Pages: 16

This chapter discusses the safety rules to be followed in clinical biochemistry laboratory. In a clinical biochemistry laboratory, all essential elements for a fire to begin are present, such as an ignition source; hence, it is important to assure the safety of self, personnel, and equipment. Fire extinguishers are used to handle fire and possess dry or wet chemicals. Laboratory glassware includes beakers, flasks, graduated (measuring) cylinders, burettes, funnels, funnels, pipettes, test tubes and desiccators. Burettes are long, graduated tubes with a stop cork at one end, available in capacities of 10 to 100 ml. These devices are used to accurately deliver known volumes of liquids into a container. Funnels are used for transferring liquids/solids into containers and for separating solids from liquids. They usually have short or long, thin stems. They are used with filter paper to remove particles from solutions. Amber-colored bottles are useful to store certain light-sensitive chemicals such as silver nitrate.

Pages: 7

Proper collection, processing, storage, and transport of samples are required to get the best quality results. The process of collecting blood is called phlebotomy, and the person who performs phlebotomy is called a phlebotomist. Biological samples such as blood, saliva, urine, and feces are analyzed for various parameters, which help clinicians to diagnose and treat various diseases. The puncture of a vein to withdraw blood for analysis or intravenous injection is called venipuncture. Venipuncture should be done from the antecubital fossa (preferred site) or from the back of hand or ankle. Venous occlusion should be attained by tourniquet/blood pressure cuff. Blood should be collected in the desired vacutainer, and the order of draw must be followed. A dry gauge pad should be used over the puncture site to stop bleeding and promote the clotting process. Vacutainers are sterile glass or plastic tubes having colored rubber stoppers, which create vacuum inside the tube.

Pages: 6

Quality control (QC) programs were usually concerned with making sure that the results furnished by test tactics are as correct as possible. A normal control product contains a normal range of analytes, whereas an abnormal control contains below or more range of analytes. Generally, in a clinical laboratory, multiple controls may be used. Internal quality control is performed on a regular basis to assess the performance of measuring systems. Performance is assessed by comparing the obtained results with those from different laboratories. When the QC results are within the acceptable range, only then the samples of the patients to be analyzed should run. The results of the patients must not be released unless QC results are below the acceptable range. Accuracy is the closeness of the estimated value to that of the true value. Precision is the closeness of the results on repeated analysis.

Pages: 29

Urine is formed by the kidneys. It is used in biochemistry laboratories for biochemical analysis. Many dangerous waste materials and water are eliminated from the body via urine. It is a regular physiological system of the body. A complete urine analysis can be done by the biochemical analysis, bacteriological analysis, and microscopic analysis. Concentrated urine has a high specific gravity, while diluted urine has a low specific gravity. It is measured by a urinometer or refractometer. Chemical analysis of urine is conducted to detect the presence or absence of certain constituents that reflect the disease state of a body. The parameters include proteins, sugar, blood, ketone bodies, bile pigments, bile salts, and urobilinogen. Urobilinogen is normally present in urine in very small amounts. The excretion of urobilinogen is increased in hemolytic jaundice and infective hepatitis. Urine containing bilirubin has a typical beer brown color when voided and produces yellow foam on vigorous shaking.

Pages: 14

Carbohydrates are aldehyde or ketone derivatives of polyhydroxy aldehydes. Glucose is the main form of carbohydrate absorbed from the gut. Humans can synthesize glucose from non-carbohydrate sources such as lactates, gluconeogenic amino acids, propionic acid, glycerol, and pyruvate by a process known as gluconeogenesis. Benedict's test is a semi-quantitative test and is the most widely employed test for the detection of reducing sugar in urine. Reducing sugars have a free aldehyde and ketone group, for example glucose, fructose, galactose, and lactose. Heating in an acidic environment leads to the hydrolysis of the glycosidic bonds present in disaccharides or polysaccharides. Many monosaccharides form a characteristic crystal but with a difference in time of formation, melting point, solubility in boiling water, and nature of crystalline structure. This test is also used to identify reducing disaccharides.

Pages: 15

Glucose, the major carbohydrate in blood, is also the major source of energy for the body’s cells. Blood sugar includes all hexoses, even though the main hexose present in the blood is glucose. Blood sugar means blood glucose. It is an essential nutrient for the brain and red blood cells. The most common disorder of glucose metabolism is diabetes mellitus, a disease in which the blood glucose is elevated because of the lack of insulin regulation. The state of having elevated blood glucose is called hyperglycemia. Decreased blood glucose is called hypoglycemia, which is a less common disorder of glucose metabolism. The glucose test is most often used to aid in diagnosing and managing diabetes and hypoglycemia. Broadly two methods are available for blood glucose estimation, namely reduction (chemical) methods and enzymatic methods. The ability of a person to metabolize a given load of glucose is referred to as glucose tolerance. The glucose tolerance test is a well standardized test.

