Dimension Vista® System Principles of Vista® Chemistry Nephelometry Online Training

Describe basic immunology terms and processes Describe how the system performs nephelometric measurements Explain the functions of key components used during the nephelometric assay Welcome to the Nephelometry online training course. After completing this course, you'll be able to: Select Next to continue. Antibodies not only provide immunity against disease, but also provide a method for quantitating a variety of proteins in body fluids because of their unique property of recognizing and distinguishing closely related antigens. In reagent manufacturing, small animals (usually rabbits) are immunized with highly purified human serum to trigger the production of specific antibodies. The resulting antisera are used as reagents in the lab to measure specific antigens in a sample.     Antiserum versus Vaccine Learn the difference between an antiserum and a vaccine. Tab TitleTextAntiserum Contains specific antibodies to an antigen Used in laboratory testing as reagents for certain immunologic tests Used in humans to confer passive immunity to diseases or toxic substances, such as a snake venom antitoxin Does not stimulate the production of antibodies because they contain no antigens Vaccine A suspension of a killed (attenuated or harmless) microorganism administered to stimulate the production of specific antibodies to that microorganism Confer active immunity. Historically, the first vaccinations were used to immunize against smallpox Now available to immunize against many diseases, such as typhoid, measles, and mumps When complete, select the X in the upper-right corner to close the window and continue. Congratulations! You’ve completed the Nephelometry course. In this online training course, you’ve been introduced to the following: Describe basic immunologic terms and processes. Immunology is the study of the body’s response to foreign substances. Immunologic processes involve: Antigens: foreign substances in the body Antibodies: specific proteins, or immunoglobulins, produced in response to the antigen that stimulated its production Immune complexes: web-like structures that form when antigens and their specific antibodies bind Immune response: interaction of antigens and antibodies to form immune complexes. In the laboratory, the interaction of antigens and antibodies to form immune complexes provides a method for quantitating a variety of proteins in body fluids. Describe how the system performs nephelometric measurements. The Dimension Vista® system uses nephelometry to measure plasma protein concentrations. In a cuvette, proteins present in the sample (antigen) react with specific antiserum (antibody) in the reagent to form immune complexes. The nephelometer measures the reaction: A light emitting diode sends a beam of light through the cuvette at a wavelength of 840 nm. Immune-complexes in the suspension scatter the light toward the photodetector. The intensity of the light scatter is proportional to the concentration of plasma protein in the sample. The photodetector converts the intensity of the light scatter to an electrical signal. The electrical signals are recorded over the entire measurement period and then the system calculates a raw value using either the Fixed-Time Kinetic or the VLinIntegral analysis methods. For a Fixed-Time Kinetic analysis, the system determines the raw value from a curve where the change in scattered light intensity is measured between a fixed start time and a fixed stop time. For a VLinIntegral analysis, the system calculates the raw value from a curve created by an algorithm that finds the area of the curve with the highest velocity (maximum rate of reaction) AND the best linearity. The system converts the raw value to a plasma protein concentration using the test method's calibration curve. When measuring small concentration of proteins, nephelometric reactions can be enhanced by the addition of latex particles, which increase the size of the immune complexes to obtain an optimal signal yield. High sensitivity CRP is an example of a latex-enhanced assay. The Heidelberger-Kendall curve shows the amount of scattered light produced at a constant antibody level as the amount of antigen in the sample increases. On the ascending part of the curve, sufficient antibody exists in the reagent to bind with the antigen in the sample. To obtain valid results, the reaction should be read in this region. On the descending part of the curve, too little antibody exists in the reagent to bind with the antigen in the sample. This is called antigen excess. To prevent antigen excess from occurring, the Vista® system performs a pre-reaction check on certain