Comparative effects of cocaine and cocaethylene on alveolar epithelial type II cells.

Abuse of cocaine (COC) and alcohol have been among the leading causes of non-prescription drug-related deaths in the USA and are known to cause acute and chronic lung diseases. The co-abuse of COC and alcohol results in the production of an active metabolite, cocaethylene (CE). The effects of COC and its metabolites on the respiratory system have been scarcely studied. This study was aimed at comparing the toxic effects of eqimolar concentration (1 mM) of COC and CE on alveolar epithelial type II cells. This was performed by measuring cell growth, viability, clonogenic activity, cell cycle and reactive oxygen species (ROS) generation. The treatment of CE and COC resulted in a significant inhibition of cell proliferation with the formation of an average of three colonies which measured about 1.74×10(-15) m each and 25 colonies each of about 5.73×10(-15) m, respectively, while untreated cells yielded 31 colonies of 8.75×10(-15) m (p<0.05). The treatments of CE and COC resulted in the reduction of the growth fraction of alveolar epithelial type II cells without significant decrease in viability. In addition, there was an approximately twofold increase in ROS generation as compared to the controls (p<0.05). Therefore, CE-induced inhibition of cellular proliferation may contribute to the pathogenesis of diffuse alveolar damage in co-abusers of COC and alcohol.

Evaluation of INK4A promoter methylation using pyrosequencing and circulating cell-free DNA from patients with hepatocellular carcinoma.

BACKGROUND: Hyper-methylation of CpG dinucleotides in the promoter region of inhibitor of cyclin-dependent kinase 4A (INK4A) has been reported in 60%-80% of hepatocellular carcinoma (HCC). As INK4A promoter hypermethylation event occurs early in HCC progression, the quantification of INK4A promoter methylation in blood sample may represent a useful biomarker for non-invasive diagnosis and prediction of response to therapy. METHODS: We examined INK4A promoter methylation using circulating cell-free DNA (ccfDNA) in a total of 109 serum specimens, including 66 HCC and 43 benign chronic liver diseases. Methylation of the individual seven CpG sites was examined using pyrosequencing. RESULTS: Our results showed that there were significantly higher levels of methylated INK4A in HCC specimens than controls and that the seven CpG sites had different levels of methylation and might exist in different PCR amplicons. The area under receiver operating characteristic (ROC) curve was 0.82, with 65.3% sensitivity and 87.2% specificity at 5% (LOD), 39.0% sensitivity and 96.5% specificity at 7% LOD, and 20.3% sensitivity and 98.8% specificity at 10% LOD, respectively. CONCLUSIONS: Our results support additional studies incorporating INK4A methylation testing of ccfDNA to further validate the diagnostic, predictive, and prognostic characteristics of this biomarker in HCC patients. The knowledge of the existence of epi-alleles should help improve assay design to maximize detection.

Combined bisalbuminemia and Bisalbuminuria: A rare finding on serum and urine electrophoresis.

BACKGROUND: Bisalbuminemia and bisalbuminuria are rarely encountered serum and urine albumin anomalies characterized by the presence of a bifid albumin band on serum/urine protein electrophoresis (SPE/UPE) and serum/urine immunofixation electrophoresis (SIFE/UIFE). They are usually detected incidentally while screening for monoclonal gammopathy with a cumulative frequency of 1:1,000---1:10,000. CASE REPORT: We report two cases of bisalbuminemia in two adult male diabetic patients. The first patient had a history of rheumatoid arthritis and strong clinical suspicion for Sjogren syndrome. The SPEP/UPEP and SIFE/UIFE in this patient showed combined bisalbuminemia and bisalbuminuria. While the second patient had chronic kidney disease due to nephrotic syndrome but showed bisalbuminemia alone. CONCLUSION: Bisalbuminemia and bisalbuminuria are rare findings with few case reports available in the English literature. These findings may occur secondary to inherited albumin variants or may be acquired. Diabetes mellitus is the medical condition most associated with acquired bisalbuminemia and bisalbuminuria. Although most cases of bisalbuminemia and bisalbuminuria are clinically insignificant, some albumin variants may have altered affinity for steroid hormones (e.g., thyroxine) and/or drugs which potentially could be clinically significant.

