percent tGHb with the fasting plasma glucose concentration in people without known diabetes. They also determined the correlation of tGHb with reticulocyte count as an index of red blood cell survival and with a carbon monoxide method for determining RBC survival. They also evaluated the stability of the tGHb, glucose, RBC mass, hemoglobin, and reticulocyte counts over a one-year period. Total glycohemoglobin, overnight fasting plasma glucose concentration, hemoglobin, RBC and reticulocyte count, and calculated percentage of RBC count represented by reticulocytes were determined monthly for at least 12 months (range, 12 to 26 months) in 48 adults (mean age, 51 years; range, 31 to 82 years). In 37 of the subjects, the authors also determined RBC survival using a carbon monoxide method. A highly significant linear correlation was noted between the fasting glucose concentration and the tGHb, while a weak correlation was found between the percent reticulocytes or with RBC survival determined by the carbon monoxide method. The tGHb, plasma glucose, RBC count, hemoglobin, and percent reticulocytes were very stable over a 12-month or longer period. The authors concluded that there is good correlation between the tGHb and plasma glucose concentration in a population without known diabetes. Furthermore, variations in RBC survival, as indicated by a reticulocyte count within the reference range, are not likely to have a clinically significant effect on interpretation of tGHb data in the context of an integrated glucose concentration.

Nuttall FQ, Gannon MC, Swaim WR, et al. Stability over time of glycohemoglobin, glucose, and red blood cell survival in hematologically stable people without diabetes. Metabolism. 2004;53:1399-1404.

Reprints: Dr. Frank Q. Nuttall, Section of Endocrinology, Metabolism and Nutrition, VA Medical Center (111G), 1 Veterans Drive, Minneapolis, MN 55147

bullet Impact of automated blood culture systems

Bloodstream infections are ranked as the tenth leading cause of death in the United States, with a recent increase in age-adjusted death rates. Bloodstream infections (BSIs) also have been associated with increased rates of hospitalization, increased lengths of hospital stay, and increased hospital costs. Identifying BSI early aids antimicrobial therapy and diminishes the need for additional diagnostic studies, which in turn may decrease length of hospital stay and associated costs. Current automated continuous-monitoring blood culture systems afford more rapid detection of bacteremia and fungemia than is possible with noninstrument-based manual methods. However, use of these systems has not been studied objectively with respect to impact on patient outcomes, including hospital charges and length of hospitalization. The authors conducted a prospective, two-center study in which they assessed the time from obtaining the initial positive blood culture until the Gram stain was called for 917 cases of bloodstream infection. Factors showing univariate associations with a shorter time to notification included higher body temperature and respiratory rate and higher percentage of immature neutrophils. Multiple linear regression models determined that the primary predictors of increased microbiology laboratory and total hospital charges for patients with bloodstream infection were nonmicrobiologic and included length of stay and host factors, such as the admitting service and underlying illness score. Significant microbiologic predictors of increased charges included the number of blood cultures obtained, nosocomial acquisition, and polymicrobial bloodstream infections. Accelerated failure-time regression analysis demonstrated that microbiologic factors, including time until notification, organism group, and nosocomial acquisition, were independently associated with length of hospitalization after bacteremia, as were the factors of admitting service, gender, and age. The data suggest that an increased time to notification of bloodstream infection is independently associated with increased length of stay. The authors concluded that time to notification is an obvious target for efforts to shorten length of stay. The newest generation of automated continous-monitoring blood culture systems, which shorten the time required to obtain a positive result, should influence length of hospitalization.

Beekmann SE, Diekema DJ, Chapin KC, et al. Effects of rapid detection of bloodstream infections on length of hospitalization and hospital charges. J Clin Microbiol. 2003;41: 3119-3125.

Reprints: S.E. Beekmann, Division of Medical Microbiology, Dept. of Pathology, University of Iowa College of Medicine, 265 MRC, Iowa City, IA 52242;

