A comparison of the analysis of 3 types of body fluids using the XN-350 hematology analyzer versus light microscopy assessment.

ABSTRACT: We evaluated the capacity of the XN-350 instrument to analyze 3 different types of body fluid samples under "body fluid mode."The performance of XN-350 was evaluated in terms of precision, carryover, limit of blank, limit of detection, limit of quantification, and linearity. Cell enumeration and differential data produced by the XN-350 were compared to manual chamber counting results in 63 cerebrospinal fluid (CSF), 51 ascitic fluid, and 51 pleural fluid (PF) samples. Comparisons between XN-350 versus Cytospin data were also performed in PF samples.The precision, carry-over, limit of blank, and linearity of the XN-350 were acceptable. The limits of detection for white blood cells (WBCs) and red blood cells were 1.0/µL, and 1,000.0/µL, respectively; the corresponding limits of quantitation (LOQs) were 5.0/µL and 2,000.0/µL, respectively. The XN-350's cell enumeration and differential counting correlated well with those of manual chamber counting for all 3 sample types (except for differential counting in CSF samples), particularly parameters involving monocytes (r = 0.33) and mononuclear cells (MO- body fluid [BF]; r = 0.26), as well as total cell (TC-BF) enumeration (r = 0.50) and WBC-BF (r = 0.50) in PF samples. The MO-BF in CSF samples differed significantly from manual chamber counting results, but neither TC-BF nor WBC-BF in PF samples did. The XN-350 also showed good correlations with Cytospin analyses for differential counting of neutrophils, lymphocytes, and monocytes in PF samples. The differential counting of eosinophils via the XN-350 and Cytospin were not significantly correlated, but the difference between them was not significant.The XN-350 is an acceptable alternative to manual fluid analysis. Samples with low cellularity around the LOQ should be checked manually. Moreover, manual differential counting should be performed on CSF samples, particularity those with low cell numbers.

Comparative Assessment of Two Strategies for Interpreting Tumor Markers in Ascitic Effusions.

BACKGROUND/AIM: There are two strategies for the interpretation of tumor markers (TM) in fluid effusions: i) high cut-off and ii) fluid/serum ratio (F/S) and low cut-off. The objective of this study is to compare these two strategies and to determine whether diagnostic accuracy improves by the identification of possible false positives using Adenosine deaminase (ADA), C reactive protein (CRP) and % of polymorphonuclear cells (%PN). PATIENTS AND METHODS: We studied 157 ascitic fluids, 74 of which were malignant. ADA, CRP and %PN were determined in ascitic fluid, and Carcinoembryonic antigen (CEA), Cancer antigen 72-4 (CA72-4), Cancer antigen CA19-9 and Cancer antigen 15-3 (CA15-3) in both fluid and serum. RESULTS: The strategy of high cut-off showed 59.5% sensitivity at 100% specificity. The F/S strategy showed 75.7% sensitivity at 95.2% specificity. Subclassifying cases with ADA, CRP and %PN negative showed 67.5% sensitivity at 100% specificity for high cut-off and for the F/S strategy was 81.7% sensitivity at 98.7% specificity. CONCLUSION: The strategy of F/S with negative ADA, CRP and %PN allow the best interpretation for TM in the ascitic fluid.

Utility of gray-scale histogram analysis in the assessment of treatment response in patients with infected cirrhotic ascites.

OBJECTIVE: To evaluate the utility of B-mode gray-scale histogram analysis in the management of patients with infected cirrhotic ascites. METHODS: A total of 97 patients (mean(SD) age : 66.8(14.2) years, 50.5% were males) diagnosed with cirrhotic ascites were included in this non-interventional study. Paracentesis for ascitic fluid analysis [culture tests, white blood cell count, albumin and protein levels, serum ascites albumin gradient (SAAG)] and gray-scale histogram analysis for ascites/subcutaneous echogenicity ratio (ASER) were performed at baseline in each patient and on Day 2 and Day 5 of treatment in patients with infected ascites. Receiver operating characteristics (ROC) curve was plotted to determine performance of ASER in identification of antibiotic resistance with calculation of area under curve (AUC) and ideal cut-off value of % change in ASER to detect antibiotic resistance. RESULTS: Treatment was associated with a significant decrease in median (min-max) ASER [from 0.005(0.0002-0.02) at baseline to 0.003(0.0001-0.01) on Day 2 and 0.0005(0.0001-0.009) on Day 5] and ascitic fluid polymorphonuclear leukocyte (PMNL) count [from 600(300-2200) at baseline to 350(50-1250) on Day 2 and 100(50-1100) on Day 5] (p<0.001 for each). ROC analysis revealed that less than 38% reduction in ASER [AUC: 0.923, 95% CI (0.797-0.982), p<0.001] was a potential marker of antibiotic resistance with a sensitivity of 90.9% and a specificity of 95.0%. CONCLUSIONS: In conclusion, our findings emphasize potential utility of gray-scale histogram based quantitative analysis of ascitic fluid echogenicity as an adjunct non-invasive method in the assessment of treatment response and early recognition of treatment failure in patients with infected ascites.

