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  Anatomic Abstracts

 

 

March 2008

Editor:

Michael Cibull, MD
Melissa Kesler, MD

Cerebrospinal fluid cell counts in meningitis
Plasma versus capillary international normalized ratios

A new candidate renal function marker

Automated parameters for neutrophils

PAPP-A as a peripheral vascular disease marker

Ability of hemoglobin A1c to predict drug treatment for diabetes mellitus

Preanalytic handling of urines for catecholamine assays

Predictive value of heart failure staging

Relationship of arteries to renal sodium handling

Role of advanced reagents in improving immunohistochemistry Cerebrospinal fluid cell counts in meningitis

The early predominance of polymorphonuclear cells among the cerebrospinal fluid white blood cells of patients who are early in the course of aseptic meningitis is well known. However, studies documenting the timing of the transition from cerebrospinal fluid (CSF) polymorphonuclear cells (PMN) to mononuclear cells report conflicting results. Some studies suggest that a rapid shift from predominance of PMN to mono­nuclear cells occurs eight to 48 hours after presentation. Other studies failed to find a correlation between duration of symptoms and CSF PMN predominance. One possible explanation for these discrepancies may be that the dynamics of transition from CSF PMN to mono­nuclear cell predominance may depend on other factors, such as the extent of menin­geal inflammation, and may be patho­gen specific. The authors conducted a study to assess the relationship between duration of symptoms and CSF mononuclear cell count in children with enteroviral meningitis. They used a binomial regression model to determine the relationship between the percentage of cerebrospinal fluid mononuclear white blood cells and symptom duration in children with proven enteroviral meningitis. The authors found that the odds of a CSF white blood cell being mononuclear increased by 15.7 percent (95% confidence interval, -3.8% to 38%; P=0.11) for each day of symptoms. Fifty percent of patients with symptoms of one day or less had predominance of mononuclear cells among CSF white blood cells. The authors concluded that factors other than symptom duration may influence the composition and evolution of CSF white blood cell response to enteroviral infection.

Shah SS, Hodinka RL, Turnquist JL, et al. Cerebrospinal fluid mononuclear cell predominance is not related to symptom duration in children with enteroviral meningitis. J Pediatr. 2006;148:118-121.

Reprints: Dr. Samir S. Shah, Division of Infectious Diseases, Children’s Hospital of Philadelphia, North Campus, Room 1526, 34th St. and Civic Center Blvd., Philadelphia, PA 19104; shahs@email.chop.edu.

Role of advanced reagents in improving immunohistochemistry Plasma versus capillary international normalized ratios

Oral anticoagulant therapy with vitamin K antagonists is monitored by the international normalized ratio, which has to be between 2.0 and 3.5 for the majority of clinical conditions. Regular controls are necessary because of the narrow therapeutic window of vitamin K antago­nists. The international normalized ratio (INR) can be determined in the laboratory using capillary blood by the Owren test or with diluted capillary blood by the Hepato Quick method (Roche Diagnostics) using various analyzers. Some more recently dedicated point-of-care tests allow INR testing to be conducted by nonlaboratory personnel and patients who are self testing. These devices offer the advantages of simple handling, smaller sample volumes, elimination of sample transport and other preanalytical errors, timely monitoring of treatment, and greater ­patient satisfaction. Point-of-care testing allows patients to manage their oral anticoagulation. Furthermore, capillary measurement of INR allows more regular monitoring and sub­sequently increases the time in the target therapeutic range, which helps diminish the complications related to vitamin K antagonists. The authors evaluated the i-Stat analyzer (Abbott Point-of-Care Division) for monitoring oral anticoagulant therapy and compared it with a well-established venous plasma method. The i-Stat also measures blood gases, glucose, and other clinical chemistry parameters. The authors first determined between-cartridge variability using two lyophilized controls with INR levels of 1.60 and 2.75 (n=10). Then they measured capillary blood INR with two i-Stat devices in 35 patients under different intensities of oral anticoagulation and compared it to central laboratory plasma INR (Innovin reagent and BCS analyzer, both from Dade Behring). The authors determined that between-cartridge coefficients of variation were five percent (95% CI, 3.4-9.1) and three percent (95% CI, 2.1-5.5) at INR levels of 1.60 and 2.75. Mean INR difference between the two i-Stat analyzers was 0.1, and the correlation coefficient was 0.98. Between i-Stat and central laboratory INR, the correlation coefficient was 0.95. Bias values were 0.04, 0.2, and -0.04 at INR levels of 2.0, 2.5, and 3.5, respectively. The authors concluded that the INR measured with the i-Stat portable clinical analyzer is precise and compares well with plasma INR performed in a central laboratory.