assays, such as serum IgM. During a pre-reaction check, the system monitors raw measurements against a reference curve using a dilution of the sample that was initially pre-diluted by the IMT probe. Based on the results, the system either performs a higher dilution, or adds the remainder of the diluted sample and allows the assay to proceed to completion. Explain the functions of key components used during the nephelometric assay. For nephelometric test methods, the reaction takes place in a cuvette. The cuvette loader loads new cuvettes into the outer section of the cuvette ring. The cuvette ring rotates to transport the cuvette to various positions during the assay. The reagent probe aspirates reagent (antibody) from the Flex® reagent cartridge and dispenses it into the cuvette. The IMT probe pre-dilutes the sample (antigen) in an aliquot plate well. Then, Sample Probe 1 dispenses the diluted sample into the cuvette. The cuvette ring rotates to move the cuvette into the nephelometer, where the reaction is read. Select Next to continue.   Because nephelometry is based on immunochemical principles, let’s briefly review some key concepts about immunology. Immunology is the study of the body’s response to foreign substances. Immunologic processes involve antigens, antibodies, and immune complexes. An antigen is a substance that the body considers to be “foreign” or unwanted. An antibody is a specific protein, or immunoglobulin that is produced to “bind” the antigen that stimulated its production. This interaction is called the immune response. Lymphocytes are the primary cells responsible for producing antibodies—in particular, the B-cell lymphocytes. The antigen-antibody immune complex formed during the immune response is a physical binding similar to a lock-and-key fit. Immune response actions can be beneficial, such as protection against viruses and other pathogens, and antagonistic, such as delayed hypersensitivity and graft or transplant rejection. Who is the founder of immunotherapy? Learn more about the founder of immunotherapy. Dr. Emil von Behring received the first Nobel Prize for Medicine and Physiology in 1901 for the discovery of passive immunity. This research led to his work in serum therapy and its use against diphtheria and tetanus. Von Behring has been called the founder of immunotherapy because of his discovery of the biological principles behind the development of specific antibodies to fight disease. When complete, select the X in the upper-right corner to close the window and continue. In the Assay Measurement area, the Dimension Vista® system uses nephelometry to measure plasma protein concentrations in serum, plasma, urine, and CSF. Let’s go to the virtual Chemistry Lab, where we’ll look inside the Dimension Vista® system to see which components are used and how the instrument performs nephelometric measurements. In this demonstration, you’ll be introduced to the following terms and processes: Latex-enhanced assays Heidelberger-Kendall curve Pre-reaction check Antigen excess Fixed-Time and VLinIntegral analysis methods What is Nephelometry? Nephelometry is the most frequently used measuring principle for immunochemical protein determination in serum, urine, and CSF.  Proteins present in the sample react with the specific antiserum or latex reagent to form insoluble complexes.  Light passes through the suspension and is scattered or reflected toward a detector that is not in the direct path of the transmitted light.  The intensity of the light scatter is proportional to the amount of antigen-antibody complex in the sample.   Plasma Proteins analyzed View a list of plasma proteins analyzed on the Dimension Vista® instrument. Which plasma proteins are analyzed on the Dimension Vista™ instrument? The following plasma proteins are examples of nephelometric test methods quantitated on the Dimension Vista™ instrument. These Dimension Vista™ methods are under development and not available for sale. a1-Acid Glycoprotein al-Antitrypsin Apo A-1 Apo B Antistreptolysin O b2-Microglobulin C3 C4 Ceruloplasmin CRP hsCRP (Cardiac) Cystatin C Haptoglobin lgA lgG lgM Prealbumin Rheumatoid Factor Soluble Transferrin Receptor Transferrin Urine Microalbumin a1- Microglobulin1 When complete, select the X in the upper-right corner to close the window and continue. Nephelometric Measurements Learn about Nephelometric Measurements. Slide NumberText BlocksCalloutsAudio ScriptImage File1Welcome to the Virtual Chemistry Lab! Let's see how the Dimension Vista® system performs nephelometric measurements and which components are involved during the assay. Select Next to continue.Note: If audio does not automatically start, select the play arrow in the top left to begin.Welcome to the virtual Chemistry Lab! Let’s see how the Dimension® Vista system performs nephelometric measurements and which components are involved during the assay. To do this, we must look inside the Dimension Vista™ system. You can only see them in our virtual laboratory! 