CC-Supplies-and-Reagents (1).pdf

Clinical Chemistry Supplies and Reagents At the end of the lesson, students should be able to: review on the different supplies and reagents according to their characteristics and uses in Clinical Chemistry laboratory; decide on appropriate supplies and reagents to use in t...

Clinical Chemistry Supplies and Reagents At the end of the lesson, students should be able to: review on the different supplies and reagents according to their characteristics and uses in Clinical Chemistry laboratory; decide on appropriate supplies and reagents to use in the performance of a laboratory procedure. Laboratory Balance A properly operating balance is vital in producing high-quality reagents. Its use has minimized because most chemistry laboratories are now purchasing commercially prepared reagents.  Laboratory Balance The most popularly used balances are analytic and electronic balances: a)Analytic balance is for the preparation of primary standard. It has a single pan enclosed by a transparent sliding door. The weight range is from 0.01 mg to 160 g. b)Electronic balance is also a single pan balance that uses electromagnetic force to counterbalance the weighed sample’s mass. Its measurements equal the accuracy and precision of any available mechanical balance, with the advantage of a fast response time ( less than 10 seconds). pH meters pH is an expression of the degree of acidity or alkalinity of a solution. The value does not actually indicate the true hydrogen ion concentration but rather hydrogen ion activity. pH values can be digitally displayed or on galvanometer type scale. Thermometers A lot of procedures in the chemistry laboratory require monitoring of accurate temperature so a chemical reaction can proceed. Reactions that are temperature dependent use some type of heating, cooling cell, heating/ cooling block, or water/ice bath to provide the correct temperature for the reaction environment. Laboratory refrigerators and current clinical chemistry analyzers have built-in thermometers for temperature maintenance. Centrifuge Centrifuge is a machine that uses centrifugal force to separate phases of different mass and densities. A centrifuge is made up of a head or rotor, carriers, or shields that are attached to the vertical shaft of a motor or air compressor and enclosed in a metal covering. Rotor is the part of the centrifuge that holds the tubes and rotates during the operation of the centrifuge. Modern centrifuges have lid for safety operation, brake or a built-in tachometer; some are refrigerated Centrifuge Principle of balance. Tubes and carriers must be of equal weight, shape, size, and must be placed in opposing positions in the centrifuge head. The principle of balance is necessary to avoid noise, vibration, or shaking of centrifuge. An unbalanced load also decreases the speed of centrifuge leading to improper separation of phases. Centrifuge Always operate centrifuges with lids closed to avoid aerosol contamination. Allow the rotor to stop spinning before opening the centrifuge lid. Use only tubes that are specified as appropriate for that particular centrifuge Tachometer (speed indicator) should be used as a guide for reproducible centrifugation. Clean any spills or debris ( glass or blood ) Centrifuge Centrifuge (traditional) horizontal-head centrifuge: the cups holding the tubes of material to be centrifuged occupy a vertical position when the centrifuge is at rest but assume a horizontal position when the centrifuge revolves. The horizontal-head is also called swinging bucket Centrifuge (traditional) fixed-angle-head centrifuge: the cups are held in a rigid position at a fixed angle. This position makes the process of centrifuging more rapid than with the horizontal- based centrifuge. There is a less chance that the sediment will be disturbed when the centrifuge stops. Borosilicate glass Commercial names: Pyrex; Kimax Low alkali content Resistant to heat, corrosion and thermal shock Balanced mechanical, thermal, and chemical strengths Subject to scratching and may cloud with strong alkali Aluminosilicate glass Commercial brand: Corex High silica content which makes it comparable to fused quartz in heat resistance, thermal stability, and electrical characteristics Strengthened chemically rather than thermally Used for high precision analytical work in radiation resistant and used for optical reflection and mirrors Acid-resistant and Alkali-resistant glass Boron –free Referred as “soft glass” because its thermal resistance is much less than that of borosilicate Low Actinic glass (amber colored