Central Line Access Is Predictive of Diagnostic Blood Loss and Transfusion in the Surgical Intensive Care Unit.

BACKGROUND: Most patients in the surgical intensive care unit (SICU) have anemia and undergo extensive diagnostic laboratory testing (DLT). Consequently, patients undergo RBC transfusion, and many are discharged with anemia, both of which are associated with poorer outcomes. OBJECTIVE: To characterize DLT blood loss in the SICU. MATERIALS AND METHODS: We performed a 1-year retrospective study of 291 patients admitted to a SICU. The number of draws, average volume, and estimated discard volume were recorded, along with clinical and laboratory findings. RESULTS: Patients who underwent greater amounts of DLT had lower hemoglobin levels at discharge (P ≤ .001). Admissions requiring central venous catheter (CVC) access (49.8%) demonstrated significantly higher DLT draws and rates of transfusion. CONCLUSION: Findings from this study suggest that DLT blood loss contributes to anemia in the SICU, and that the presence and duration of CVC leads to increased testing, anemia, and RBC transfusion.

Cannabis positivity rates in 17 emergency departments across the United States with varying degrees of marijuana legalization.

BACKGROUND: Many states in the United States have progressed towards legalization of marijuana including decriminalization, medicinal and/or recreational use. We studied the impact of legalization on cannabis-related emergency department visits in states with varying degrees of legalization. METHODS: Seventeen healthcare institutions in fifteen states (California, Colorado, Connecticut, Florida, Iowa, Kentucky, Maryland, Massachusetts, Missouri, New Hampshire, Oregon, South Carolina, Tennessee, Texas, Washington) participated. Cannabinoid immunoassay results and cannabis-related International Classification of Diseases (ninth and tenth versions) codes were obtained for emergency department visits over a 3- to 8-year period during various stages of legalization: no state laws, decriminalized, medical approval before dispensaries, medical dispensaries available, recreational approval before dispensaries and recreational dispensaries available. Trends and monthly rates of cannabinoid immunoassay and cannabis-related International Classification of Diseases code positivity were determined during these legalization periods. RESULTS: For most states, there was a significant increase in both cannabinoid immunoassay and International Classification of Diseases code positivity as legalization progressed; however, positivity rates differed. The availability of dispensaries may impact positivity in states with medical and/or recreational approval. In most states with no laws, there was a significant but smaller increase in cannabinoid immunoassay positivity rates. CONCLUSIONS: States may experience an increase in cannabis-related emergency department visits with progression toward marijuana legalization. The differences between states, including those in which no impact was seen, are likely multifactorial and include cultural norms, attitudes of local law enforcement, differing patient populations, legalization in surrounding states, availability of dispensaries, various ordering protocols in the emergency department, and the prevalence of non-regulated cannabis products.

Methods matter - Tailoring SARS-CoV-2 antibody targets to vaccination status.

Individuals who have been vaccinated for COVID19 should have IgG antibody in response to the specific antigen that is the target in the vaccine development. There are several options for targeted COVID19 antigen, but most manufacturers have focused on the spike protein. Using our understanding of the targeted antigen for vaccine development, we can develop testing algorithmic scheme for anti-spike and anti-nucleocapsid antibody assays to aid delineation of infection versus vaccination in our patient population. Clear communication from laboratories specifying the specific SARS-CoV-2 antibodies (i.e., anti-spike, anti-nucleocapsid, or both) in their antibody tests at both the ordering and reporting levels will play crucial role in the development of this approach and is essential to avoid potential provider/patient confusion in the interpretation of serologic testing.

Urinary NT-proBNP as a potential noninvasive biomarker for screening of pulmonary hypertension in preterm infants: a pilot study.

OBJECTIVE: This pilot study aimed to determine the feasibility of urinary NT-proBNP (NT-proBNP) as a potential noninvasive screening marker for pulmonary hypertension (PH). STUDY DESIGN: A prospective cross-sectional study was conducted. Preterm infants (PI) (birthweight <1500 gm and <30 weeks gestational age (GA)) were enrolled. Serial urinary NT-proBNP measurements and echocardiograms (ECHO) were performed at 28, 32, and 36 weeks. RESULTS: Thirty-six patients were included in the final analysis (BPD-PH group = 6, BPD group = 20, control = 10). Urinary NT-proBNP levels were higher in the BPD-PH group compared with BPD and control groups at all study intervals. A urine NT-proBNP cutoff level of 2345 pg/ml at 28 weeks of GA had a sensitivity and specificity of 83.3% and 84.2%, respectively, for detection of BPD-PH (AUC 0.816, p = 0.022). CONCLUSION: Urinary NT-proBNP measurement is feasible in preterm infants and appears to be a good noninvasive screening tool for PH.