2The Nephelometric Process The reaction takes place in a hard plastic cuvette. Select Next to continue.For nephelometric test methods, the reaction takes place in a hard plastic cuvette. 3The Nephelometric Process Reagent probe dispenses reagent into the cuvette. Select Next to continue.CalloutsCuvette RingReagent ProbeThe reagent probe dispenses reagent from a Flex® reagent cartridge into the cuvette. 4The Nephelometric Process Cuvette Ring rotates to move cuvette near the aliquot plate. Select Next to continue.CalloutsAliquot PlateThe cuvette ring rotates to move the cuvette into position near the aliquot plate.5The Nephelometric Process   IMT probe pre-dilutes the sample in an aliquot well Sample Probe 1 dispenses sample from aliquot plate to the cuvette Select Next to continue.CalloutsIMT ProbeSample Probe 1After the IMT probe pre-dilutes the sample in an aliquot well, sample probe 1 dispenses sample from the aliquot plate into the cuvette.6The Nephelometric Process As the immune chemical reaction occurs, the cuvette ring rotates to move the cuvette in front the nephelometer which takes measurements and generates a result. Select Next to continue.CalloutsNephelometerAs the immune chemical reaction occurs, the cuvette ring rotates to move the cuvette in front of the nephelometer. The nephelometer takes measurements and generates a numeric result. 7The Nephelometric Process The cuvette ring rotates to the cuvette unload position to be discarded. Select Next to continue..CalloutsCuvette UnloaderAfter the assay is completed, the cuvette ring rotates to the cuvette unload position, where the cuvette is discarded. 8Cuvettes For nephelometric test methods, the reaction takes place in a hard plastic cuvette. Select Next to continue.As you saw in the animation, for nephelometric test methods, the reaction takes place in a cuvette. 9Loading Cuvettes The cuvette loader loads cuvettes into the cuvette ring, where they are available for testing. Select Next to continue.Callouts Cuvette ring Cuvette loaderThe cuvette loader loads new cuvettes into the outer section of the cuvette ring, where they are available for testing. 10Dispensing Reagents When a nephelometric test is ordered, the cuvette ring rotates to one of five reagent probes—depending on where the Flex® reagent cartridge used for the assay is stored. Select Next to continue.CalloutsR2 Reagent probeThe cuvette ring rotates the cuvette to the reagent probe. 11Dispensing Reagents The reagent probe dispenses reagent from a Flex® reagent cartridge into a cuvette loaded in the cuvette ring. Select Next to continue.Slide QuestionAnswer TextNephelometry uses the principle of immunochemistry. Does the reagent serve as antigen or antibody?Antibody!Callouts Flex® reagent cartridge The reagent probe aspirates reagent from the Flex® reagent cartridge and dispenses it into the cuvette.12What’s in the cuvette? For nephelometric test methods, the antiserum in the reagent serves as the antibody. Select Next to continue.CalloutsAntibodyThe cuvette now contains reagent. The antiserum in the reagent serves as the antibody for the immunochemistry reaction.13Dispensing Sample The cuvette ring rotates to move the cuvette into position near the sample aliquot plate. Select Next to continue.CalloutsAliquot platesFrom the reagent probe, the cuvette ring rotates to move the cuvette into position near the sample aliquot plate.14Dispensing Sample The IMT probe pre-dilutes the sample in an aliquot plate well. Select Next to continue.CalloutsIMT probeFor nephelometric reactions, the IMT probe pre-dilutes the sample in an aliquot plate well. 15Dispensing Sample Sample Probe 1 aspirates the diluted sample from the aliquot plate well and dispenses it into the cuvette. Select Next to continue.Slide QuestionAnswer TextDoes the sample contain antigen or antibody?Antigen!CalloutsSample Probe 1Then, Sample Probe 1 aspirates the diluted sample from the aliquot plate well and dispenses it into the cuvette.16Reading the Reaction The cuvette moves in front of the nephelometer to read the reaction. Select Next to continue.CalloutsNephelometerTo read the reaction, the cuvette ring rotates to move the cuvette into the nephelometer. The cuvette is moved in front of the nephelometer to be measured. 