Pages: 22

Proteins are defined as sequence-specific polymers of Lα amino acids linked by peptide bonds. Reactions of proteins are studied as precipitation reactions and color reactions. Proteins are large molecules with variable sizes, shapes, and charges. Protein solubility depends on the distribution of polar hydrophilic groups and nonpolar hydrophobic groups in the molecule. The stability of a protein in a solution will depend mainly on the charge and the degree of hydration (shell of water molecules around the particles). Any factor that neutralizes the charge or removes the shell of hydration will cause precipitation of proteins. Precipitation is used to separate proteins from biological fluids such as blood, plasma, and CSF before the estimation of important chemical constituents such as urea, sugar, and creatinine, because proteins interfere in their estimation. The negatively charged ions of the alkaloids neutralize the positive charge on the protein causing denaturation, which results in precipitation.

Pages: 7

Lipids are esters of fatty acids with alcohol, which are soluble in organic solvents and insoluble in water. Lipids are insoluble in water (polar solvent) and soluble in organic solvents (nonpolar solvents). When oil and fat are boiled with alkali, both are hydrolyzed and fatty acids are liberated from salts with alkali, called soap. The process is known as saponification. When oil and water are shaken together, the oil is broken down into small droplets, which are dispersed in the water. This is acknowledged as an oil-in-water emulsion. Water, due to its high surface tension, has a tendency to close together and separate as a layer. Emulsification is used in the analysis of lipids. Borax is hydrolyzed into NaOH (strong acid) and boric acid (weak acid) after the addition of glycerol. It reacts with boric acid and forms a strong acid (glyceroboric acid).

Pages: 9

Cholesterol is one of the causative factors of heart diseases, thrombosis, and cerebral hemorrhage. The cholesterol present in serum gets precipitated with uranyl acetate, ferric chloride, and glacial acetic acid (uranyl acetate reagent). This eliminates all the interfering chromogens, including bilirubin. The absorbance of chromophore quinoneimine is directly proportional to the concentration of cholesterol in the sample. Therapeutic reduction is seen during the administration of lipid-lowering drugs such as clofibrate and cholestyramine and drugs such as nicotinic acid and D-thyroxine. Sometimes it may accompany chlorpropamide and phenformin therapy. In primary biliary cirrhosis, the increase is observed almost entirely in the free cholesterol and may be as high as 90%. Estimation of HDL is done by phosphotungstic acid method. First precipitate LDL, VLDL, and chylomicrons by polyanions (Phosphotungstate) in the presence of metal ions like magnesium ions to leave HDL in the solution.

Pages: 17

The liver is a critical organ that carries out many functions. It is referred to as the metabolic factory of the body. The liver function test routinely assesses the functioning of liver. Bilirubin is the breakdown product of hemoglobin present in RBCs, from its heme part. It is produced in the reticuloendothelial system present in the spleen and liver. This bilirubin is unconjugated and insoluble in water. Unconjugated bilirubin gets bind to albumin and then it is taken up by liver through carrier-mediated process. The secretion of conjugated bilirubin in bile throughout the biliary canalicular membrane is rate limiting and is sensitive to liver damage. Bile is saved in the gall bladder from where it passes into the duodenum through the cystic and common bile duct. Blood contains a larger number of proteins that perform so many functions. Most enzymes, hormones, and clotting factor are proteins in nature.

Pages: 13

Urea is an end product of protein metabolism. Breakdown of proteins forms amino acids, which is precursor for the formation of ammonia (NH3). NH3 further get converted into urea in the liver, and the kidney eliminates it in urine. Interference from hemolysis, bilirubin, lipemic serum, and ammonia in reagents or atmosphere gives rise to false high values. So, it must be removed by adsorption on permutit or sodium aluminum silicate. Blood urea can be estimated by the diacetyl monoxime method. Blood urea estimation is important for the assessment of kidney function since the route of urea excretion is through the kidneys. Ammonia generated with the assistance of the enzyme urease is made to react with Nessler’s reagent (potassium mercuric iodide), which gives rise to a brown compound, which is studied at 450 nm. Increased level of blood urea is termed azotemia. When it is associated with clinical symptoms, it is called uremia.