Comparison of POCT and central laboratory blood glucose results using arterial, capillary, and venous samples from MICU patients on a tight glycemic protocol.

BACKGROUND: Point of care (POC) glucose meters are routinely used to monitor glucose levels for patients on tight glycemic control therapy. We determined if glucose values were different for a POC glucose meter as compared to the main clinical laboratory for medical intensive care unit patients on a tight glycemic protocol and whether the site of blood sampling had a significant impact on glucose values. METHODS: Eighty-four patients (114 paired samples) who were on a tight glycemic protocol in the period November 2005 through August 2006 were enrolled. After simultaneous blood draws, we compared the glucose levels for the glucose meter (arterial/venous/capillary), blood gas (arterial/venous), and central clinical laboratory (serum/plasma from arterial/venous samples). RESULTS: The mean glucose levels of all arterial/venous/fingerstick samples using the glucose meter demonstrated a positive bias of 0.7-0.9 mmol/l (12.6-16.2 mg/dl) (p<0.001) relative to central laboratory venous plasma. There was also a smaller positive (0.1-0.3 mmol/l or 1.8-5.4 mg/dl, p<0.05) bias for arterial/venous blood gas samples and laboratory arterial serum/plasma glucose samples. Using Parkes error grid analysis we were able to show that the bias for arterial or venous POC glucose results would have not impacted clinical care. This was not the case, however, for fingerstick sampling where a high bias could have significantly impacted clinical care. Additionally, in 3 fingerstick samples a severe underestimation (<46% of the central laboratory plasma result) was found. CONCLUSION: Glucose meters using arterial/venous whole blood may be utilized in the MICU; however, due to the increased variability of results we do not recommend the routine use of capillary blood sampling for monitoring glucose levels in the MICU setting.

Critically low sodium levels due to concentration gradients formed in patient samples after undergoing a freeze-thaw cycle.

BACKGROUND: Concentrations gradients that form in plasma as a result of freezing and thawing is a well-known phenomenon. As the water fraction converts into ice, plasma constituents diffuse from the freezing front by natural convection in the liquid phase. This process can lead to erroneous lab results, if the sample is not thoroughly mixed prior to testing. METHODS: A series of patient samples received at the clinical chemistry core lab were found to have low sodium levels that normalized after tube inversion. We suspected that the samples may have frozen during shipping and therefore examined the effects of freezing and thawing on serum. RESULTS: Our investigation revealed that prior to arriving at the core lab, samples from one of our satellite clinics were undergoing a freeze-thaw cycle during shipping, which resulted in the formation of concentration gradients and spurious lab results on arrival. CONCLUSIONS: Large hospitals that have a central core lab and receive patient samples from satellite clinics need to be aware of this phenomena, which can contribute to erroneous lab results being posted in a patient's electronic medical record, resulting in a misdiagnosis.

NTproBNP as a surrogate biomarker for early screening of pulmonary hypertension in preterm infants with bronchopulmonary dysplasia.

OBJECTIVE: Pulmonary hypertension (PH) is a known complication of bronchopulmonary dysplasia (BPD). This study aimed to determine the utility of serial N-Terminal pro-Brain Natriuretic Peptide (NTproBNP) levels in the screening of BPD associated PH (BPD-PH) in preterm infants. STUDY DESIGN: Infants with birth weight <1500 g and <30 week corrected gestational age (CGA) were followed with serial NTproBNP levels and echocardiograms (ECHO). They were divided into control, BPD and BPD-PH groups. Statistical analyses included repeated measures analysis of variance and receiver operator curve (ROC) generation. RESULTS: Infants in the BPD-PH and BPD group had significantly elevated NTproBNP levels as compared to the control group. ROC curves for NTproBNP at 28 weeks CGA provided a cut-point of 2329 pg/ml and 578.1 pg/ml for detection of BPD-PH and BPD, respectively. CONCLUSIONS: NTproBNP appears to be a good screening tool to determine the onset of BPD-PH as early as 28 weeks CGA.

