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(BQ) Part 2 book Concise book of medical laboratory technology - Methods and interpretations presents the following contents: Enzymology, blood gases and electrolytes, diagnostic immunology, the endocrine system, microbiology and bacteriology,...
CHAPTER 22 Serology/Immunology BASIC IMMUNOLOGY The immune system offers protection against invading microorganisms, viruses and other foreign materials Somehow, it must distinguish between Valuable what “belongs” and what doesn’t “belong” Failure to detect and expel foreign materials can lead to problems due to immunodeficiency (i.e AIDS) and misidentifi cation of “self” (autoimmunity) Antigen-Immunogen Antigen is a molecule that binds with an antibody or T cell receptor (antigenicity is the ability to bind to the antibody) Immunogen is a molecule that can elicit an immune response (immunogenicity is the ability to elicit an immune response) Epitopes (Fig 22.1) For a molecule such as a protein, a given antibody will “be directly against” only one of all the possible parts of the entire molecule This part is known as an EPITOPE A molecule may have several epitopes Also, a complex antigen (such as a cell) will have many molecules, each of which will contain several epitopes An epitope is also known as an antigenic determinant Some epitopes are better able to elicit antibodies than others They are known as Immunodominant Epitopes How Big is an Epitope? About units of a polysaccharide chain, or about 6–8 amino acids For a protein epitope, it is the shape of the epitope, rather than the specific amino acid sequence that is Antigenicity Several factors influence how “antigenic” a molecule is Most important is how foreign it is, with molecules that are most unlike self-being the most antigenic There are also numbers of physi cal and chemical determinants, which also matter molecular size — the larger the better, generally 1000 Daltons are about the lower limit ¾¾ Complexity: The more complex the better For example, simple repeating polysaccha rides like starch aren’t very good, while proteins with a constantly changing sequence of 20 or so different amino acids are good ¾¾ Structural stability: A fixed shape is helpful For example, gelatin (which wobbles) is a poor antigen unless it is stabilized ¾¾ Degradability ¾¾ Foreignness FIG 22.1: Sites for obtaining blood by venipuncture from forearm 564 Concise Book of Medical Laboratory Technology: Methods and Interpretations important For example, a few amino acids, which come from different parts of the chain, can come together in one physical spot to create an epitope When an antibody directed against one epitope can bind to another epitope, this is known as “cross-reactivity” If this happens, it will be because the two epitopes ‘look alike” in some way Some intestinal bacteria possess antigens that look like blood group A and B antigens which can be absorbed through the intestinal wall into the bloodstream; therefore, people of blood group A will have antibodies against the B antigens even if they never have been exposed to B-type blood cells Antibodies directed against human serum will crossreact with serum from chimpanzees, gorillas, orangutans and spider monkeys to an increasingly lesser extent What are the Different Kinds of Epitopes? Conformational Discontinuous Some Examples of Antigens Proteins: Most antigens are proteins, such as the ones on the outer coverings of microorganisms Antibodies themselves: Human immuno globulin G, which contains human antibodies, is immunogenic in experimental animals, because it is foreign to them Polysaccharides: Simple ones are not good Longer ones, especially if they are complex and/or associated with proteins, can be good Blood Group Antigens: A, B, AB and O LPS or Lipopolysaccharides: From cell wall of gramnegative bacteria Lipids are generally poor antigens Nucleic acids are generally poor antigens Antibody A class of proteins that migrate in the gamma fraction They are classified on the basis of heavy chains ¾¾ IgG — Eighty percent plasma immunoglobulin, present in all body fluids, transplacental, ¾¾ IgM — large molecule, pentameric in structure, present in vascular system, activates complement ¾¾ IgA — present in body secretion, respiratory and GI tract ¾¾ IgE — involved in hypersensitivity and allergic reactions ¾¾ IgD — present in B cell surfaces FIG 22.2: Antibody structure What is the Structure of Antibody? Basic model consists of polypeptide chains small/light chains large/heavy chains Heavy chains are structurally different for different class of antibodies (Fig 22.2) What is the Kinetics of Antigen–Antibody Reaction? The reaction complies with the law of mass action The higher the K, the stronger the reaction The forces governing the reaction are: ¾¾ Hydrogen bonds ¾¾ Hydrophobic bonds ¾¾ Electrostatic bonds ¾¾ van der Wall’s bonds (Ag.Ab) K = (Ag)(Ab) Immunological Reactions What are the different ways of detection of antigen– antibody reaction? ¾¾ Immunodiffusion ¾¾ Electrophoresis ¾¾ Flocculation ¾¾ Complement assays ¾¾ Flow cytometry ¾¾ Immunohistochemical techniques Serology/Immunology ¾¾ Binder–ligand assays ¾¾ A clinical laboratory performs different kinds of tests for detection of antigen–antibody reactions; ¾¾ Agglutination blood grouping, Widal test ¾¾ Latex agglutination—CRP, RF test ¾¾ Flocculation—VDRL test for syphilis ¾¾ Electrophoresis—protein biochemistry ¾¾ Chromatography—pregnancy tests What are the Different Indicators Used in Immunoassay? Indicator Example Technology Enzyme Horse radish peroxidase EIA Radio isotope 131 RIA Fluorescence IFA How is Binder-ligand Assays Classified? Fluorescein iso thiocynate (FITC) Chemiluminescent dyes Acridinium ester CLIA • Isotopic assays—radioimmunoassays • Non-isotopic assays—enzyme Immunoassays, fluorescence polarization immunoassays Chromogen Colloidal gold Chromatography Microparticles Latex Latex agglutination What is the Difference Between All these Reactions? All are basically antigen-antibody reaction The indicator used will differentiate the technology (Fig 22.3) What form of Reaction Takes Place in HLA Typing? It is also antibody reaction in which the end product is visualized by using a dye in a phase contrast microscope The reaction can also be visualized using fluorescent dyes in a fluorescent microscope What is the Principle of HLA Typing? It is called ad mixed lymphocytotoxicity test (MLT) In this the antibody (antisera) is coated in the microwell The patient’s B or T lymphocytes containing HLA antigens is added and incubated Complement proteins are added which will destroy the complex, if they are formed The dead and viable cells are differentiated and graded using an appropriate dye The principle is same for both cross-matching and tissue typing FIG 22.3: Indicators used to differentiate immunological reactions 565 I Interferences in Immunoassays Despite advances in the design of immunoassays, the problems of unwanted interference have yet to be completely overcome An ideal immunoassay should have the following attributes: ¾¾ The immunochemical reaction behavior should be identical and uniform for both the reference preparation and the analyte in the sample ¾¾ The immunochemical reaction of the antibody reagent is uniform from batch to batch ¾¾ The immunochemical method is well standardized to ensure that the size of measurement signal is caused only by the antigen-antibody product ¾¾ For macromolecules the results declared in arbitrary units (IU – International Units), the conversion to (SI) units is not constant and depend on many factors Definition of Interference Interference may be defined as “ the effect of a substance present in an analytical system which causes a deviation of the measured value from the true value, usually expressed as concentration or activity.” IFCC (International Federation of Clinical Chemistry) offers the following d efinition – “Analytical interference is the systematic error of measurement caused by a sample component, which does not, by itself, produce a signal in the measuring system” Assay interference can be “Analyte depen dent or Analyte independent” It can increase or decrease the measured result Increase (positive interference) is due to lack of specificity Decrease (negative interference) is due to lack of sensitivity Assay interference can be of different types: 566 Concise Book of Medical Laboratory Technology: Methods and Interpretations Preanalytical Variables All factors associated with the constituents of the sample are termed as preanalytical variables They can be of two types: Patient based: Such as incorrect sampling times and environmental factors such as smoking, etc may change analyte concentration and consequently interpretation Specimen based: There are many factors that constitute this ¾¾ Blood collection ¾¾ Nature of the sample: For all immunoassays, serum is the matrix of choice Samples collected into tubes containing sodium fluoride may be unsuitable for some enzymatic immunoassay methods; preservation with sodium fluoride may affect results Impurities in tracers interfere with direct dialysis methods for free hormones ¾¾ Hemolysis and hyperbilirubinemia ¾¾ Lipemia — may cause interference with assays for fat soluble compounds such as steroids ¾¾ Stability and storage Matrix Effects A fundamental problem with the analysis of components in biological materials is the effect of the extremely complex and variable mixture of proteins, carbohydrates, lipids, and small molecules and salts constituting the sample The effect of these compounds on analytical techniques is termed as matrix effect It can be defined as “ the sum of the effects of all the components, qualitative or quantitative, in a system with the exception of the analyte to be measured” The Effect of Reagents Assay buffers: The ionic strength and pH of buffers are vitally important, particularly in the case of monoclonal antibodies with pI values of 5–9 The use of binding displacers (blockers) may change the binding characteristics of antibodies, particularly those of low affinity Detergents used in the buffers may contain peroxides, which inhibit antigen-antibody reaction Immunoassay labels: Labels have a profound effect on assays The structure of most molecules, especially haptens, may be dramatically changed by labeling, e.g by attachment of a radioactive iodine atom to a steroid Labeling antibodies with enzymes is less of a problem because of their large size Separation of the antibody-bound and free fractions: The proportion of free analyte in the bound fraction and vice versa is known as the “misclassification error” Antibody bound fraction may be efficiently separated from the free analyte using solid-phase systems in which the antibody is covalently linked to an inert support, e.g the reaction tube, a polystyrene bead, a cellulose or nylon Effect of Proteins Interfering proteins of general relevance include Albumin: May interfere as a result of its comparatively huge concentration and its ability to