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Ovid: Oxford Handbook of Medical Sciences

Editors: Wilkins, Robert; Cross, Simon; Megson, Ian; Meredith, David Title: Oxford Handbook of Medical Sciences, 1st Edition Copyright ©2006 Oxford University Press, 2006, except ‘Clinical aspects’ section of Chapter 2 (Copyright by Keith Frayn) > Table of Contents > Chapter 14 – Techniques of medical sciences Chapter 14 Techniques of medical sciences Genomics Techniques involving DNA Genomics = the application of automated oligonucleotide sequencing and computerized data retrieval and analysis to sequence an organism’s entire genetic complement.

  • Southern blot analysis involves cutting DNA with restriction enzyme(s), running the fragments on an agarose gel, transferring onto a membrane, and probing with a labelled complementary DNA probe to the gene of interest (Fig.14.1)
  • DNA fingerprinting is a technique used in forensic science to identify whether a sample found at a crime scene is that from a particular individual
    • DNA is isolated and digested with restriction enzymes
    • The fragments are separated by electrophoresis on an agarose gel and visualized by ethidium bromide staining
    • Due to the sequence variability (polymorphisms) and differences in the length of multiple repeat sequences in introns, the band pattern is unique to an individual
      • Closely related individuals will have less polymorphisms in their genomes
      • This allows a similar approach to be used for paternal testing
    • A polymerase chain reaction (PCR) based technique (pp.892–3) can also be used with primers that hybridize with strongly conserved sequences but spanning variable number repeat regions
  • Where a disease is known to be caused by a specific mutation in a gene, it is possible to use PCR to screen for carriers and even from cells taken from a foetus in utero
  • A good example of this is the most common mutation causing cystic fibrosis (CF), the loss of three bases resulting in an amino acid deletion (δF508)
  • A PCR product made from primers spanning this region of the gene will be three bases shorter in CF than wild-type
  • This difference can be seen when the products are run on a suitable gel.
Fig. 14.1 Diagram of the Southern blot technique showing site fractionation of the DNA fragments by gel electrophoresis, denaturation of the double-stranded DNA to become single-stranded, and transfer to a nitrocellulose filter. (Reproduced with permission from Mueller RF and Young ID (2003), Emery’s Elements of Medical Genetics, 11th edn, Churchill Livingstone.)

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Techniques involving RNA

  • Northern blotting is essentially the same in principle as Southern blotting (p.868), except that the starting material is isolated mRNA. mRNA can be isolated as its poly(A) tail will bind to oligo-d(T) based resins
  • Most other techniques involving RNA require conversion of mRNA into cDNA. This can be done using an oligo-d(T) primer and the enzyme reverse transcriptase (RT)
  • Once mRNA has been converted into cDNA, it is possible to amplify specific sequences using PCR. The whole process is often called RT-PCR. This can be used to see if a particular gene is expressed in a cell type or tissue
    • Normal PCR is not quantitative
    • However, PCR can be used to quantify the level of mRNA expression using a variation of the technique known as semi-quantitative PCR
      • Known amounts of an engineered DNA that binds the same primers but gives a different sized product to the wild-type are added to the sample
      • Comparing the intensity of the reaction bands on an ethidium bromide agarose gel allows estimation of the original mRNA concentration
    • A newer technique, called real-time PCR, uses an extra primer labelled with a fluorescent dye to accurately measure the amount of product formed during the PCR reaction. When compared to a set of standard reactions, this allows a very accurate estimation of the number of copies of mRNA in the starting sample
  • The genes expressed by a particular cell or tissue can be seen using an expressed sequence tag (EST) library
    • RT-PCR is carried out to make a cDNA copy of all the genes expressed
    • The resulting cDNAs are sequenced and put into a database.
    • This allows the genes to be identified, and the number of clones of a gene indicate its relative frequency of expression
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  • A technique for looking for changes in gene expression at the mRNA level in cells involves the use of DNA microarrays, also known as ‘gene chips’
    • DNA microarrays have oligonucleotides representing up to the entire gene compliment spotted on them in an ordered array
    • cDNA is prepared (e.g. from two sets of the same cells grown under different conditions), with one set labelled with red dye (condition 1) and the other green (condition 2)
    • Both cDNA samples are hybridized to the gene chip
    • Scanning with lasers reveals which genes are expressed under which condition
      • Red spot signifies only condition 1
      • Green spot, only condition 2
      • Yellow spot, both conditions
      • Variations in between—(e.g. orange) would indicate expression in condition 1 > in condition 2
    • Computer analysis allows analysis of which genes are up- and downregulated.

