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Basic Skills in Interpreting Laboratory Data. 4th ed. Bethesda, MD: American Society of Health-System Pharmacists. pages; $ (paperback). Basic Skills in Interpreting Laboratory Data - Ebook download as PDF File .pdf), Text fourth edition, the book was well received and was becoming a popular. Here is the ideal place to get Basic Skills In Interpreting Laboratory Data 4th Edition Pdf by. Marina Bosch Learning for free. Everyone enables to review online.
The book contains 22 chapters, a glossary of terms, and 3 appendices that contain the reference value tables, SI unit conversions, and blood collection tube information. Every chapter is referenced fully; the 4 th edition has added learning objectives, and retains the mini-cases designed to reinforce the material presented.
There are 3 new chapters in the 4 th edition: Several of the chapters have added graphics and illustrations, and the format has been updated. The 5 introductory chapters definitions and concepts; introduction to common laboratory assays and technology; primer on drug interferences and test results; substance abuse and toxicological tests; and interpretation of serum drug concentrations are full of background knowledge and clinical pearls gained from experienced practitioners.
The chapter on laboratory assays and technology could use an expanded section on point-of-care testing, but flow cytometry, polymerase chain reaction PCR and nanotechnology are included. The chapter on laboratory interferences includes methods to minimize these events.
Chapters 6 through 22 employ a systems-based approach electrolytes; other minerals and trace elements; kidneys; arterial blood gasses and acid base balance; pulmonary function tests; heart, liver, and gastroenterology; endocrine; lipids; hematology—anemia; hematology-coagulation; infectious disease; rheumatic diseases; oncology; pediatrics; women's health; aging males; and pharmacogenomics. The chapters begin with a description of the physiology of the system, and then introduce the laboratory tests in the sequence that they would be ordered in a patient diagnostic work-up.
Relating the pathophysiology of disease to its evaluation with laboratory testing is one of the strong points of this book. A consideration of all of the diverse methodologies used in these procedures is beyond the scope of this chapter.
Although these clinicians themselves do not usually perform laboratory tests.
Basic Skills in Interpreting Laboratory Data
These advances have enabled a more detailed understanding of the impact of genetics in disease and have led to new disciplines: And as these tests are automated.
Clinical laboratory tests represent a vast array of diverse procedures ranging from microscopic examination of tissue specimens histopathology to measurement of interactions of cellular and molecular components typically clusters of atoms.
Antibiotics are based on the observation that microbes produce substances. It can involve consolidated analyzers. It is anticipated that discoveries in these areas will change the practice of traditional clinical medicine into a more personalized medicine approach and will also impact pharmaceutical development. The common goals of clinical laboratory mechanization and automation result in increased efficiency. Agents have been developed that are aimed at improving insulin release from the pancreas and sensitivity of the muscle and fat tissues to insulin action.
Many of the traditional laboratory procedures and tests that are described in the following parts of this chapter will create the framework upon which these potential advances will be based—researchers are simplifying them. Hypotensive medications that lower blood pressure have typically been designed to act on certain pathways involved in hypertension such as renal salt and water absorption.
This understanding is essential for the proper ordering of tests and the correct interpretation of results. The past 30 years has seen remarkable progress in the role of laboratory in personalizing medicine. Total laboratory automation systems are currently capable of performing only some of the above. The use of flushing systems prevents carryover between specimens. Modern laboratory information systems have the capability of analyzing data in a variety of ways that enhance patient care.
In parallel with the development of the highly automated core laboratory. Automation does not end at this stage. Determining whether.
Standardization within the laboratory automation arena is an essential means of assuring QC. In addition to the more commonly requested serum chemistry levels.
Using the example of a chemistry analyzer. Many results generated by automated chemistry analyzers rely on reactions based on principles of photometry. All modern automated analyzers rely on computers and sophisticated software to perform these processing functions. Informatics in the laboratory involves the use of collected data for the purposes of problem solving and healthcare decision-making. As generators and collectors of information. The ability to transmit and share such information via the Internet is becoming as indispensable a function of the laboratory as performing the tests themselves.