bind as well as to release large quantities of ligands Rheumatoid factors: These are autoantibodies usually IgM class, and directed against the Fc portion of IgG They are not specific to rheumatoid arthritis and are found in other autoimmune diseases, including systemic lupus erythematosus, scleroderma and chronic active hepatitis Complement: These proteins bind to the Fc fragment of immunoglobulins, blocking the analyte specific binding sites Lysozyme: Strongly associates with proteins having low isoelectric points (pI) Immunoglo bulins have a pI of around and lysozyme may form a bridge between the solid–phase IgG and the signal antibody Endogenous hormone-binding proteins: These are present in varying concentrations in all serum and plasma samples and may markedly influence assay performance For example: SHBG (sex hormone binding globulin) inter feres in immunoassay of testosterone and estradiol TBG, (thyroxine binding globulin) and NEFA (non esterified fatty acid) interfere with the estimation of free T4 Abnormal forms of endogenous binding proteins: These are present in the plasma of some patients They are present in familial dysalbuminemic hyperthyroxinemia (FDH) in which albumin molecules bind to thyroxine (T4) Individuals with FDH can be diagnosed as thyrotoxic, in spite of being normal Heterophilic antibodies: They may arise as a consequence of intimate contact, either intentional or unintentional, with animals The most familiar effect of heterophilic antibodies is observed in two-site sandwich reagent-excess assays, in which a ‘bridge’ is formed between the two antibodies forming the sandwich Assays that are affected by heterophilic antibodies include for CEA, CA 125, hCG, TSH, T3, T4, free T4, Prolactin, HBsAg and Digoxin Serology/Immunology Mechanical Interference Fibrinogen from incompletely clotted samples interferes with sampling procedures on automated immunoassay instruments and may produce spurious results Paraproteinemia causes interferences in many assays by increasing the viscosity of the sample They may also nonspecifically bind either analytes or reagents that may affect the result Nonspecific Interference Non-specific interference may arise from excessive concentrations of other constituents of plasma Free fatty acids affect some assays for free T4 by displacement of T4 from endogenous binding proteins Hook Effect The “Hook Effect” is characterized by the production of artefactually low results from samples that have extraordinarily high concentrations of antigen (analyte), far exceeding the concentration of the upper standard in the assay concerned The Hook effect is most commonly found in single-step immunometric assays, a popular format, chosen for its specificity and speed, particularly with high-throughput immunoassay analyzers The assays most affected are those that have analyte concentration that may range over several orders of magnitude For example, α Feto protein (AFP), CA 125, hCG, PSA, TSH, prolactin and Ferritin are most affected by Hook effect Reduction of Hook Effect The incidence of Hook effect can be reduced (but not eliminated) by careful assay design – incorporating a wash step prior to addition of the second antibody, thereby avoiding simultaneous saturation of both antibodies Despite attempts to eliminate or reduce the Hook effect by careful assay design the only reliable method of routinely eliminating the effect is to test the samples that are likely to be affected by Hook effect in undiluted and also at a suitable dilution Such samples should be diluted using either the assay diluent or serum from a normal subject until a stable quantitative response is achieved Assay Specificity It is one of the most important requirements of immunoassays Interference occurs in all situations in which the antibody is not absolutely specific for the analyte Consequently, assessment of specificity is a vital step in the optimization of every new immunoassay Poor specificity results in interference from compounds of similar 567 molecular structure or which carry similar immunoreactive epitopes In determining the overall specificity of an assay, a major factor is the cross reactivity of the antibody Some the major specificity problem areas are related to measurement of steroids and struc turally related compounds All commonly used testosterone assays, cross react in varying degrees with 5α-dihydrotestosterone, and all cortisol assays cross react with prednisolone Assessment of the specificity of immunometric assays is complex and quite different from that used for singlesite assays In most assays, two different antibodies are employed, each having unique specificity for a different epitope on the antigen It is usual practice to employ at least one monoclonal antibody, which can be selected by epitope mapping to react only with predetermined sites on the antigen molecule Use of two monoclonal antibodies can introduce extreme specificity What is the difference between an antigen and immunogen? The word “antigen” is conventionally used to describe as antibody generators, i.e that can generate antibody against itself Also, anything that is foreign to the body is also known as antigen This definiton of foreignness has become irrelevant with autoantigens Antigen can be defined as those that bind with the antibody They need not be foreign in nature Some antigens also require a carrier/helper to bind with the antibody Immunogens, as the name goes are those that can elicit an immune response It may be either T-cell or B-cell response All immunogens can be antigens But all antigens need not be immunogens What are the different types of epitopes? There are two different types — sequential and con formational Sequential epitopes are made of linear region of peptides Conformational epitopes are formed when the protein chain is folded Disulfide bonds are important for maintaining the conformational integrity What is Hook effect? Sometimes, the value of an analyte obtained by laboratory testing will be very low in spite of suspicion that it will be high This false low values derived in spite of it being very high is known as Hook effect This is due to very high concentration in the blood The levels are so high that they actually mask the binding sites available in the immunoassay system, leading to very low values (Imagine one hundred persons fighting to sit in chairs Even though there were hundred the actual number of people who sat were only 5) This is observed in parameters like PSA,hCG, CEA, etc The solution for this is to dilute the sample and run the assay 568 Concise Book of Medical Laboratory Technology: Methods and Interpretations What is the difference between chemiluminescence and fluorescence? Which is better? Fluorescence is a phenomenon where molecule absorsbs light in one wavelength and emits in another wavelength In this, there is a source of excitation Chemiluminescence is the production of light by a chemical reaction The main difference is that there is no radiation is absorbed The energy required to emit light comes from the energetics of chemical reaction Definitely chemiluminescence is a better technology for use in immunoassays What is meant by apoptosis? In an organism such as a human, the number of new cells created must be balanced by an equal number of cells dying Sometimes cell death occurs as a result of injury; most often, however, it is a planned, natural process called apoptosis Apoptosis is sometimes called cellular suicide because it is a cell’s own gene products that carry out its death While it kills a cell, apoptosis is beneficial to the host as a whole - it is important, for example, in development, in the immune response, etc How does secondary response differ from primary response? It differs mainly in three ways: ¾¾ It involves an amplified population of memory cells ¾¾ The response is more rapid ¾¾ Higher levels of antibodies are formed than primary response What are primary and secondary lymphoid organs? Primary lymphoid organs are the bone marrow and thymus These organs function as sites for B-cell and T-cell maturation, respectively Secondary lymphoid organs are spleen, lymph nodes and various mucous associated lymphoid tissues All these trap antigens and provide sites lymphocytes can interact with antigen What is the difference between active and passive immunity? Active immunity Passive immunity Produced actively by the host Received passively by the host Induced by infection Conferred by introduction of readymade antibodies Durable and effective protection Protection transient and less effective Immunity effective only Immunity effective immediately after a lag time Immunological memory present No immunological memory Negative phase may occur No negative phase Not applicable in immunodeficient host Applicable in immunodeficient hosts What is the difference between analytical and functional sensitivity? Analytical sensitivity refers to intra assay precision, whereas functional sensitivity refers to inter assay precision TECHNOLOGIES Rapid Immunochromatographic Techniques Perspective on Membrane-based Rapid Diagnostic Tests The need for a rapid, reliable, simple, sensitive in vitro diagnostic assay for use at point-of-care, have lead to the commercialization of in vitro Rapid Diagnostic Tests based on the principle of immunochromatography Rapid Diagnostic Tests are membrane-based immunoassays that allow visual detection of an analyte in liquid specimens In clinical assays, specimens such as urine, whole blood, serum or plasma, saliva and other body fluids may be employed What are the Principles of Membrane-based Rapid Diagnostic Tests? Currently available Rapid Diagnostic Tests comprise of a base membrane such as nitrocellulose A detector reagent (antigen/antibody-indicator complex) specific to the analyte, impregnated at one end of the membrane A capture reagent is coated on the membrane at the test region When the specimen is added to the sample pad, it rapidly flows through the conjugate pad Analyte if present in the specimen, binds to the detector reagent As the specimen passes over the test band to which the capture reagent is coated, the analyte-detector reagent complex is immobilized A colored band proportional to the amount of analyte present in the sample, develops The excess unbound detector reagent moves further up the membrane and is immobilized at the control band What are the Components of Membrane-based Rapid Diagnostic Tests and how are they Constructed? Rapid Diagnostic Test consists of (Fig 22.4) Sample pad Detector reagent/conjugate: Antigen/antib odyindicator complex specific to the analyte, impregnated in the conjugate pad but remains unbound Test band: Coated on nitrocellulose membr ane; specific to the analyte Control band: Usually antidetector antibodies coated on the membrane, served to validate the test results Soak pad Serology/Immunology 569 FIG 22.4: Construction of rapid diagnostic tests Currently, immunochromatography tests are available in two formats; “lateral flow” and “transverse flow or