glass) Amber or red color to reduce the amount of light through the substance inside the glassware Used for substances that are particularly sensitive to light, like bilirubin and Vit A Flint glass (Soda lime glass) Composed of a mixture of the oxides , calcium, and sodium Less expensive Less resistant to high-temperature and sudden change in temperature Chemical resistance is fair Can cause contamination because it is releases alkali into solutions Comparison of plasticware and glassware Containers and Receivers are of high quality material; they are not calibrated to hold an exact volume. These include beakers, test tubes, Erlenmeyer flask, and reagent bottles. Pipettes types of volumetric plasticware or glassware used commonly in the laboratory. They are used to transfer or measure aliquots of a liquid. They come in different sizes and transfer different volumes of liquid less than 20 mL. Pipettes (according to design) To Contain (TC): TC pipette holds a particular volume but does not dispense that exact volume. To Deliver (TD): TD pipette dispense the volume indicated Pipettes (according to type) Transfer: Volumetric Ostwald-folin Pasteur Automatic macropipettes or micropipettes Pipettes (according to type) Measuring/Graduated: Serologic Mohr Bacteriologic Ball, Kolmer, or Kahn Micropipette Pipettes (according to drainage characteristics) Blowout: has continuous etched ring or two small, close, continuous rings located near the mouth or top of the pipette. This indicates that the remaining liquid at the tip must be expelled into the receiving container. Pipettes (according to drainage characteristics) Self Draining: the fluid is allowed to drain by gravity. With the exception of Mohr pipette, the tip must remain in contact with the vessel with the side of the receiving container for some second after the liquid has drained. Pipettes Serological pipet is known as a “to deliver(TD)/blowout pipet ”. Calibration is until the tip. Mohr pipet has the same measuring technique as the serological pipet. It is however, a TD/”no blowout” pipet. The end-calibration line for measuring the full volume of the pipet is before the tip of the pipet. The pipet is held vertical and the liquid is allowed to drain from the beginning calibration line to the end calibration line to measure the full volume. Pipettes Volumetric pipet has a bulb in the middle of the pipet and two slender pieces of glass on either side. It is not graduated. It measures only one volume. It is a TD/”no blowout” pipet. It is more accurate than serological and is used to add diluent to a lyophilized control or measure standards and reagents. Ostwald-Folin Pipet is a TD/blowout” pipet. It’s use for measuring viscous solutions such as whole blood, but is not used much in laboratory anymore. The same pipetting technique applies. Pipettes Micropipettes are pipets that measure small amount of a liquid, less than 1 mL. They can be deigned either as Mohr or a serologic but they are usually “to contain” (TC) pipets, Unopette is a special micropipette used in Hematology laboratory. It is self-filling pipet. Capillary pipets (TC) are other disposable micropipette. It is inexpensive and is made of capillary tubing with a calibration line marking a specified volume. Pipettes Pasteur and Disposable Transfer pipets are used for transferring liquid from one receptacle to another. Disposable pipets are made of plastic, while, Pasteur usually have a reusable suction top and disposable glass pipet bottom.  Pipetting with manual pipets Check the pipet to ascertain its correct size, being careful also to check for broken delivery or suction tips. Volume indication and type of pipette can be seen near the mouth of the pipette. Wearing protective gloves, hold the pipette lightly between the thumb and the last three fingers. Place the tip of the pipet well below the surface of the liquid to be pipetted.  Pipetting with manual pipets 4. Using mechanical suction or an aspirator bulb, carefully draw the liquid up into the pipette until the level of the liquid is well above the calibration mark. 5. Quickly cover the suction opening at the top of the pipet with the index finger 6. Wipe the outside of the pipet dry with a piece of gauze or tissue to remove excess fluid.  Pipetting with manual pipets 7. Hold the pipette in a vertical position with the delivery tip against the inside of the original vessel. Carefully allow the liquid in the pipet to drain by gravity until the bottom of the meniscus is exactly at the calibration mark. (The meniscus is the concave or convex surface of a column of liquid as seen in a laboratory pipette, buret, or other measuring devise). To do this, do not entirely remove the index finger from the suction-hole end of the pipette; rather, by rolling the finger slightly over the opening allow slow drainage to take place.  