17Key Areas of the Nephelometer The nephelometer includes: Electronics   Optical unit: Sender unit—sends a light beam through the cuvette   Receiver unit—measures light scattered by immune complexes Select Next to continue.Select each number to review the corresponding text.CalloutsElectronics Optical unit Sender unitReceiver unitLet’s take a look at key areas of the nephelometer. The nephelometer includes an electronics area with an optical unit underneath. The optical unit sends a light beam through the cuvette, and a receiver unit that measures the amount of light scattered by the immune complexes. 18Reading the Reaction Inside the nephelometer, you can see how the measurement is obtained: 1. A light-emitting diode senses a beam of light through the cuvette at a wavelength of 840 nm. 2. Immune complexes scatter the beam of light. 3. The stop diaphragm traps light—only scattered light gets through. 4. The photodetector captures the scattered light and converts the intensity to an electric signal. 5. The intensity of the light scattered is proportional to the concentration of the protein in the sample. Select Next to continue.CalloutsPhotodetectorStop DiaphragmMirrorCuvette840 nm light-emitting diode (LED)Inside the nephelometer, you can see how the measurement is obtained: 1. A light-emitting diode senses a beam of light through the cuvette at a wave length of 840-nm. 2. Immune complexes scatter the beam of light. 3. A stop diaphragm traps light from the primary beam so that only scattered light can get through. 4. A Photodetector captures the scattered light and converts the intensity to an electric signal. 5. The intensity of the light scattered is proportional to the concentration of the protein in the sample. 19Key Components to Nephelometry-the Chemistry! What's in the cuvette now? In the cuvette, antiserum reagent (antibody) binds with the specific protein being measured in the sample (antigen). Forming the Immune Complex As the reaction proceeds, agglutination occurs, resulting in the formation of immune complexes. Select Next to continue.   CalloutsAntigenAntibodyImmune ComplexIn the cuvette, the antiserum binds with the specific protein being measured in the sample. When antigen and antibody bind, agglutination occurs, resulting in the formation of immune complexes.20Latex Enhanced Reaction When measuring small concentrations of proteins during an immunoassay, often the immune complexes that form are too small to be detected by the optical system. These reactions can be enhanced by the addition of latex particles, which increase the size of the immune complexes and allow us to obtain an optimal signal yield. Select Next to continue.Slide QuestionAnswer TextDid you know?The Siemens Healthcare Diagnostics CardioPhase™ hsCRP assay is considered by many to be the Gold Standard and that it was the first to have a cardiac specific intended use.CalloutsSmall immune complexesLatex-particles bind to antibodyWhen measuring small proteins during an immunoassay, often the immune complexes that form are too small to be detected by the optical system. These reactions can be enhanced by the addition of latex particles, which increase the size of the immune complexes and allow us to obtain an optimal signal yield. An example of a latex-enhanced assay is high sensitivity CRP. 21Heidelberger-Kendall Curve The Heidelberger-Kendall curve shows the relationship between the: Amount of antigen in the sample (X axis)   Amount of scattered light produced at a constant antibody level (Y axis) Select Next to continue.CalloutsAgglutination/Measurement SignalExcess of Antibody Ascending curveAntigen concentrationZone of Equivalence RangeDescending curveExcess of Antigen The Heidelberger-Kendall curve displayed here shows the relationship between the amount of antigen in a sample and the amount of scattered light produced at a constant antibody level. For antigen-antibody reactions, you must have an adequate antibody concentration in the reagent to bind with the antigen in the sample. Let’s take a closer look at the Heidelberger-Kendall curve… 22Heidelberger-Kendall Curve Ascending curve: Antibody excess—all antigen bound by antibody Zone of Equivalence range: Unstable—equal concentrations of antigen and antibody Descending curve: Antigen excess—all antibody bound by antigen; additional unbound antigen remaining Select Next to continue.CalloutsAgglutination/Measurement SignalExcess of AntibodyAscending curveAntigen concentrationZone of Equivalence rangeDescending curveExcess of Antigen Ascending portion of the H-R curve—the amount of antibody exceeds the amount of the antigen in the sample. Zone of Equivalence—the amount of antibody and antigen are equal, but the region is unstable. Descending portion of the H-R curve—the amount of antibody is not sufficient to bind all existing antigen in the sample. This is called antigen excess. 