Clinical and Laboratory Standards Institute. The centralized clinical laboratory of the future will house automated laboratory systems capable of performing high volume and esoteric testing. In many divisions of the centralized laboratory. Calculations statistics on patient or control values. Each specimen passes through the same continuous stream and is subjected to the same various analytical reactions. Community clinics.
Schematic of single-beam upper portion and double-beam lower portion spectrophotometers. These test procedures are based on one or more of the following methodologies. Biosensor systems consist of two components: Common transducers used in these systems are based on amperometry.
The transducer detects this change and converts it into a measurable signal that is proportional to the concentration of the analyte. In contrast to conventional assay methods that involve multiple steps and liquid reagents. In the future. The principle technology that underlies the function of these smaller instruments usually involves biosensor systems. Point-of-care testing may be thought of as any specimen testing that exists outside the walls of the large. The interaction between the bioreceptor and the target analyte generates either a specific molecular species or results in a physiochemical change that can be measured by electrochemical methods.
The bioreceptor is a molecule such as an antibody. The result is then compared with a standard curve to yield a specific concentration of analyte. The first stage involves the absorption of radiant energy by an electron in the ground state creating an excited singlet state. If substances are known to interfere at this wavelength. The technology found in these instruments is based on the principle of luminescence. Molecular Emission Spectrophotometers Molecular emission spectrophotometry is usually referred to as fluorometry.
The high specificity and accuracy are obtained by isolated analytes reacting with various substances that produce colorimetric reactions. Spectrophotometers are easy to use.
It utilizes a mirror VI to split the light from a single source into two beams. The greatest sensitivity is achieved by selecting the wavelength of light in the range of maximum absorption.
Most measurements are made in the visible range of the spectrum. Single-beam instruments have a light source I e. Three types of fluorescence phenomena—fluorescence. After passing through the test solution the light strikes a detector. By doing so.. In clinical laboratory instruments. Light of a specific wavelength then passes through the exit slit and illuminates the contents of the analytical cell cuvette III.
Molecular Absorption Spectrophotometers Molecular absorption spectrophotometers. During the very short lifetime of this state order of nanoseconds. This modified procedure allows detection or measurement of the analyte with minimal interference from other substances. This tube amplifies the electronic signal. Fluorescence results from a three-stage process that occurs in certain molecules known as fluorophores. Specific wavelengths of light are selected by the use of a monochromator II.
The basic components of two types of spectrophotometers single and double beam are depicted in Figure 2—1. Four types of photometric instruments are currently in use in laboratories: The double-beam instrument.
The electronic energy of a triplet state is lower than a singlet state. The energy is typically derived from the oxidation of an organic compound. As in the case of an excited singlet state. This is followed by relaxation to the electronic ground state by the emission of radiation fluorescence. When a pair of electrons occupies a molecular orbital in the ground or excited state. Light is derived from the excited products that are formed in the reaction.
Because the various forms of radiationless energy transfer compete so effectively. Because energy is dissipated. Unlike fluorescence. In this state. Small molecules that rotate faster will emit light that is depolarized relative to the excitation plane.
The probability of this type of transition is much lower than a singlet-singlet transition fluorescence. These devices use similar basic components along the following pathway: An important example is fluorescent polarization in fluorometers. Fluorescent molecules fluorophores become excited by polarized light when the plane of polarization is parallel to their absorption transition vector.
When the electrons are no longer paired. If the molecules rotate rapidly. The difference between these two energies is known as Stokes shift. Different instruments have been developed that use these basic principles of luminescence.
This principle is the basis for the sensitivity of the different fluorescence techniques since the emission photons can be detected at a different wavelength band than the excitation photons. Large molecules move slowly during the excited state and will remain highly polarized. The degree to which the emission intensity varies between the two planes of polarization is a function of the mobility of the fluorophore.
