An enzyme converts a substrate to a reaction product that emits photons of light instead of developing a visible color. Luminescence is described as the emission of light from a substance as it returns from an electronically excited state to ground state. The various forms of luminescence (bioluminescence, Chemiluminescence, photoluminescence) differ in the way the excited state is reached.
Chemiluminescence is light produced by a chemical reaction. The chemiluminescent substance is excited by the oxidation and catalysis forming intermediates. When the excited intermediates return back to their stable ground state, a photon is released, which is detected by the luminescent signal instrument.
Chemiluminescent assays, in particular enhanced luminescent assays, are very sensitive and have a wide dynamic range. It is believed that luminescence is the most sensitive detection method currently in use due to the ability of signal multiplication and amplification. Luminescent reactions are measured in relative light units (RLU) that are typically proportionate to the amount of analyte present in a sample.
Principle of luminescent immunoassays
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Enzyme-linked Immunosorbent Assays (ELISAs) combine the specificity of antibodies with the sensitivity of simple enzyme assays, by using antibodies or antigens coupled to an easily-assayed enzyme. ELISAs can provide a useful measurement of antigen or antibody concentration. There are two main variations on this method: The ELISA can be used to detect the presence of antigens that are recognized by an antibody or it can be used to test for antibodies that recognize an antigen.
A general ELISA is a five-step procedure:
1) coat the micro titer plate wells with antigen;
2) block all unbound sites to prevent false positive results;
3) add primary antibody (e.g. rabbit monoclonal antibody) to the wells;
4) add secondary antibody conjugated to an enzyme (e.g. anti-mouse IgG);
5) reaction of a substrate with the enzyme to produce a colored product, thus indicating a positive reaction.
The process involves the interaction of the compounds in the analyte (which travels along with a mobile phase) across an immobile surface (stationary phase).
The compound bind at specific regions of stationary phase based on certain physical and chemical properties. These bound molecules are then eluted with a suitable buffer and the same are collected with time.
These are –
Principle of D-10
A dual piston, low pulsation HPLC pump and a proportioning valve deliver the buffer solution to the analytical cartridge and detector. Whole blood samples undergo an automatic two-step dilution process and are then introduced into the analytical flow path. Prediluted samples are identified based upon the use of a sample vial adapter in the sample rack, and the automatic dilution step is omitted. Pre-diluted samples are aspirated directly and introduced into the analytical flow path. Between sample injections, the sample probe is rinsed with Wash/Diluent Solution to minimize sample carryover.
A programmed buffer gradient of increasing ionic strength delivers the sample to the analytical cartridge, where the hemoglobins are separated based upon their ionic interactions with the cartridge material. The separated hemoglobins then pass through the filter photometer flow cell where changes in the absorbance are measured at 415 nm.
The determination of electrolytes (sodium, potassium, and chloride) is one of the most important functions in the clinical laboratory.
Electrolytes affect most metabolic processes. They serve to maintain osmotic pressure and hydration of various body fluid compartments, proper body pH, and regulation of appropriate heart and muscle functions. Electrolytes are also involved in oxidation-reduction reactions and participate as essential parts or cofactors in enzyme reactions.
Photometry is the science of measuring visible light and is based on a relationship between adsorption of light and the properties of the material through which the light is travelling (Beer Lambert’s Law).
The relationship between absorption of light by a solution and the concentration of that solution has been described by Beer and others. Beer’s law states that the concentration of a substance is directly proportional to the amount of light absorbed or inversely proportional to the logarithm of the transmitted light. Percent transmittance (% T) and absorbance (A) are related photometric terms that are explained in this section. Figure 5-3A shows a beam of monochromatic light entering a solution. Some of the light is absorbed. The remainder passes through, strikes a light detector, and is converted to an electric signal. Percent transmittance is the ratio of the radiant energy transmitted (T) divided by the radiant energy incident on the sample (I). All light absorbed or blocked results in 0% T. A level of 100% T is obtained if no light is absorbed.
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Fluorescence flow cytometry (FFC) is used to analyse physiological and chemical properties of cells. It can also be used to analyse other biological particles in urinalysis analysers. It provides:
First a blood sample is aspirated and proportioned, then diluted to a pre-set ratio and labelled with a proprietary fluorescence marker that binds specifically to nucleic acids.
Next the sample is transported into the flow cell. The sample is illuminated by a semiconductor laser beam, which can separate the cells using three different signals:
The intensity of the forward scatter indicates the cell volume. The side scatter provides information about the cell content, such as nucleus and granules. The side fluorescence indicates the amount of DNA and RNA present in the cell.