Electrolytes and pH changes in pre-eclamptic rats.

BACKGROUND: Intracellular free calcium [Ca2+]i and magnesium [Mg2+]i ions play major roles in the mechanism of vascular smooth muscle (VSM) contraction. Although essential hypertension and abnormal intracellular homeostasis of these ions have long been recognized as major icons in the pathogenesis of pre-eclampsia, the underlying mechanism(s) remain poorly understood. METHODS: Alterations of vascular smooth muscle and platelet intracellular cations [Ca2+]i, [Mg2+]i and [H+]i relative to plasma concentrations of these ions in nitric oxide synthase (NOS) blockade-induced models of pre-eclampsia have been evaluated in the present study. RESULTS: Pregnant rats injected with the NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) developed a significantly elevated arterial blood pressure, proteinuria and other clinical parameters characteristic of pre-eclampsia compared to age-matched pregnant and non-pregnant rat controls that received the L-NAME vehicle only. Plasma total calcium concentration was significantly lower in pre-eclamptic rat models compared to normal pregnant rats (10.29+/-0.08 vs 10.67+/-0.18 mg/dl, p<0.05). A significant increase in plasma calcium was observed in pregnant controls compared to non-pregnant rats (10.67+/-0.18 vs 10.14+/-0.09 mg/dl, p<0.01). Plasma Ca2+ levels in pre-eclamptic rats were consistently lower than those of pregnant controls (5.69+/-0.09 vs 5.98+/-0.06 mg/dl, p<0.05). Resting levels of [Ca2+]i was significantly higher in pre-eclamptic rats than in pregnant controls. (351+/-45.2 vs 196+/-23.2 nmol/l, p<0.01). Blood pH was significantly increased in pre-eclamptic rats as compared to pregnant controls (7.16+/-0.02 vs 7.05+/-0.03, p<0.05). There was no significant difference in plasma and intracellular magnesium concentrations between the three rat groups. CONCLUSIONS: These findings suggest that a significantly decreased plasma level of Ca2+ coupled with a concomitant increase in VSM [Ca2+]i concentrations and an altered blood pH are associated with pre-eclampsia in the pregnant rat. Routine monitoring of serum pH, Ca2+ and Mg2+ especially in the late third trimester, may have potential in the early detection of patients at risk for pre-eclampsia, and monitoring the progress of diverse therapeutic regimens during clinical management.

Skin Rash and Microscopic Hematuria in a 10-Year-Old Caucasian Male.

OBJECTIVE: Henoch-Schonlein purpura (HSP) is an acute, systemic, vasculitis with IgA-dominant immune deposits. With the emphasis on educational value of HSP, which is the most common form of vasculitis in children, we report an actual case from a 10-year-old boy. METHOD: The patient presented with the chief complaint of a skin rash. His illness history, family medical history, physical examination, and relevant laboratory findings were summarized, followed by a question and possible answer format discussion. RESULTS AND CONCLUSION: With the significant elevation of red blood cells in his urine and moderate to severe deposition of IgA in kidney biopsy, the patient was diagnosed with HSP nephritis. Renal symptoms, such as proteinuria and hematuria, are mostly the last to develop and determine the long-term prognosis in HSP patients. The patient is currently undergoing steroid treatment, which is the primary intervention for HSP as it spontaneously resolves in most of affected children.

Evidence for injurious effect of cocaethylene in human microvascular endothelial cells.