When an unknown quantity of an unlabeled analyte is added to the mixture. Large concentrations are necessary because this test measures small differences in large numbers.
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The main differences are that 1 the light source is usually a laser. The technique is based on the elementary quantum principle that electrons in an atom are excited to a higher energy level by heat. Turbidimetry is the technique for measuring the percent light absorbed. When conditions are held constant. The labeled analyte will emit depolarized light because its motion is not constrained. Like many modern laboratory instruments.
One of the most common applications of fluorescence polarization is competitive immunoassays.
Atomic absorption spectrophotometry procedures are currently associated mainly with toxicology laboratories where poisonous substances. Atomic Emission and Atomic Absorption Spectrophotometers Atomic emission flame photometry and atomic absorption spectrophotometry have limited use in modern laboratories. The electrons. Unlike flame photometry. In doing so. By using standard curves of known drug concentrations versus polarization.
In the past. Errors associated with this method usually involve sample and reagent preparation. A major advantage of turbidimetry is that measurements can be made with laboratory instruments. The length of time between sample preparation and measurement.
This important methodology involves the addition of a known quantity of fluorescent-labeled analyte molecules to a serum antibody specific to the analyte mixture. Beam light scattered by particles is a function of the size and number of the particles. In the refractometer. These are simple devices consisting of a sample chamber with a stirrer and a thermistor.
By keeping the first three parameters constant. In the eyepiece. Because of the large molecular weight of plasma proteins. The sample is stirred to initiate freezing of the super-cooled solution. Although proteins are the predominant chemical species. Nephelometric measurements are more precise than turbidimetric ones since the smaller signal generated for low analyte concentrations is more easily detected against a very low background.
The ability of a liquid to bend light depends on several factors: The sample is rapidly cooled several degrees below its freezing point in the cooling chamber. Since each property is related. In the case of plasma samples.
This procedure is particularly useful. The scale is calibrated in grams per deciliter for serum protein. The refracted light is projected on an eyepiece scale. When the freezing point of the solution is reached the point where the rate of the heat of fusion released by ice formation comes into equilibrium with the rate of heat removal by the cooling chamber.
When osmotically active particles are dissolved in a solvent water. Primary testing method hepatitis B. When the plasma or serum is placed in the sample.
Primary testing method lithium. Primary testing method glucose. One chamber is filled with a colloid-free physiologic saline solution that is in contact with a pressure transducer. The COP osmometer. Primary and secondary vanilmandelic acid. Since a low COP favors a shift of fluid from the intravascular compartment to the interstitial compartment.
Primary testing method aminoglycosides. This is known as the redox potential difference since the reaction involves the transfer of electrons between substances that accept electrons oxidant and substances that donate electrons reductant.
These produce membrane or diffusion potentials. Junctional potentials rather than redox potentials occur when either a solid state or liquid interface exists between the ion conductive phases. The electrode selectively binds the ion to be measured. The ISE method. A redox potential occurs when the two electrolyte solutions in the electrochemical cell are brought into contact with each other by a salt bridge so that the two solutions can achieve equilibrium. Potentiometry Potentiometry involves the measurement of electrical potential differences between two electrodes in an electrochemical cell at zero current flow.
This electrochemical method is based on the Nernst equation. These analytic techniques are based on the fundamental electrochemical phenomena of potentiometry. One of the electrodes is a reference electrode with a constant electric potential. In each case the concentration of an ion in solution can be measured using the Nernst equation. The boundaries between the ion conductive phases in the cell determine the type of potential gradients that exist between the electrodes and are defined as redox oxidation reduction.
To measure the concentration. The three types of electrodes are 1. A potentiometer may be used to measure the potential difference between the two electrodes. The principle of ISE involves the generation of a small electrical current when a particular ion comes in contact with an electrode.
