College of American Pathologists
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May 2001

Plasma-reduced homocysteine levels in end-stage renal disease
Increased levels of homocysteine in serum are a strong, independent risk factor for atherosclerotic vascular events in end-stage renal disease patients. This association has been described epidemiologically, but it has not been confirmed in prospective trials of homocysteine-lowering treatments. The current hypothesis accounting for the effect of homocysteine on vascular or thrombotic events depends on the reactivity of its “free” or reduced sulfhydryl group. Approximately two percent of circulating homocysteine is in this reduced form. The authors developed a chemical method for measuring plasma-reduced homocysteine concentrations. The method involves incubating freshly drawn blood with sodium iodoacetate. The s-carboxymethylhomocysteine is then analyzed by gas chromatography and mass spectrometry. The authors found that plasma-reduced homocysteine concentrations in end-stage renal disease patients were two- to four-times higher than normal and that these concentrations were lowered by hemodialysis. The reduced plasma homocysteine levels were proportional to plasma total homocysteine levels over the range of plasma homocysteine levels that have been associated with increased cardiovascular risk.

Hoffer LJ, Robitaille L, Elian KM, et al. Plasma reduced homocysteine concentrations are increased in end-stage renal disease. Kid Int. 2001;59:372-377.

Reprints: Dr. L. John Hoffer, Lady Davis Institute for Medical Research, Jewish General Hospital, 3755 Cote-Ste. Catherine Rd., Montreal, Quebec H3T 1E2, Canada

Guidelines for the rejection of stool cultures
Stool cultures performed in hospitalized patients have a very low diagnostic yield. This is especially true when cultures are obtained more than three days after admission. Furthermore, the test is very expensive to produce. A simple “three-day rule” has been adopted by as many as 25 percent of U.S. microbiology laboratories. However, enteropathic bacteria other than Clostridium difficile may be associated with nosocomial illness that would be missed by strict use of this rule. The authors performed a five-part study of stool culture guidelines in four European health care centers. The derivation sample consisted of 1,735 adult patients who provided a total of 3,416 stool cultures between 1995 and 1997 and 68 inpatients who had grown enteropathogenic bacteria from stool cultures between 1988 and 1998. Based on their study of this group, the authors developed the following modified three-day rule: Obtain stool culture in the presence of community-acquired diarrhea if the onset is 72 hours or less after admission, for nosocomial diarrhea if the onset is more than 72 hours after admission, and for at least one of the following: age of 65 years or older with pre-existing comorbidity, HIV infection, neutropenia or suspected nosocomial outbreak, and suspected nondiarrheal manifestations of enteric infections. The modified rule also says to obtain a maximum of two repeat examinations in a given patient. When the criteria were applied post hoc to a series of 1,025 stool cultures, the number of stool cultures would have been reduced by 52 percent and no clinically significant cases would have been missed. If the traditional three-day rule had been applied, 52 cases would not have been identified, 28 of which required antibiotic treatment.

Bauer TM, Lalvani A, Fehrenbach J, et al. Derivation and validation of guidelines for stool cultures for enteropathogenic bacteria other than Clostridium difficile in hospitalized adults. JAMA. 2001;285:313-319.

Reprints: Dr. Tilman M. Bauer, Dept. of Internal Medicine II, University Hospital Freiburg, Hugstetterstrasse 55, D-79106 Freiburg, Germany;

The renin-angiotensin-aldosterone system in chronic metabolic acidosis
Chronic acidosis is associated with complex derangements in endocrine and metabolic function, including sodium wasting in the kidney and extra cellular fluid volume depletion with activation of the renin-angiotensin-aldosterone axis. Cortisol secretion is also increased in association with renal nitrogen wasting. Plasma angiotensin II concentration likely increases in parallel with plasma renin activity in chronic metabolic acidosis and it exerts complex and opposing nephron segment-specific effects on renal acidification mechanisms. The plasma angiotensin II response to chronic metabolic acidosis may be an important element of the renal response to this disorder and, therefore, may affect the severity of acidosis in response to acid loads. The authors conducted studies to evaluate the role of angiotensin II and aldosterone in regulating acid base equilibrium in normal, acidotic humans. They did this by assessing the separate effects of chronic mineralocorticoid and angiotensin II antagonist administration on the acid-base and endocrine responses to chronic metabolic acidosis. Oral ammonium chloride was administered to eight normal subjects for seven days, followed by a seven-day dosage of spironolactone. This was followed by a four-day period of spironolactone and losartan. Ammonium chloride was continued during all study periods. Spironolactone resulted in exacerbation of the acidosis because of a large increase in indigenous acid production, which caused a significant increase in the net acid excreted (from 116 to 185 mmol/day). The urinary anion gap and sulfate excretion rates were also markedly increased. Plasma potassium increased secondary to decreased urinary potassium excretion, and plasma angiotensin II, cortisol, aldosterone, urinary aldosterone, urinary tetrahydrocortisol, free cortisol, and nitrogen excretion all increased significantly. The acidosis was further exacerbated when losartan was added to spironolactone. A renal mechanism accounted for the exacerbation of acidosis, as indicated by the decreased net excretion of acid and the unchanged, unmeasured anion and nitrogen excretion in the urine. The authors concluded that losartan exacerbates acidosis by inducing a distal tubular acidification effect, that angiotensin II is an important modulator of the renal acid excretory response to chronic metabolic acidosis in humans, and that inhibition of aldosterone action by spironolactone results in an increase in acid production and exacerbates chronic metabolic acidosis by a nonrenal mechanism.