Population Pharmacokinetics and Target Attainment of Meropenem in Plasma and Tissue of Morbidly Obese Patients after Laparoscopic Intraperitoneal Surgery.

Meropenem serves as a clinically important, broad-spectrum antibiotic. While meropenem is commonly used in obese patients, its pharmacokinetics in this patient group is not well known. Our aim was to characterize the population pharmacokinetics and target attainment in plasma, subcutaneous tissue, and peritoneal fluid for meropenem in morbidly obese patients. Four doses of 1g meropenem were given as 15-min infusions every 8 h to five morbidly obese patients (body mass index [BMI], 47.6 to 62.3 kg/m(2)). After the fourth dose, serial meropenem concentrations were determined in plasma and, via microdialysis, in subcutaneous tissue and peritoneal fluid. All concentrations were analyzed simultaneously via population modeling, and target attainment probabilities predicted via Monte Carlo simulations using the target of unbound meropenem concentrations above the MIC for at least 40% of the dosing interval. For patients with 53 kg fat-free mass, total clearance was 18.7 liters/h and volume of distribution at steady state was 27.6 liters. The concentrations in subcutaneous tissue and peritoneal fluid largely paralleled those in plasma (equilibration half-life, <30 min). The area under the curve (AUC) in subcutaneous tissue divided by the plasma AUC had a mean of 0.721. For peritoneal fluid, this AUC ratio had a mean of 0.943. Target attainment probabilities were >90% after 1 g meropenem every 8 h as a 15-min infusion for MICs of up to 2 mg/liter in plasma and peritoneal fluid and 0.5 mg/liter in subcutaneous tissue. Meropenem pharmacokinetics in plasma and peritoneal fluid of obese patients was predictable, but subcutaneous tissue penetration varied greatly. (This study has been registered at ClinicalTrials.gov under registration no. NCT01407965.).

A potential novel strategy, inhibition of vasopressin-induced VEGF secretion by relcovaptan, for decreasing the incidence of ovarian hyperstimulation syndrome in the hyperstimulated rat model.

OBJECTIVE: To investigate the effects of V1A receptor antagonist through inhibition of vasopressin-induced VEGF secretion in an experimental model. STUDY DESIGN: Thirty rats were randomly divided into five groups. Four groups were given 10IU pregnant mare serum gonadotropin/day (sc) at 8:00-8:30am on days 22-25 of life. They were administered 30IU hCG at 8:00-8:30am on day 26 of life. On days 26 and 27 of life at 8:00am and 4:00pm, (ip) per animal, 50µg/kg/day GnRH antagonist in the GnRH antagonist group, 0.3mg relcovaptan in the high dose relcovaptan group, and 0.15mg relcovaptan in the low dose relcovaptan group were administered. The control group was given the same dosage of 0.9% saline solution (ip) on days 22-26 day of life. The main outcomes were weight gain, ovarian weights, peritoneal fluid VEGF values, corpus luteum count, and atretic follicle count. RESULTS: Weight gain was highest in the OHSS group; it was almost twice as much in the OHSS group than it was in the control group. Ovarian weights were significantly lower in all treatment groups (p=0.03). There was no statistically significant difference in ovarian weights between the GnRH antagonist and relcovaptan groups (p=0.176). The evaluation of peritoneal fluid VEGF-A levels revealed statistically significant differences between levels in the treatment groups and in the OHSS group (p=0.005). Atretic follicle count in the OHSS group was significantly lower (p=0.048). In all treatment groups, CL counts were prominently lower than they were in the OHSS group (p=0.002). CONCLUSION: Relcovaptan may be a novel strategy for decreasing risk of OHSS by inhibition of vasopressin-induced VEGF secretion through V1A receptor antagonist.

Biochemical assessment of canine body cavity effusions using three bench-top analysers.