The resultant pressure is the colloidal osmotic pressure. Ion-selective electrodes ISEs consisting of a membrane that separates the reference and test electrolyte solutions are very selective and sensitive for the ions that they measure. For this reason further discussion on potentiometry will focus on these types of electrodes. Ion-selective glass membranes.
Liquid ion-exchange membranes As shown in Figure An electrical potential is created when these ions diffuse across the membrane.
Since the liquid junction generates its own voltage at the sample interface. This is accomplished through a form of titration where a standardized concentration of the titrant is reacted with the unknown analyte. Coulometry Coulometry is an analytical method for measuring an unknown concentration of an analyte in solution by completely converting the analyte from one oxidation state to another. Solid-state electrodes 3. The pH meter is an example of a test that uses ISE to measure the concentration of.
Ions outside the membrane produce a concentration-related potential with the ions bound to the carrier inside the membrane. They are also very useful in biomedical applications because they can measure the activity of the ion directly in addition to the concentration. An example is the silver—silver chloride electrode for measuring chloride. This error is overcome by adjusting the composition of the liquid junction. The point at which all of the analyte has been converted to the new oxidation state is called the endpoint and is determined by some type of indicator that is also present in the solution.
Solid-state electrodes consist of halide-containing crystals for measuring specific ions. This instrument may be used to measure the chloride ion Cl— concentration in sweat. By monitoring the current of an electrochemical cell at varying electrode potentials.
Conductometry Conductometry is the measurement of current flow proportional to conductivity between two nonpolarized electrodes of which a known potential has been established. An electric current is generated when hydrogen ions come in contact with the ISE A. Voltammetric techniques use an externally applied force potential to generate a signal current in a way that would not normally occur.
This technique is based on the Faraday law. The ability to apply different types of potential functions or waveforms has led to the development of different voltammetric techniques: The electrode arrangement is also quite different between the two techniques. F is the Faraday constant The three electrodes include the working. At more positive potentials. I is the current. The circuit is completed through the use of a reference electrode B submerged in the same liquid as the ISE also known as the liquid junction.
In order to analyze both the potential and the resulting current. By varying the potential of an electrode. The concentration can then be read on a potentiometer C. Voltammetry Voltammetry encompasses a group of electrochemical techniques in which a potential is applied to an electrochemical cell with the simultaneous measurement of the resulting current.
The measurement of the resulting current can yield results about ionic concentrations. The chloridometer is a common instrument that employs this method. Clinical applications include. At lower potentials. The technique is limited at low concentrations because of the high conductance of biological fluids. Supports for the buffer include filter paper. These factors are related by the following equation: The mobility of the molecules will be affected locally by the charge of the electrolytes.
The movement of molecules in this electrical field is dependent on molecular charge. Electrophoretic systems are usually combined with highly sensitive detection methods to monitor and analyze the separations that suit the specific application. Electrophoresis tests. When an electrostatic force EOF is applied across the electrophoresis apparatus.
This method is discussed in detail in the cytometry section. E is the electromotive force. The primary application of electrophoresis is the analysis and purification of very large molecules such as proteins and nucleic acids. The force that acts on these molecules is proportional to the net charge on the molecular species and the applied voltage electromotive force. Through the proper selection of the medium for electrophoretic separations.
Because of a large number of clinical applications. Perhaps the most important application of impedance inversely proportional to conductance measurements in the clinical laboratory involves the Coulter principle for the electronic counting of blood cells.
The main types of electrophoresis techniques used in both clinical and research laboratories include cellulose acetate. Molecules to be separated must be dissolved in a buffer that contains electrolytes.
Electrophoresis also can be applied to the separation of smaller molecules. Densitometry is typically used to quantify each band. These techniques differ in the target molecules that are separated. This technique is often used for separation of isoenzymes and hemoglobin variants.
Each method involves a series of steps that leads to the detection of the various targets. Three common techniques used are Southern. Electrophoresis at both pH conditions is performed for optimal resolution of comigrating hemoglobin bands that occur at either of the pH conditions. When a monoclonal immunoglobulin pattern is identified.