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Proteomics Introduction The sequencing of the human genome by the year 2000 was a major scientific achievement which has provided a huge amount of invaluable information. We now know approximately how many functional genes there are in the human genome (about 25,000), their location, and, in some cases, their function. However, this knowledge now needs to be integrated with knowledge about the proteins which these genes produce, how these proteins interact, and how their expression varies in different types of cells and in disease. This has led to a need for techniques that can identify and quantitate proteins in cellular samples—the science of proteomics. The number and scope of these techniques is increasing all the time. The following is a short description of the most popular current techniques—protein extraction and mass spectrometry. Protein extraction To analyse the proteins in a sample, they must be extracted from the cells within which they are contained. Two-dimensional gel electrophoresis This is a simple and popular technique for separating a mixture of proteins into its constituents:

  • The protein extract is mixed with a non-charged detergent, β-mercaptoethanol and urea to solubilize, denature, and disassociate all the proteins
  • The resulting mixture is put in a tube of polyacrylamide gel (PAGE) with a gradient of pH across it, and an electric current is applied
  • Each protein migrates to its isoelectric point where the pH of the surrounding gel means that the protein has no overall positive or negative charge and so does not move
  • This can produce resolution into about 50 protein bands
  • The tube of gel is soaked in a solution containing the detergent, sodium dodecyl sulphate (SDS), which is highly negatively charged and so masks the native charge of a protein, making the overall charge now proportional to molecular weight
  • The tube of gel is attached to a slab of polyacrylamide gel and an electric current is applied across the gel in a direction at 90° to the isoelectric focusing
  • The proteins now migrate according to molecular weight
  • This can produce resolution into more than 1000 proteins
  • Proteins may be visualized by stains (such as Coomassie or silver) or by autoradiography (if the proteins have been radioactively labelled in vivo).
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  • A sample will typically produce thousands of individual spots and it would be difficult to identify any difference in pattern between samples by simple inspection. Images of these gels are scanned into a computer system which then compares gels from two different samples and identifies those spots which are different in the two
  • In this way, for example, the differences between proteins in normal tissue and a cancer arising from that tissue could be identified.

When these differential spots have been located, the specific proteins within these spots need to be identified. This is usually done by cutting the spot out of the gel and subjecting it to a further analytical step such as mass spectrometry (Fig. 14.2).

Fig. 14.2 Two-dimensional gel electrophoresis.

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Mass spectrometry A protein has a typical precise mass (if allowance for processes such as glycosylation is made) but it is possible that more than one protein has the same mass (or the same mass within the resolution of the measuring system). However, if a protein is cleaved using a specific enzyme, such as trypsin, then the resultant peptide fragments will have a specific range of molecular weights that can be used to precisely identify the whole protein by mass spectrometry—a peptide ‘fingerprint’. The rather lengthily named ‘matrix-assisted laser desorption ionization-time-of-flight spectrometry’ (MADLI-TOF) is the most common method:

  • The enzyme-digested peptide mix is dried onto a slide (ceramic or metal)
  • A laser heats the mixture causing formation of an ionized gas
  • The ionized particles are accelerated in an electric field towards a detector
  • The time taken for a peptide to reach the detector is a function of its mass and charge
  • The data from the detector is matched with information on protein databases to give definitive identification of the protein (a task requiring considerable computing resources).

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Cytology Definition The examination of isolated cells rather than cohesive tissues (p.876). Methods of cytological sampling

  • Exfoliative cytology—uses cells that are shed or scraped from body surfaces (e.g. bronchial epithelial cells that are coughed up in sputum, epithelial cells from the uterine cervix removed using a wooden spatula)
  • Aspiration cytology—uses cells which have been aspirated from a solid organ using a fine gauge needle and syringe (e.g. cells from a breast lump).

Uses

  • Diagnosis of malignancy—e.g. detection of bronchial carcinoma by exfoliated cancer cells in sputum
  • Screening for malignancy—e.g. detection of cervical intra-epithelial neoplasia (CIN) in cells scraped from the cervix in the UK national cervical screening programme
  • Detection of micro-organisms—e.g. Trichomonas vaginalis in cervical smears, Pneumocystis carinii in bronchoalveolar lavage from immunocompromised patients.

Technique

  • Smearing of cells directly onto glass slides or cytocentrifuging from a transport medium
  • Brief fixation of cells using an alcohol
  • Staining of cells by a histochemical method such as Papanicolaou
  • Interpretation by a cytopathologist or cervical screener
  • Issuing of written report.