Pages: 5

The pancreas is 12-15 cm long and lies across the posterior wall of the abdominal cavity. Its head is found within the curve of the small intestine, and the body and tail are directed toward the left, extending to the spleen. Pancreatic digestive enzymes containing bicarbonate-rich juice enter the small intestine. It is located behind the peritoneal cavity. Test of pancreatic function test includes tests for detection of malabsorption, tests for exocrine function, test to assess extrahepatic obstruction, endocrine-related tests and imaging/noninvasive tests. Gastrointestinal fluid aspiration by incubation is required. After an overnight fasting, a stimulus is given to the pancreas, followed by the measurement of he total volume of pancreatic juice, the concentration of secreted carbonate, and the activities of duct gland enzymes. Amylase is the main enzyme for pancreatic function.

Pages: 6

An acid can be defined as a proton donor that increases the concentration of hydrogen ions in a solution. A base can be defined as a proton acceptor, electron pair, or hydroxide ion donor that decreases the concentration of hydrogen ions in a solution. pH is the measurement of the concentration of hydrogen ions (H+) present in a dilute solution. It is the measure of the level of acidity or basicity of a solution. The pH measurement system consists of a pH-measuring electrode, reference electrode and high input meter. The pH-measuring electrode is a glass electrode, which utilizes a thin glass membrane responsive to change in H+ activity. A standard is used to calibrate analytic instruments. Before taking a pH measurement, it is essential to calibrate the pH using standard solutions of known pH. This standardization is necessary to maintain a reference state of pH.

Pages: 6

Photometry is the measurement of the propagation of light through various materials. The color of light is a function of its wavelength and a change in the color of light can be detected in the visible region with a change in wavelength. In photometry, measurement of the color intensity of a solution is governed by two laws, namely Beer’s law and Lambert’s law. Colorimetry is the measurement of color. It measures the concentration of substances that are colored or can be converted into colored compounds by a suitable reaction. This technique is very sensitive and requires minute amount of samples. A colorimeter is an instrument used to measure the concentration of colored substances. The spectrophotometer is based on the principle of Beer–Lambert law and is used to measure the amount of light absorbed by the sample. Spectrophotometers are of two types: single beam and double beam.

Pages: 16

Chromatography is a biophysical technique, which allows the separation and purification of a molecule of interest from a crude mixture for quantitative and qualitive analysis. The stationary phase in reverse phase HPLC is nonpolar, while the mobile phase is polar. In a chromatography, separation of the mixture is performed on a paper strip which may be a stationary phase and a liquid solvent act as a mobile phase. The principle of thin-layer chromatography is the same as that of chromatography (partition). It also separates and analyzes complex biological and non-biological samples into their constituents. It is most popular for the estimation of a substance in a mixture and monitoring the progress of a chemical reaction. In adsorption chromatography, the absorbent is used as the stationary phase. Affinity chromatography is a powerful chromatographic technique used in the isolation of proteins, vitamins, enzymes, nucleic acids, drugs, antibodies, and hormone receptors.

Pages: 7

Electrophoresis is the movement of charged particles through an electrolyte when exposed to an electric field. Electrophoresis literally means walking in an electric field. Negatively charged particles (anions) move to the anode, and positively charged particles (cations) move to the cathode. The speed of movement of ions in the electric field depends on the following factors: particle size and shape, net charge of the molecule, electric field strength, characteristics of carrier medium, and operating temperature. Polymer or acrylamide crosslinked with methylene bisacrylamide is used as a support medium. A thin slab of polyacrylamide, a glass plate, or a short column is used. The resolution is far better in this technique. The gel is mounted between two buffer chambers containing separate electrodes in most electrophoresis units so that the only electrical connection between the two chambers is through the gel.

Pages: 10

Enzyme-linked immunosorbent assay (ELISA) is an analytical method for detecting a substance through a specific interaction between an antibody and its antigen. This extremely sensitive technique is used for detecting and quantifying substances such as antigens, antibodies, hormones, and proteins. In direct ELISA, the first step is immobilization of the antigen on the 96 wells ELISA plate, followed by incubation at 37°C for 1 h or 4°C overnight. Direct ELISA is a simple process but less specific than sandwich ELISA. Sandwich ELISA begins with a capture antibody coated onto the wells of the plate. It is termed a “sandwich” because the antigens get sandwiched between two layers of antibodies (capture and detection antibodies). Competitive ELISA has a low specificity and cannot be used in dilute samples. However, its benefit is that less sample purification is needed. Detection is carried out by the addition of a substrate that can generate a color. Many substrates are used in ELISA.