OBJECTIVES: To assess the performance of three bench-top chemistry instruments for the analysis of canine effusions. Acceptable results were compared with those obtained by a reference chemistry analyser. METHODS: Total protein, albumin, creatinine and bilirubin concentrations were measured in 74 effusions using the VetScanVS2, VetTest8008 and SpotchemEZ analysers. Cholesterol and triglyceride concentrations were also measured by the VetTest and Spotchem. Results were analysed using Westgard's error analysis, Spearman's correlation, Bland-Altman plots and Deming regression. Results were considered acceptable when observed total error (TE(obs) ) was less than allowable total error (TE(A) ). RESULTS: VetScan error analysis revealed acceptable results for total protein (TE(obs) =1.11, TE(A) =4.7) and creatinine (TE(obs) =42.2, TE(A) =78.1). Correlation was fair for protein (r(s) =0.66) and creatinine (r(s) =0.76), but poor and not significant for bilirubin (r(s) =0.01, P=0.08), precluding error analysis. VetTest error analysis was acceptable for creatinine only (TE(obs) =5.55, TE(A) =25.5). Correlation was good (r(s) =0.81). The difference plot revealed a bias (95% confidence interval) of -1.5 (-37 to 40) and four outliers. The Spotchem did not generate a precise arithmetic value in most (56.9 to 73.6%) samples, precluding further analysis. CLINICAL SIGNIFICANCE: Acceptable results were obtained for total protein (VetScan) and creatinine [Vetscan, Vettest (with good correlation)]. The Spotchem is of limited value in canine effusion analysis.

Assessment of HB-EGF levels in peritoneal fluid and serum of ovarian cancer patients using ELISA.

BACKGROUND: Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a rational target for ovarian cancer therapy. The aim of this study was to examine HB-EGF levels in the peritoneal fluid and serum of ovarian cancer (OVCA) patients. PATIENTS AND METHODS: Samples were collected from six healthy women, 21 OVCA patients, and 21 ovarian cyst patients. HB-EGF levels were measured using a sandwich ELISA kit and calculated using a parallel line assay. RESULTS: No significant difference between the slopes of the standard and sample curves was observed at an anti-HB-EGF antibody concentration of 1.6 µg/ml. HB-EGF levels in the peritoneal fluid and serum of OVCA patients were significantly higher than those in patients with ovarian cysts or controls. Serum HB-EGF levels were also significantly correlated with levels in peritoneal fluid in OVCA patients. CONCLUSION: We developed an assay for the exact measurement of HB-EGF levels in peritoneal fluid and serum.

Peritoneal vascular density assessment using narrow-band imaging and vascular analysis software, and cytokine analysis in women with and without endometriosis.

The development and onset of endometriosis is associated with angiogenesis and angiogenic factors including cytokines. We analyzed intrapelvic conditions in women with endometriosis via vascular density assessment of grossly normal peritoneum and determination of cytokine levels in peritoneal fluid. Seventy-three patients underwent laparoscopic surgery because of gynecologic disease including endometriosis in our department using a narrow-band imaging system. Each patient was analyzed for peritoneal vascular density using commercially available vascular analysis software (SolemioENDO ProStudy; Olympus Corp, Tokyo, Japan). Each patient was also subjected to analysis of interleukin 6 (IL-6), IL-8, tumor necrosis factor-alpha, and vascular endothelial growth factor concentrations in peritoneal fluid. We defined 4 groups as follows: group 1, endometriosis: gonadotropin-releasing hormone (GnRH) agonist administration group (n=27); group 2, endometriosis: GnRH agonist nonadministration group (n=15); group 3, no endometriosis: GnRH agonist administration group (n=18); and group 4, no endometriosis: GnRH agonist nonadministration group (n=13). No significant differences in peritoneal vascular density between the 4 groups were found under conventional light; however, under narrow-band light, vascular density in the endometriosis groups (groups 1 and 2) was significantly higher. Cytokine analysis of the 4 groups determined that IL-6 and IL-8 concentrations were significantly higher compared with the no endometriosis groups (groups 3 and 4). Tumor necrosis factor-alpha and vascular endothelial growth factor concentrations were not significantly different between groups. In endometriosis, peritoneal vascular density was significantly higher as assessed using the narrow-band imaging system and SolemioENDO ProStudy, whereas GnRH agonist did not obviously decrease vascular density but IL-6 concentration was lower in the GnRH agonist administration group.

Evaluation of an abdominal fluid scoring system determined using abdominal focused assessment with sonography for trauma in 101 dogs with motor vehicle trauma.