BACKGROUND: Cocaethylene (CE) is a conjugate of cocaine and ethanol that may contribute to the pathogenesis of systemic vascular diseases. This study was conducted to investigate the effect of CE on human microvascular endothelial cells (HMEC-1) in culture. METHODS: Proliferating and confluent monolayers of HMEC-1 were used for assessing growth kinetics, viability, cytotoxicity, and morphologic/barrier alterations after CE treatment (0-1 mmol/l) for up to 7 days. The Trypan blue exclusion, lactate dehydrogenase (LDH) release assay, manual cell counts, and silver nitrate staining technique were used. RESULTS: The doubling times of 30.0 and 31.4 h for the 0.5 and 1.0 mmol/l CE-treated HMEC-1, respectively, were significantly longer than the 28.6 h for the control group (p < 0.05). The viabilities of 90.4 +/- 3.8% (control) and 93.1 +/- 1.9% (CE-treated) from the Trypan blue exclusion-staining experiments indicated non-lethality of CE. LDH activities of 173 +/- 33 U/l (control) and 157 +/- 43 U/l (CE-treated) confirmed the absence of CE cytotoxicity. Silver staining results indicated increased monolayer permeability as demonstrated by the formation of intercellular gaps after 1 h of exposure. CONCLUSIONS: HMEC-1 exposure to CE induced cellular injury that could affect the permeability of small blood vessels. These cellular changes could in part be the pivotal point for studies to explain the edema and inflammation in surrounding tissues of individuals exposed to CE.

Capillary electrophoresis and its application in the clinical laboratory.

Over the past 10 years, capillary electrophoresis (CE) is an analytical tool that has shown great promise in replacing many conventional clinical laboratory methods, especially electrophoresis and high performance liquid chromatography (HPLC). The main attraction of CE was that it was fast, used small amounts of sample and reagents, and was extremely versatile, being able to separate large and small analytes, both neutral and charged. Because of this versatility, numerous methods for clinically relevant analytes have been developed. However, with the exception of the molecular diagnostic and forensic laboratories CE has not had a major impact. A possible reason is that CE is still perceived as requiring above-average technical expertise, precluding its use in a laboratory workforce that is less technically adept. With the introduction of multicapillary instruments that are more automated, less technique-dependent, in addition to the availability of commercial and cost effective test kit methods, CE may yet be accepted as a instrument routinely used in the clinical laboratories. Thus, this review will focus on the areas where CE shows the most potential to have the greatest impact on the clinical laboratory. These include analysis of proteins found in serum, urine, CSF and body fluids, immunosubstraction electrophoresis, hemoglobin variants, lipoproteins, carbohydrate-deficient transferrin (CDT), forensic and therapeutic drug screening, and molecular diagnostics.

Fatty acid ethyl esters: markers of alcohol abuse and alcoholism.

Chronic alcoholism, which is associated with hepatic, pancreatic, and myocardial diseases, is one of the major health problems in the United States with high morbidity and mortality. Many individuals who abuse alcohol chronically die even before reaching the clinical stage of the disease. Reliable biomarkers of the diseases induced by chronic alcohol abuse, as well as for alcoholism, currently are not available. In the current study, we measured plasma concentrations of fatty acid ethyl esters [(FAEEs), nonoxidative metabolites of ethanol] in 39 patients with a detectable concentration of alcohol in their blood samples. In turn, we determined the relation of FAEE concentrations with blood alcohol concentration (BAC). Of 39 patients in whom we evaluated this relation, only five had a history of chronic alcohol abuse, and six had a history of acute alcohol abuse. Patients' age ranged from 25 to 71 years. Within this age range, greater concentrations of FAEEs were found in the plasma samples obtained from patients in the 41- to 50-year age group. There were no sex-related differences in BAC, nor in FAEE concentrations. Thirteen patients had a BAC greater than 300 mg%. For 11 patients, the BAC ranged between 200 and 299 mg%, and, for 12 patients, the BAC ranged between 100 and 199 mg%. In comparison with findings for patients with a BAC that ranged between 100 and 299 mg%, the FAEE concentrations were approximately twofold higher in patients with a BAC greater than 300 mg%. Ethyl palmitate and ethyl oleate were the main FAEEs detected in most patients. In general, FAEE concentrations increased with increasing BAC. However, in comparison with patients with a history of acute alcohol abuse, a greater increase in total FAEE concentrations was observed in patients with a history of chronic alcohol abuse (4,250 ng/ml and 15,086 ng/ml, respectively). Fatty acid ethyl esters were either detected in trace amounts or not detectable in the plasma of control subjects with no known alcohol ingestion. These results support our hypothesis that nonoxidative metabolism of ethanol to FAEEs is an important pathway of ethanol disposition during chronic alcohol abuse, and that FAEE concentrations can be a more reliable biomarker of chronic alcohol abuse than a history of acute alcohol abuse.

Serum lipid profiles and risk of cardiovascular disease in three different male populations in northern Nigeria.