Cells with similar physical and chemical properties form a cluster in a graph known as a scattergram.
The principle of fluorescence flow cytometry is used in different analysers for haematology and urinalysis. For blood cell counts we use fluorescence flow cytometry, e.g. for the WBC and differential, for NRBC counting and reticulocyte measurement.
In urinalysis analysers, fluorescence technology is also used for counting bacteria, red blood cells, white blood cells and other elements.
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The VITEK® 2 system has everything healthcare laboratories need for fast, accurate microbial identification, and antibiotic susceptibility testing.The innovative VITEK® 2 microbial identification system includes an expanded identification database, the most automated platform available, rapid results, improved confidence, with minimal training time.
The VITEK® 2 system next-generation platform provides greater automation while increasing safety and eliminating repetitive manual operations. The rapid response time means results can be provided more quickly than with manual microbial identification techniques.
The VITEK® 2 system can:
VITEK® 2 systems use Advanced Colorimetry™, an identification technology that enables identification of routine clinical isolates. Advanced Colorimetry provides:
The system includes an Advanced Expert System (AES) that analyzes MIC patterns and detects phenotypes for most organisms tested. This helps optimize laboratory efficiency for lean laboratory management. Rapid results allow clinicians to discontinue empiric therapy and prescribe targeted therapy, resulting in improved patient outcomes and enhanced antibiotic stewardship.
With its ability to provide accurate “fingerprint” recognition of bacterial resistance mechanisms and phenotypes, the Advanced Expert System (AES) is a critical component of VITEK® 2 technology.
VITEK® 2 technology with the Advanced Expert System™ offers:
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The BacT/ALERT® 3D instrument is a state-of-the-art, automated microbial detection system. The BacT/ALERT® system offers advantages in every dimension of microbial detection testing.
From its space-saving modular design to its easy touch-screen operation and flexible data management options, every size laboratory can perform microbial detection with the BacT/ALERT® 3D.
BacT/ALERT® 3D is used for detecting the presence or absence of microorganisms in:
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Because it's so easy to use, BacT/ALERT® 3D saves time, facilitates cross-training and helps prevent errors. This system offers immediate bottle recognition, putting you in control of bottle loading and unloading and virtually eliminating bottle handling errors during microbial detection testing. The system's automatic, built-in quality control, along with a low false positive rate and a rapid response time of this system, means you do more in less time with greater accuracy.
The latest generation of BacT/ALERT® Culture Media brings the most advanced, innovative microbial growth and detection technology to your laboratory. BacT/ALERT® media provide unsurpassed performance, in the detection of a wide variety of microorganisms including bacteria, fungi, and yeasts.
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Traditional Gram staining is a cumbersome procedure that lacks reliability and reproducibility. PREVI® Color Gram is an automated Gram staining system that utilizes patented spray technology to provide rapid, standardized results for all types of specimen while adding efficiency to the microbiology process.
Histopathology- Definition it is a branch of pathology which deals with the study of disease in a tissue section.
The tissue undergoes a series of steps before it reaches the examiners desk to be thoroughly examined microscopically to arrive at a particular diagnosis. To achieve this it is important that the tissue must be prepared in such a manner that it is sufficiently thick or thin to be examined microscopically and all the structures in a tissue may be differentiated.
The term histochemistry means study of chemical nature of the tissue components by histological methods. The cell is the single structural unit of all tissues. The study of cell is called cytology. A tissue is a group of cells specialized and differentiated to perform a specialized function. Collection of different type of cells forms an organ.
Definition It is a complex series of chemical events which brings about changes in the various chemical constituents of cell like hardening, however the cell morphology and structural detail is preserved. Unless a tissue is fixed soon after the removal from the body it will undergo degenerative changes due to autolysis and putrefaction so that the morphology of the individual cell will be lost. Principle of fixation- The fixative brings about crosslinking of proteins which produces denaturation or coagulation of proteins so that the semifluid state is converted into semisolid state; so that it maintains everything in vivo in relation to each other. Thus semisolid state facilitate easy manipulation of tissue. Aims and Effects of fixation If a fresh tissue in kept as such at room, temperature it will become liquefied with a foul odour mainly due to action of bacteria i.e. putrefaction and autolysis so the first and fore most aim of fixation is
1. To preserve the tissue in as lf like manner as possible.
2. To prevent postmortem changes like autolysis and putrefaction.
Autolysis is the lysis or dissolution of cells by enzymatic action probably as a result of rupture of lysosomes. Putrefaction The breakdown of tissue by bacterial action often with 7 formation of gas.