Boehlen F, Reber G, de Moerloose P. Agreement of a new whole-blood PT/INR test using capillary samples with plasma INR determinations. Thrombosis Res. 2005;115:131-134.

Reprints: Philippe de Moerloose, Hemostasis Unit, Dept. of Medicine, University Hospital, 1211 Geneva 14, Switzerland; Philippe. deMoerloose@hcuge.ch

Role of advanced reagents in improving immunohistochemistry A new candidate renal function marker

Creatol is a creatinine oxidative metabolite originally isolated from the urine of patients with chronic renal failure, and it may represent a uremic toxin. Creatol (CTL, 5-hydroxycreatinine) has been recognized as a creatinine (Cr) metabolite directly produced through oxidation with an active oxygen species, probably the hydroxyl radical, and also as a key precursor of the uremic toxin methylguanidine. The change in CTL reflects kidney function more sharply than Cr, suggesting in vivo oxidative stress. Renal damage may be caused by its repeated circulation. It has been reported that the ­ratio of serum (s-) CTL to s-Cr represents a possible oxidation stress marker. The effectiveness of CTL as an indicator of oxidative stress after kidney transplantation has not been reported. Therefore, the authors examined the relation between changes in oxidative stress (using s-CTL) in renal transplant patients and the patients’ change in renal function (using s-Cr). They examined the serum Cr and serum and urine CTL in five renal transplant patients going back to June 2003. The patients were a mean age of 33.6 years and three were male. Three recipients (Nos. 1, 3, and 4) received a kidney from their mothers, one (No. 2) from her grandmother, and one (No. 5) from his son. The authors found that serum CTL closely correlated with serum Cr. Serum CTL also correlated with urine CTL, but in some cases there was a time lag. Both the ratio of CTL/Cr and serum CTL observed in the second patient were slow to improve after transplantation. The process through which oxidative stress was reduced was shown by the index of renal damage correlated with kidney function oxidative stress (using s-CTL) after transplantation. The authors concluded that CTL/Cr may be a good indicator of graft prognosis.

Matsumoto S, Hanai T, Matsuura T, et al. Creatol, an oxidative product of creatinine in kidney transplant patients, as a useful determinant of renal function: a preliminary study. Transplant Proc. 2006;38:2009-2011.

Reprints: Dr. Seiji Matsumoto, Dept. of Urology, Kinki University School of Medicine, 377-2 Ohno-Higashi, Osaka-Sayama, Osaka 589-8511, Japan; matsums@med.kindai.ac.jp

Role of advanced reagents in improving immunohistochemistry Automated parameters for neutrophils

