Amin A. Mohammad

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.

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.

Hyperbilirubinemia and Transcutaneous Bilirubinometry

BACKGROUND Neonatal jaundice or hyperbilirubinemia is a common occurrence in newborns. Although most cases of neonatal jaundice have a benign course, severe hyperbilirubinemia can lead to kernicterus, which is preventable if the hyperbilirubinemia is identified early and treated appropriately. CONTENT This review discusses neonatal jaundice and the use of transcutaneous bilirubin (TcB) measurements for identification of neonates at risk of severe hyperbilirubinemia. Such a practice requires appropriate serial testing and result interpretation according to risk level from a nomogram that provides bilirubin concentrations specific for the age of the neonate in hours. In this context, we have evaluated the potential impact on clinical outcome and limitations of TcB methods in current use. SUMMARY TcB measurement is a viable option in screening neonates to determine if they are at risk for clinically significant hyperbilirubinemia. Total serum bilirubin should be measured by a clinical laboratory if a newborn is shown to be at higher risk for clinically significant hyperbilirubinemia. In addition, external quality assessment to identify biases and operator training issues should be part of any TcB monitoring program.

Development of a urinary free cortisol assay using solid-phase extraction-capillary electrophoresis.

In clinical practice, the measurement of urinary free cortisol (UFC) provides the most sensitive and specific diagnostic information for excess adrenal production of cortisol. The existing methodologies (RIA and HPLC) are time consuming, costly, involve tedious extractions, derivatizations and problems with non-specific interactions with cortisol metabolites in urine. In the present study, we describe the development of an SPE-CE method for the rapid analysis of UFC. UFC was concentrated using SPE C18 cartridges (3M Empore) under a vacuum and eluted with acetonitrile-SDS. The use of 10% acetone to wash cartridges before final elution with acetonitrile-SDS showed significant improvements in the free cortisol recovery. The complete extraction was accomplished in 10-15 min with a recovery of 89-94%. CE analysis was done on a Beckman P/ACE 5010 with detection at 254 nm using a neutral capillary. Detection limits of free cortisol in urine was improved to 10 microg/l with SPE compared to 500 microg/l without SPE. No interferences either from BSA or other urinary cortisol metabolites affected the free cortisol determinations. The results showed the feasibility of a rapid UFC detection with improved sample handling capacity.

Anionic-zwitterionic mixed micelles in the micellar electrokinetic separation of clinically relevant steroids on a fused-silica capillary

Abstract The structural similarities of different clinically relevant steroids make their routine determination in biological samples an analytical challenge. Determination of steroidal hormones in urine and serum is of great interest in clinical endocrinology. A large number of disorders is associated with hypo- or hypersecretion of one or more steroids, for example, Addisons disease, Cushings syndrome, hirsutism and virilism, adrenal adenomas, hypertension, congenital adrenal hyperplasia etc. A SDS/SB3-12 mixed micellar system was used to achieve baseline separation of eight steroids in less than 10 min. Five different zwitterionic surfactants were evaluated at different concentrations, pH and temperature conditions. The SB3-12/SDS mixed micellar system gave highest resolution and precision. No significant capillary wall interaction or coating effects were observed with the zwitterionic surfactants as is evident from the reproducible migration times (relative standard deviation ≤2%). A detection limit of 1–5 μm/ml was obtained for pure standards.

Clinical and forensic applications of capillary electrophoresis

Section I. Introduction Capillary Electrophoresis: General Overview and Applications in the Clinical Laboratory Manjiri Lele, Subodh M. Lele, John R. Petersen, and Amin A. Mohammad Basic Principles and Modes of Capillary Electrophoresis Harry Whatley Section II. Protein Electrophoresis Capillary Coatings for Protein Analysis Kannan Srinivasan, Chris Pohl, and Nebojsa Avdalovic Clinical Serum Protein Capillary Zone Electrophoresis Carl R. Jolliff Serum and Urine Paraprotein Capillary Electrophoresis Carl R. Jolliff Cerebrospinal Fluid Protein Electrophoresis John R. Petersen and Amin A. Mohammad Lipoprotein Analysis Rainer Lehmann Clinical Analysis of Structural Hemoglobin Variants and Hb A1c by Capillary Isoelectric Focusing James M. Hempe, Alfonso Vargas, and Randall D. Craver Section III. Metabolic Diseases Amino Acid Analysis, Mary Kathryn Linde. Organic Acids Kern L. Nuttall and Norberto A. Guzman Steroids, Cecilla Youh Amin A. Mohammad, and John R. Petersen Section IV. Immunoassay Capillary Electrophoresis Based Immunoassay Bode Adesoji, Amin A. Mohammad, and John R. Petersen Section V. Molecular Diagnostics Quantitation of Viral Load Jill M. Kolesar The Application of Capillary Electrophoresis in the Analysis of PCR Products used in Forensic DNA Typing Bruce R. McCord and John M. Butler Capillary Electrophoresis with Electrospray Ionization Mass Spectrometry Samir Cherkaoui Section VI. Mass Spectrometry, Therapeutic Drug Monitoring, and Toxicology Capillary Electrophoresis-Mass Spectrometry of Biologically Active Peptides and Proteins Stephen Naylor and Andy J. Tomlinson Serum Drug Monitoring by Capillary Electrophoresis Zack K. Shihabi Applications of Capillary Zone Electrophoresis in the Analysis of Metal Ions of Clinical Significance Lokinendi V. Rao, John R. Petersen, Amin A. Mohammad, and Anthony O. Okorodudu Clinical and Forensic Drug Toxicology: Analysis of Illicit and Abused Drugs in Urine by Capillary Electrophoresis Wolfgang Thormann and Jitka Caslavska Screening Biological Specimens for Drugs of Forensic Significance John C. Hudson, Murray J. Malcolm, and Mauro Golin