23Antigen Excess If antigen excess occurs: Immune complexes no longer form Signal decreases Inaccurate analysis results Select Next to continue.Slide QuestionAnswer TextDid you know?The optimal measurement should be taken on the ascending portion of the H-K curve.CalloutsAgglutination/Measurement signalExcess of AntibodyAscending curveAntigen concentrationZone of Equivalence RangeDescending curveExcess of Antigen If antigen excess occurs, the signal starts to decrease because immune complexes can no longer formthis can cause inaccurate analysis results! 24Pre-reaction Check The Dimension Vista® instrument automatically runs a pre-reaction check when antigen excess is likely—such as serum IgM assays. During a pre-reaction check, the assay is run using: Complete amount of reagent (antibody) Small amount of diluted sample (antigen) Select Next to continue.The Dimension Vista instrument automatically runs a pre-reaction check when antigen excess is possiblesuch as serum IgM assays. During a pre-reaction check, the assay is run using the complete amount of reagent (antibody) and a small amount of diluted sample (antigen).25Pre-reaction Check During the pre-reaction check, the system monitors the reaction signal: If the signal exceeds a specific threshold, the system will automatically perform a higher sample dilution and repeats the reaction. This continues until the raw value is within the acceptable range, or dilution limits are met.   If the signal does not exceed the threshold, the remainder of the diluted sample is added. Then the assay proceeds to completion. Select Next to continue.CalloutsAgglutination/Measurement signalAscending CurveExcess of AntibodyZone of Equivalence RangeExcess of AntigenDescending Curve During the pre-reaction check, the system monitors the reaction signal for a fixed amount of time and evaluates the raw value on a reference curve. • If the signal exceeds a specific threshold, the system will automatically perform a higher sample dilution and repeats the reaction. This continues until the raw value is in the acceptable range or dilution limits are met. • If the signal does not exceed the threshold, the remainder of the diluted sample is added. Then the assay proceeds to completion. 26Fixed-Time Kinetic Method Using the fixed-time kinetic method, the system calculates the signal from the difference between two measured values that are obtained at different times.   The increase, or for some assays, the decrease, in scattered light is calculated from these measured values and converted to the concentration using the calibration curve. Select Next to continue.CalloutsScattered light intensityTime Some nephelometric assays use the fixed-time kinetic analysis method to obtain results. During the reaction, the system calculates the difference between two measured values that are obtained at different times during the assay. The increase, or for some assays, decrease, in scattered light is calculated from these measured values and converted to the concentration using the calibration curve. 27VLinIntegral Method VLinIntegral is a mathematical algorithm for evaluating agglutination reactions (such as particle-enhanced immunoassays or precipitation tests) via the maximum rate of reaction.   Use of this algorithm can have one or more of the following effects, depending on the particular assay: The analytical sensitivity is increased. The measuring range is extended. The antigen excess reliability is improved. The measurement time is reduced.   This method determines the change in scattered light intensity per time interval. The kinetics are used to ascertain how many measuring points are to be included for the calculation of the line of regression. Select Next to continue.CalloutsScattered light intensityCuvette blank valueTime Other nephelometric assays involve agglutination reactionssuch as particle-enhanced immunoassays and precipitation tests. These assays use the VLinIntegral method to obtain results. VLinIntegral is a mathematical algorithm that evaluates the maximum rate of reaction during an assay. This analysis method increases sensitivity, extends the assay range, improves antigen excess reliability, and reduces measuring time. This method determines the change in scattered light intensity per time interval. The kinetics are used to ascertain how many measuring points are to be included for the calculation of the line of regression. 28Now you know how the Dimension Vista® system performs nephelometric measurements.When complete, select the X in the upper-right corner to close the window and continue. That’s it! Now you know how the Dimension Vista™ system performs nephelometric measurements and which components are involved during the assay.