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Cytology and Histology Histology Definition Literally, examination of tissue (from the Greek histos = tissue). However, in current usage, it refers to the examination of tissue by light microscopy after it has been processed into thin sections. Although the histological appearances of tissue are altered by many artefact-inducing processes, these artefacts are reproducible, and there is a huge body of knowledge about the histological appearances of disease which has built up over the past 150 years. Uses

  • Tumour diagnosis—histology is the primary method of definitive tumour diagnosis in current medical practice. Although imaging (such as CT or MRI scanning) may provide very clear views of definite masses which are presumed to be tumour (e.g. multiple masses in the liver which are most likely to be metastases), there are some non-neoplastic processes which can produce masses on imaging (e.g. abscesses), so a definite tissue diagnosis by histology is still required
  • Tumour staging—the extent of tumour spread (e.g. metastasis to local lymph nodes) is an important determinant of an individual patient’s prognosis. Although modern imaging is, again, very valuable in this process, histology is still the main method of assessing tumour stage in surgically resected tumours (e.g. colorectal cancer)
  • Non-neoplastic diagnosis—the histological appearances of many non-neoplastic diseases are reproducible and specific to a particular diagnosis. Inflammatory conditions of the skin are a good example of a spectrum of diseases which can be diagnosed by histology
  • Assessment of the body’s response to disease—inflammatory and fibrotic processes are easily assessed by histology, and so histology may play an important role in determining the extent of a nonneoplastic disease. An excellent example is hepatitis C—histological examination of a liver biopsy can determine the amount of inflammatory activity (and, therefore, the predicted response to treatment such as interferon therapy) and also the amount of damage that has already been caused to the liver (by assessing the amount of fibrosis from none through to frank cirrhosis)
  • Detection of micro-organisms—although microbiological culture is generally more sensitive than histology, there are some circumstances where histology is an effective technique for detecting micro-organisms (e.g. Helicobacter pylori in endoscopic gastric biopsies).

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Technique Histology is relatively simple technology that has changed little over the decades. The most significant advance has been the use of specific antibody stains (p.878). The basic steps are:

  • Fixation of the fresh tissue by a chemical solution (usually formaldehyde) which cross-links proteins
  • Selection of a sample of the tissue if it large; small biopsies are examined in their entirety
  • Processing of the tissue from the formaldehyde solution through progressive dehydration by alcohol into 100% liquid alcohol
  • Embedding of the tissue into paraffin wax
  • Cutting of thin (7 micrometre thick) sections from the wax block
  • Mounting of the wax sections onto a glass slide
  • Staining of the section on the glass slide, usually by haematoxylin and eosin dyes which stain nuclei, dark purple, and cytoplasm, pink
  • Interpretation of the sections by a histopathologist (a medically qualified doctor with postgraduate training in histology)
  • Issue of a written report of the histological findings.

Although relatively simple, the process in labour intensive and takes 1 to 2 days to complete. Frozen sections If an immediate histological diagnosis is required (e.g. an unexpected finding of disseminated tumour at an exploratory laparotomy), then a small tissue sample can be frozen in liquid nitrogen and sections cut and stained from this within a few minutes. P.878
Immunohistochemistry Immunohistochemistry allows the identification of proteins in cells and tissue samples (or thin sections of tissue), using specific antibodies and microscopy (Figs. 14.3, 14.4 and 14.5). Once the tissue has been incubated with the primary antibody (i.e. the one raised to the protein of interest), the antibody binding pattern can be visualized in a number of ways, usually involving the use of a secondary antibody (2° Ab) that recognizes the primary one:

  • The 2° Ab can be conjugated to an enzyme such as horseradish peroxidase, which can be used to catalyse a colourimetric reaction to stain the area(s) of the cell/tissue where the protein is expressed. This is a relatively old-fashioned approach, largely replaced by fluorescent staining (Fig. 14.3).
Fig. 14.3 (a) A specific protein (shown as triangle) is present on the surface of a cell; (b) a specific primary antibody binds to this protein; (c) a secondary antibody binds to the tail end (Fc portion) of the primary antibody; (d) the secondary antibody has an enzyme attached to it which catalyses a reaction from a colourless substrate to a colourless substrate to a coloured dye product which can be seen by light microscopy.

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Fluorescent microscopy uses a similar approach to above, except instead of being conjugated to an enzyme, the 2° Ab is conjugated to a fluorescent chromophore.

  • Usual chromophores include FITC or fluorescein (green) and TRITC or rhodamine (red)
  • Dual labelling of two proteins allows identification of areas of co-expression (red + green = yellow staining)
  • An enhancement on fluorescent microscopy is confocal microscopy
    • This allows the production of an optical section through a sample by using a pinhole to exclude light that is out of focus
    • A 2D picture can be built up by scanning the sample, and a 3D image, by focusing and scanning at different levels in the sample.
Fig. 14.4 Same intact gastrula-stage Drosophilia embryo, stained with a fluorescent probe for actin filaments: (a) conventional, unprocessed image is blurred by presence of fluorescent structures above and below the plane of focus; (b) this out-of-focus information is removed in confocal image. (Reproduced with permission from. Alberts B et al. (2002), Molecular Biology of the Cell, 4thedn, Garland Science, Taylor & Francis LLC.)
Fig. 14.5 Immunogold labelling for growth hormone (GH) in secretory granules in the anterior pituitary. The cell on the right does not contain GH. (Scale bar = 400nm) (Image courtesy of Helen Christian).

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Much higher levels of detail can be imaged using an electron microscope (at least 100-fold higher resolution than with a light microscope).

