College of American Pathologists
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  Clinical Abstracts






November 2007

Michael Bissell, MD, PhD

Improved thyroid test reference intervals
Preoperative N-terminal proBNP as a cardiac marker for surgery
Transaminases as markers for intracerebral hemorrhage
Using the biuret method for urinary peptides
Thyroid function, insulin sensitivity, and lipid concentrations in diabetes
Beta-human chorionic gonadotropin and tubal obstruction
Secretion of human kallikrein 4 into biological fluids
Serum ferritin as a risk factor for stroke in postmenopausal women

bullet Improved thyroid test reference intervals

The National Academy of Clinical Biochemistry proposed that only euthyroid healthy volunteers be used to establish new thyrotropin reference intervals. These volunteers should be free from detectable autoantibodies against thyroid peroxidase (TPOAbs) or thyroglobulin (TgAbs) and a personal or family history of thyroid dysfunction. In addition, no visible or palpable goiter and no medication, except estrogens, should be allowed. The authors conducted a study to establish new reference intervals for thyrotropin based on the National Academy of Clinical Biochemistry (NACB) criteria and regular thyroid ultrasonography. In addition, the authors studied whether these criteria could be helpful for establishing reference intervals for total and free triiodothyronine (T4 and FT4) and total and free thyroxine (T4 and FT4). They investigated 870 apparently healthy subjects and excluded those with a family history of thyroid disease, pathologic thyroid ultrasonography results, and increased anti-thyroid peroxidase or anti-thyroglobulin antibodies. This left 453 people as the reference collective. The authors measured serum concentrations of thyrotropin, T4, FT4, T3, and FT3 of the whole and the reference collective on Roche Diagnostics' Elecsys system assays and calculated the 2.5th and 97.5th percentiles for comparison. The authors found that the calculated lower limit for thyrotropin differed significantly between the reference intervals for healthy people whose normal thyroid gland was assessed versus a nonselected group of healthy blood donors. Age was the only independent factor and was significantly inversely associated with thyrotropin (P<0.0001). Use of oral contraceptives was a significant predictor for variation in T4 concentrations (P<0.001). Age and oral contraceptives were independently associated with T3 variations (P<0.05). For FT4 versus FT3 variation, gender and, inversely, age (P<0.01) were independent modulating factors. The authors concluded that selecting healthy people according to NACB criteria, combined with sonographic confirmation of a normal thyroid gland, provides a valid basis for the reference interval for thyrotropin. Factors indicating a preclinical disease state, such as family history, pathologic ultrasonography result, or increased antithyroid peroxidase and anti-thyroglobulin antibodies, can be associated with normal hormone concentrations. Furthermore, patient age and gender, as well as use of contraceptives, should be considered in the diagnostic evaluation of thyroid diseases.

Kratzsch J, Fiedler GM, Leichtle A, et al. New reference intervals for thyrotropin and thyroid hormones based on National Academy of Clinical Biochemistry criteria and regular ultrasonography of the thyroid. Clin Chem. 2005;51:1480-1486.

Reprints: Juergen Kratzsch, Institute of Laboratory Medicine, Clinical Chemistry and Molecular Diagnostics, University Hospital Leipzig, Paul-List-Strasse 13-15, D-04103 Leipzig, Germany;