3. Preservation of chemical compounds and microanatomic constituents so that further histochemistry is possible.
4. Hardening : the hardening effect of fixatives allows easy manipulation of soft tissue like brain, intestines etc.
5. Solidification: Converts the normal semifluid consistency of cells (gel) to an irreversible semisolid consistency (solid).
6. Optical differentiation - it alters to varying degrees the refractive indices of the various components of cells and tissues so that unstained components are more easily visualized than when unfixed.
7. Effects of staining - certain fixatives like formaldehyde intensifies the staining character of tissue especially with haematoxylin.
Preparation of the specimen for fixation
1. For achieving good fixation it is important that the fixative penetrates the tissue well hence the tissue section should be > 4mm thick, so that fixation fluid penetrates from the periphery to the centre of the
tissue. For fixation of large organs perfusion method is used i.e. fixative is injected through the blood vessels into the organ. For hollow viscera fixative is injected into the cavity e.g. urinary bladder,
2. Ratio of volume of fixative to the specimen should be 1:20.
3. Time necessary for fixation is important routinely 10% aqueous formalin at room temperature takes 12 hours to fix the tissue. At higher temperature i.e. 60-65°C the time for fixation is reduced to 2
Tissue Fixative of choice Time for fixative Routine Formalin 10-12 hours.
GIT biopsies buffered formaldehyde 4-6 hours
Testicular biopsy Bouin's fixative 4-6 Hours.
Liver Biopsy Buffered formaldehyde 4-12 hours.
Bone marrow biopsy Bouin's fixative in running 2½ hours followed by washing in running water overnight
Spleen and blood filled cavities
Zenker's fluid 1-6 hours
Lymph node B5 12-18 hours
phosphatides and Nissil
Carnoy's fluid 1-2 hours
Chromosome / cell
Clarke's fluid 1-2 hours
Specific Objective - The aim of the study is to ensure staining of hard bony lesions so that the study of pathological lesions is possible. Definition Decalcification is a process of complete removal of calcium salt
from the tissues like bone and teeth and other calcified tissues following fixation.
1. Bancroft, J.D. and Stevens, A.: theory and practice of histological techniques ed.3, Churchill livingstone inc. 1990. Edinburgh. London, Melbourne and New York.
2. Lillie, R.D.: Histopathologic technique and practice histochemistry ed. 3, New York, 1965 McGraw Hill Book co.
3. Manual of histologic and special staining techniques ed. 2, New York, 1960, The Blakiston Division McGraw Hill Book Co.
4. Pearse A.G.E.: Histochemistry, ed. 2, Boston 1960, Little Brown and Co.
5. H.J. Conn's Biological Stains (1969) Lille, R.D. 8th edn, Baltimore; Williams and Wilkins
Nephelometry is a technique used in immunology to determine the levels of several blood plasma proteins. For example the total levels of antibodies isotypes or classes:Immunoglobulin M, Immunoglobulin G, and Immunoglobulin A. It is important in quantification of free light chains in diseases such as multiple myeloma. Quantification is important for disease classification and for disease monitoring once a patient has been treated (increased skewing of the ratio between kappa and lambda light chains after a patient has been treated is an indication of disease recurrence).
It is performed by measuring the turbidity in a water sample by passing light through the sample being measured. In nephelometry the measurement is made by measuring the light passed through a sample at an angle.
This technique is widely used in clinical laboratories because it is relatively easily automated. It is based on the principle that a dilute suspension of small particles will scatter light (usually a laser) passed through it rather than simply absorbing it. The amount of scatter is determined by collecting the light at an angle (usually at 30 and 90 degrees).
Capillary electrophoresis (CE) is a special case of using an electrical field to separate the components of a mixture. Electrophoresis in a capillary is differentiated from other forms of electrophoresis in that it is carried out within the confines of a narrow tube. To understand the behavior of molecules under the influence of an electrical field inside a capillary it is essential to understand the phenomena that result from the geometry of a capillary.
Protein electrophoresis is used to identify the presence of abnormal proteins, to identify the absence of normal proteins, and to determine when different groups of proteins are present in unusually high or low amounts in blood or other body fluids.
Proteins do many things in the body, including the transport of nutrients, removal of toxins, control of metabolic processes, and defense against invaders.
Protein electrophoresis separates proteins based on their size and electrical charge. This forms a characteristic pattern of bands of different widths and intensities on a test media and reflects the mixture of proteins present in the body fluid evaluated. The pattern is divided into five fractions, called albumin, alpha 1, alpha 2, beta, and gamma. In some cases, the beta fraction is further divided into beta 1 and beta 2.