Review of peripheral blood smears can yield important diagnostic information by identifying morphologic changes characteristically seen in reactive neutrophils during infection. However, this approach is labor intensive and time consuming because it requires manual examination by an experienced medical technologist. Furthermore, the results are subjective, and only a few hundred cells can be analyzed for any given sample. The VCS technology of the Coulter LH 750 hematology analyzer (Beckman Coulter) can obtain data directly from more than 8,000 white blood cells using direct current impedance to measure cell volume (V) for accurate size of all cell types, radio frequency opacity to characterize conductivity (C) for internal composition of each cell, and a laser beam to measure light scatter (S) for cytoplasmic granularity and nuclear structure. These data are used to identify each cell as a neutrophil, lymphocyte, monocyte, eosinophil, or basophil, generating an automated differential count. However, the use of VCS technology to evaluate morphologic changes within the same cell population, such as the previously described neutrophil changes during acute bacterial infection, has never been well studied. Band forms and other immature granulocytes (metamyelocytes and myelocytes), as well as reactive segmented neutrophils, tend to be larger and have lower nuclear complexity than their normal "resting" counterparts. The authors proposed that the morphologic changes seen in the left-shifted and reactive segmented neutrophils could be analyzed quantitatively using the Coulter LH 750 hematology analyzer with VCS technology. They evaluated the clinical usefulness of these morphologic parameters as possible indicators of an acute infectious process. The authors studied peripheral blood samples from 69 patients with positive blood cultures for bacteria and 35 control subjects. They observed a significant increase in the mean channel of neutrophil volume (MNV) from septic patients compared with control subjects (156 ± 13.5 versus 143 ± 4.8; P<0.001). The mean channel of neutrophil light scatter was decreased significantly in patients (140 ± 10.1 versus 146 ± 7.3; P=0.002). An elevated MNV was associated with a higher WBC count and percentage of neutrophils and was present even in patients who did not have leukocytosis or neutrophilia. With a cutoff of 150 for the MNV, a specificity of 91 percent and sensitivity of 70 percent were achieved. The authors concluded that the MNV potentially could be an additional indicator for acute bacterial infection.

Chaves F, Tierno B, Xu D. Quantitative determination of neutrophil VCS parameters by the Coulter automated hematology analyzer: new and reliable indicators for acute bacterial infection. Am J Clin Pathol. 2005;124:440-444.

Reprints: Dr. Dongsheng Xu, Dept. of Pathology and Laboratory Medicine, H3600, Boston Medical Center, 88 E. Newton St., Boston, MA 02118

Role of advanced reagents in improving immunohistochemistry PAPP-A as a peripheral vascular disease marker

Pregnancy-associated plasma protein A (PAPP-A) is a high-molecular-weight glycoprotein first identified in the sera of pregnant women. It is also present in ­unstable atherosclerotic coronary plaques. Circulating concentrations are higher in patients with acute coronary syndrome than in patients with chronic stable angina and healthy people. Furthermore, PAPP-A concentrations are associated with the presence and extent of stable coronary heart disease. Increased serum PAPP-A concentrations are predictive of future ischemic cardiac events and the need for percutaneous coronary intervention or coronary artery bypass graft surgery. PAPP-A is a zinc-binding metalloproteinase that ­degrades insulin-like growth factor-binding proteins, thereby allowing unbound/active IGF to bind to cell-surface IGF receptors. Unbound IGF and metalloproteinases are thought to be mediators of atherosclerosis. In contrast to the proposed proatherogenic role of IGF and, subsequently, of the PAPP-A molecule, an increasing body of evidence indicates that IGF protects against ischemic vascular diseases by preventing plaque formation and disruption. Because IGF may be cardioprotective, increased PAPP-A should also offer cardiovascular protection. Athero­scler­o­sis-­related PAPP-A is different from pregnancy-related PAPP-A and is thought to circulate as a homodi­mer­ic active form, uncomplexed with the proform of eosinophil major basic protein (proMBP). The authors conducted a study to test the hypothesis that PAPP-A is associated with atherosclerotic peripheral arterial disease (PAD). The study comprised 433 patients with symptomatic atherosclerotic PAD-that is, chronic limb ischemia-and 433 control subjects matched to the patients with PAD in a 1:1 design by gender, age (±2 years), and diabe­tes mellitus status. The authors measured serum PAPP-A concentrations using an enzymatically amplified two-step sandwich-type immuno­assay. The study sample included 612 male and 254 female patients who were a median age of 68 years. The median PAPP-A value was higher in the patients with PAD than in the controls (0.81 versus 0.64 mU/L; P<0.001). After adjusting for several possible confounding variables with multivariable logistic regression, the odds ratios for PAD were 1.59 (95% confidence interval, 1.00-2.52; P=0.049), 2.28 (1.45-3.61; P<0.001), and 2.86 (1.78-4.59; P<0.001) in the second, third, and fourth quartiles of serum PAPP-A concentrations compared with the first quartile. The authors found that PAPP-A added to the predictive value of other commonly used markers. They concluded that it was associated with atherosclerotic PAD in the sample of elderly patients studied. Because atherosclerotic PAD is considered an indicator of systemic atherosclerotic disease in elderly patients, the results indicate that PAPP-A may be a marker for systemic atherosclerotic disease.