bullet Preoperative N-terminal proBNP as a cardiac marker for surgery

Brain natriuretic peptide, a cardiac natriuretic peptide, was discovered in porcine brain, although the left ventricle is now known to be the major source of BNP. BNP is secreted by the left ventricle in response to wall stress. It is synthesized as a pro-hormone called proBNP and, upon secretion, is cleaved into the N-terminal proBNP (NTproBNP) and bioactive BNP. NTproBNP is useful in clinically assessing left ventricular dysfunction and heart failure and aids in risk stratification of patients with acute coronary syndromes. Serum NTproBNP levels are raised in patients with asymptomatic left ventricular dysfunction before symptoms become overt. Cardiac complications are a major cause of mortality and morbidity in patients undergoing major surgery. Heart failure is an important risk factor. Patients with a history of heart failure tend to be intolerant of hemodynamic changes and prone to pulmonary congestion. The underlying causes of ventricular dysfunction may also contribute to cardiac complications. It can be difficult to diagnose heart failure because some patients remain asymptomatic. Furthermore, patients with left ventricular diastolic dysfunction may have normal cardiac size and left ventricular ejection fraction. Because NTproBNP is a sensitive marker of left ventricular dysfunction, the authors conducted a study to test the hypothesis that the preoperative level of NTproBNP could be useful in predicting cardiac complications in patients undergoing noncardiac surgery. The authors studied 190 consecutive patients who underwent noncardiac surgery that required general anesthesia. In addition to routine preoperative evaluation, the authors took a blood sample to estimate plasma NTproBNP concentration. Postoperative cardiac complications were defined as cardiac death, acute coronary syndrome, heart failure, and hemodynamic compromise from cardiac arrhythmias. The authors found that 15 of the 190 patients had a cardiac complication—four had acute coronary syndrome and 13 had congestive heart failure. NTproBNP concentration was significantly higher in patients with a cardiac complication; a level greater than 450 ng/L was predictive of cardiac complications with a sensitivity of 100 percent and specificity of 82.9 percent. Other factors associated with cardiac complications were a higher American Society of Anesthesiologists grade, age, and clinical cardiac impairment. But in multivariate analysis, NTproBNP level was the only independent factor. The authors concluded that preoperative plasma NTproBNP concentration may be an independent predictor of cardiac complications in patients undergoing noncardiac surgery.

Yeh H-M, Lau H-P, Lin J-M, et al. Preoperative plasma N-terminal pro-brain natriuretic peptide as a marker of cardiac risk in patients undergoing elective non-cardiac surgery. Brit J Surgery. 2005;92:1041-1045.

Reprints: Dr. L.-P. Lai, Institute of Pharmacology, National Taiwan University, 1 Jen-Ai Rd., Section 1, Taipei, Taiwan;

bullet Transaminases as markers for intracerebral hemorrhage

Serum aspartate aminotransferase and alanine aminotransferase assays are commonly used to screen for liver diseases. Epidemiologic studies have reported positive associations between serum aminotransferase levels and various conventional cardiovascular risk factors. Nevertheless, a direct association between serum aminotransferase levels and risk of cardiovascular disease has not been fully studied. Some patient studies reported a positive association between hemorrhagic stroke and history of liver dysfunction, which was defined by elevated liver enzymes. However, there are no data on the association between aminotransferase levels and incidence of stroke. Consequently, the authors prospectively investigated the relation between serum aminotransferase levels and the 10-year incidence of stroke by subtypes. They measured serum aspartate and alanine aminotransferase levels and traditional cardiovascular risk factors in 108,464 Korean men, aged 35 to 59 years, in 1990 and 1992. Serum aminotransferase levels were classified into three categories: less than 35 IU/L, 35 to 69 IU/L, and 70 IU/L or more. The outcomes were hospital admissions and deaths from stroke subtypes—ischemic stroke, intracerebral hemorrhage (ICH), and subarachnoid hemorrhage (SAH) —from 1993 through 2002. The authors found that during the 10 years, 1,728 ischemic, 1,051 hemorrhagic (718 ICH and 222 SAH), and 243 unspecified stroke events occurred. After adjusting for age and other traditional risk factors and according to Cox proportional-hazards models, serum aminotransferase level had an independent positive association with ICH. Ischemic stroke and SAH were not associated with aminotransferase levels. Compared with the level of less than 35 IU/L, the adjusted relative risks (95% confidence interval) of ICH for an aspartate aminotransferase level of 35 to 69 IU/L and 70 IU/L or more were 1.49 (1.21-1.83) and 4.21 (3.06-5.77), respectively. The corresponding risks for alanine aminotransferase were 1.34 (1.09-1.65) and 2.89 (2.09-4.01), respectively. These associations were consistent regardless of the level of obesity, blood pressure, fasting glucose, alcohol intake, and length of followup. The authors concluded that these findings suggest that an elevated aminotransferase level is a predictor of ICH. The biologic significance of aminotransferase level relative to developing ICH merits further study.

Kim HC, Kang DR, Nam CM, et al. Elevated serum aminotransferase level as a predictor of intracerebral hemorrhage: Korea Medical Insurance Corporation study. Stroke. 2005;36:1642-1647.