  • In this case, the 2° Ab can be conjugated to a small particle of gold, which is electron-dense and, therefore, will appear black
  • Use of different size particles (e.g. 5nm and 10nm) allows more than one protein to be localized.

Although not strictly immunohistochemistry, one modern approach has been to genetically engineer proteins that are endogenously fluorescent. This can be done by adding the coding sequence for the ~30KDa protein, green fluorescent protein (GFP), originally isolated from the jellyfish Aequoria Victoria.

  • Experimental mutations in the gene for GFP has allowed the development of an enhanced GFP (EGFP) and other colours e.g. cyan (CFP) and yellow (YFP)
  • The use of these GFP-fusion proteins has allowed imaging of live samples.

Therapeutic uses Immunohistochemistry has a very wide application in diagnostic histopathology to determine the precise nature of a tumour. This, in turn, allows the optimal therapy to be planned for the patient. Increasingly, individualized tumour therapies are being developed which rely on a specific protein being present in a tumour—the therapy itself is often an antibody, so the tumour of an individual patient will require immunohistochemical staining to identify the presence or absence of this protein. Common clinical uses of immunohistochemistry include:

  • Estrogen receptor assay in breast cancer: all breast cancers are immunohistochemically stained for the estrogen receptor protein and if a tumour does contain estrogen receptor on its surface (‘ER positive’), then the patient will be treated with the anti-estrogen drug, tamoxifen, or one of its derivatives
  • HER2 receptor assay in breast cancer
    • If a breast cancer overexpresses the epidermal growth factor receptor 2 (HER2) protein, then it may respond to the drug, Herceptin (trastuzumab), which is a monoclonal antibody directed against this receptor. Again, immunohistochemistry is required to determine the presence of this protein
    • In the UK, at present, Herceptin is used in the palliative treatment of metastatic breast cancer
  • Typing of lymphomas: lymphomas (malignant tumours of lymphoid cells) are a very heterogeneous group of tumours with widely differing prognoses. Most lymphocytes have a relatively similar appearance by conventional light microscopy, so immunohistochemistry is widely used to detect specific cell surface proteins which allows classification of the tumour into a specific lymphoma type.

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In situ hybridization (Fig. 14.6) This technique is used to look for particular mRNA species in a section of fixed tissue. It reveals which cell is expressing the mRNA (unlike PCR where a tissue/cell sample is homogenized).

  • Thin sections of tissue are mounted onto microscope slides
  • They are then treated with proteinase K to allow the labelled probe access to the cellular mRNA
    • The probe is an antisense RNA made by in vitro transcription
    • Labelling can be either with 32P (with radiography used to detect signal) or biotin (detected with a 20Ab conjugated with horseradish peroxidase which can be used in a colourimetric assay)
    • A fluorescently labelled probe is used in fluorescent in situ hybridization (FISH)—usually a DNA probe to identify specific chromosomes or chromosomal regions (e.g. can check the number of copies of chromosome 21 as a test for Down’s syndrome).
Fig. 14.6 Expression of the amino acid transporter gene, path, in a late-stage whole-mount Drosophilia melanogaster embryo (dorsal view), analysed by in situ hybridization. Path is expressed ubiquitously, but at higher levels in specific tissues (as highlighted here in the midgut, brain, and sensory nervous sytem). (Image courtesy of Deborah Goberdhan, Department of Physiology, Anatomy and Genetics, University of Oxford).

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Analysing protein structure and function Protein structure X-ray crystallography

  • ‘Gold standard’ for protein structure
  • X-rays are diffracted by atoms in the protein
    • In a well-ordered crystal, the scattered waves reinforce each other at certain points to give spots
    • This can be converted into a 3D atomic structure by computer if one knows the amino acid sequence
  • At highest resolution, can identify the position for every atom, except hydrogen
  • Main problem is getting good-quality crystals of pure protein
    • Membrane proteins have proved especially difficult.

Nuclear magnetic resonance (NMR)

  • Does not require large amounts of protein to be crystallized
    • Useful for hard to crystallize proteins
    • As protein is in solution, can visualize structure changes (e.g. on binding substrate)
  • Small volume of solution is placed in strong magnetic field
    • This causes nuclei with magnetic moment (spin) to line up with the magnetic field (e.g. protons)
    • Pulses of radiofrequency electromagnetic radiation excite the nuclei and misalign them
    • As they realign, they release radiofrequency electromagnetic radiation. This radiation release is affected by adjacent atoms
    • By knowing the protein sequence, it is possible to compute a 3D structure from these data.