flow through” The lateral flow formats are available in device or dipstick format The lateral flow formats are commonly employed where rapid detection of pregnancy, drug abuse, infectious disease or parasitology is required, and serve as qualitative screening assay at laboratories, physician’s office or at homes due to their simplicity and ease of performance The flow through format is less common as the assay requires greater operator involvement However, some of these assays enable semi-quantitative estimation of the analyte by visual comparison with an inbuilt reference Regardless of the format used, the desired specificity, sensitivity and assay performance depends upon reliable formulation and proper assay assembly What are the Limitations and Effects of Various Components on the Performance of Membrane Rapid Diagnostic Tests? This section highlights the role of various components of Rapid Diagnostic Tests and their effect on attaining the desired performance characteristics How does the Nitrocellulose Membrane Affect the Sensitivity of Rapid Diagnostic Tests? Rapid Diagnostic Tests are fabricated on a solid support membrane, usually made of nitrocellulose Membranes employed in Rapid Diagnostic Tests are porous Depending upon the porosity, some membranes are better suited for applications with certain specimens than others This is because, the pore size of the membrane has significant effect on the capture reagent binding properties and the lateral flow rate The combined effects of these two phenomena in turn determine the sensitivity and performance of the test assay Pore Size and Capture Reagent Binding Properties It has been observed that as the pore size decreased the effective surface area available for binding of capture reagent increases Greater effective surface area available for binding, results in optimal coating of the capture reagent, which is essential for attaining the desired sensitivity of the assay Pore Size and Lateral Flow Rate It has been observed that as the pore size increases, the lateral flow rate increases How ever, slower flow rate increases the effective concentration (concentration required for interaction) of the analyte, since a slower flow rate allows the analyte and the capture reagent to be in close proximity for a longer times As it is well known, immunological reactions are time-dependent and prolonged exposure of the analyte with the capture reagent allows better interaction and thus, results in increased sensitivity The flow rate is important when the analyte is present in low concentrations, such as borderline samples The relationship between lateral flow rate and effective analyte concentration is: Effective analyte α concentration (Flow rate)2 Thus, it is important to optimize the membranes such that Rapid Diagnostic Tests can achieve rapid results which are also reliable and accurate Why are Colloidal Gold Sol Particles Commonly Employed in the Detector Reagent in Membranebased Rapid Diagnostic Tests? Interpretation of results in Rapid Diagnostic Tests depends upon the development of a signal at the stipulated time A signal is generated when capture reagent—analytedetector reagent complex is formed The detector reagent/ 570 Concise Book of Medical Laboratory Technology: Methods and Interpretations conjugate consists of an antibody or antigen bound to the indicator The indicator imparts color to the signal, enabling visual interpretation of results Colored latex particles, colloidal gold sol particles, dyes, enzymes and carbon particles are some of the indicator used in immuno chromato graphic assays However, stability, protein-binding properties, and particles’ size are critical factors that determine their use in immunochro matographic assays The most popular indicators used in immunochromatographic assays is the colloidal gold sol particle FIG 22.5: Graph of particle’s size v/s signal color of colloidal gold sol Colloidal Gold Sol Particles as Indicator Homogeneous colloidal gold sol particles are inert and can couple with antibody/antigen, which is stable in dry as well as in liquid forms All the above-mentioned parameters are determined by the particles’ shape and size of colloidal gold Effect of Shape of Colloidal Gold Sol Particles on Stability Colloidal gold sol particles have a net negative charge called “zeta potential” This zeta potential maintains the minimal distance between two particles resulting in longterm stability Ideally, colloidal gold sol particles should be spherical in shape, since, this shape allows uniform distribution of zeta potential at the surface In case of nonhomogeneous particles, the zeta potential is not uniformly distributed, thus the particles may come together to form aggregates These aggregates may permanently get impregnated into the conjugate pad, or during the test assay may deposit on the nitrocellulose membrane leading to discrepant results Such nonhomogeneous colloidal gold is usually blue/black in color Effect of Shape of Colloidal Gold Sol Particles on Sensitivity Spherical, homogeneous colloidal gold sol particles also allow uniform coating of the detector reagent at their surface Whereas non-homogeneous colloidal gold sol particles not allow uniform coating of detector reagent, resulting in decreased assay sensitivity and specificity Effect of Size on Color of Colloidal Gold Sol Particles It has been observed that as the colloidal gold sol particles increase in size, the color turns from light pink to cherry red to red-purple to blue-black to gray-black Darker colored particles are preferred in Rapid Diagnostic Tests since darker colors allow easy interpretations of results However, as the colloidal gold sol particles increase in FIG 22.6: Two-site sandwich immunoassay size, these particles are less stable and aggregate together Secondly, due to the steric hindrance, the larger colloidal gold sol particles tend to dwarf the coated antigen/antibody making interaction with the analyte difficult (Fig 22.5) Ideally, the colloidal gold sol used in immuno chromatographic assay is ~40 nm in size and imparts a cherry red color, which enables optimal visualization of results against a clear white background and is stable in dry and liquid forms However, purple colored colloidal gold sol particles if properly stabilized, can also be used in Rapid Diagnostic Tests Why are Variations in Band Appearance Commonly Observed in Membrane-based Rapid Diagnostic Tests Employed for Antigen Detection? The sensitivity/specificity of Rapid Diagnostic Tests primarily depends upon the detector and capture reagent pair Ideally, the detector reagent should be specific to one epitope of the analyte and the capture reagent specific to another epitope of the same analyte, thereby enabling two-site sandwich immunoassay To illustrate the same, please refer to Figure 22.6 Serology/Immunology FIGS 22.7A and B: A Band appearance due to avid antibodies B Band appearance due to less avid antibodies For higher analyte sensitivity, manufacturers of commercial Rapid Diagnostic Tests for antigen detection depend on the use of various combinations of capture reagents at the test and control band Avid capture reagents have a high affinity for the analyte When the sample containing the analyte reaches the avid capture reagent at the best band, due to high affinity, the avid reagent at the edge of the band captures most of the analyte Thus, resulting in a distinct thin colored line at the edge of the test band (Figs 22.7A and B) On the other hand, use of less avid capture reagent (lesser affinity for the analyte) results in capture of the analyte uniformly across the test or control band Thus, broader bands are generated by less avid antibodies Variations in band appearance in different assays is due to use of varying avidity of the antibodies at the test/ control band What is the Role of Sample Pad in Membranebased Rapid Diagnostic Tests? Rapid Diagnostic Tests enable detection of the analyte in several specimens such as urine, whole blood, serum or plasma However, the pH, viscosity, ionic concentraction, turbidity, and total protein content may vary from specimen to specimen Variations in these factors can cause alterations in the colloidal gold particles or the capture reagent leading to non-specific results For example, highly turbid specimens can cause invalid results since the particles from the specimen may block the membrane preventing sample flow Urine specimen becomes acidic on storage due to bacterial growth Due to a shift in the pH, the colloidal gold particles come together to form aggregates which may interfere in the performance of the test Rapid Diagnostic Tests incorporating serum as specimen may give false-positive results due to the presence of heterophillic antibodies These antibodies have multispecificity and bind the capture reagent to the detector reagent leading to false positive results Use for Rapid Diagnostic Tests incorporating heterophillic blocking reagents (HBR) is recommended to avoid this intereference 571 A sample pad with a bed volume of minimum retention capacity facilitates transfer of the entire specimen dispensed This not only ensures minimal wastage of specimen but also the excess specimen can be used to wash away unbound conjugate from the test region for better visualization of results Thus, use of sample pad that allows incorporation of buffer salts, stabilizers and HBR, to a large extent eliminates variation in pH, ionic concentration and interference of heterophillic antibodies What is the Role of Soak Pad in Membrane-based Rapid Diagnostic Test? Use of a soak pad with high bed volume is preferred in Rapid Diagnostic Tests because the total volume of specimen that enters the test assay can be increased This increased volume can be used to dislodge the conjugate as well as wash away the unbound/unreacted conjugate from the test region contributing to clearer background and better visualization of results Why “Faint Ghost Bands” Appear at the Test Region if the Device is Left Out on the Worktable? A common phenomenon observed in the device format is appearance of faint ghost bands at the test region after some time After completion of the test, if the device is exposed to warm ambient temperatures, evaporation occurs from the result window Due to evaportion, the excess sample along with unreacted/unbound conjugate from the soak pad flows back to reaction area This unreacted or unbound conjugate may then get deposited on the test band resulting in appearance of a “Faint Ghost Band” after sometime (Fig 22.8) Results must be recorded at the end of the recommended reaction time for correct interpretation FIG 22.8: Appearance of “Faint Ghost Band” 572 Concise Book of Medical Laboratory Technology: Methods and Interpretations How We Interpret “Broken Bands” at the Test/ Control Region? To prevent evaporation of the specimen from the test window, the membrane of the device is laminated with the help of a thin transparent tape Sometimes, during the process of lamination, air pockets may be formed