Laser induced resonance energy transfer - A novel approach towards achieving high sensitivity in capillary electrophoresis I. Clinical diagnostic application

Most of the procedures currently performed by capillary zone electrophoresis (CZE) with laser induced fluorescence detection requires prior derivatization. This increases cost, the turn-around-time and chances of extraneous contaminations. CZE with laser induced resonance energy transfer is demonstrated as a viable alternative for detecting non-fluorescent compounds with no prior derivatization. The feasibility of this approach is demonstrated by separating and directly detecting salicylic acid (2,4-dihydroxybenzoic acid), gentisic acid (o-methoxybenzoic acid), salicyluric acid (o-hydroxyhippuric acid) and 4-aminosalicylic acid in urine. The detection of salicylate in serum is also shown. The method is highly sensitive with detection limits in the 1.10(-7) M range. Importantly it requires no prior preconcentration or sample preparation and can be used with complex sample matrices such as serum and urine.

Apparatus and method for interfacing capillary electrophoresis and chemiluminescence nitrogen detection for the analysis of nitrogen-containing compounds.

We have developed an interface that allows the specific detection of nitrogen-containing compounds by using a chemiluminescence nitrogen detector. The feasibility of using this interface was demonstrated by separating and detecting two nitrogen-containing compounds, p-aminosalicylic acid and L-phenylalanine. Although baseline separation was achieved, the theoretical plates were lower when compared to UV detection (25000 vs. approximately 85000). A sensitivity of 75 ng (approximately 500 pmol) per injection was achieved with this system which is adequate for pharmaceutical and biotech applications.

Multiple site analytical evaluation of a portable blood gas/electrolyte analyzer for point of care testing.

Objective To evaluate the analytical performance of the SenDx 100 portable blood gas and electrolyte analyzer (SenDx Medical, Carlsbad, CA). Design Accuracy was evaluated by correlation of whole blood patient samples with the Nova Stat Profile 5 (Nova Biomedical, Waltham, MA) and the Ciba Corning 865 (Chiron Diagnostics, Medford, MA). Precision was evaluated using quality control materials (RNA Medical, Acton, MA). Setting Critical care laboratories and operating rooms in two institutions. Measurements and Main Results Precision studies performed at three different concentration levels for each analyte demonstrated intra-assay precision of ≤2.5% coefficient of variation and interassay precision of ≤4.0% coefficient of variation in all cases. Analysis of patient specimens in general showed good to excellent correlation to reference analyzers. Regression variables are tabulated. Conclusions The SenDx 100 portable blood gas and electrolyte analyzer is a simple and easy to use analyzer demonstrating acceptable performance compared with reference methods.

Laser induced resonance energy transfer — a novel approach towards achieving high sensitivity in capillary electrophoresis Part B: Applications for environmental testing☆

Abstract Capillary electrophoresis with laser induced resonance energy transfer detection is used for direct determination of ethylenediamine tetraacetic acid (EDTA), 1,2-diaminopropane-N,N,N′,N′-tetraacetic acid (DPTA) and (±)- trans -1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (DCTA) in aqueous samples. The method involves CZE separation of salicylate-Tb-EDTA, salicylate-Tb-DPTA and salicylate-Tb-DCTA ternary complexes at alkaline pH. The highly efficient transfer of energy from salicylate to terbium in these complexes facilitated a sensitive detection. Detection limits in the 1·10 −7 M range are easily achieved. A 325 nm HeCd laser is used for excitation and terbium luminescence is monitored at 547 nm. The potential application of this method in analyzing these acids in a complex radioactive waste is discussed.