Henger A, Tutt P, Riesen WF, et al. Acid-base and endocrine effects of aldosterone and angiotensin II inhibition in metabolic acidosis in human patients. J Lab Clin Med. 2000;136:379-389.

Reprints: Dr. Reto Krapf, Medizinische Universitätsklinik, Kantonsspital Bruderholz, 4101 Bruderholz/Basel, Switzerland

The plasma isoforms of coagulation factor V
Blood coagulation factor V exists in two molecular forms in blood—FV1 and FV2. These two forms differ in their degree of glycosylation at residue Asn2181 in their caboxyterminal C2 domain. Activated FV1 (FVa1) has a lower affinity for negatively charged phospholipid vesicles than does FVa2. FVa1 is, therefore, a less potent cofactor in prothrombin activation than is FVa2. Plasma from individuals carrying the R2 haplotype (a polymorphism involving amino acid changes in FV and that is associated with an increase risk of thrombosis) shows a ratio of FV1 to FV2 that is shifted in favor of the more thrombogenic FV1. The authors described an assay they developed that enables the quantification of the plasma levels and FV1 and FV2. The first assay is performed at saturating amounts of FXa and phospholipid vesicles, with a high mole fraction of phosphatidylserine FVa1 and FXa2. This assay quantifies the total FV level, FV1 plus FV2, present in plasma. The second assay is performed with limited amounts of FXa and phospholipid vesicles with a low mole fraction of phosphatidyl serine, in which FVa2 has an approximately eight-fold higher activity than FVa1. Thus, the assay response depends on the relative amounts of FV1 and FV2 in the plasma sample.

Hoekema L, Rosing J, Tans G. An assay to quantify the two plasma isoforms of factor V. Thromb Haemost. 2000;84:1066-1071

Reprints: Dr. J. Rosing, Dept. of Biochemistry, Cardiovascular Research Institute Maastricht (CARIM), Maastricht University, P.O. Box 616, 6200 MD Maastricht, Netherlands

Serum cystatin C concentration as a renal function test in elderly patients
Glomerular filtration rate decreases markedly with age. The effect is increased by atherosclerosis, hypertension, and heart failure. In elderly patients, the plasma creatinine level is an unreliable indicator of glomerular filtration rate since its daily production is diminished as a result of the reduced muscle mass occurring in these individuals. Serum cystatin C concentration is a new marker for renal function that may be superior to plasma creatinine in this regard. Serum cystatin C is a proteinase inhibitor with a low molecular weight that is produced in all nucleated cells. Its appearance rate in blood is, therefore, constant and independent of muscle mass and gender. The authors compared the measurements of cystatin C and creatinine in a group of 12 normotensive men and women in young adulthood and a group of 41 male and female subjects who were normotensive and hypertensive and in their sixth and seventh decades of life. The glomerular filtration rate was also estimated in all individuals using the inulin clearance technique. The mean plasma creatinine concentration was identical in both groups and, with only one exception, all of the elderly subjects had plasma creatinine concentrations that were within the 95 percent confidence interval for young patients. The correlation between serum cystatin C concentration and inulin clearance was considerably better for cystatin C than for plasma creatinine. The elderly patients with significantly reduced glomerular filtration rates were those who had serum cystatin concentrations greater than the upper 95 percent confidence interval of the young subjects.

Fliser D, Ritz E. Serum cystatin C concentration as a marker of renal dysfunction in the elderly. Am J Kid Dis. 2001;37:79-83.