between the membrane and the tape These air pockets prevent uniform sample flow, which may result in appearance of broken bands at the test/control region However, appearance of even a broken band at the test region indicates positive results In the following section, we shall discuss the role of hCG as a marker for diagnosing pregnancy and certain conditions that may give discrepant results Excess Sample Volume Dispensed Adding excess sample in no way improves the performance of the test The excess sample added, cannot be absorbed by the sample pad and thus flows out through the sides of the device Sometimes, the excess sample may flow out along with the conjugate The amount of the conjugate left in the device is insufficient to perform the assay, leading to invalid results Secondly, once the specimen flows through the device, the soak pad cannot retain the excess volume of the sample, which then may flow out through the sides of the device or may also flow back to the membrane along with unreacted/unbound conjugate This unreacted/ unbound conjugate may then deposit onto the membrane resulting in apparently discrepant results ENZYME IMMUNOASSAY Introduction An immunoassay can be defined as a qualitative or quantitative assay, which relies on the reaction between an antigen and its specific antibody The antigen being bound is called “ligand” and the antibody is the “binder” of the ligand Enzyme labeled conjugates were introduced first in 1966 for localization of antigens in tissues, as an alternative for fluorescent conjugates In 1971, enzymelabeled antigens and antibodies were developed as serological reagents for assay of antibodies and antigens Their versatility, sensitivity, simplicity, economy and absence of radiation hazard have made EIAs the most widely used procedure in clinical serology The availability of test kits and facility of automation have added to their popularity The enzyme-linked immunosorbent assay (ELISA), [Enzyme immunoassay (EIA) or solid-phase immunosorbent assay (SPIA)] is a sensitive laboratory method used to detect the presence of antigens (Ag) or antibodies (Ab) of interest in a wide variety biological sample Many variations in the methodology of the ELISA have evolved since its development in the 1960s, but the basic concept is still the immunological detection and quantitation of single or multiple Ag or Ab in a patient sample (usually serum) Classification of ELISA ELISA can be classified in different ways (Fig 22.16): Direct ELISA Direct ELISA is the most basic of ELISA configurations It is used to detect an Ag after it has been attached to the solid phase (e.g a membrane or dipstick) An Ab conjugated with a label (e.g HRPO, AP, FITC) is then incubated with the captured antigen After washing off excess conjugate and incubating with a substrate and chromogen, the presence of an expected color indicates a specific Ab-Ag interaction The conjugate could be a commercial preparation specific for the Ag of interest, or an in-house conjugated monoclonal or polyclonal Ab, or even patient serum (Fig 22.9) Indirect ELISA This is extensively used for the detection and/or titration of specific antibodies from serum samples The specificity of the assay is directed by the antigen on the solid phase, which may be highly purified and characterized The first, or primary Ab is incubated with the Ag, and then the excess is washed off A second or secondary Ab conjugate is then incubated with the samples The excess is again removed by washing For color to develop, a primary Ab that is specific for the Ag must have been present in the sample (e.g human serum, CSF or saliva) This indicates a positive reaction It is important, during assay FIG 22.9: Direct ELISA Index for pipettes 33 for screw-capped bottles 33 for selection 32 for test tubes 33 GLDH kinetic method 470 Globulin test 384 Glomerular filtration rate (GFR) 107 proteinuria 62 Glow 586 GL-related symptoms 645 Glucagon 447, 758 normal values 447 Glucocorticoids 738, 739, 775 Glucose 385, 490 chemical principles of procedure 72 homeostasis, impaired 438 oxidase methods 65 test strips, active 448 tests for 65 tolerance test 121, 435 Glutamyl transferase 536 Glycated hemoglobin 438, 439 Glycerin jelly solution 802 Glycerol 32 Glycine-HCL/EDTA elution 350 Glycogen and brunner-gland mucin 797 Glycohemoglobin 439, 442 in blood, determination of 443 Glycosuria with hyperglycemia 65 without hyperglycemia 65 Glycosylated haemoglobin (GHB) 438, 439, 442-444 kit 443 separation 444 Gold chloride solution 801, 802 Gomori’s aldehyde fuchsin stain 797 chromaffin stain 797 iron reaction 798 methenamine-silver nitrate technique 798 one-step trichrome stain 796 trichrome stain 202 Gonadotropin disorders 738 Goodpasture’s syndrome 103 Gradocol membranes See Collodion Gram stain 821, 872, 881 Gram-negative bacilli 865 cocci 839 intestinal bacilli 865 Gram-positive bacilli 842 cocci 837 Granular casts 99 to waxy cast 96 Granulocyte decreased 233 increased 233 Granulosus 167 Graph of particle’s size v/s signal color of colloidal gold sol 570f Graves’ disease 744 Gravity method, specific 211 Grayish marrow particles 231 Gridley’s stain for fungi 798 Grocott’s application to fungi 798 Grof’s method 475 Group O cells 319 reagent screen cells 319 Growth hormone (GH) 732 chemiluminescence immunoassay 733 disorders 738 GS junior applications 1015 system 1014 Guaiac test 70, 115 method 115 Guillain-Barré syndrome 385 Guinea worm 166 H H gene 318 H influenzae 410 Haematocrit, causes of reduced 213 Haemophilia, diagnosis of 305 Haemophilus 409, 410 aegyptius 869 ducreyi 869 influenzae 386, 869 Hair 885 Halogens 32 Ham’s serum test 253 Hand care 34 centrifuge 22 Handling roche reagent strips, procedures for 73 Hanging drop method 825 Hansen’s bacillus 845 Haptoglobin (HP) 697, 717 Harris’s alum hematoxylin 795 hematoxylin 813 for counterstain 802 Hartnup disease 84 Hashimoto’s thyroiditis 436 HB Bart’s 257 HB pattern (electrophoresis) 257 HB pipette 210f 1043 HBH disease blood picture, diagnosis of 257 diagnosis of 257 HBH inclusions, demonstration of 257 HBSAG II quant 926 virucheck device, test for 664 hCG 682 assays, strategies of 682 estimation 682 forms of 682 serum/plasma, normal values of 411 hCV flavicheck device 665 hCV infection 667 HDL cholesterol 484 PPT set 485 HDN, illustration of 341f HE4 935 Healthcare personalized 897 personnel 15 professionals, coagulation monitoring for 969 Health-disease-health cycle 2f Heart disease 410 Heat and sulfosalicylic acid 61 dry heat, sterilization by 29 elution 351 instability test 256 moist heat, sterilization by 29 sterilization by 29 Heavy metals description 91 Heidelberger-Kendall curve 707f Heidenhain’s aniline blue stain 796 iron 796 hematoxylin staining method for intestinal protozoa 202 Heinz bodies demonstration of 256 presence of 238 Hemacytometers 401 Hemagglutination 700 Hematocrit definition 212 methods 212 Hematocrit/packed cell volume (PCV) 212 Hematologic investigations, routine 268 Hematology 43t, 466 analyzers in nutshell, basics of 225 automation in 225 bleeding disorders 272 clinical 205 control chart 270 cusum analysis 270 duplicate tests 270 1044 Concise Book of Medical Laboratory Technology: Methods and Interpretations external quality assessment 270 inbuilt quality control 270 internal quality control 270 plain tube 466 quality control in 270 sodium citrate 466 testing control sample 270 Hematoma 206, 365 Hematopathology 1004 Hematoxylin 794 and eosin stain, routine 795 shorr S3 stain for inclusion bodies 798 stain 202 counterstains for 795 Hematuria 371 Hematuria See Blood in urine Hemoglobin (Hb) 210, 231 electrophoresis 254, 257 estimation 210 fraction 444 structure, diagnosis of 254 values, normal 211 Hemoglobinopathy blood picture 256 special tests 256 Hemograms Hemolysate, preparation of 444, 245f Hemolysis 375, 580 cause of 372 evidences of 252 presence of 371 Hemolytic anemias 212, 252 causes of 252 classification of 252 Hemolytic attacks in MT malarias, acute 144 Hemolytic disease of newborn (HDN) 331, 341, 348 diagnosis of 372 Hemopexin 697 Hemophilia B 305 Hemorrhagic disease of newborn 305 pleural fluid 390 Hemostar 305, 305f XF 306, 306f Hemostasis testing 949 Hemothorax 390 Henderson-hasselbalch equation 557 Heparin 207 therapy 277 Hepatic disease (alcoholic) 545 Hepatitis A profile 459 B profile 459 B surface antigen, slide test for 662 C profile 459 C virus (HCV) 665 D profile 459 E profile 459 Hepatobiliary system 456 Hepatocellular disease 535 Hereditary elliptocytosis 253 metabolic disorders 84 spherocytosis 252 Herpes simplex virus (1 + 2) infections 667 Heterogeneous ELISA 576 immunoassays 701 Heterophilic antibodies 566, 583 blocking reagents (HBR) 571 Hickey-Hare test 737 Hippuric acid test 458 Hiss’s method 824 Hiss’s serum water sugars 828 Histamine provocative test 778 Histocenter (shandon) 806 Histopathology 791 automation in 805 Histoplasma capsulatum 408, 884 infection with 890 Histoplasmin test 890 HIV combi PT 4th generation (AG+AB test) 927 HIV-AIDS and severe acute respiratory syndrome HLA typing, principle of 565 Hodgkin’s disease 270, 523, 524, 525, 725 Homogeneous ELISA 576 fluorescence polarization immunoassay (FPIA) 576 immunoassays 701 Homogentisic acid See Alkaptonuria Hook effect 583 Hookworm 156, 160 infection 154 Hormonal tests for diagnosing cause of anovulation 767 Hormone 759, 760 balancing act 760 free 743 from testicles, feedback 760 secreted by pituitary gland 732 therapy 262 Horseradish peroxidase (HRP) 603 Hospital aquired infections 984 Hospital information systems, communication with 919 Hospitality industry 36 Hot air oven 26, 27f Howell-Jolly bodies 234f, 238 presence of 238 HPRL levels decreased in 787 increased in 787 Human AB, class of 573 antimouse antibodies (HAMA) 787 chorionic gonadotropin (hCG) 787 cytogenetics 891 D (rho) antigen 333 male karytype 893f plasma proteins 695 saliva, status in 324 Hyaline 95 cast 96, 98 in urine 96f normal value 98 to finely granular cyst 96 Hybridomas 319 Hydatid disease causing of 172 diagnosis of 890 echinococcus granulosus 168 Hydrochloric acid solution, normal 803 Hydrolases 522 Hydrops fetalis 257 Hymenolepiasis diminuta 173 hymenolepis nana 168 nana 167 Hyperaldosteronism primary 777 secondary 777 Hyperbilirubinemia 455, 580 causes of 68, 477 Hypercalcemia (increased total calcium) 496 Hypercenter (shandon) 805 Hyperchromatism 236 Hyperchromia 236 Hypercut (shandon) 806 Hyperglobulinemia 274 Hypergranular promyelocytic leukemia 265 Hyperkalemia 560 Hypernatremia 560 Hyperparathyroidism 754 Hyperphosphatemia 499 Hyperprolactinemia 761, 774 causes for 774 symptoms of 774 Hyperthyroid 744 Hypertonic saline test 737 Hyperventilation 367 Hyphae 883 Hypoalbuminemia, causes of 480 Hypocalcemia 496 Hypochromatism 236 Hypochromia 236 Hypochromic anemia 428 Index Hypoglycemia 448 causes of 448 induced 448 Hypogonadotropic hypopituitarism 761 Hypokalemia 561 Hyponatremia 560, 737 Hypoparathyroidism 754 Hypophosphatemia 499 Hypopituitarism resulting in multiple deficiencies, causes of 