Protein function Fluorescence resonance energy transfer (FRET)

  • This is a specialist application of confocal microscopy (p.879).
  • Proteins believed to be interacting are labelled with different colour fluorescent proteins (e.g. GFP (green) and CFP (cyan))
  • The proteins are co-expressed in the same cell
  • Excitation of the CFP fluorochrome usually results in cyan light out; however, if the proteins are close enough (1–10nm), then the GFP will be excited by the CFP emission and green light will be seen instead (Fig. 14.7). This energy transfer is called FRET.
Fig. 14.7 Fluorescence resonance energy transfer (FRET). To determine whether (and when) two proteins interact inside the cell, the proteins are first produced as fusion proteins attached to different variants of GFP: (a) protein X is coupled to a blue fluorescent protein; (b) if protein X and Y do not interact, illuminating the sample with violet light yields fluorescence from the blue fluorescent protein only; (c) when protein X and Y interact, FRET can now occur. (Reproduced with permission from Alberts B et al.(2002), Molecular Biology of the Cell, 4thedn, Garland Science, Taylor & Francis LLC).

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Affinity chromatography/co-immunoprecipitation

  • The protein of interest is covalently bound to a column matrix
  • Cell extract is run through this column and interacting protein(s) non-covalently stick to the immobilized protein
  • Any proteins adhering to the protein of interest can be eluted and identified using mass spectrometry
  • Co-immunoprecipitation is similar, except that an antibody to one of the proteins is used to precipitate out the protein complex
  • This can then be probed with antibodies to candidate associating protein(s)
    • Method requires proteins to be associated together tightly enough for the complexes to survive co-immunoprecipitation.

Yeast two-hybrid assay

  • ‘Bait’ protein DNA is fused to DNA-binding domain of a gene activator protein
  • ‘Prey’ protein DNA is fused to transcriptional activation domain— usually make from a cDNA library
  • If bait and prey associate, then the gene activator and transcription activation domains will interact with each other and turn on a reporter gene, Fig. 14.8
  • From this, the positive prey protein DNA can be selected and sequenced
  • Can also perform reverse two-hybrid assay—used to look for mutations in DNA or chemicals that disrupt a proven association through the two-hybrid system.
Fig. 14.8 The yeast two-hybrid system for detecting protein–protein interactions (see text). (Reproduced with permission from Alberts B et al. (2002), Molecular Biology of the Cell, 4thedn, Garland Science, Taylor & Francis LLC.)

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Microbiology Microscopy Micro-organisms can be viewed in samples by microscopy after treatment with special stains.

  • Gram stain. Allows detection of Gram-positive and negative bacteria, cocci, and rods. Also stains yeast. Some bacteria stain poorly
  • Ziehl—Neelsen stain. Used to detectMycobacterium tuberculosis and related mycobacterial species that are acid-fast and stain poorly with the gram stain. Can be modified to detect Nocardia sp. Other stains such as fluorochromes (e.g. auramine-rhodamine) are more sensitive for screening specimens for Mycobacteria sp
  • Potassium hydroxide (KOH). Used on wet mounts to detect fungi. Calcofluor white staining and fluorescence microscopy is more sensitive. In biopsy specimens, silver stains such as periodic acid-Schiff are used to detect fungi
  • Direct fluorescent antibodies (DFA). A monoclonal antibody specific to a micro-organism may be tagged with a fluorochrome and used to detect the micro-organism in a specimen
  • Giemsa stain. Used on blood films to detect parasites (malaria in particular) and, occasionally, other intra-cellular pathogens
  • Electron microscopy. Detection of viruses in certain samples
  • Direct microscopy on unstained samples. Used on unicellular parasites and the larger ectoparasites
  • Histologic specimens. Special stains and pathologic features (e.g. granulomata) aid diagnosis

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Culture Bacteria

  • Media. Cultured on solid or liquid media that can be enriched to optimize growth of specific bacteria, selective to allow only growth of certain bacteria or to contain an indicator so that bacteria with a certain characteristic (e.g. the ability to ferment a specific sugar) are detected
    • Blood agar is an enriched all-purpose media
    • MacConkey agar is selective for common gram-negative bacteria and contains an indicator so that lactose fermentors appear red
    • Broth cultures allow inoculation of greater volumes and are more sensitive
  • Conditions. Automation allows early detection of bacterial growth by a characteristic such as microbial production of CO2. Growth conditions (temperature and atmospheric conditions) are adjusted depending on the requirements of specific micro-organisms. Anaerobes require culture without O2
  • Identification. Bacteria are speciated by colony morphology on solid media (including hemolysis pattern on blood agar), gram stain, biochemical tests, serology, motility pattern, flagella, and spores. Multiple tests require computer-based programs for precise identification.