Reprints: Dr. Il Suh, Dept. of Preventive Medicine, Yonsei University College of Medicine, 134 Shinchon-dong, Seodaemun-gu, Seoul 120-752, Republic of Korea;

bullet Using the biuret method for urinary peptides

A variety of compounds, including proteins, can complex with copper(II) (cupric) ions under alkaline conditions. Formation of this complex between protein and cupric ions shifts the color of solutions from blue to purple and has been termed the biuret reaction after one of the first compounds that was recognized to yield this color reaction. Subsequently, many variations of this reaction were developed to measure total protein in serum or other specimens. A major advantage of this method is the relatively equivalent reactivity of all proteins, leading to a proposal for a biuret method to serve as a way of standardizing the measurement of total serum protein. The authors conducted a study to examine the cross-reactivity of amino acids, short peptides, and a variety of other compounds in the biuret reaction to develop a greater understanding of the specificity of the reaction and to examine its suitability for quantifying low-molecular-weight peptide components in urine. The authors found that the biuret assay cross-reacted with several amino acids, dipeptides, and other organic compounds able to form five- or six-member ring chelation complexes with copper. Reactions with amino acids and dipeptides had higher absorbance maxima (blue color) than with larger peptides and proteins (purple). Compounds forming potential four-, seven-, eight-, or nine-member ring complexes with copper had low reactivity. Amino acid amides, dipeptides, and longer peptides, with the exception of those containing proline, had substantial reactivity. Proteins and polypeptides had similar biuret reactivities per peptide bond, but reaction kinetics were slower for proteins than peptides. Urine specimens ultrafiltered through 3-kDa-cutoff membranes had substantial biuret reactivity, but absorbance maxima were consistent with cross-reactive amino acids rather than peptides. The authors concluded that many compounds, including amino acids, amino acid derivatives, and dipeptides, cross-react in biuret assays. These studies improve understanding of the specificity of endpoint and kinetic biuret assays widely used in clinical laboratories. Amino acids, urea, and creatinine contribute to biuret assays overestimating urinary peptide content.

Hortin GL, Meilinger B. Cross-reactivity of amino acids and other compounds in the biuret reaction: interference with urinary peptide measurements. Clin Chem. 2005;51:1411-1419.

Reprints: Glen L. Hortin, Dept. of Laboratory Medicine, National Institutes of Health, Bldg. 10, Room 2C-407, Bethesda, MD 20892-1508;

bullet Thyroid function, insulin sensitivity, and lipid concentrations in diabetes

The association between thyroid status and dyslipidemia continues to generate interest. A positive association between overt hypothyroidism and hypercholesterolemia is well recognized, but evidence for such a relationship in subclinical thyroid disease is inconsistent. In euthyroid nondiabetic adults, the relationship between serum thyrotropin and cholesterol appears to be modified by insulin resistance, such that those with higher serum thyrotropin and relative insulin resistance are at greatest risk of dyslipidemia. More severe insulin resistance is a cardinal characteristic of most patients with type 2 diabetes. Such patients are at increased risk of dyslipidemia, and mild thyroid dysfunction is also a relatively frequent finding. The authors, therefore, conducted a community-based cross-sectional observational study to determine whether insulin sensitivity modifies the association between thyroid dysfunction and lipid parameters in diabetic patients. The study subjects were 117 females with type 2 diabetes who were not receiving oral hypoglycemic therapy, insulin, or lipid-lowering therapy. The authors measured the subjects' serum thyrotropin, insulin, total and high-density lipoprotein cholesterol, and triglycerides. They also conducted age-adjusted multiple linear regression analysis of serum lipid concentrations and derived parameters as functions of serum thyrotropin and homeostasis model assessment-derived insulin sensitivity (HOMA-S). The authors found that the relationship among serum lipid concentrations, serum thyrotropin, and HOMA-S was significantly modified by an interaction term ln(TSH)*ln(HOMA-S). In three-dimensional graphs, strong positive associations were noted between thyrotropin and lipid parameters with adverse cardiac risks at low insulin sensitivity, which were absent at higher insulin sensitivity. The effect was strongest for lipid risk factors associated with insulin resistance. The authors concluded that the interaction between thyroid function and insulin sensitivity is an important contributor to diabetic dyslipidemia and may justify free thyroxine T4 replacement in some patients.

Chubb SA, Davis WA, Davis TME. Interactions among thyroid function, insulin sensitivity, and serum lipid concentrations: the Fremantle Diabetes Study. J Clin Endocrinol Metab. 2005;90:5317-5320.