Mueller T, Dieplinger B, Poelz W, et al. Increas­ed pregnancy-associated plasma protein-A as a marker for peripheral atherosclerosis: results from the Linz peripheral arterial disease study. Clin Chem. 2006; 52: 1096- 1103.

Reprints: Thomas Mueller, Dept. of Laboratory Medicine, Konventhospital Barm­her­zige Brueder, Seilerstaette 2-4, A-4020 Linz, Austria; thomas.mueller@bs-lab.at

Role of advanced reagents in improving immunohistochemistry Ability of hemoglobin A1c to predict drug treatment for diabetes mellitus

Hemoglobin A1c has been used as a measure of average glucose concentration and correlates with 24-hour integrated glucose, but it has not generally been included in the diagnostic criteria of diabetes mellitus. Studies have shown that higher HbA1c correlates with metabolic syndrome, of which the unifying pathophysiology is insulin resistance. Because metabolic syndrome is a strong predictor of type 2 diabetes mellitus, HbA1c may be useful in predicting the disease. Therefore, it is important to examine HbA1c as a possible predictor of type 2 diabetes and as the drug treatment for diabetes when left without intensive intervention. To this end, the authors evaluated HbA1c to determine whether it would predict drug treatment for type 2 diabetes mellitus using routine clinical data in a Japanese university hospital. Adolescent and adult patients who were not prescribed anti-diabetic drugs before the study period (three years) were followed to establish the association between the level of HbA1c and future drug treatment for diabetes mellitus. The authors also evaluated a more specific association between the level of HbA1c and initial drug treatment for type 2 diabetes mellitus in a small group of middle-aged patients by examining the medical records of individual patients. The authors studied 38,628 adolescent and adult patients (?15 years old) who use routine medical care services provided by the hospital and who were not prescribed anti-diabetic drugs in the 12 months before the three-year study period. The rates of starting anti-diabetic drugs during the study period were calculated for subgroups divided by the level of HbA1c followed by Kaplan-Meier survival analysis and Cox proportional hazards regression. In addition, the medical rec­ords of middle-aged patients with borderline (5.6% to 6.4%) and high (?6.5%) levels of HbA1c were examined to estimate the rates of initial drug treatment for type 2 ­diabetes mellitus. The rate (95% confidence interval; CI) of beginning anti-diabetic drug therapy was 1.5 (0.9-2.5) per 1,000 patient-years in patients with a normal level of HbA1c (?5.6%), which is simi­lar to 2.1 (1.8-2.5) per 1,000 patient-years in patients with no HbA1c data, but lower than the value of 23 (18.6-28.6) and 161.8 (144.8-180.7) per 1,000 patient-years in those with borderline and high levels of HbA1c, respectively (P<0.001). In a small group of 513 middle-aged patients, the rates of initial drug treatment for type 2 diabetes mellitus were 39.4 (28.1-55.1) per 1,000 patient-years and 270.4 (209.4-349) per 1,000 patient-years in those with borderline and high levels of HbA1c, respectively. The authors concluded that a borderline (5.6% to 6.4%) or high (?6.5%) level of HbA1c was found to strongly predict future drug treatment for diabetes mellitus.

Shimazaki T, Kadowaki T, Ohyama Y, et al. Hemoglobin A1c (HbA1c) predicts future drug treatment for diabetes mellitus: a follow-up study using routine clinical data in a Japanese university hospital. Transl Res. 2007; 149: 196-204.