Pages: 4

Radioimmunoassay (RIA) is a method developed by Rosalyn Yalow and Solomon Berson in 1959 to measure insulin in plasma. In 1977, Yalow won the Nobel Prize for the development of RIA. It is an important technique in clinical biochemistry for the estimation of hormones, steroids, and drugs, which are present in minute quantities and cannot be detected by the colorimetric or other chemical methods. It is used for measuring the antigen concentration of a patient’s sample. RIA is a combined technology of nuclear medicine (tracer technique) and immunology. RIA is a competitive assay, where a competition between unlabeled and labeled antigen is seen to quantify and detect an antigen. In this assay, a fixed amount of antibody is used, then sample containing antigen are allowed to react with the constant and fixed amount of antibody which is already bound with radio-labeled antigen. The labeled antigen will get displaced by the unlabeled antigen from sample (if sample contains more antigens).

Pages: 6

Amplification techniques can be classified into two types, namely thermal cycling, where temperature of the reaction varies [polymerase chain reaction (PCR) and ligase chain reaction], and isothermal cycling in which temperature of the reaction remains constant [nucleic acid sequence-based amplification and branched DNA technology]. PCR involves a series of cycles of three successive reactions. The product of the DNA acts as a template strand for further DNA synthesis. Primers are short nucleotide sequences, which can bind to the target DNA. So prior knowledge of the DNA sequence (to be amplified) is mandatory. PCR methods based on mitochondrial genes have been used in forensic science because of their high copy number per cell and lack of recombination. DNA profiling (DNA typing, genetic fingerprinting, DNA testing) is a technique used in forensic science to identify criminals based on their DNA profile. PCR is widely used for the DNA analysis of archaeological specimens.

Pages: 10

This chapter covers the different sorts of transducers that are utilized in various biosensors, their characteristics, a couple of important applications, and also new trends in transducers found in biosensors. Based on the transducing mechanism used in biosensors, transducers can be classified into the following types: electrochemical transducers, optical transducers, calorimetric (thermometric) transducers, piezoelectric transducers, and magnetic transducers. Electrochemical transducers detect various kinds of electrochemical species, either generated or consumed, during the interaction of the biosensing element (enzyme/antibody) with the analyte (or substrate). Based on the mechanism used for the detection of electrochemical species, electrochemical transducers used in biosensors are mainly classified as amperometric, potentiometric, conductometric, and photoelectrochemical. During some chemical reactions, different reactants react with each other and undergo an excited state. Piezoelectric biosensors are analytical devices that sense the affinity interaction between analytes and biorecognition elements, and they utilize crystals that undergo an elastic deformation when an electric potential is applied to them.

Pages: 4

Glycated hemoglobin (HbA1c) is an additional marker besides the standard glucose and glycemia analyses, which has become a relevant marker in new analytical methods. Point-of-care testing of HbA1c appears to be a suitable approach to timely and accurately revealing diabetes mellitus, and it demonstrates a better quality of diagnosis compared to the standard determination of glycemia. There are three major HbA1c testing methods currently available to clinical laboratories, namely chromatography-based HPLC assay, antibody-based immunoassay, and enzyme-based enzymatic assay. Boronate affinity chromatography is a glycation-specific method based on boronate binding to the unique cis–diol configuration formed by stable glucose attachments to Hb. Recent innovations have yielded a Direct Enzymatic HbA1c AssayTM, which uses a single channel test and reports %HbA1c values directly, without the need for a separate THb test or a calculation step.

Pages: 8

Enzymes are biocatalysts involved in various chemical reactions in the biological system. They have become increasingly important these days due to their widespread acceptance in the industry, chemical laboratories, dairy products, and other disciplines. The most desirable character observed in enzymes is that unlike other catalysts in aqueous solutions, they can catalyze a reaction in mild conditions at normal temperature and pressure with very high reaction specificity, bond specificity, and optical specificity. Various kinetic properties of xanthine oxidase enzymes have been studied such as effect of pH, incubation temperature for maximum activity, incubation time, and effect of substrate (xanthine) concentration on optimal working conditions. Immobilization means physical confinement or localization of enzyme molecules. This can be done through the physical adsorption of enzymes onto water-insoluble organic or inorganic supports, the entrapment of enzymes within gel matrices or semipermeable microcapsules, and the covalent attachment of enzymes to the water-insoluble supports.

Pages: 4

DNA is a genetic material and the basic hereditary unit of each and every cell. DNA isolation is the process of purification of DNA by using a combination of physical and chemical methods. DNA was first isolated in 1869 by Friedrich Miescher. The purification of nucleic acids broadly involves the following steps: breaking or opening of cells to expose nucleic acids, separation of nucleic acids from other cellular components, and recovery of nucleic acids in pure form. The nucleic acid absorbs light strongly in the UV region at 260 nm due to the conjugated bond present in purine and pyrimidine bases. DNA concentration is measured using a double cell UV spectrophotometer. To extract DNA from tissue/cell of interest, the cells have to be separated, and cell membranes have to be extracted by an extraction buffer. DNA is concentrated by mostly ethanol precipitation. In the presence of salt, ethanol efficiently precipitates polymeric nucleic acid.