732 single deficiencies, causes of 732 Hyposthenuric 59 Hypothalamic-hypophyseal portal system 732 Hypothalamus 738 Hypothyroid 744 patient 745 Hypothyroidism 751, 761 primary 744 secondary 744 subclinical 744 I IAT, applications of 343 Icons used 34 ICT pregnancy test 417 techniques for urine 418 urine 419 Icterus index 477 Idiopathic thrombocytopenic purpura (ITP) 272 IEP analysis of human serum 695 IG classes 724 structure 724 IGD class 717 IGE class 717 IGG antibodies to dengue virus 639 IGM and IGG antibodies, distinguish between 349 class 717 response, detectable 634 IHC detection 1001 IHC/ISH detection 1001 Immune haemolytic anemia (IHA) 376 diseases 341 Immunoassays 700 for FSH 768 for LH 768 for PRL 768 Immunochromatographic device 118 techniques 568 Immunodiffusion of oudin, single linear 698 technique double 700 single 700 Immunodominant epitopes 563 Immunoelectrophoresis 700 application of 697 Grabar and Williams’ method of 693 Immunoenzymometric assay 599, 604, 734, 788 sandwich sequential (streptavidin-biotin) ELISA 602 streptavidin-biotin, ELISA 598, 604 sequential assay 602 Immunofluorescence 818 direct 818 indirect 818 Immunoglobulins 716, 724 A (IGA) 725 G (IGG) 725 M (IGM) 725 Immunohematology See Blood banking Immunologic basic for skin tests 886 methods 412 tests for pregnancy 412 Immunological reactions 564 used to differentiate 565f Immunology 563 basic 563 diagnostic 693 Immunosuppresive drugs See Antiinflammatory drugs Immunoturbidimetry tests 729 Immutex 643 Impulse 9.0 enhanced pulse chemilunescence system 587f Inappropriate cellular metabolism 560 India ink method 824 Indoxyl acetate 697 Infections 721 Infectious diseases 272, 898 continuum of care in 898 Infectious mononucleosis diagnosis of 264 test for 643 Infective material, contamination from 14 Inform her2 dual ISH DNA probe cocktail assay 1003 Inhibin 764 Inoculation, method of 831 INR system advantages of 291 disadvantages of 292 Insulin 445, 757 dependent diabetes mellitus 436 functions of 758 microplate CIA 788 1045 normal values 445 test significance 445 Intensive management improves glycemic control 453 Interferences in immunoassays 565, 579 Intermittent heat 30 International unit 42 Intestinal canal, Amebae of 181 cilliate 132 flagellates 131 protozoa cultivation of 203 diseases of man 125t of man 124 roundworms of man, common 154 Intracellular growth 675 Intracorpuscular defects 252 Intracutaneous injection 886 Intradermal tests 204 Intrathoracic masses liver, aspiration of 810 lung, aspiration of 810 aspiration of 810 Intrinsic pituitary disease 732 Iodamoeba butschlii 124 Iodine solution stain 881 use of 121 Ion exchange resin method 443 Ionic strength 708 Ionized calcium decreased 496 increased 496 Ionizing radiations 31 Iron binding capacity deficiency anemia 248 Iscan coreo slide scanner 1009 Isolated hypoaldosteronism 777 Isopropanolol precipitation test 256 Isotope 54 ITO-reenstierna test 889 IV insulin tolerance test 442 tolbutamide test 442 IVYS’s method 274 J Jamshidi’s marrow aspiration needles 231 Jaundice absent 458 classification of causes of 454 present 458 Jenner’s stains 223 1046 Concise Book of Medical Laboratory Technology: Methods and Interpretations K Kala-azar 146, 148 Kallmann’s syndrome 762 Kaplow’s method 265, 266 Karyotyping 893 Kato cellophane thick smear technique 201 Kelling’s test 426 Ketogenic steroids 775 Ketone bodies in urine 66 tests for 66 Ketone, chemical principles of procedure 72 Ketonuria, causes of 66 Ketosteroid excretion 775 Ketur test, boehringer 66 Kidney, functions of 104 Killing organisms by heat 29 Kinetic methods, biochemical reactions measurements of 703 of antigen–antibody reaction 564 reaction 710 Kinyoun’s acid-fast stain 798 Kit composition 303 Klebsiella 34, 409, 833 ozaenae 867 pneumonia See Klebsiella species rhinoscleromatis 867 species 866 Klima’s needle 231 Klinefelter’s ovarian failure 786 syndrome 397, 399, 762, 780, 785 Koch’s postulates 821 steamer 30 Koch-week bacillus See Haemophilus aegyptius Koneff’s stain for bone and cartilage 797 Kveim-siltzbach test 890 L L braziliensis 146 Laboratory instruments 17 Laboratory safety, basic Laboratory set-up requirements for 812 Lactic dehydrogenase 538 Lactobacillaceae 653 Lactophenol cotton blue 824 Lactose fermenters 831 Laennec’s cirrhosis 697 Lambert’s law 461 Lange’s colloidal gold test 385, 386f Langerhans’ islet cells 789 Larvae in focal lesions 154 Latex agglutination inhibition method 412 tests 700 Latex particle agglutination immunoassay (LPAIA) 576 Latex slide test for VDRL syphfinal 615 Latex VDRL reagent 615 Latum 167 LDL cholesterol calculation of 485 fully enzymatic, colorimetric test 487 precipitation of 484 Le (shandon) 806 Lead poisoning, diagnosis of 254 Lecithinase 431 Lee-brown’s modification 796 Legal’s test 66 Legionellae 38 Leishman’s stain 223, 224 Leishmania braziliensis 145 donovani 145, 148 tropica 145 caused by 149 Leishmaniasis 147 causing 183 Lepra 678 bacilli 674 cells 845 Leprosy 845 Leptocheck-WB 642, 645 Leptocytes/target cells 236 Leptocytosis 236, 239 Leptospira 645, 871 genus 645 icterohaemorrhagiae 871 species of 645 Leptospirosis 645 Leucine 99 aminopeptidase 460 spheres 100f Leukemia acute 268 diagnosis of 265 from leukemoid reaction 268 type Leukemoid reactions 262 eosinophilic 263 lymphocytic 262 neutrophilic 262 Leukocyte 98 casts 96, 97f count, differential 231 Leukocytic morphology 207 Leukopoiesis 228, 229f Levaditi’s method for staining spirochetes in blocks 798 Leydig cells 779 Light exposure to 464 green solution 800, 803 source 462, 464 Lightcycler® 2.0 instrument 991 Lightcycler® MRSA advanced test 993 Lightcycler® septifast test 992 Lightcycler® systems 990 Linistain GLX (shandon) 806 Lipid-protein double staining 697 Lipolytic enzymes 431 Lipopolysaccharides 564 Lipoprotein 716, 728 A 716 stains 696 Liquefied and compressed gases Liquid handling systems 591 plasma 371 Liver battery serum 459 disease 524, 526, 536, 546, 725 chronic 726 function tests 454, 458 classification 454 synthesis in 457 tests of excretion 454 Lobe, anterior 732 Loeffler’s method 824 methylene blue 821 serum slopes 828 Loiasis 162 Long-acting thyroid stimulator (LATS) 744 Low ionic medium polybrene indirect anti human globulin test 345 salt solution for serological applications 359 strength solution phase indirect anti human globulin test 345 Low prothrombin in absence of jaundice 457 presence of jaundice 457 Lowenstein-Jensen media panel MTB sensitivity tests 861 medium 831 slant 858 Lugol’s solution 791 Lumbar CSF in adults, normal values for 382 Lumbar puncture 382 Index needle 382 complications of 383 Luna’s mast cell stain 797 Lung abscess 409 cancer diagnostic solutions 1005 differential diagnosis in 936 disease, obstructive 551 erythematosus cell 264 stones See Broncholiths Luteinizing hormone (LH) 89, 763, 785 Lymphoblast 229 Lymphocyte 261, 384 increased 233 large 230 morphologic forms of 261 small 230 Lymphocytic leukemia (CLL), chronic 268 series 229, 230f Lymphocytosis 260 acute infections 260 endocrine disorders 260 neoplasms 261 Lymphocytotoxicity test, mixed 565 Lymphogranuloma venereum, diagnosis of 890 Lymphoid organs, secondary 568 Lymphopenia 262 Lymphoreticular tissue, disorder of 270 Lysozyme 566 effect of 583 M M tuberculosis 836 MacConkey’s agar 829 medium 831 Macrocytes 237 Macrocytosis 237 Macrogametocyte (female) 134 Magnesium 506 calculations 507 calmagite method 506 clinical relevance 507 deficiency symptoms 507 in urine 508 normal values 507 ribonucleate 821 sample material 507 toxic level symptoms 507 values 507 Malabsorption methods of assessing 121 tests for 432 Malaria 184 acute phase, pathogenesis of 141 blackwater fever, pathogenesis of 144 chronic phase, pathogenesis of 142 complications and sequelae, pathogenesis of 143 infections in human 647 negative for 649 pathogenesis of 140, 141, 142, 143, 144 positive for 649 species identification in mosquito— pigment in oocysts 185 Malarial parasites life cycle of 139 of man 134 stained by leishman of giemsa, morphology of 135 stained in thin films, morphology of 136 Malarial pigment 794 Malayan filariasis 162 Male fertility 760 hormone system 760 Malignant tumor 722 definitions of 679 Mallory’s aniline blue collagen stain 796 blue stain 796 phosphomolybdic acid 796 phosphotungstic acid 796 hematoxylin stain 799 reaction for iron, modification of 798 Mallory-Weiss syndrome 114 Mandler filters 32 Mantoux test 889 MAPLAB plus 512, 512f technical features of 512 Maple syrup disease 84 Marrow film preparation 231 Massive metabolic derangement 65 Masson’s trichrome stain 800 Matrix effects 582 Mature neutrophil, development of 228 Maximal tubular capacity 108 Mayer’s egg albumin 793, 808 hematoxylin 265, 402, 795 mucicarmine stain 797, 804 May-Grünwald-Giemsa (MGG) stain 813 Mean cell hemoglobin (MCH) 217 concentration (MCHC) 217 Mean cell volume (MCV) 216 Media for growth of anaerobes 830 for identification of fungi 828 quality control of 872 sources of 872 Medical laboratories, signs for Medical parasites 124 Medicolegal aspects of clinical practice 17 Megakaryocytes 230, 232, 249 decreased 233 increased 233 Megaloblastic anemias 251 macrocytic anemias 249 Meiotic cell division 892f Melanin pigment 794 Melanocyte-stimulating hormone 736 Melitensis 635, 638 Membrane rapid diagnostic tests, performance of 569 Membrane-based rapid diagnostic tests 568 employed for antigen detection 570 principles of 568 Meningitis 721 Menstrual cycle 765f patterns, types of 765 periods, irregular 766 Menstruation 272 Menu driven operation 559 Mercury 91, 92 precipitate 794 Merthiolate-iodine-formalin (MIF) 201 Metabolic acidemia 555 alkalemia 555 alkalemia See Nonrespiratory alkalemia Metallic salts 32 Metamyelocyte 229 Metanil yellow solution 804 Metastasis, definitions of 679 Metastatic See Bone cancer Metastatic bone disease 526 cancer 458 Methemoglobin reduction test 240 method 240 reagents 240 Methylene blue solution 805 Metyrapone, tests with 741 Microalbuminuria 62, 63 definition 62 detection 63 Microbiology 819 general instructions for 837 Microcell methods 401 Microcytes 237 Microcytosis 237 Microfilaria 103f concentration of 204 1047 1048 Concise Book of Medical Laboratory Technology: Methods and Interpretations Microgametocyte (male) 134 Microhematocrit 212 centrifuge 23 parts of 23f Microparticle enzyme immunoassay (MEIA) 576 Microprocessor-based automation 558 Microscope objectives 19f Microscope optics 19 Microscope, parts of 18 Microscopic examination of stool specimens 121 Microsedimentation (Landau) method 220 Microsporum 883 Microtainers 208 Microtatobiotes (smallest living things) 819 Miliary tuberculosis 675 Milk-alkali syndrome 526 Milky fluid 390 Mineral salt solution 831 Mineralocorticoids 738, 775 disorders of 777 Mitotic cell division of 891 MNPT 291 Mod Gomorri’s method 498 Mod jendrassik method 475 Mod King’s method 527 Modular® pre-analytics EVO 912 Module sample buffer 903 Moist heat boiling 29 inspissation 31 steam 30 temperature 29 Moisture 464 Molar solutions 53 Mole (mol) 