Reprints: Dr. Kiyoshi Kubota, Dept. of Pharmacoepidemiology, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-8655 Japan; kubotape-tky@umin.ac.jp

Role of advanced reagents in improving immunohistochemistry Preanalytic handling of urines for catecholamine assays

Pheochromocytomas are rare neuroendocrine, catecholamine—producing tumors that arise from the chromaffin cells of the adrenal medulla or from sympathetic ganglia, or paraganglioma. Excessive production of the catecholamines epinephrine (EPI) and norepinephrine (NE) and their metabolites meta­nephrine (MN) and nor­meta­neph­rine (NMN) is considered a hallmark in the biochemical diagnosis of cate­cholamine-producing tumors. Although evidence indicates that plasma concentrations of the free (unconjugated) meta­neph­rines MN and NMN are better indices than other manifestations of catecholamine excess for detecting pheochromocytomas, measurements of fractionated meta­neph­rines and catecholamines in urine are still commonly used to diagnose pheo­chromo­cytoma. Because data are partly contradictory or scarce, the authors studied the stability of urinary (fractionated) meta­neph­rines and catecholamines at room temperature, during and up to one week after the first 24 hours of collection, with and without preservative measures-that is, acidifying to pH 4 or adding Na2EDTA and Na2S2O5 during collection and acidification to pH 4 after collecting 24-hour urine portions. For the study, spot urine samples were collected from eight healthy volunteers. Aliquots were immediately frozen at -20°C or acidified to pH 4 and then frozen directly or after 24 hours at room temperature. The remaining urine was left at room temperature for 24 hours and then split into one portion that was acidified and one portion that was not. Aliquots were frozen or allowed to stand at room temperature for an additional 24, 48, 72, 96, and 168 hours before freezing. The authors also tested the efficacy of adding Na2EDTA and Na2S2O5 as an alternative to acidification for preserving the catecholamines. The authors did not observe clinically relevant degradation (<5%) for the fractionated meta­nephrines under any of the storage conditions. In contrast, in approximately 50 percent of the untreated samples, catecholamines were partially degraded during the first 24 hours at room temperature. However, immediate acidification prevented degradation, whereas acidification after 24 hours prevented further decay. Adding Na2EDTA and Na2S2O5 prevented degradation of catecholamines during the first 24 hours in four of five cases. In the remaining case, degradation did not exceed 10 percent. The authors concluded that preserving samples for measuring urinary fractionated metanephrines is not necessary if samples are assayed or frozen within one week, which is an important advantage if it is necessary to transport samples. In contrast, urinary catecholamines require preservation measures during collection.

Willemsen JJ, Ross HA, Lenders JWM, et al. Stability of urinary fractionated meta­neph­rines and catecholamines during collection, shipment, and storage of samples. Clin Chem. 2007;53:268-272.

Reprints: H. Alec Ross, 479 Dept. of Chemical Endocrinology, Radboud University Nijmegen Medical Center, P.O. Box 9101, 6500 HB Nij­megen, Netherlands; a.ross@ace.umcn.nl

Role of advanced reagents in improving immunohistochemistry Predictive value of heart failure staging

Evidence indicates that heart failure is usually a progressive condition that begins with risk factors for cardiac dysfunction, proceeds to asymptomatic changes in cardiac structure and function, and then evolves into clinically overt heart failure, disability, and death. Recog­nizing the importance of this concept, the American Heart Association and American College of Cardiology released a joint statement proposing a novel model that classifies heart failure into stages. These are stage A, heart failure risk factors; stage B, asympto­matic cardiac structural or functional abnormalities; stage C, symptomatic heart failure; and stage D, end-stage heart failure. This model emphasizes progressive pathophysiology and underscores the importance of early detection and prevention of symptomatic heart failure. The pre­valence of the proposed heart failure stages in the community has not been determined, and the prognostic implications of such a classification are unknown. The authors conducted a three-pronged study to: estimate the prevalence of heart failure stages in a population-based cohort of 2,029 adults aged 45 years or older; provide neurohumor­al validation of the staging model by measuring the association between B-type natriuretic peptide concentration and heart failure stages; and determine the prognostic significance of heart failure stages. They identified a population-based, cross-sectional, random sample of 2,029 Olmsted County, Minn., residents aged 45 years or older. Participants were classified by medical record review, symptom questionnaire, physical examination, and echocardiogram as stage 0, healthy; stage A, heart failure risk factors; stage B, asymptomatic ­cardiac structural or functional abnormalities; stage C, heart failure symptoms; and stage D, severe heart failure. In the cohort, 32 percent of participants were stage 0, 22 percent stage A, 34 percent stage B, 12 percent stage C, and 0.2 percent stage D. Mean B-type natriuretic peptide concentrations (in pg/mL) increased by stages: stage 0 was 26; stage A, 32; stage B, 53; stage C, 137; and stage D, 353. Five-year survival was 99 percent in stage 0, 97 percent in stage A, 96 percent in stage B, 75 percent in stage C, and 20 percent in stage D. The authors concluded that their study provides prevalence estimates and prognostic validation for heart failure staging in a community cohort. Of note, 56 percent of adults 45 years of age or older were classified as stage A (risk factors) or B (asymptomatic ventricular dysfunction). Heart failure staging underscores the magnitude of the population at risk for progression to overt heart failure.