Mycobacteria Grow on specific solid (e.g. Lowenstein—Jensen) and liquid media. Identification is by growth rate, colony morphology, biochemistry, and the use of molecular techniques. Fungi Grow on specific agar and liquid media (e.g. brain-heart infusion). Identification includes detecting yeasts or hyphal elements, the pattern of sporing bodies, and biochemical tests. Culture of mycobacteria and fungi is slow (often requiring weeks) and dangerous (necessitating a biosafety level 3 laboratory). Viruses Cultured in cell lines and identified by the pattern of cytopathic effect or reaction with specific fluorescent antibody. P.890
Serology Used for detection of viruses, parasites, fungi, and fastidious (difficult to culture) bacteria. Requires identification of specific IgM or a four-fold increase in IgG antibody titre. Antibody is detected by using a secondary antibody with a tag that allows detection of bound antibody—antigen complexes. Detection may be by agglutination, precipitation, complement fixation, or radioactivity, but the majority of commercial assays now use indirect fluorescence or enzyme-linked immunosorbent assay (ELISA) (see Fig. 14.9 and p.896). ELISA can be adapted to detect IgG or IgM antibody or antigen. Western blotting allows microbial proteins to be separated by SDSPAGE and transferred electrophoretically to a nitrocellulose membrane. Reaction of patient sera with the proteins is detected by a specific pattern of bands when treated with a secondary antibody.

Fig. 14.9 Principles of ELISA. Specific antigen (Ag) is bound to the wells of the test plate and blocking protein (BP) is added to stop non-specific antibody binding to the wells. The patient sample is added and antibody binds to antigen. A secondary antibody, with an attached enzyme, is added. Addition of substrate results in cleavage of a chemical and release of light. The optical density is measured in a microplate reader.

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Molecular techniques

  • Polymerase chain reaction (PCR). Allows detection of a variety of micro-organisms. Microbial DNA is amplified using primers (short sequences of DNA) that bind to a specific sequence of microbial DNA (Fig. 14.10). Analysis can be qualitative or quantitative in which case the amount of target DNA in a sample is estimated by measurement of a signal such as the production of light. RNA can also be amplified using reverse transcription PCR (RT-PCR)
  • Molecular probes. Chemiluminescent DNA probes target sequences of ribosomal RNA and aid speciation ofMycobacterial sp. in culture. In situ hybridization allows the detection of nucleic acid derived from micro-organisms in biopsy specimens
  • Restriction patterns. Microbial DNA is cleaved using endononucleases (cleave at specific sites). The pattern of fragments on a gel provides a molecular fingerprint, useful in epidemiological studies.
Fig. 14.10 Principles of PCR. (A) The three stages of the PCR cycle. (B) There is exponential amplification of the region of interest, whereas longer PCR products undergo linear amplification. Thus after several cycles the correct sized product predominates. (C) There is a linear region of amplification, followed by non-linear region as reagents are exhausted.

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Resistance testing

  • Disk diffusion susceptibility testing. An antimicrobial agent diffuses from an impregnated disc onto an agar plate containing bacteria or fungi
  • Minimun inhibitory concentration (MIC) testing. Micro-organisms are grown in a liquid media with a known concentration of antimicrobial agent. Can be adapted to mycobacteria, fungi, and viruses, to solid media, or to use with a strip impregnated with drug (Etest)
  • Molecular techniques. Detection of genes associated with resistance (e.g. the mec A gene for methicillin resistance in Staphy lococcus aureus). Resistance mutations in mycobacteria or viruses are detected by PCR (genotypic assays) and are compared with MIC estimates (phenotypic assays).

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Photometry and spectrophotometry Flame photometry Flame photometry has long been used to detect metal ions (Na+, K+, Mg2+, and Ca2+). The principle underlying the technique is that the different metal ions burn with different coloured flames. Thus, a solution containing such ions can be sprayed through a flame, and the change in flame colour detected by a spectrophotometer. This technique used to be routinely used to establish Na+ and K+ levels in the blood, although nowadays, ion-selective electrodes are often used for this purpose. Spectrophotometry The colour that a substance appears to our eye is determined by those wavelengths of white light that are reflected by it—all the other wavelengths are absorbed. A spectrophotometer is an instrument that determines the amount of light of different wavelengths—(usually between ultraviolet (~200nm) and infrared (~600nm)—that is absorbed by a dissolved compound of a given depth (usually 1cm—the pathlength). The amplitude of the peaks in the ‘absorption spectrum’ (measured in an arbitrary scale of absorption units from 0.0–3.0) is directly proportional to the concentration of the compound present. This is a simple means of measuring the concentration of highly coloured compounds that are found in high concentrations in biological samples (e.g. haemoglobin). Colourimetric, luminescence, and fluorescence assays However, the range over which spectrophotometry is effective is very limited (e.g for haemoglobin, concentrations of 0.5–5µM can easily be measured; higher concentrations require dilution to be measurable by this means; lower levels are undetectable). Unfortunately, the majority of substances that we want to measure are not coloured (although most absorb in the ultraviolet spectrum) or do not lend themselves to direct spectrophotometric analysis. However, a number of colourimetric, luminescence, and fluorescence assays have been developed for specific compounds. These assays rely on highly specific reactions of the substance of interest with an added reagent. The reaction gives rise to a highly coloured, luminescent, or fluorescent compound that can be detected in a spectrophotometer, luminometer, or fluorometer. Many of these assays are now available in kit form for use with 96 well plates that can be read in a plate-reader for very rapid throughput of large numbers of samples. P.896
Biochemical Assays Radioimmunoassay, ELISA, chromatography Radioimmunoassay (RIA) RIA is often the technique of choice for determining the concentration of specific peptides and proteins in blood or urine samples. Kits are now available for a huge range of peptides and proteins. The principle involves introducing the sample into a tube that is coated with a known amount of the specific antibody for the antigen of interest. An excess of radiolabelled antigen is then added to the tube, which will bind to any antigen that is still available. It is then washed out, leaving the antibody bound to a mixture of unlabelled ligand from the sample and radio-labelled ligand. The amount of the antigen in the sample is inversely proportional to the amount of radioisotope present, as determined by measuring the radioactivity in γ-counter and comparing the reading to those of standards of known amount. It is important to note that the standard curve for RIAs is not linear—it is usually fitted with a cubic spline curve. Enzyme-linked immunosorbant assay (ELISA) ELISA (pp.890–1) is a popular means of detecting peptides and proteins that does not require the use of radioisotopes. ELISAs are generally purchased in kits consisting of pre-formed 96-well plates coated with an antibody specific to the antigen of interest.