Reprints: Dr. Danilo Fliser, Dept. of Internal Medicine, Medical School of Hannover, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany

Autoantibodies to p53 as tumor markers in small cell lung cancer
The most common genetic mutation in cancer is the p53 mutation. The normal gene has an important tumor-suppressor function, which is lost with the mutation. Abnormalities of the p53 gene have been associated with short survival or resistance to chemotherapy or radiotherapy. The gene product of the mutation tends to lead to an accumulation of a protein that acts as an auto-antigen with subsequent development of an antibody (p53-Ab). When measured by standardized enzyme-linked immuosorbent assay, these p53 antibodies are present in fewer than 0.5 percent of healthy controls but are elevated in various types of cancer and have been associated with the worst prognosis in breast, head and neck, and colon cancer. In small cell lung cancer, the rate of p53 mutation is 50 to 75 percent. The authors conducted a retrospective study of patients from the Royal Marsden Hospital in England that focused on serum p53 antibody expression and survival. They banked serum from 231 patients, of whom 63 percent were male and an overall median age of 65. The patients had been diagnosed and treated for small cell lung cancer between 1987 and 1994. Fifty-four of the patients were positive for p53 antibodies, representing 23 percent of the total. A high titer of p53 antibody is a favorable prognostic sign, as those without the antibody have a relative risk of death of 1.71 relative to the p53 antibody-positive patients.

Murray PV, Soussi T, O’Brien MER, et al. Serum p53 antibodies: predictors of survival in small-cell lung cancer? Br J Cancer. 2000;83:1418-1424.

Reprints: M. Tavassoli, Oral Oncology Group, The Rayne Institute, Guys, Kings and St. Thomas School of Dentistry, 123 Coldharbour Lane, London SE5 9NU, England

Prospects for a diagnostic screening test in Creutzfeldt-Jakob disease
The infectivity of blood from patients with various transmissible spongiform encephalopathies is a controversial subject. New interest in this area has been sparked by reports of the new variant form of Creutzfeldt-Jakob disease, in which the blood and body fluids of those with the variant form may present a greater danger than those of patients with classic Creutzfeldt-Jakob disease. Though eight different screening test methods are in various stages of development, none has yet been shown to detect the prion protein in the blood of humans who are incubating Creutzfeldt-Jakob disease. Whether any of these approaches will eventually be successful depends on whether the prion protein to infectivity level in blood follows the same proportionality as it does in the brain. An ideal system would combine a very sensitive detection method with the largest test volume of crude or extracted blood, and it would be inexpensive and adaptable to large numbers of specimens. Until such a system is developed, however, we must depend on current donor screening and referral methods.

Brown P, Cervenáková L, Diringer H. Blood infectivity and the prospects for a diagnostic screening test in Creutzfeldt-Jakob disease. J Lab Clin Med. 2001;137:5-13.

Reprints: Dr. Paul Brown, Building 36, Room 4A-05, National Institutes of Health, 36 Convent Dr., MSC 4122 Bethesda, MD 20892

Calcitonin precursors as sepsis markers
Making an unequivocal diagnosis of sepsis in ICU patients can be difficult because traditional markers of infection can be misleading. Precursors of the hormone calcitonin (procalcitonin and the calcitonin-calcitonin carboxy peptide 1 complex) have been reported to be elevated in adults and children with infection. The authors measured serum calcitonin precursors, C-reactive protein, interleukin-6, and lactate in 101 consecutive patients with anticipated lengths of stay in a medical ICU of 24 hours or more. The patients were classified daily as manifesting systemic inflammatory response syndrome, sepsis, severe sepsis, or septic shock using standardized diagnostic criteria. All markers showed significant increases with increasing severity of infection. Comparing the assays directly with one another using receiver operating characteristic analysis showed that calcitonin precursors were the most reliable. The calcitonin precursors showed 89 percent sensitivity and 94 percent specificity versus ranges of 40 to 71 percent sensitivity and 77 to 79 percent specificity for the other assays. Greater concentrations of calcitonin precursors were highly correlated with poorer prognosis.

Müller B, Becker KL, Schächinger H, et al. Calcitonin precursors are reliable markers of sepsis in a medical intensive care unit. Crit Care Med. 2000;28:977-983.

Reprints: Dr. Beat Müller, Div. of Endocrinology, Diabetology and Metabolism, Dept. of Internal Medicine, University Hospital, Petersgraben 4, CH-4031 Basel, Switzerland;