Reprints: Dr. S. A. P. Chubb, Dept. of Biochemistry, Fremantle Hospital, P.O. Box 480, Fremantle, Western Australia 6959, Australia;

bullet Beta-human chorionic gonadotropin and tubal obstruction

The incidence of ectopic pregnancy has increased in the last two decades, and it remains the leading cause of pregnancy-related death in the first trimester. However, early awareness coupled with increased sensitivity of serum b-human chorionic gonadotropin (b-hCG) immunoassay and an improved quality of transvaginal ultrasonography, which allows ectopic pregnancy to be detected early, has changed the goal of management from a life-saving intervention to tubal and fertility preservation. Early detection of unruptured tubal pregnancy has led to less invasive methods of treatment, which have almost reduced the need for laparotomy. Therefore, conservative laparoscopic surgery, medical treatment with methotrexate, and expectant management offer extremely positive results when their criteria for use are respected. The fertility outcome of patients treated conservatively with methotrexate or expectant management of unruptured ectopic pregnancy can be evaluated by hysterosalpingography (HSG) or future pregnancy. HSG represents important diagnosis methods after treating ectopic pregnancy. If the results demonstrate tubal patency, there is a possibility of spontaneous pregnancy. In cases demonstrating obstruction in both tubes, the only treatment is assisted reproduction with in vitro fertilization. Because there is little information about the fertility outcome of women treated conservatively with methotrexate or expectant management, the authors presented the subsequent reproductive performance in women treated according to the authors' methotrexate and expectant management protocol. The authors analyzed tubal permeability using HSG after clinically treating ectopic pregnancy. They evaluated the possible risk of tubal obstruction through initial titers of b-hCG, size of adnexal mass, aspects of the image at ultrasonography, and color Doppler of the mass. Eighty patients were submitted to HSG after being treated for tubal pregnancy from April 1994 to February 2002. Fifty received expectant management and 30 were treated with single-dose methotrexate (50 mg/m2 intramuscularly). The authors found that the patency of the ipsilateral tube was 84 percent and 78 percent after methotrexate and expectant treatments, respectively (P>0.05). After logistic regression was performed, it was observed that levels of b-hCG greater than 5,000 mUI/mL were directly related to tubal obstruction risk (odds ratio, 11.79; 95% confidence interval, 2.27-61.32). Other variables were not directly related to tubal obstruction risk. The authors concluded that the probability of ipsilateral tubal obstruction depends on b-hCG levels. The increase in b-hCG levels is followed by enhanced tubal obstruction risk. Therefore, b-hCG may be effective in predicting the reproductive future of these patients.

Elito J, Han KK, Camano L. Values of b-human chorionic gonadotropin as a risk factor for tubal obstruction after tubal pregnancy. Acta Obstet Gynecol Scand. 2005; 84:864- 867.

Reprints: Julio Elito Jr., Rua Maria Carolina, 68 CEP-01445-000, Jardim Paulistano, Sao Paulo, Brazil;

bullet Secretion of human kallikrein 4 into biological fluids

Serine proteases are the most intensely studied group of enzymes in biology, reflecting their diverse physiologic functions in digestion, coagulation, immune regulation, fibrinolysis, reproduction, and development. The activity of most serine proteases is governed by one or more regulatory mechanisms through transcriptional control, by translation into zymogen forms that need activated at their sites of action, and through complex formation with protease inhibitors. Deregulated, abnormal serine protease activity can lead to disorders as diverse as asthma, clotting abnormalities, emphysema, and cancer. Closely related serine proteases appear to contribute to the pathogenesis of various malignancies. The best-known cancer-associated proteases are those belonging to the matrix metalloprotease family, although others belonging to the human kallikrein family have recently received attention. One of these is the human kallikrein 4 (KLK4) gene. The expression of this gene, discovered independently by two groups, was initially deemed to be restricted to human prostate based on Northern blotting data. However, subsequent reverse transcription-PCR studies demonstrated that KLK4 mRNA is also expressed in other tissues, although at much lower concentrations than in the prostate. But the function of KLK4 in malignant ovarian tissue or healthy prostate tissue remains to be determined. To investigate the concentrations of human kallikrein 4 (hK4) in tissues and biological fluids, the authors developed a new hK4-specific sandwich-type immunoassay using a monoclonal antibody as the capture reagent. The assay has a detection limit of 0.02 μg/L and less than 0.1 percent cross-reactivity toward any of the other 14 human kallikreins. Twelve of 40 tissue extracts prepared from various human tissues contained detectable hK4 concentrations (0.68-7,143 ng/g of total protein), with healthy prostate tissue containing the highest amount of hK4. Examination of 16 malignant and 18 benign prostate tissues revealed no significant differences in hK4 protein content, and the tissues contained a wide range of values (benign, less than 0.02-801 ng/g; malignant, less than 0.02-824 ng/g). Among the biological fluids tested, seminal plasma and urine contained widely varying amounts of hK4; concentrations in 54 urine samples were less than 0.02 to 2.6 µg/L, whereas concentrations in 58 seminal plasma samples were 0.2 to 202 μg/L. Affinity purification of hK4 from seminal plasma and subsequent mass spectrometry demonstrated the secreted nature of hK4 in seminal plasma. The authors concluded that hK4 is found primarily in prostate tissue and is secreted in seminal plasma. Its value as a novel prostatic biomarker needs to be defined further.