41 Molecular diagnostics 974 Molybdate UV method 497 Monoclonal antibodies, use of 681 Monoclonal blood typing antibodies for slide and modified tube tests 336 tube tests 335, 338 Monoclonal capture 772 gammopathies, causes of 481 Monocular microscope with substage lamp 18f Monocytes 384 Monocytic leukemia 265 series 230 Monocytosis 261 collagen diseases 261 infections 261 neoplasms 261 Morax-axenfeld bacillus See Moraxella lacunae Moraxella 833 lacunae 869 Morphologic identification, importance of 124 Motility of bacteria 825 semisolid agar 881 Motor-driven centrifuge 23 with RPM 23f Mounting and preservation of films 225 Mouth-to-mouth respiration 14f Mucicarmine stain solution 804 Mucin clot test 388 Mucolytic 848 reagent, preparation of 849 Mucoproteins, stains for 797 Mucor 885 Mucoraceae 38 Mucoviscidosis 433 Mucus 114 in stool, causes of 114f Mueller hinton agar 829 Multi drug resistant TB (MDRTB) 676 Multidimensional modularity 902 Multiplate® analyzer 949 Multiple marker testing (MMT) 681 Multiplex PCR 590 Multistix® configurations 77t Multistix® urinalysis strips 77 Multivalent antisera 695 Mumps and herpes simplex tests 890 Muscle biopsy for trichinella spiralis 204 Myalgia symptoms 645 Mycobacteria 406, 843 atypical 674 balnei 845 causing skin ulcers 674 cultures 843 leprae 845 lepraemurium 845 morphology 843 paratuberculosis 845 strains of 843 susceptibility testing of 860 ulcerans 845 Mycobacterial culture 429 Mycobacterial infections, serodiagnosis of 678 Mycobacterium 673 balnei 845 butyricum 845 fortuitum 844 kansasii 853 leprae 845 lepraemurium 845 paratuberculosis 845 smegmatis 845 tuberculosis 38, 673, 678, 832, 833, 836, 845, 846, 857, 859, 982 infection, diagnosis of 846 isolation, medium for 855 positive for antibodies to 677 ulcerans 845 Mycological methods 885 Mycology 883 Mycoses, deep or systemic 884 Mycotic (fungal) disease 408 Myeloblast 228 Myeloblastic leukemia with maturation 264 without maturation 264 Myelocytes 229, 268 Myeloid leukemia, chronic 268 series 228 Myeloma, multiple 269 Myelomonocytic cells 268 leukemia 265 classification of acute 264 Myeloperoxidase 266 Myeloproliferative disorders 274 Myelosclerosis 269 Myocardial infarction 722 diagnosis of 967 indication of 543 N N catarrhalis 839 N meningitidis 839 Nail 885 NaOH reagent 531 Naphthol ASBI phosphate method for acid phosphatase 815 Napier’s aldehyde test 125 Nasal smear 836 Necator americanus 154 Needles 34 choice of 33 Neisser’s method, modified 823 Neisseria 839 catarrhalis 836, 839 gonorrhoeae 839 meningitidis 839 reaction of 828 Nematoda 188 Neonatal thyroxine 743 Neoplasm, definitions of 679 Nephrotic syndrome 540, 560 Neubauer hemacytometer 402 Neurohypophysis See Posterior pituitary Neuromuscular disease 552 Index Neutral formaldehyde 215 Neutropenia 262, 263 causes of 263 drugs, causes of 263 Neutrophil alkaline phosphatase 265 Neutrophilic leukocytes 94 Neutrophils 384 Nicotine stimulation 737 Nimblegen sequence capture 1015 Nine-unit laboratory cell counter 231f Nipple 808 Nitric acid method 791 Nitrite procedure 71 Nitrite/bacteria 71 Nitroblue tetrazolium 578 Nitrocellulose membrane affect sensitivity of rapid diagnostic tests 569 Nocardia 884 asteroides 408 species See Mycobacterium kansasii Noise ratio, signal 594 Non-B hepatitis (NANBH) 665 Nonbacterial regional lymphadenitis test 890 Nondiabetic 67 Nonglucose sugars in urine 66 Noninsulin-dependent diabetes mellitus 436 Nonpathogenic intestinal amebae 133 Non-prostatic ACP assay 529 Nonrenal causes of proteinuria 61 Nonrespiratory acidemia metabolic acidemia 554 alkalemia 554 Nonspecific esterase 267 method for 816 Nonthyroidal illness (NTI) 744, 750 Non-treponema antilipoidal antibodies 623 Normoblast 234f decreased 233 increased 233 intermediate 228 late 228 Normocytic anemias 234 NT-probnp 932 Nuclear fast red, kernechtrot solution 802 stain 801 solution 805 Nucleases 431 Nucleic acid 564 purification made simple 978 Nutrient agar 827, 828 broth 827 Nutritional megaloblastic anemia 428 O P O group red blood cells, sensitization of 352 O2 capacity 551 Obstructive disease, chronic 552 Obstructive lung disease, chronic 552 Occult blood 116 tests for 115 OCPC method 493 Oil immersion objective 20f red O fat stain 797, 802 method for lipids 817 solution 802 Oliguria 59 Onchocerciasis 162 Oncology tests 984 Operation, range of 559 Optical density 462 Optiview DAB IHC detection 1002 Oral glucose tolerance test (OGTT) 440 Orange stain 881 Organisms, storage of 837 Oriental sore 146, 149 Orthostatic proteinuria, collecting specimen for 62 Orthotolidine 116 Osmium tetroxide stain for fat 797 Osmometry 59 Otorrhea 383 Ouchterlony, double diffusion method of 693, 694 Oval fat body 99f and fatty casts 99 Ovalocytes 236 Ovarian cancer 685 care 935 dysgenesis 89, 785 failure, detecting 767 Ovary 758 Owren’s buffer 299 Oxalates 207 bulbs, making double 207 double 207 Oxalate-fluoride 208 Oxalic acid method 844 Oxygen content 552 partial pressure of 552 saturation 551 Oxytocin 737 Oxyuris vermicularis 157 P falciparum 647 malaria 649 P malariae 647 P ovale 647 P vivax 647 malaria 647, 649 P westermani 122 Paget’s disease 526, 527, 528 Pancreas 757 carcinoma of 431 Pancreatic cancer 432, 685 disease 65 function tests 430 juice chemical characteristics of 430 composition of 430 Pancreatitis acute 431, 432 chronic 431, 432 tests in acute 432 Pancytopenia 259 blood picture 259 causes 259 Pandy’s test 384 Panhypopituitarism 762 Papanicolaou’s method of staining smears 808 stain 808 with EA-36 813 Paper strip method 60 strips 70 Paracolon bacilli 867 Paradysenteriae 868 Paragonimiasis 178 Paragonimus westermani 177, 406 Paraproteinemias 269 causes 269 Pararosanilin HCl-stock solution 816 Parasites 101, 201 Parasitic diseases 260 ova 101 worms, extracts of 889 Parathyroid 754 hormone 756 Paratyphoid 867 Parenteral administration of epinephrine 442 glucagon 442 Paroxysmal nocturnal hemoglobinuria 253 Pars intermedia See Intermediate lobe Parson’s initiation 37 1049 1050 Concise Book of Medical Laboratory Technology: Methods and Interpretations pipttes 26f stain for negri bodies 798 Pasteurella pestis 868 tularensis 636, 638, 869 Patch tests 886 Pathogenic clostridia 840 mycobacteria 844 Paul bunnel test for heterophile antibody 264 PCR differential 590 types of 590 PCT and tina-quant® CRP 945 Pediatric fever 721, 723 PEG precipitation method 485 PEG/CHOD-PAP method 484 PEG-enhanced indirect anti-human globulin test 345 Penicillin hypersensitivity 889 Penicillium 885 species 38 Pentose 66 Pentosuria 84 Peptone water 827 Percent solutions 52 Pericardial fluid (PF) 391 aspiration 392 indications for 391 presence of 391t Periodic acid 266 schiff (PAS) reaction 266, 797, 803 Periodic table of elements 54, 55 Peripheral blood 205, 248 findings in vitamin B12 249 smear 223f Peripheral smears 207, 248, 253 Peritoneal fluid 392 presence of 392t Pernicious anemia See Vitiligo Peroxidase 266 reaction 697 stain 817 system 584 Personalized lab automation 909 Petroff’s method 844 pH 114 blood 533 chemical principles of procedure 72 normal values 114 reaction 708 reagent 73 semen 400 urine 58t Phenol group 32 red test 105 red test for residual urine 106 Phentolamine blocking test 779 Phenylketonuria 84 Philadelphia chromosome 891 Phloxine toluidine blue stain for malarial parasites 798 Phosphatases 522 buffer solution, preparation of 893 specimen 523 Phospholipase 431 Phosphomolybdic acid solution 801 phosphotungstic acid solution 800 Phosphorus 497, 498 levels decreased 499 increased 499 Phosphotungstic acid hematoxylin 799 Photochromogens 820 Photoelectric cell 463 Photometer 462 Photometry, sources of error in 463 Photons 585 Phycomycetes 408 Phytohemagglutinin 892 Pickwickian syndrome 551 Pigments and minerals, stains for 798 Pin worm 157 Pineal gland 759 Pinworm infection 154 Pipette basics 591 classification of 591 combined 592 fixed 592 techniques 592 variable 592 Pirquet reaction 889 Pitfalls of laborartory diagnosis 676 Pituitary anterior 732 disorders, extrinsic 732 gland 731, 732f hormones 761 anterior 735 hyposecretion, anterior 545 system, disorders of 738 Plasma 371, 472 cells, increased 233 cortisol 739 factors 222 fibrinogen 205 layer 213 normal values 472 trepolisa 3.0 624 Plasmid nematodes 156 Plasmodium falciparum infection 246 Plastic pipette 591 Plasticine 212 Platelets 215, 231 abnormal distribution of 216 action of 277 count for reesecker method 215 count, raised 216 disorders 272 functional 273 free plasma See Platelet poor plasma increased destruction of 216 mature 230 method 215 on RBCs 238 poor plasma 278 production failure, causes of 216 production, control of 230 Pleural fluid 389 presence of 390t Pneumococci 839 morphology 839 Pneumoconiosis 410 Pneumonia 409 PNPP kinetic method See Alkaline phosphatase Poikilocytes 237 Poikilocytosis 234f, 237 Poisoning symptoms 92 treatment 92 Pollen 101f Polychromatophils 236 Polychromatosis 236 Polyclonal antibodies, use of 680 blood typing antibodies for slide and modified tube tests 333 gammopathies, disorders associated with 480 reagent 319 Polycystic ovarian disease 785 Polycythemia 212 causes 212 primary 212 secondary 212 vera 269 Polymerase chain reaction 587, 588f Polymyalgia rheumatica 721 Polyploidy 895 Polysaccharides 564 Polyuria 59 Polyvinyl alcohol method of brooke and goldman 201 Pork tapeworm 170 Porphyria, acute 84 Porphyrias 69 Porphyrin metabolism, abnormal 84 Porphyrins 69 Index causes 69 interfering factors 70 levels of 70 normal values 69 porphyria 70 Postanalytical variables 581 Postcoital test 403 Posterior pituitary 736 Postexposure care 16 collection of specimen 17 containing spills 16 first aid 16 initial consultation 16 report 16 transport of specimen 17 Postglomerular proteinuria 62 Postoperative surgery, adults 721 Potassium 89 cyanide method 89 normal values 89 oxalate 207 Potency titration, complement 353t Povidone-iodine 206 solution 395 Power on display of instrument 451f P-phenylenediamine 697 Prausnitz-Kustner reactions 888 Preanalytical variables 566, 579 Precautionary measures 14 Precision autoclave 30f Preeclampsia 943 Pregnancy direct latex agglutination method, slide test for 414 interpretation of results 414 material provided with kit 413 accessories pack 413 reagent pack 413 slide test for 412, 414 specimen collection and preparation, slide test for 415 test 3, 411, 766 procedure 414 qualitative method 414 semi-quantitative method 414 Pregnanediol 89 method 90 normal values 89 Pregnanetriol 90 method 90 normal values 90 Pretesticular function 760 Procoagulant deficiency, diagnosis of 303 Proficiency surveillance 271 Progesterone 782 abnormalities 759 normal values 782 withdrawal test 766 PROGRP 936 Proinsulin products 757f Prolactin 736 disorders 738 hormone (PRL) 787 sequential method 602 Prolonged APTT, causes of 296 Promegakaryocyte 230 Promyelocyte 229, 268 Pronormoblast 228 Proper pipetting skills 592 Propionibacteria species 35 Prostate cancer 685 diagnostics 1003 specific antigen (PSA) 681, 685 Prostatic acid phosphates (PAP) 686 Protein