bullet Coagulation abnormalities in Ebola hemorrhagic fever

Among viruses that cause hemorrhagic fever, Ebola virus stands out for its impressive lethality. Along with Marburg virus, the four species of Ebola virus (EBOV)—Zaire, Sudan, Reston, and Ivory Coast—make up the negative-stranded, enveloped RNA virus family Filoviridae. In outbreaks among humans, acute mortality caused by the Zaire species of EBOV has been approximately 80 percent. EBOV infection is thought to involve inappropriate or maladaptive host responses, including disseminated intravascular coagulation (DIC). A number of studies have found positive correlations between plasma levels of coagulation inhibitors and the degree of DIC. During DIC, the extrinsic tissue factor-dependent pathway is the dominant route to thrombin generation that leads to fibrin deposition. Overexpression of tissue factor, a 47-kDA transmembrane glycoprotein that functions as the primary cellular initiator of the coagulation protease cascades, is a leading cause of DIC and thrombosis-related organ failure. Despite progress during the last decade in identifying key modulators of EBOV pathogenesis, there is no universal concept of the triggering mechanism of the hemorrhagic diathesis of EBOV infection. The authors conducted a study to investigate the processes that trigger the coagulation abnormalities that are characteristic of human and nonhuman primate EBOV infections and to identify key targets for potential chemotherapeutic interventions. The in vivo portion of their study focused on longitudinal evaluation of various coagulation parameters in macaques that were experimentally infected with EBOV. In addition, the authors inoculated primary human monocytes/macrophages to further evaluate the potential triggers for DIC, including expression of tissue factor. They reported that tissue factor plays an important role in triggering the hemorrhagic complications that characterize EBOV infections. Analysis of the samples obtained from 25 macaques showed increased levels of tissue factor associated with lymphoid macrophages, whereas analysis of peripheral blood-cell RNA showed increased levels of tissue factor transcripts by day three. Plasma from macaques contained increased numbers of tissue factor-expressing membrane microparticles. Dysregulation of the fibrinolytic system developed during the course of infection, including a rapid decrease in plasma levels of protein C. Infection of primary human monocytes/macrophages was used to further evaluate the role of tissue factor in EBOV infections. Analysis of primary human monocyte/macrophage RNA at one to 48 hours showed increased tissue factor transcripts, whereas levels of tissue factor protein were dramatically increased by day two. The authors concluded that chemotherapeutic strategies aimed at controlling overexpression of tissue factor may ameliorate the effects of EBOV hemorrhagic fever.

Geisbert TW, Young HA, Jahrling PB, et al. Mechanisms underlying coagulation abnormalities in Ebola hemorrhagic fever: overexpression of tissue factor in primate monocytes/macrophages is a key event. J Infect Dis. 2003;188:1618-1629.

Reprints: Dr. Thomas W. Geisbert, USAMRIID, Attn: MCMR-UIV, 1425 Porter St., Ft. Detrick, MD 21702-5011;

bullet Rethinking the impaired fasting glucose cutoff

The American Diabetes Association recommended the recognition of impaired fasting glucose as a category of glucose tolerance analogous to impaired glucose tolerance. It was recommended that the impaired fasting glucose (IFG) be diagnosed in those with fasting plasma glucose (FPG) between 6.1 mmol/L and 6.9 mmol/L. Since then, many analyses have examined the equivalence of FPG and the two-hour post-challenge glucose in predicting diabetes and cardiovascular disease. Several key findings have emerged from these studies. First, the association between impaired glucose tolerance (IGT) and cardiovascular disease events and mortality is stronger than that for IFG. Second, although IFG and IGT identify some of the same individuals, the degree of overlap is variable. As a consequence of the recommendations to use FPG rather than the two-hour post-challenge glucose to diagnose diabetes, many subjects with IGT who are at risk of future cardiovascular disease events would not be identified. On the basis of these findings, the American Diabetes Association recently recommended that the lower limit for the diagnosis of IFG be changed from 6.1 mmol/L to 5.6 mmol/L. The authors conducted a study to identify the optimal level of fasting glucose that would allow one to identify those with IGT and those at risk of incident diabetes, as well as to determine the effect on disease prevalence of lowering the criterion for the diagnosis of IFG and to determine the risk of future diabetes and cardiovascular disease associated with the different levels of FPG and compare them against those associated with IGT. Three studies were used: the 1998 National Health Survey (NHS98), a randomly selected cross-sectional sample of 4,723 subjects; the Singapore Impaired Glucose Tolerance Follow-up Study, a cohort study involving 295 IGT and 292 normal glucose tolerance subjects (frequently matched for age, gender, and ethnic group) followed up from 1992 to 2000; and the Singapore CVD Cohort Study, which included 5,920 subjects from three cross-sectional studies in whom the first ischemic heart disease event was identified through linkage to registry databases. Risk of diabetes (Singapore IGT Follow-up Study) was estimated using logistic regression adjusted for age, gender, and ethnicity. Risk of ischemic heart disease (Singapore CVD cohort) was estimated using stratified (by study, from which data were derived) Cox’s proportional hazards models adjusted for age, gender, and ethnicity. The authors found that lowering the criterion for diagnosing IFG to 5.6 mmol/L increased the prevalence of IFG from 9.5 percent to 32.3 percent in the NHS98 study. The lower cutoff identified more subjects at risk of diabetes and ischemic heart disease, but the relative risk was lower than that for IGT. The authors concluded that greater efforts to identify those with IGT, or a group at similar risk of diabetes and cardiovascular disease, may be a more efficient public health measure than lowering the FPG criterion for diagnosing IFG.

Tai ES, Goh SY, Lee JJM, et al. Lowering the criterion for impaired fasting glucose. Diabetes Care. 2004;27:1728-1734.

Reprints: Dr. E. Shyong Tai, Dept. of Endocrinology, Block 6, Level 6, Room B35, Singapore General Hospital, Outram Rd., Singapore 169608, Republic of Singapore;

Dr. Bissell is professor and director of clinical services and vice chair, department of pathology, Ohio State University Medical Center, Columbus. Dr. Domen is professor of pathology, medicine, and humanities, Penn State University College of Medicine, Hershey, Pennsylvania.