  • After addition of sample to the wells, a secondary enzyme-linked antibody is added to the wells, which binds to the antibody—antigen complex
  • Addition of the colourless substrate for the enzyme that is linked to the antibody results in the formation of a coloured product, the amount of which is measured spectrophotometrically in a plate-reader and is proportional to the amount of enzyme bound and, consequently, the amount of antigen present.

Chromatography Chromatography works on the principle that substances can be separated according to their specific chemical characteristics (e.g. charge, molecular size, and/or partition in aqueous/organic solvents). In this way, we can separate and measure the concentrations of the components of very complex mixtures, including blood plasma, tissue, and cell extracts.

  • Chromatography usually utilizes a ‘mobile phase’—a solvent in the case of liquid chromatography (high-performance liquid chromatography; HPLC) and an inert gas for gas chromatography (GC)—that passes through or over a ‘stationary phase’ in a column
  • The greater the interaction of the substance of interest with the stationay phase, the longer it will take to pass through the column, giving it a longer ‘retention time’
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  • The effluent from the column is passed through a detector, which can work by either measuring the absorbance in the ultraviolet region of the spectrum or by electrochemical means. Compounds appear as ‘peaks’ on a chart—the higher the peak, the more of the compound is present. The precise amount present can be analysed by comparing the peak height or area to those of standards of known concentration. Sometimes, the specificity of HPLC can be improved by exposing the components of the mixture to a reagent that will fluoresce when it reacts with a particular target molecule. The amount of the fluorescent product can be detected downstream of the column using a fluorescence detector
  • A recent refinement to chromatography techniques is their combination with mass spectrometers (MS) that can be used to determine the molecular mass of the substance after separation by gas chromatography (GC/MS) or HPLC (LC/MS). This is particularly useful in identifying unknown substances.

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Functional Studies In vitro, ex vivo, in vivo In order to examine a physiological or pathological process, or to determine the characteristics of drug action, it is necessary to work with live cell cultures or tissue samples. A range of different techniques is available to help elucidate biological processes. In vitro Literally, this term means ‘in glass’ and can equally apply to experiments with cell cultures, tissue homogenates, or functionally intact pieces of tissue. These are very useful techniques for providing clues as to the cellular mechanisms involved in physiological, pathological or pharmacological processes, without the complications of the highly complex physiology of a whole organism. This simplicity is, however, also the major limitation of in vitro investigation: extrapolation of in vitro results to the in vivo situation is inherently dangerous on the basis that the complex integrated systems and metabolic processes of the whole body are likely to have a bearing on the processes involved in vivo. Table 14.1 summarizes the benefits and drawbacks of in vitro experiments. Ex vivo This term is often confused with in vitro. Ex vivo should only be applied to experiments that are conducted on cells or tissue that have been removed from an animal or human, after having been subjected to a drug treatment in vivo. Under these conditions, the therapeutic intervention is subject to all the metabolic processes that occur in a fully functional animal or human, but the end-point of the experiment is a specific test that is carried out on excised tissue. In humans, these experiments are only possible when a realistic end-point can be achieved from easily obtainable tissue (e.g. skin biopsy, blood samples), but in animals, these experiments might be terminal (i.e. the animal is killed prior to tissue removal), allowing any tissue sample to be used to determine the drug effect (e.g. blood vessel, brain tissue, liver homogenate). The in vivo element of these experiments (human or animal) means that they require ethical permission from the respective authorities. In vivo: animal experiments Clearly, where possible, the most accurate reflection of a drug effect on a physiological or pathological process will be obtained from experiments in vivo. However, this is a highly emotive issue because it usually involves vivisection in laboratory animals. In almost all cases of drug development, this is an essential step, prior to ethical approval for clinical studies, to highlight any unforeseen side-effects or problems. As with any experiments, in vivo studies should set out to test a specific hypothesis (or hypotheses), with clear, achievable end-points. Ethical review panels have to be assured that any animal suffering will be minimized, or preferably avoided altogether, before permission will be granted. Consideration must be given to the best species to use as a model for the human condition in question, and power calculations should be conducted to determine how many animals are likely to be P.899
needed in each group to ensure a reasonable chance of observing a statistically significant difference between placebo and treatment groups. Where possible, randomized, blinded, crossover studies should be considered, to minimize the number of animals used.