binding 746 chemical principles of procedure 72 effect of 583 electrophoresis 460 of CSF 385 in urine, quantitative estimation of 60 loss 121 role of carrier 743 stains 696 Proteinase inhibitor 717 Proteinuria 61 interpretation of 61 mechanisms of 62 minimal 61 moderate 61 Proteolytic enzymes 398, 431 Proteus 833 identification of 825 vulgaris 866 Prothrombin concentration 457 determination (two-stage method) 293 group 305 time 283, 310 Protocols and blood coagulation tests 277 Protophyta 819 Prozone effect 707 phenomenon 610 PSA diagnosis, issues of 681 ratio testing, advantages of 682 Pseudoagglutination See Rouleaux Pseudomonadaceae 870 Pseudomonas 35, 409, 410, 833 aeruginosa 38, 870 pyocyaneus 821 Pseudopelger cells 268 Puberty, delayed 762 Pulmonary alveolar proteinosis 410 cancer 685 embolism 410 infection 722, 723 TB, clinical manifestation of 675 Puncture injury 209 Pus 114, 885 cells See Neutrophilic leukocytes cells in urine 94f swabs 836 Q Q banding 894 Queckenstedt’s test 383 Quinacrine banding technique See Q banding R RA cells 391 Rabbit monoclonal 1006 antibody 1003 Rack rotor 905 Radial immunodiffusion 701 single 698, 699 Radioimmunoassay 585, 586, 590 measurement of radioactivity 591 Random access autoanalyzer 510, 546 Rapid diagnostic tests, construction of 569f Rapid plasma regain card test/carbon antigen for syphilis testing 618 interpretation of test results 619 material provided with RPR kit 619 qualitative method 619 quantitative method 619 reagent storage and stability 618 sample collection and storage 618 Rapid test for IGM 639 antibodies to leptospira 645 malaria PAN/PV/PF 647 simultaneous 671 Rat ovarian hyperemia test 411 Rayleigh scattering 704 Rayleigh-Debye scattering 704 RBC abnormalities 236t cast 96f in urine 96f content, causes of abnormal 237 morphological alterations in 234f morphology 234 1051 1052 Concise Book of Medical Laboratory Technology: Methods and Interpretations normal values 234 usage 235 pipette 215f size, causes of abnormalities of 237 Reagent 72, 377 bilirubin 72 blood 72 for chequerboard titration 351 for quantitative estimation of fibrinogen 299 for RH(D) grouping 332 glucose 72 ketone 72 manager 903 preparation 600 procedure 352 protein 73 specific gravity 72 urobilinogen 73 Real sample blanking system 710t Real-time PCR 590 Recapitulation 187 morphological differentiation 186 protozoa inhibiting intestine 182 worm infections pathogenesis of 191 pathology of 191 Red blood cell (RBC) 93, 98, 205, 214, 215, 217, 348 abnormalities on stained smear 238 diluting fluid 214 in urine 94f interpretation 215 method 215 morphological types of 234 Red blood cells See Hemolysis Red cell 97 anemia, increased destruction of 212 casts 95, 97 damage to 252 factors 222 for serological applications 328 fragility test 239 increased 98 lifespan of 252 reagents 319 suspensions, preparation of 319 Reflotron® plus system 971f sprint system 971f plus system 971 sprint system 971 Regular menstrual period 765 Reiter cells 389 Reitman and Frankel’s method 530 Renal diseases 105 function 104 impaired 104 tests glance 107t tests of 107 physiology in brief 104 plasma flow (RPF) 107 tubular epithelial cell 94, 95f casts in urine 97f tubule, casts in 95f Renin-angiotensin system 738 Repetitive pipetting 592 Respiratory acidemia 554, 555 alkalemia 554, 555 disorders, common 406 Reticulocyte 228 count 235 method 235 normal values 235 stain 235 large basophilic 234f Retroquick-HIV 669 test 670 Retroscreen-HIV 671, 672 device, pouch of 673 kit 672 Reverse cholesterol transport (RCT) 728 Reverse pipetting 592 Reverse transcriptase-PCR 590 Reye’s syndrome 535, 545 RF calibration curve, method for preparation of 719 Rh antibodies 332 blood group system 331 DU antigen 332 factor 331 importance of 331 typing 376 delayed 377 false results negative 376 positive 376 hemolysis of red blood cells 377 Rh-D grouping procedures 332 negative 332 positive 332 Rheumatic diseases 723 disorders with latex agglutination 660 Rheumatoid arthritis 428, 718 adult 721 Rheumatoid factors 566 effect of 583 slide test for 656 Rheumatology 721 Ribonuclease 431 Ring test 693 Robertson’s cooked meat medium 831 Roche dialog 1017 for molecular diagnostics, solutions from 974 hitachi 911 chemistry analyzer 546, 547f pipeline 898 Ropes’ test See Mucin clot test Ross jones test 384 Rotary microtome 793f Rothera’s test 66 Rouleaux 372 causes of 372 formation 238 Round bottomed flask 24 Roundworm See Ascaris lumbricoides Routine hemostasis laboratory, quality assurance for 278 Rubella IGM and IGG 930 infection 667 Rumpel-Leede sign 272 S S paratyphi 624 A 624, 631 AO 624 BO 624 S typhi 624, 631 H 624 O 624 S viridans 839 Sabouraud’s agar 883, 885 soft 884 Sabouraud’s dextrose agar 829 glucose agar 828 Sahli’s hemoglobinometer 210, 210f method 210 pipette 216 Saline cross-match 368 phase indirect anti-human globulin test 344 tube method 368 use of 121 Saliva constituents, normal 425 Salmonella 833 antigen suspensions 631 enteritidis 868 food poisoning, diagnosis of 868 paratyphi 867 typhi 867 typhimurium 868 Sand and paper pulp filter 32 Sandwich immunoassay, two-site 570f Saprophytic Index acid-fast bacilli 853 mycobacteria 674 Sarcoidosis, diagnosis of 890 Saturated solution 54 Schaudinn’s fixative 202 Schiff’s leuko-fuchsin solution 803 test for 803 Schiff’s reagent 266 Schistocytes 237 Schistocytosis 239 Schistocytosis See Schizocytosis Schistosoma haematobium 101, 103f, 177 japonicum 177 mansoni 177 Schistosomiasis 178 Schizocytosis 237 Schlesinger’s test 68 Schültz-Charlton reaction 887 Schumm’s test 372 Sciences, imaging Screenmaster 3000 511 Seat worm 157 Secretin test 432 Sedimented cells 370 Segmented neutrophil 229 Seitz 29 filter 31, 31f Selenium 91, 92 Sella turcica 731 Semen analysis 398, 400 pH 400 volume 400 Semi-autoanalyzers 510 Semiautomated analyzers 110, 714 Semi-quantitative ELISA 576 method 303 Sensitivity to horse serum, test for 887 Sensitivity, assay 584 Separation of LDH See Electrophoresis Sequencing solutions 1013 Sera from boehringer, control 467 Sera, control 467 Serocheck-MTB kit 677 Serology 563, 827 plain tube 466 Serotonin 5-hydroxytryptamine 83 carcinoids 83 test 83 Sertoli cell 763 Serum 205, 474, 519, 776 abnormalities of 560 acid phosphatase 530 administration 888 albumin 479, 716 determination of 479 alkaline phosphatase 458, 529, 530 amylase 431 bilirubin 475 normal values 475 biochemistry 248 calcium 755 cholesterol 481 concentration of thyrotropin 598 creatinine 472 levels, causes of raised 475 enzyme patterns values 546 folate assays 251 glutamic oxaloacetic transaminase 530 half-life of tumor marker 680 hepatitis, transmission of 206 immunoglobins, reduced concentration of 268 immunoglobulin changes in various diseases 725 iron 501 LD activity 538 parathyroid hormone 756 pregnancy test 418, 419 protein 457 changes in selected diseases 458 in different clinical conditions 716t sample 418 sickness 888 T3 concentration 749 testing, advantages of 418 thyroid hormones 753 transaminases 458 uric acid levels 493 versus plasma 372 vitamin B12 assay 250 work area tests 921 β2-microglobulin 717 Severe acute respiratory syndrome (SARS) Severe burns 13 Sex hormone binding globulin (SHBG) 566, 583 Sexually transmitted (venereal) disease 622, 624 SGOT/AST/ASAT, clinical relevance of 536 SGOT/SGPT comparison 535 SGPT (ALT) catalyzes 534 SGPT/ALT/ALAT, clinical relevance of 535 Sheard-sanford oxyhemoglobin method 211 Sheep liver fluke 179 Shigella 36 flexneri 868 shigae 867, 868 1053 Sickle cell 234f, 236 anemia blood picture 256 diagnosis of 256 trait, diagnosis of 256 Sickling, tests for 245 Siderocytes/pappenheimer bodies 238 Siderocytosis/pappenheimer bodies 238 Silver method for spirochetes and donovan bodies 798 nitrate solution 805 fontana 801 Simeon’s citrate agar 881 modification of Boye’s and Sterenal’s method 224 stain 224 Sims-Huhner test 403 Sinopulmonary disease, chronic 726 Sintered glass 32 Sinus 808 Six-tube method 329 Sjögren’s syndrome 697, 737 Skeletal muscle disease, chronic 544 Skin 885 disease 540 infections 36 preparatives 36 test 888 common 887 delayed reaction type of 889 diagnostic 886 for allergens, direct 888 immediate reaction type of 887 positive 888 technique of 886 Sleeping sickness 150 Slide ABO grouping test 329 and tube tests, additional material required for 337 cleaning and preparation of 893 tests 336 Small cell lung cancer (SCLC) 936 Smearing techniques 812 Smegma bacillus 845 Soak pad in membrane-based rapid diagnostic test, role of 571 Sodium 88 alterations of 560 lauryl sulfate method 211 method 88 nitrite solution 816 normal values 88 thiosulfate hypo solution 800, 801 solution 802, 805 urate 100f 1054 Concise Book of Medical Laboratory Technology: Methods and Interpretations Spaulding classification system 39t Spaulding scheme (dental), modified 39t Specific gravity, chemical principles of procedure 72 Specimen collection 112, 832 Spectrometry 551 Spectrophotometer See Colorimeter Spectrophotometers Beer’s law 701 Beer-Lambert law 702 Lambert’s law 701 Spectrophotometry 701 Sperm count 398, 400 morphology 398, 402 motility 398 movement 400 shape 400 Spermatozoa, morphological forms of 403f Spherocytes 237 Spherocytosis 237, 239 Spin tube test 336 Spinal fluid 203 exudates 885 Spirillum minus 872 Spirochetales 819 Spirochetes 871 staining of 824 Spontaneous (fasting) hypoglycemia 448 Spores, staining of 824, 881 Spread, direction of 223f Spurs 694 Sputum 203, 405, 835, 855, 885 color 406 culture 406 examination 405 macroscopic examination 405 normal 406 rust colored 406 specimen collection 405 Squamous cells in urine 95f epithelial cells 94 Staff safety and facilities Stain direct fecal smear examination, negative 201 Staining procedures, routine 794 Staining thick smears, procedure for 225 Staining, negative 825 Standard air compressor for flame photometers 560f Staphylococci 837 coagulase test 838 culture 838 morphology 837 pathogenicity 838 Staphylococcus 833 aureus 35, 374, 409, 821, 838 epidermidis 34 Starch 101f Steatorrhea 428 Sterilization, chemical 32 Sterilization, methods commonly used for 29 Steroids 781 Stirrer 210f Stomatocytes 237 Stomatocytosis 237, 239 Stool analysis, normal values in 113, 113t concentration methods 122 examination 112 Stray light 464 Streak plate method 831 Streptavidin advantages of 574 biotin assay system 768, 772f coated microplate 599 reaction wells 605 Streptavidin-biotin based LEMA systems 594 based systems 594 ELISA 574, 575, 575f, 733 systems 594 Streptococci 838 culture 838 morphology 838 pathogenicity 838 Streptococcus 653 faecalis 833, 839 hemolyticus 409 pyogenes 838 viridans 838 Streptomyces avidinii 574 Strongyloidiasis 154 Sub-leukemic leukemia 213 Substrate solutions 816 Sucrose hemolysis test 253 Sudan black B stain 267 for fat 797 Sugar fermentation 827 in urine, significance of 65 media 828 Sulfadiazine See Sulfonamide crystals Sulfobromophthalein 456 sodium 456 Sulfonamide crystals 99 Sulfosalicylic acid test 60 Sulfur 32 granules 884 Sulfuric acid water solution 805 Sulkowitch