Pipetting with manual pipets 8. While still holding the pipette in a vertical position, touch the tip of the pipette to the inside wall of the receiving vessel. Remove the index finger from the top of the pipette to permit free drainage. Remember to keep the pipette in a vertical position for correct drainage. In TD pipettes, a small amount of fluid will remain in the delivery tip.  Pipetting with manual pipets 9. To be certain that the drainage is as complete as possible, touch the delivery tip of the pipette to another area on the inside wall of the receiving vessel. 10. Remove the pipette from the receiving vessel, and place it in the appropriate place for washing  Semiautomated pipettes Offer more convenience and efficacy to pipetting. No pipetting bulb is needed. It makes use of plastic tips which are disposable and autoclavable. Plastic tips retain less inner surface film than the glass tips The pipets can be single or multichannel. A plunger or trigger is used to aspirate the liquid into the pipet. These pipets use air or positive displacement to draw up the fluid.  Steps in using semi- automated micropipettors: Attach the proper tip to the pipettor, and set the delivery volume. Depress the piston to a stop position on the pipettor. Place the tip into the solution, and allow the piston to rise slowly back to its original position. Some tips are wiped with a dry gauze at this step, and some are not wiped. Follow the manufacturer’s directions.  Steps in using semi- automated micropipettors: 5. Place the tip on the wall of the receiving vessel, and depress the piston, first to a stop position where the liquid is allowed to drain, then to a second stop position where the full dispensing of the liquid takes place. 6. Dispose of the tip in the waste disposal receptacle. Some pipettors automatically eject the used tips, thus minimizing biohazard exposure. Reagents Reagent is defined as any substance employed to produce a chemical reaction. With automation, most instruments use their own ready-to-use form or “kit”. Chemicals Reference materials Reagent Water Buffers Reagents: chemicals ·Analytic reagent (AR) grade are of a high grade of purity and are used often in the preparation of reagents. ·Ultrapure grade - for specific procedures such as chromatography, AAS, immunoassays, molecular diagnostics, and standardization techniques Reagents: chemicals ·Chemically Pure (CP) Grade the impurity limitations are not stated and that the preparation of these chemicals is not uniform. Clinical laboratories cannot use these chemicals in reagent preparation unless with reagent blank. However, the CP designation does not reveal the limits of impurities that are tolerated. Reagents: chemicals ·United Sates Pharmacopeia (USP) and National Formulary (NF) grade are chemicals that are generally less pure than CP-grade chemicals, because the tolerances specified are such that USP and NF chemicals are not injurious to health, rather than chemically pure. Reagents: chemicals ·Technical or commercial grade are chemicals used only for industrial purposes and are generally not used in the preparation of reagents for clinical laboratory. Reagents: reference materials Primary standard is a highly purified chemical that can be measured directly to produce a substance of exact known concentration of purity. National Institute of Standards and Technology (NIST) develops certified reference materials for clinical chemistry use. Reagents: reagent water type 1 reagent water is the most pure and should be used for procedures that require maximum water purity. It must be used immediately after it is produced and not to be stored. Uses: flame Photometry, AAS, blood gases and pH, enzyme studies, electrolyte testing, HPLC, trace metal and iron studies Reagents: reagent water Type II reagent water is used for qualitative chemistry procedures and for most procedures done in Hematology, immunology, microbiology, and other clinical test area. Type III reagent water can be used for some qualitative laboratory tests, such as done in general urinalysis. It is used as a source for type 1 or type II water. It is also used for washing and rinsing glassware. Reagents: buffers Buffers are weak acids or bases and their related salts, that, as a result of their dissociation characteristics, minimize changes in the hydrogen ion concentration.