Gel Electrophoresis Cellulose Acetate and Agarose Gel Electrophoresis Cellulose acetate and agarose gel electrophoresis are commonly used for both serum protein and hemoglobin separations. The above cited factors affecting the process of electrophoresis are controllable and provide optimal resolution for each specific application. Hemoglobin electrophoresis is the most common method for the screening of hemoglobin variants.
When the molecules have been separated into bands. Following electrophoresis. Southern blots separate DNA that is cut with restriction endonucleases and then identified with a labeled usually radioactive DNA probe. Variant hemoglobins are separated on a cellulose acetate membrane at an alkaline pH 8. The porosity of these media will.
Media used include polysaccharides cellulose and agarose and synthetic media such as polyacrylamide. The choice of support media is determined by the resolution of the hemoglobin bands that are achieved. Once these proteins are separated on an agarose gel.
Each protein moves to its isoelectric point i. Western blots separate proteins that are probed with radioactive or enzymatically-tagged antibodies. Serum protein electrophoresis is often used as a screening procedure for the detection of disease states.
The sample is then fixed and stained to visualize and quantify the bands. The probe-target hybrids are detected following a wash step to remove any unbound probe. The overall movement of the solvent is called electroosmotic flow.
CE is being adapted for various applications in the clinical laboratory because of its rapid and high-efficiency separation power. This prevents the probe from randomly sticking to the paper during hybridization.
During the hybridization stage. Two-Dimensional Electrophoresis Two-dimensional electrophoresis 2-D electrophoresis is a powerful and widely used method for the analysis of complex protein mixtures extracted from cells. The DNA. In the former method the molecules.
The separated proteins are eluted from the. Following the transfer. When the sample solution is injected into the apparatus. Proteins are sorted according to two independent properties: The negatively charged surface of the silica capillary attracts positively-charged ions in the buffer solution. The latter method involves layering the gel on wet filter paper with the nitrocellulose paper on top. Capillary Electrophoresis Capillary electrophoresis CE includes diversified analytical techniques.
Capillary electrophoresis apparatus consists of a small-bore. The possibility of CE becoming an important technology in the clinical laboratory is illustrated by its use in the separation and quantification of a wide spectrum of biological components ranging from macromolecules proteins. In a CE separation. While historically a research tool. Each spot on the resulting two-dimensional array corresponds to a single protein species in the sample.
This technology also has many research applications especially in the field of proteomics. Dry filter paper is placed on the nitrocellulose paper and the molecules are transferred with the flow of buffer from the wet to dry filter paper via capillary action. Densitometers can perform measurements in an absorbance optical mode and a fluorescence mode.
The fluorescence method is used in the case of electrophoretic patterns that fluoresce when radiated by UV light nm.
The absorbance is proportional to the sample concentration. Capillary Gel Electrophoresis Capillary gel electrophoresis CGE is the CE analog of traditional gel electrophoresis and is used for the size-based separation of biological macromolecules such as oligonucleotides. The separation is performed by filling the capillary with a sieve-like matrix such as polyacrylamide or agarose to reduce the EOF.
The light is focused by a collection of lenses onto a UV blocking filter and then to a photomultiplier tube where the visible light is converted into an electronic signal that is proportional to the intensity of the light.
Capillary gel electrophoresis is primarily used in research. When the pattern located on the carriage moves across a focused beam of UV light. In CZE. After the light passes through the pattern. The filter system provides a narrow band of visible light to provide better sensitivity and resolution of the different densities.
Current densitometry systems employ sophisticated software to provide. DNA restriction fragments. In this way the negative. Quantitative detectors such as fluorescence. Densitometers used in this mode include a UV light source and a photomultiplier tube instead of the silicon photocell.
This mode of operation is commonly used to evaluate hemoglobin and protein electrophoresis patterns and applications of molecular diagnostics MDx. An absorbance optical system consists of a light source.