test See Calcium in urine Superficial deep mycoses, intermediate 884 Suprarenal gland See Adrenal gland Svain’s bile pigment stain 798 Swallowing acids 12 alkalis 12 Sweat electrolytes pilocarpine iontophoresis 432 testing, application of 433 Symphony platform 995 system 996 Syndrome of inappropriate ADH secretion (SIADH) 737 Synovial analysis in arthritis 389t Synovial fluid (SF) 388 presence of 388t Synthesis disorders, diagnosis of 254 Syphfinal 615 Syphicheck reading 623f Syphilis assays 929 immunoassay 929 introduction 621 one-step test for 621, 622 tests for 608 Syphilitic spinal fluid, grades of 388t Syringes 33 by boiling disinfection of 34 disposable sterile 34 glass barrel and metal plunger 33 infected 33 new 33 used 33 Systemic diseases affecting renal medulla 105 Systemic lupus erythematosus 389, 659 T T3 thyrotoxicosis 747 Tacrolimus 947 Taenia saginata 167, 171 solium 167, 168, 170 Tailormade solutions 900 Tapeworm diseases of man 168t of man 167, 167t Taqman 590 Target cells 234f Tartrate inhibited 529 Tay-Sachs disease 396 TB infection and disease 675 Teniasis solium 168 Test value readout 450f Testes 758, 779 Index actions 779 deficiency 779 gonadotropin stimulation test 779 physical examination 779 Testicular enzyme defects 763 failure, causes of 762 function 760 Tetanus 840 Tetra-methyl benzidine (TMB) 578, 756 solution of 679 TG II 939 Thalassemias 256 Thallium 91, 92 Therapeutic ranges for oral anticoagulant therapy 290 Thermophilic bacteria 826 Thiazide therapy 434 Thick films, staining of 224 Thick smears, making 223 Thiocyanate method 500 Third generation thyrotropin (TSH), development of 595 Thoracentesis complications of 390 indications for 390 Thorn test 740 Three-D intelligence in lab automation 914 Throat smear 836 Thrombasthenia 274 Thrombocytopenia, causes of 216 Thrombocytosis 216 Thrombophob 206 Thromboplastin reagent for prothrombin time (PT) determination 283, 287 Thrombopoiesis 230 Thyroid 742 diagnosis 752 disease 598 function tests for 744-746, 749 interpretation of 748 function, different markers of 744 gland, anatomical position 742f hormone 767 binding-proteins, affinity of 748 hormones 743f and TBG 748f levels in different disease conditions 754 peroxidase antibodies 785 peroxidase antibodies See Anti-TPO stimulating hormone (TSH) 761, 762 testing 753 treatment 752 tumors in 744 Thyrotropin 598, 604, 784 calibrators 599, 605 Thyroxine 784 binding globulin (TBG) 566, 583 concentration 783 test 743 free 784 index, free 745, 783 TIBC assay 502 Tina-quant® cystatin C gen 942 hemoglobin A1C 942 immunoglobulin 941 lipoprotein 940 Tinea capitis 36 Tissue attaching sections to slides 793 decalcification 791 diagnostics 995 diseases, connective 721 fixation 791 flagellates 183 preparation of 791 processing of 792 processing unit, automatic 792f roundworms of man 161, 162 Titrimetric method 120 Toluenesulfonic acid 61 Toluidine blue metachromasia stain 797 Toluidine red unheated serum test for rapid serodiagnosis of syphilis redgen 613 Topfer’s test 426 Torch panel 930 Total CK decreased 545 increased 545 Total proteins 478, 480 Tourniquet test 272 Toxic drugs 49t level symptoms 504 shock syndrome 545 symptoms 92 treatment 92 Toxin produced 840 Toxin-antitoxin neutralization 886 tests 887 material 887 schick test 887 technique 887 Toxins 31, 821 Toxocara canis 155, 406 Toxoid sensitivity, test for 887 Toxoplasma antibodies 668 gondii 204, 667 antigens 668 infections 667, 668 skin test 890 Trace elements 503 1055 Transaminases 530 Transcutaneous administration 886 aspiration of palpable lesions 809 Transient hyperglycemia 491 hypoglycemia 491 Transitional epithelial cells in urine f 95 Trepolisa 3.0 624 Treponema 621, 871 antibodies 622, 624 calligyrum 871 carateum 871 infection 610 microdentium 871 pallidum 364, 621, 622, 624, 871 antigen 621 pertenue 871 Trichinella 889 skin test 890 spiralis 155 Trichomonas 404 hominis 124 vaginalis 103f, 124 Trichophyton 883 Trichuriasis 154 Trichuris trichiura 154 Triglyceride levels, classification of 490 Triglycerides 488 Tri-iodothyronine 597, 783 free 783 Tripartigen plates 699 Triple phosphate in urine 100f Trisodium citrate 207, 215 phosphate method 844 Troponin T high sensitive (TNT HS) 931 Trypanosoma cruzi 145 gambiense 145, 146 rhodesiense 145 Trypanosomiases 150, 152 Trypanosomiasis 183 Trypsin 431 TSH CIA test system, expected values for 608t disorders 738 enzyme reagent 599 estimation thyroglobulin antibodies 785 free T4 relationship 748 FT4 relationship 748f FT4/FT3 discrepancies, causes of 749 in humans 753 induced thyrotoxicosis 748 tracer reagent 605 Tsuchiya’s regent 60 1056 Concise Book of Medical Laboratory Technology: Methods and Interpretations Tube test 325, 336 Tubercle bacilli 674 Tuberculin equivalents 889 test 889 type of hypersensitivity (delayed reactions) 887 Tuberculosis 673, 678 Tuberculous skin testing 676 Tularemia skin test 890 Tulip’s eryclone range 335, 336 Tumor cell, representation of 680f definitions of 679 markers 679 definitions of 679 diagnosis 681 diagnostically important 680 different 681 features of 679 portfolio 933 Turbidimetric 699 assays, quality control of 707 immunoassay 718 for determination of antistreptolysin ‘O’ in human serum 724 C-reactive protein 722 microalbuminuria 724 rheumatoid factors 718 for estimation of antithrombin III in human serum 727 complement C3 in human serum 726 complement C4 in human serum 727 immunoglobulin IGA in human serum 725 immunoglobulin IGG in human serum 726 immunoglobulin IGM in human serum 726 lipoprotein in human serum 727 for ultrasensitive determination of C-reactive protein 723 method 521 Turbidity 59 Turke’s fluid 213 Turner’s syndrome 89 Two-hours postprandial blood glucose 435 Tydal antigen 628 Tyndallization 30 Typhoid 867 Tyrosine 99, 100f Tyrosinosis 84 U Ultra sensitive assay technology 681 Ultrasensitive assays 595 TSH assay 786f Ultrasound-guided fine-needle aspiration cytology 811 Ultraviolet radiation 31 Unconjugated (indirect) hyperbilirubinemia 477 Unconjugated bilirubin 454 Unique immunoassay technology 925 Universal donor 369 blood, method of cross-matching 369, 370f Unstable hemoglobinopathy, diagnosis of 256 Uranium nitrate solution 801 Urea 468-470 calculations 468 dithionite test 246 linearity 469 normal reference values 468, 469 principle 469 procedure 468 reagent preparation 468 sample material 468 storage/stability 468 summary 469 Uremia, causes of increased 471 Ureteral catheterization method 105 Uric acid 85, 99, 100f, 492 normal values 85 Uricase/pap method 492 Urinalysis 104 Urinalysis, automation in 78 Urinary 17-hydroxycorticosteroids 775 albumin excretion 715 sediment, microscopy of 92 squamous cells 95f urobilinogen decrease in 69 increase in 68 volume 59 Urine 203, 519, 776, 833, 855 Urine See Serum analysis 56 casts in 97f, 101 chemical examination of 60 collection of 57 color 57 composition in disease, abnormal 102t composition of 56 culture 868 epithelial cell, casts in 103 glucose 435 granular, casts in 103 gross examination of 57 heat 60 hyaline, casts in 101 LDH levels, elevated 540 lead poisoning 254 pH 58t physiochemical characteristics of 56 red cell, casts in 101 sample 418 specific gravity 105 specimen, preservation of 57 test 82, 418 for protein 60 urobilinogen 454f, 455 absent 456 increased 454f waxy casts, casts in 103 white cell, casts in 103 Urinometer 58, 59f Urisys 1100® analyzer 952 Urobilin 68 Urobilinogen 68 chemical principles of procedure 72 UWP, components of 15 V Vacutainer color codes 209t systems 208 Vaginal secretions 203 van Gieson’s solution 799 van Gieson’s stain 800 for collagen fibers 796, 799 Vancomycin-resistant enterococcus (VRE) 35 Vanillylmandelic acid (VMA) 85 Vantage software connection 1008 workflow solution 1009 Varicella zoster virus 38 Varicocele 762 Varistain 12, shandon 807 24, shandon 806 Vascular bleeding disorders, diagnosis of 272 Vasopressin 736 Vasopressin-resistant diabetes See Chronic nephritis VDRL reagent trepolipin, modified 610 syphfinal additional material required 616 interpretation of test result 616 Index material provided with kit 616 principle 615 qualitative method 616 quantitative method 616 reagent 615 storage and stability 615 sample collection and storage 616 test procedure 616 troubleshooting 617 test 608, 615 conventional 608 Veillonella 840 Venepuncture, quality of 276 Venous blood (venipuncture) 205 Ventana basal cell cocktail 1003 iscan HT slide scanner 1009 special stains reagents 998 Verhoeff’s elastic stain 797, 800 Vertical autoclave 30f Vibrio cholerae 636, 638, 870 Viral infections 410 Virales 819 thallophyta 819 Virocyte 261 Virucheck result reading 665f Virulence factors 675 Virus isolation 386 Virutex HBsAG 662 Visceral larva migrans 155 leishmaniasis 148 Viscosity 388 Vitamin B12 251 absorption test, radioactive 250 deficiency 250 causes of 250 D total 945 H 574 K 293 Vitiligo 436 VLDL, precipitation of 484 Volatile antiseptic See Chloroform Volumetric flasks 24f von Kossa’s method for demonstrating calcium 798, 804 von Willebrand’s disease 274, 305 W Warm antibodies 317 AIHA, diagnosis of 253 Warthin-Starry method for staining spirochetes 798 Wash solution concentrate 605 Washing, normal 581 Waste disposal 15 Water bath, serological 25, 27f excretion test (soffer) 740 restriction 736 test See Dilution test Waxy casts 97, 99 WBC pipette 214f Weak agglutination 375 Weigert’s hematoxylin solution 799 Weigert’s iron hematoxylin 799 solution 800, 804 Weigert’s modification See Lugol’s solution Weigert’s resorcin-fuchsin elastic stain 796 Westergren’s ESR pipette with stand 220f pipette 220 White blood cells (WBC) 213, 231, 263, 370, 400 in urine 94f White cell casts 98 count 213 pathological variations in 259 values 259 Widal antigen set 627 reduced 630 reaction 868 test positive control for 631 procedure 631 reagent 631 slide test method 631 Wilder’s reticulin stain 797, 801 Willebrand’s disease 295, 305 Wilson’s disease 84 Wintrobe’s ESR tube with stand 221f method 206, 220 tube 212, 213 Worm infections, local effects of 195 Wuchereria bancrofti 164 Z Zenker’s fluid 791 Zenker-fixed material 794 Zeta potential 570 Zeta sedimentation rate (ZSR) 221 Ziehl-Neelsen (ZN) stain 406, 673, 822, 881 Ziehl-Neelsen method 853 modified 824 Ziehl-Neelsen stain for acid-fast bacteria 798, 805 modified 823 Ziehl-Neelsen techniques 821 Zinc 91, 92, 503 colorimetric method 503 sulfate concentration method 122 Zoonotic disease 645 1057 ... only low-grade inactivation of reagent and enzyme 578 Concise Book of Medical Laboratory Technology: Methods and Interpretations ¾¾ Long-term stability without loss of immunological and enzymatic... Appearance of “Faint Ghost Band” 5 72 Concise Book of Medical Laboratory Technology: Methods and Interpretations How We Interpret “Broken Bands” at the Test/ Control Region? To prevent evaporation of. .. with direct dialysis methods for free hormones 580 Concise Book of Medical Laboratory Technology: Methods and Interpretations FIG 22 .18: Factors affecting EIA Hemolysis and Hyperbilirubinemia