OBJECTIVE: Evaluate an abdominal fluid scoring (AFS) system using an abdominal focused assessment with sonography for trauma (AFAST) protocol. DESIGN: Prospective study. SETTING: Private veterinary emergency center. ANIMALS: One hundred and one client-owned dogs with motor vehicle trauma. Interventions- AFAST performed on admission and 4 hours post-admission. MEASUREMENTS AND MAIN RESULTS: An AFS was assigned to each dog based on the number of AFAST fluid-positive quadrants identified using a 4-point scale: AFS 0 (negative for fluid in all quadrants) to AFS 4 (positive for fluid in all quadrants). Free abdominal fluid was identified in 27 of 101 dogs (27%). Dogs with AFS scores of 3 or 4 (14/27 [52%] AFS-positive dogs) experienced more marked decreases in packed cell volume and total plasma protein, increases in alanine aminotransferase, and needed more blood transfusions than dogs with lower AFS scores and AFS-negative dogs. Serial AFAST was performed in 71% of dogs (71/101); 17% (12/71) of these cases changed AFS score, and 75% (9/12) of the changes were higher (worsened) AFS, correlating with increasing amounts of free abdominal fluid. Ninety-eight percent of the study population was a primary presentation. Overall, median time from trauma to initial AFAST was 60 minutes, and median AFAST examination time was 3 minutes. CONCLUSIONS: Initial and serial AFAST with applied AFS allowed rapid, semiquantitative measure of free abdominal fluid in traumatized patients, was clinically associated with severity of injury, and reliably guided clinical management. Where possible, AFAST and AFS should be applied to the management of blunt trauma cases.

Development of breakpoints of cephems for intraabdominal infections based on pharmacokinetics and pharmacodynamics in the peritoneal fluid of patients.

We developed breakpoints for cephem antibacterial agents for intraabdominal infections based on pharmacokinetics (PK) and pharmacodynamics (PD) at the target site. Cefepime (CFPM), cefotiam (CTM), cefozopran (CZOP), and flomoxef (FMOX) were each administered to 8-10 patients before abdominal surgery, and venous blood and peritoneal fluid (PF) samples were obtained. The drug concentrations in plasma and PF were determined and analyzed using population pharmacokinetic modeling. Using the pharmacokinetic model parameters, a Monte Carlo simulation was conducted to estimate the probabilities of attaining the bacteriostatic and bactericidal targets (40% and 70% of the time above the minimum inhibitory concentration (T > MIC), respectively) in PF. The bacteriostatic and bactericidal breakpoints were determined as the highest MIC values at which the bacteriostatic and bactericidal probabilities in PF were > or =80%, which values varied with drug and dosing regimen. Site-specific PK-PD-based breakpoints for CFPM, CTM, CZOP, and FMOX are proposed, and should help us to select appropriate cephems and design their dosing regimens for intraabdominal infections.

Peritoneal penetration and pharmacodynamic exposure of intravenous cefozopran in abdominal surgery patients.

This study aimed to examine the peritoneal penetration and pharmacodynamic exposure of intravenous cefozopran. Cefozopran 1 g was administered to 10 patients before abdominal surgery. Venous blood and peritoneal fluid (PF) samples were obtained at the end of the 0.5-h infusion and at 1, 2, 3, 4, 5 and 6 h thereafter. Drug concentrations in plasma and PF were determined, analysed pharmacokinetically and used for a Monte Carlo simulation with minimum inhibitory concentration (MIC) data. Cefozopran penetrated well into PF, with a mean (+/-standard deviation) maximum drug concentration in PF/plasma ratio of 0.65+/-0.17 (n=10) and area under the concentration-time curve ratio of 0.92+/-0.13. The probabilities of attaining the pharmacodynamic exposure target (PTA), defined as 70% of the time above the MIC, in PF were almost identical to those in plasma. The PTAs were 95-100% against Escherichia coli, Enterobacter spp. and Staphylococcus aureus with 0.5 g every 12 h; however, 1 g every 8 h or 1 g every 6 h was required for 93-98% PTA against Pseudomonas aeruginosa. These results should help us to understand the peritoneal pharmacokinetics of cefozopran whilst also helping to choose the appropriate dosage for surgical intra-abdominal infections.

Spectrophotometric assessment of peritoneal fluid haemoglobin in colic horses: an aid to selecting medical vs. surgical treatment.