Ammar KA, Jacobsen SJ, Mahoney DW, et al. Prevalence and prognostic significance of heart failure stages: application of the American College of Cardiology/American Heart Association heart failure staging criteria in the community. Circulation. 2007; 115: 1563-1570.

Reprints: Dr. Khawaja Afzal Ammar, Division of Cardiovascular Diseases, Mayo Clinic, 200 First St. SW, Rochester, MN 55905; ammar.khawaja@mayo.edu

Role of advanced reagents in improving immunohistochemistry Relationship of arteries to renal sodium handling

The kidneys play a central role in the pathogenesis of essential hypertension. Blood pressure starts to rise when the kidney requires a higher than usual blood pressure to maintain extracellular fluid volume within normal limits. Measuring the clearance of endogenous lithium provides a way to estimate sodium handling in the nephron. Lithium ions are freely filtered at the glomerulus and reabsorbed in the proximal tubule in parallel with sodium and water. Although lithium may be partially reabsorbed in the loop of Henle, distal tubular handling of lithium is minimal. The fractional excretion of lithium (FELi) and fractional distal reabsorbtion of sodium (RNadist) are noninvasive markers of proximal tubular sodium handling and the proportion of sodium escaping reab­sorption in the proximal tubule that is not eliminated in urine, respectively. The authors investigated the functional and structural properties of three large arteries in relation to renal sodium handling as assessed by the clearance of endogenous lithium. They also measured plasma renin activity and the urinary aldosterone excretion rate. Using ultrasonography, the authors measured diameter, cross-sectional compliance (CC), and distensibility (DC) of the carotid, brachial, and femoral arteries in 1,069 untreated subjects (mean age, 41.6 years; 50.1% women; 18.8% hypertensive subjects). While accounting for covariates and standardizing for the sodium excretion rate in both genders, CC and DC of the femoral artery increased with higher fractional distal sodium reabsorption. Differences associated with a 1-standard deviation (SD) change in fractional distal reabsorption of sodium were 51.7 mm2/kPa 10-3 (95% CI, 23.9-79.5; P=0.0002) and 0.56 10-3/kPa (95% CI, 0.17-0.94; P=0.004) for femoral CC and DC, respectively. In women and men, a 1-SD increment in fractional proximal sodium reabsorption was associated with decreases in femoral and brachial diameter, amounting to 111.6 µm (95% CI, 38.2-185.1; P=0.003) and 52.5 µm (95% CI, 10.0-94.9; P=0.016), respectively. There was no consistent association between the properties of the elastic carotid artery and renal sodium handling. The authors concluded that higher fractional sodium reabsorption in the distal nephron is associated with higher femoral CC and DC, and higher proximal sodium reabsorption is associated with decreased brachial and femoral diameters. These findings demonstrate that renal sodium handling may influence arterial properties or vice versa, or that common mechanisms might influence arterial and renal function.

Seidlerová J, Staessen JA, Maillard M, et al. Association between arterial properties and renal sodium handling in a general population. Hypertension. 2006;48:609-615.

Reprints: Jan A. Staessen, Studies Coordination Centre, Laboratory of Hypertension, Campus Gasthuisberg, Herestraat 49, Box 702 B-3000 Leuven, Belgium; jan.staessen@med.kuleuven.be