Obiezu CV, Shan SJC, Soosaipillai A, et al. Human kallikrein 4: quantitative study in tissues and evidence for its secretion in biological fluids. Clin Chem. 2005;51:1432-1442.

Reprints: Eleftherios P. Diamandis, Dept. of Pathology and Laboratory Medicine, Mount Sinai Hospital, 600 University Ave., Toronto, Ontario, Canada M5G 1X5;

bullet Serum ferritin as a risk factor for stroke in postmenopausal women

It has been hypothesized that the lower incidence rates of ischemic heart disease in premenopausal women compared with men and the increase in ischemic heart disease rates in postmenopausal women result from the rise in iron stores after cessation of menses, with oxidative imbalance as the central biologic mechanism. In the Fenton reaction, Fe(II) catalyzes the formation of extremely reactive hydroxyl radicals. Interaction with lipids may initiate the formation of oxidized low-density lipoprotein that ultimately leads to the development of foam cells and progression of atherosclerosis. Iron could also play a role in vascular disease by activating platelets via a protein kinase C mechanism. Although its initial focus was on ischemic heart disease, the hypothesis may apply to cerebrovascular disease as well. Another proposed mechanism by which iron may play a role in ischemic vascular disease, which might be more relevant to stroke risk, is through ischemia/ reperfusion injury. During reperfusion after cerebral infarction, oxygen-radical production markedly increases and iron ions are released, leading to progressive tissue damage and cellular death. There are few articles about the association between iron and risk of stroke in population-based studies. The authors conducted a study in which they investigated the relation between high body iron stores, indicated by serum ferritin levels, and risk of stroke in a cohort of healthy, postmenopausal women. They studied the association between iron status and stroke risk in a population-based cohort of 11,471 Dutch postmenopausal women between 49 and 70 years of age. Women were included between 1993 and 1997 and followed up for cerebrovascular events until Jan. 1, 2000. The authors conducted a case-cohort study by using all stroke cases (n=63) and a random sample of the baseline cohort (n=1,134). They measured serum ferritin, serum iron, and transferrin saturation as markers of iron status. The authors used a weighted Cox proportional-hazards model to estimate crude and multivariate-adjusted hazard ratios for tertiles of different iron parameters in relation to stroke. In a multivariate model, the highest tertile of serum ferritin concentration was associated with an increased risk of stroke (hazard ratio, 1.45; 95% confidence interval, 0.87-2.42) compared with the lowest tertile. For ischemic stroke, the increase was more pronounced (hazard ratio, 2.23; 95% confidence interval, 1.05-4.73) and reached statistical significance. The authors concluded that neither serum iron nor transferrin saturation was associated with an increased risk of stroke. However, higher serum ferritin concentrations in postmenopausal women are associated with an increased risk of ischemic stroke.

Van der A DL, Grobbee DE, Roest M, et al. Serum ferritin is a risk factor for stroke in postmenopausal women. Stroke. 2005;36:1637-1641.

Reprints: Dr. Yvonne T. van der Schouw, Julius Center for Health Sciences and Primary Care, University Center Utrecht, HP Str 6.131, P.O. Box 85500, 3508 GA Utrecht, Netherlands;

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