When a densitometer is operated in the absorbance mode. In each case. Common clinical applications include high throughput separation of serum and urine protein and hemoglobin variants. These factors affect the separation of a mixture into the different components.
After separation is complete usually 12—24 hours. The choice of sorbent depends on the specific application since compounds have different relative affinities for the solvent mobile phase and the stationary phase.
Chromatographic assays do not require premanufactured antibodies and. Quantification of various substances is possible with TLC. Silica gel is the most commonly used sorbent because it may be used to separate a broad range of compounds. Thin layer chromatography is used for identification and separation of multiple components of a sample in a single step.
The sorbent may be composed of silica. The paper is then hung in a chromatography jar so that the bottom edge contacts a solvent. GC uses an inert gas e. Another disadvantage. Various fractions are then identified by how far they migrated on the paper. The procedure involves placing a drop of the sample near the bottom of a piece of chromatography paper and allowing it to dry.
Instead of a solvent. In this method. Chromatographic assays require more time for specimen preparation and performance.
Table of Contents
Three types of chromatography are currently and routinely used in the clinical laboratory: Each component. Paper Chromatography Paper chromatography. Since quantification of a substance is not possible with this method. This technique is also based on the principles of paper and TLC.
A column packed with inert material. Gas Chromatography Gas chromatography GC is used to identify and quantify volatile substances such as alcohols. The recorder produces a chromatogram with various peaks being recorded at different times. Area under the curve or peak height of an analyte e. This ratio is compared to a standard curve of peak area ratios to give the concentration of the analyte.
The mobile phase is pumped through the column under high pressure to decrease the assay time. When the sample leaves the column. This technique has many advantages. Because each sample component is retained for a different length of time.
The amount of each component present is determined by the area of the characteristic peak or by the ratio of the peak heights calibrated against a standard curve. The gas carries the sample through the column where it contacts the liquid phase.
The sample is injected into the column contained in a heated compartment where it is immediately volatilized and picked up by the carrier gas. Its basic principles are similar to those of GC.
This current is amplified by an electrometer. In cases of such interference. HPLC utilizes a liquid solvent mobile phase and a column packed with a stationary phase. Gas chromatogram. Analytes with lower boiling points migrate faster than those with higher boiling points.
When the sample is exposed to the flame. The most common detector consists of a hydrogen flame with a platinum loop mounted above it. Heating at precise temperature gradients is essential for good separation of the analytes. Instead of gas.
Compounds are identified by their retention times and quantified either by computing the area of the peak or by comparing the peak height or area to an internal standard to obtain a peak height or peak area ratio. This ratio is then used to calculate a concentration by comparison to a predetermined standard curve.
A signal from the detector is sent to a recorder or integrator. Various components move at different rates. Although HPLC offers both high sensitivity and specificity. The sample is injected onto the column at one end and migrates to the other end in the mobile phase. Another concern is that since many assays require a mobile phase composed of volatile and possibly toxic solvents.
Appearance of this chromatogram is similar to the gas chromatogram. The detector is usually a spectrophotometer with variable wavelength capability in the ultraviolet and visible ranges. Once again. HPLC chromatogram. As the mobile phase leaves the column. As with GC. The numbers as well as the specificities of the antibodies depend on the size and number of antigenic sites on the antigen.
It is also a great resource for students, especially those starting the pharmacotherapy course sequence, or during advanced professional practice experiences. National Center for Biotechnology Information , U. Am J Pharm Educ. Reviewed by William R. Wolowich , PharmD.
Author information Copyright and License information Disclaimer. College of Pharmacy, Nova Southeastern University. Corresponding author. Corresponding Author: William R.Electrophoretic systems are usually combined with highly sensitive detection methods to monitor and analyze the separations that suit the specific application. This error is overcome by adjusting the composition of the liquid junction. The result is a high degree of cross-reactivity with similar substances.
Predictive Value The predictive value. J Clin Chem Clin Biochem. Many factors influence the reference range.