The Fulani of northern Nigeria are indigenous semi-nomadic pastoralists whose diet consists largely of dairy products. Despite their consumption of relatively large amounts of saturated fats, an earlier study showed that their total cholesterol, high-density lipoprotein (HDL), low-density lipoprotein (LDL) and serum triglyceride levels fall within the reference range of values for North Americans. Men in the cities of Jos and Abuja, two populations who also reside in northern Nigeria, differ from the Fulani with regard to diet and activity level. Males in both Jos and Abuja have diets consisting of high protein or carbohydrate and are more sedentary than the Fulani subjects. The main aims of the study were to measure the concentrations of various lipids in the blood serum of male urban dwellers in Jos and Abuja and to compare their blood lipid profiles with those of the rural Fulani (mean age 33.9 years). Blood serum samples from 118 men in Jos (mean age 37.9 years) and 77 men in Abuja (mean age 34.4 years) were analyzed for total cholesterol, triglycerides, LDL, HDL, homocysteine, folate, and vitamin B12. In addition to height and weight, systolic and diastolic blood pressures were measured. The mean total cholesterol, triglyceride, HDL and LDL values for the three groups of subjects fell within or close to the accepted range of values for North Americans. However, the Fulani males had HDL values (mean, 33.9 mg/dL) below the range of values prescribed for North Americans (>40 mg/dL). Moreover, the Fulani men and the men in Abuja had a total cholesterol/ HDL ratio of 4.2 and 4.0 respectively, which exceed the accepted value (< or =3.5) prescribed by the Columbia University. In all three populations, the incidence ofhomocysteinaemia (serum homocysteine > 12.4 micromol/L) was very high. Their mean homocysteine levels ranged from 14.7 to 16.7 pmol/L and could not be accounted for by folate or vitamin B12 status. The mean blood pressures of the Abuja (127/77 mm Hg) and the Fulani (120/74 mm Hg) men were within the normotensive range (<130/85 mm Hg). However, the mean blood pressures of the Jos males (131/85 mm Hg) indicated borderline hypertension. These data indicate that, with regard to serum lipids, urban and rural adult Nigerian males have generally favourable risk factors for cardiovascular disease when compared with healthy North Americans. All three sub-populations, however, have levels of homocysteine that are cause for concern vis-à-vis their overall health status.

Evaluation of the performance of urine albumin, creatinine and albumin-creatinine ratio assay on two POCT analyzers relative to a central laboratory method.

BACKGROUND: The evaluation of microalbumin, creatinine and albumin-creatinine ratio is very important in patients with diabetes for the early detection of kidney disease and the identification of patients at risk for complications from diabetes or hypertension. METHODS: A total of 88 spot urine samples previously analyzed using the Vitros 5,1 FS (creatinine) and Beckman Coulter Immage (microalbumin) located in the central laboratory and having microalbumin and creatinine values within the Afinion and DCA Vantage reportable ranges were run on 2 point of care (POC) instruments (Siemens DCA Vantage and Axis-Shield Afinion). RESULTS: The mean values for the DCA Vantage were: 42.6 mg/l for albumin, 10.3 mol/l for creatinine, and 5.4 mg/mol for ACR. For the Afinion AS100, the mean values were: 48.5mg/l for albumin, 9.5 mol/l for creatinine, and 6.7 mg/mol for ACR. The mean values obtained for CL were: 40.8 mg/l for albumin, 10.0 mol/l for creatinine, and 5.4 mg/mol for ACR. All POC analyzers showed good correlation to the central laboratory tests for microalbumin, creatinine and albumin creatinine ratio (ACR) for Afinion (R(2)=0.954, 0.974, and 0.964, respectively) and DCA Vantage (R(2)=0.989, 0.987, and 0.991, respectively). With the exception of the DCA Vantage ACR (p=0.53), the levels of microalbumin, creatinine and ACR obtained for the Afinion and DCA Vantage instruments as compared to the CL were statistically different (p<0.05). The inter and intraday imprecision for both POC instruments was <2.9% and total imprecision <8.7%. CONCLUSIONS: The 2 instruments evaluated in this study were in good agreement with the quantitative laboratory results and thus can be used for microalbumin, creatinine and ACR assays at the POC. However, facilities using Afinion will have to use different normal range for ACR.