Table 14.1 Advantages and disadvantages of various in vitro techniques
  Advantages Disadvantages
Cultured ‘immortalized’ cell lines
  • Relatively cheap (purchase of only one sample of seed cells required for an indefinite number of culture generations)
  • Rapid results
  • Human cell lines available
  • Real-time changes in ion flux or morphology can be observed using fluorescent markers
  • Immortalization will change the phenotype—results may not reflect the response of the ‘mortal’ cells in vivo
  • Measures tend only to be of biochemical markers rather than the real physiological response
Primary cultures
  • Human cells can be harvested (ethical approval required) or bought commercially
  • In some cases, functionality can be observed (e.g. isolated cardiac myocycte contraction)
  • Real-time changes in ion flux (e.g. Ca2+) or morphology (e.g. cell cytoskeleton) can be observed using fluorescent markers
  • Requires harvesting of cells from tissue
  • Bought cells are expensive
  • Cells may progressively change phenotype through progressive generations
  • A cell of a particular type might require interaction with different cell types to produce a normal physiological effect
Isolated tissues or organs
  • Real-time functional responses can be observed easily (e.g. muscle contraction, blood vessel function) and correlated with changes in biochemical mediators
  • Whole organ function can be investigated (e.g. heart, kidney)
  • Impact of drugs on function can be easily measured under equilibrium conditions (useful for determining efficacy and affinity)
  • Fluorescence imaging more difficult than with cell cultures
  • Human specimens are difficult to obtain, except skin biopsies or ‘surgical debris’ (e.g. discarded organs after transplant, excess tissue that has been biopsied for diagnostic purposes, tissues from amputated limbs)—experiments with human tissue require ethical approval

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Clinical studies Ethical approval should be sought at the earliest opportunity—evidence of in vitro and in vivo studies in animals is usually required, together with toxicology data. Clinical trials proceed in a standardized fashion:

  • Phase I: a small study (20–80 subjects) to help evaluate the correct dosing range, the safety of the drug, and any side-effects. First-in-man studies are often carried out in healthy volunteers before trying them in patients from the target group
  • Phase II: a larger study group is involved (100–300 patients). A clear end-point is identified, which must be achieved for the drug to be taken further
  • Phase III: a very large study (1000–3000 patients) to confirm the drug efficacy, perhaps in comparison to an existing therapy and usually also compared to a placebo group (‘dummy’ treatment with the same appearance as the study drug but without the active agent). Side-effects are closely monitored and information collected as to the safety of the drug prior to marketing
  • Phase IV: post-marketing studies that help to determine the optimal use of the drug and to clarify the risks and benefits of the drug
  • Expanded access protocols: clinical trials (phases I–IV) are necessarily conducted on a very restricted group of patients, minimizing any confounding factors that might compromise the findings of the studies. As a result, a large number of potential patients who might benefit from the drug on trial are excluded. In some cases, the pharmaceutical company might apply for ‘expanded access’ to allow patients who do not meet the strict criteria for the trials (e.g. wrong age, wrong gender, complex medical history) to gain access to the trial drug. This is usually only granted in cases where there is no credible alternative therapy, particularly if the disease is life-threatening. Furthermore, there should be no evidence from the trials that precede the application to suggest that there might be detrimental effects.

As with in vivo animal studies, great consideration must be given to minimize any potential pain or suffering of the subject of clinical trials, with the added caveat that the subject must be fully informed of the procedure and possible implications. Patients must sign a consent form to that effect. End-points must be clearly stated prior to the experiment and, unless the nature of the experiment dictates otherwise, the trial should be of a double-blind (both patient and researcher are unaware of whether the administered agent is placebo or test drug), randomized, crossover (some patients receive drug first, followed at a later date by placebo or vice versa) style. This is not always possible—for example, it is sometimes necessary to run parallel groups, one of which receives placebo whilst the other receives drug treatment. In these cases, it is essential that the groups be matched as closely as possible (e.g. medical history, age, gender). In very large trials, an interim analysis is normally carried out: if the drug is found to be detrimental, or indeed highly beneficial, the trial may be stopped on the grounds that it is unethical to continue.

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