In a case-control study in colic horses the ability of spectrophotometric measurement of the haemoglobin concentration in the peritoneal fluid supernatant and visual assessment of the colour of peritoneal fluid supernatant to differentiate between surgical and medical treatment of colic was assessed. Based on previous studies, which have found anda association between peritoneal fluid colour and the kind of treatment required, our hypothesis was that the peritoneal fluid haemoglobin concentration would be higher in horses requiring surgical intervention than in horses amenable to medical treatment. Seventy-four horses admitted to a teaching hospital were included. Thirty-five horses were classified as requiring surgery and 39 medical treatment. Logistic regression revealed a significant (P < 0.0001) association between the haemoglobin concentration measured with the spectrophotometer and the need for surgical treatment. Odds ratio for an increase in haemoglobin concentration of 0.01 mmol/l was 6.4, which means that the odds for 'need for surgical treatment' increased when peritoneal fluid haemoglobin concentration increased. When used as a diagnostic test with a threshold of 0.01 mmol/l haemoglobin for selecting surgical vs. medical treatment, sensitivity was 80% and specificity 82%, whereas simple visual assessment had a sensitivity of only 51% and a specificity of 95%.

Antiviral activity of mice with chronic renal failure--an assessment using peritoneal effluents.

Antiviral activity (AVA) determined by the inhibition of the cytopathic effect (CPE) of vesicular stomatitis virus (VSV) on mice fibroblasts, was measured in the peritoneal effluent of mice. Four groups of animals (each group numbering 30 mice) were studied. Group 1 consisted of sham operated mice and served as the control group. Group 2 underwent implantation of silastic matter (of which the Tenckhoff catheter is made). In group 3, chronic renal failure was induced. Group 4 comprised those mice in which both chronic renal failure was induced and silastic matter implanted. Optical density readings, directly related to the inhibition of CPE were 0.66 +/- 0.01, 0.59 +/- 0.08, 0.85 +/- 0.06 and 0.86 +/- 0.13 for groups 1, 2, 3 and 4, respectively (p < 0.01 for groups 1 and 2 versus 3 and 4). Virus control readings indicative of the CPE of VSV without the presence of peritoneal effluent were 0.58 +/- 0.07, not significantly different from those obtained in groups 1 and 2. They were, however, significantly below values from groups 3 and 4 (p < 0.05). These data show that AVA is undetectable in the peritoneal effluent of normal mice. Chronic renal failure produces an enhancement of AVA. Silastic matter (Tenckhoff catheter) implantation does not play a role in the production of AVA.

Use of monoclonal antibody for assessment of estrogen and progesterone receptors in malignant effusions.

An immunocytochemical assay utilizing specific monoclonal antibodies against estrogen receptor (ER) and progesterone receptor (PgR) has been shown to be highly reliable for the detection of hormone receptors in hormone sensitive tumors. To assess the usefulness of this technique in malignant effusions, CytospinR (Shandon, Inc., Pittsburgh, PA 15275), preparations of 41 pleural and ascitic fluid were studied. The findings from the malignant cells employing estrogen and progesterone receptor immunocytochemical assay were compared with the results obtained from primary tumors by biochemical (dextran-coated charcoal) assay. The results agreed in 88% for ER and 83% for PgR. This study supports the potential value of cytochemical technique in detection of hormone receptors in malignant effusions. Assessment of hormone receptors in malignant effusions may be of clinical significance, particularly in situations where the hormone receptor status of the original tumor is not known. This information may also have some diagnostic and therapeutic importance in assessment of patients presenting with metastatic tumors of unknown origin.

[Evaluation of the usefulness of the physico-chemical study of ascitic fluid in patients with chronic hepatopathies, neoplasms and spontaneous peritonitis]. / Valoración de la utilidad del estudio fisicoquímico del líquido, ascítico en pacientes con hepatopatías crónicas, neoplasias y peritonitis espontánea.

In order to separate malignancy from chronic liver disease, the ascitic fluid (AF) of 45 in-patients was studied prospectively. Protein, cholesterol, triglycerides, LDH and glucose concentrations were determined in AF. Ascites/Serum (A/S) protein and LDH ratios were also established. Spontaneous bacterial peritonitis (SBP) incidence was studied in the cirrhotic group performing the pH, PMN count and culture of the AF. The 45 patients were classified in three groups: 29 with chronic liver disease, 10 with malignancy and 6 with miscellaneous pathology. Af protein concentration, its A/S ratio and AF cholesterol concentration were statistically significant (p less than 0.001) to differentiate malignancy from chronic liver disease. However, the AF glucose, triglycerides and LDH concentrations and the A/S LSH ratio were not useful in the differential diagnosis. The SBP incidence was 13%, its mortality rate 75% and the cultures were positive only in 25%.