Pages: 7

Mass spectrometry has become an attractive molecular histology tool in pharmaceutical and medical research. It is a widely used instrumental technique in physics, analytical chemistry, and forensic laboratories. After the ionization of the analytes of interest into various fragments, the spectrum generated is interpreted based on mass- to-charge ratios of the different ions. An important part of this technique is sample preparation prior to mass analysis. Quadrupole mass spectrometer is the most common mass spectrometer in use today. It consists of four cylindrical rods, which are placed parallel to each other. Quadrupole ion trap mass analyzers work on the same physical principles as quadrupole mass analyzers. The only difference is that in this type, the ions are trapped in stable orbits inside a three-dimensional chamber. These trapped ions are then sequentially ejected based on their m/z. Mass spectrometry is coupled with liquid chromatography or gas chromatography, which makes it a powerful technique.

Pages: 10

Flow cytometry is a technique used to detect, identify, and count specific cells. This method can also identify specific components within cells. So, it is used for the detection and measurement of physical and chemical characteristics of cells or particles in a heterogeneous fluid mixture. Lasers as light sources are used to produce both scattered and fluorescent light signals, which are then read by detectors such as photodiodes or photomultiplier tubes. Multiparametric flow cytometry provides rapid and accurate determination of antigen expression profiles in leukemias. By detecting different types of markers, one can easily differentiate between different types of leukemias. Cell sorting or fluorescence-activated cell sorting allows the physical separation of a cell or particle of interest from a heterogeneous population. A mixture of fluorescently stained cells is passed through a small aperture. A fluorescent-labeled antibody specific to a particular cell surface protein is added to the mixture of cells.

Pages: 5

Centrifugation is the process of using centrifugal force to separate lighter components of a solution, mixture, or suspension from heavier components. A centrifuge is a device that separates various components of a solution based on shape, size, density, viscosity of the medium, and rotor speed. It works on the principle of centrifugal force. A body moving in a circular motion with an angular velocity omega is subjected to a centrifugal force acting in the outward direction. The centrifuge works on the principle of gravity and generation of a centrifugal force to sediment different fractions. The centrifuge works on the sedimentation principle, where the centripetal acceleration causes denser substances and particles to move outward in the radial direction.

Pages: 8

Laboratory automation is a complex integration of robotics, computers, liquid handling, and some other technologies. It has dramatically changed both analytical and non-analytical aspects of clinical laboratory operations. Automation enables anyone to perform many tests with the help of analytical instruments without an analyst. Like a modular kitchen, each part of a modular system is designed in such a way that it easily fits in different systems. Batch analysis is beneficial if one parameter is to be analyzed on multiple samples, e.g., blood sugar in multiple specimens. In centrifugal analyzer, only single test can be done at one time, so it is a type of batch analyzers in which analysis is sequential, discrete, and parallel. Centrifugal force is used for transferring and mixing the samples and reagents.

Pages: 3

Cerebrospinal fluid (CSF) is formed by the selective dialysis of plasma by the choroid plexus present in the ventricles of the brain. CSF is present in the cavity that surrounds the brain in the skull and the spinal cord in the spinal column. The total volume of CSF is about 150 ml. CSF diagnoses central nervous system (CNS) abnormalities.

Pages: 4

Oxalate is a metabolic breakdown product of the Krebs cycle in eukaryotes and the glyoxylate cycle in other microorganisms. Normal range of oxalic acid Urine: 10–30 mg/24 h Plasma: 0.8–2.50 µmol/L Oxalate concentration is higher in whole blood than in serum. Ammonium, lithium oxalate, sodium, and potassium inhibit blood coagulation by forming an insoluble complex with Ca2+. Oxalate oxidase is of great importance in the measurement and diagnosis of primary and secondary hyperoxaluria. Hyperoxaluria leads to the formation of calcium urinary oxalate stones, malabsorption, steatorrhea, ethynylene glycol poisoning, and ileal disease. Crystals of oxalate are sharp and can be large enough to irritate the body, which leads to the formation of kidney stones. A decreased level of oxalate in urine is also associated with hyperglycosuria and hyperglycinemia. Oxalate is extracted from urine and subsequently assayed by measuring the amount of hydrogen peroxide produced in an oxidation reaction catalyzed by oxalate oxidase.