Clinical Chemistry Introduction Since the earliest attempts to develop procedures for diagnosing diseasesfrom testing urine for

glucose by tasting it or fi nding it attractive for ants, to boiling and cooling specimens to identify various proteins in body secretionsno aspect of the medical laboratory has become more automated than the chemistry area. A panoply of instruments that are phenomenally accurate, cost-effi cient, and fast is commonly used today to perform chemistry procedures that evaluate the physiological functions of cells, tissues, organs, and entire organisms such as the human body. Many aspects of early testing procedures to measure constituents of the blood are still utilized in the modern clinical chemistry laboratory. The original end-point reaction (also called colorimetric determination), where a reaction develops to the greatest extent possible, then ceases, is a methodology fashioned more than a century ago. It is still used but is now performed more effectively and in a shorter time frame. A second method now determines many enzymes and even some nonenzymatic analytes (constituents being tested for) by kinetic reaction, which measures the amount and speed of change occurring in the reagent and sample mixture over a specifi c period of time. The end-point reaction that required perhaps 30 minutes to reach completion can now be determined by kinetic reaction in only a few seconds. Calculations from basic physics and chemistry principles are also used to provide test results from processes such as spectrophotometry (where a reaction is read by the amount of light transmitted or absorbed in a test reagent) and electrochemistry (chemical reactions that involve an electron transfer). In fully automated systems, all the functions and steps are computer driven. The technician or technologist often has little to do except troubleshoot the functions of the instrument after all the preliminary information has been entered and the instrument has been set up to perform a work list of procedures. An automated system is able to move the provided samples about the instrument before, during, and after the procedures are completed. To provide results from a given sample in an automated system, the sample must fi rst be identifi ed. Following identifi cation, usually from a bar code affi xed at the time the sample is collected, the specimen volume is measured to ensure a suffi cient sample is available for the number of procedures requested. After making sure there is a sample in the proper position, the system will begin the actual procedures. Samples sometimes require pretreatment for certain procedures (this is fully explained in courses dedicated to clinical chemistry). Reagents and samples will be mixed in the proper proportions and then subjected to certain conditions that may be necessary for the chemical reaction to take place. Some of these conditions include acidity, alkalinity, temperature, and reaction times. The reaction that occurs, for the most part in an instrument called a spectrophotometer, will then be analyzed, results will be calculated, and a valid report will be shown on a visible screen. A printed report can be provided if required. The Laboratory Information System (LIS) is often interfaced with the chemistry analyzers and results are then automatically transmitted to the physicians offi ce or to the area of the hospital where the patient is hospitalized. Methods of Analysis Most larger chemistry laboratories have a number of instruments that are used for certain groups of tests. When batches of tests are performed only periodically, there may be an instrument dedicated to only that particular set of tests. Batteries (related groups of tests) may be performed on one type of instrument and another type of panel or profi le (other names for batches or batteries of tests) on another. Certain tests may be more effi ciently performed on one type of instrument while general testing of broad groups may be more cost effective, accurate, or give a shorter turnaround time on another type of machine. A great deal of thought and organization must go into providing the proper instrument for certain tests, as no one instrument will be the best for all single procedures as well as for a grouping of procedures related to a given disease state. There are several terms by which the various automated chemistry instruments are known. Some instruments are known as single channel and others as multichannel. The difference between the two lies in the ability of the multichannel instrument to perform a number of calculations simultaneously. This does not always mean that one is faster than another in its analysis of samples. Other types of analytical processes are common. In discrete analysis, each test reaction takes place in a separate compartment that requires either cleaning or disposal of an element used in the reaction, before the next procedure begins. Random access instruments are able to be programmed to accept samples out of order, even when a large number of tests are already in progress. This is important if a large

batch of routine tests is being performed and a stat (immediate need) request is made by the physician. Sequential analysis means that all tests are performed in the order they were entered into the analyzer, and batteries of tests are performed in a certain order. History of Analyzer Development The original multiple analyzers developed in the 1950s and 1960s were either continuous-fl ow analyzers or used centrifugal analysis to mix the samples and reagents. The fi rst sequential multiple analyzer instruments were developed by Technicon. Over a period of a few years, these instruments were improved until they could perform a larger number of tests in a shorter time. Subsequent chemistry analyzers employed a range of testing methodologies, using built-in computersto handle large volumes of data and store patient results and quality control results.Micropipettes are also now available to measure very small volumes of patient samples and dispense them into cuvettes (reaction chambers) through which spectrophotometers read the reactions. This also nearly eliminates the problem of carryover (new samples being affected by the remnants of previous samples and reagents). Centrifugal analysis used a wheel-shaped device with two portsone where the sample, or unknown, was introduced by pipette into a well and another larger port or well where the reagent was placed. When the wheel was completed with samples and reagent, the instrument spun the samples and the reagent parcels into the end of the wheel, which was of high-grade, clear plastic. The reading device or spectrophotometer was able to read the reaction in the plastic cuvette for each section of the wheel. This system is rarely used today. Automation in the Chemistry Laboratory Almost total automation of the clinical chemistry laboratory has now been achieved. Preanalytical steps have been largely automated through use of robotic or other mechanical devices to identify samples by bar code and enter prescribed workloads into the LIS, which in turn generates identifi cation labels for each sample. The prompt delivery and safe handling of infectious specimens may require protective transportation techniques and containers to ensure personnel safety. Traditionally, specimens are delivered to the laboratory by human transporters and, in some facilities, by a pneumatic tube system. In some areas, robotic tracking and transport systems are available that transport samples from the receiving area to the correct laboratory department. More advanced systems identify the sample, program the analyzers, and even centrifuge the samples to provide serum or plasma for testing. Total automation is designed primarily for larger laboratories as it not feasible or cost effective for smaller laboratories. Commercial laboratories that complete tens of thousands of tests per day all use fully automated systems. The miniaturization of analyzers has paralleled that of computers. Ionselective electrodes that are extremely small and perform procedures almost instantaneously are being used for a number of procedures, and others are on the way. Smaller electronic components with microchips use electrophoresis to perform minute separations of proteins into their divisions based on size and weight. Nanotechnology is a term used to describe the manipulation of materials that are extremely small and are measured in nanograms (billionths of a gram), such as atoms or molecules, including biochemical components like hormones that are found at very small levels in the human body. Signifi cance of Specimen Preparation and Testing Chemistry procedures available for treating diseases or assessing health are many and varied. The sample collection process for chemistry is the fi rst and most important step in performing clinical chemistry tests. Subtle changes or errors in collection will invalidate the results of many procedures, causing delays in treatment or perhaps administration of the wrong treatment. There are also many different types of specimens that are required for certain analyses. Some instruments require a particular type of specimen, such as plasma using only heparin as an anticoagulant (a substance to prevent clotting), although there are several other anticoagulants. Others may accept whole blood, plasma, serum, capillary blood, and other body fl uids. When drawing lists are provided for specimen collection, the type of tube and any anticoagulant required will sometimes be specifi ed to avoid errors leading to erroneous results from preanalytical error (mistakes that occur through mishandling or misidentifi cation of a sample). Remember also that arterial blood is used for blood gases but not for other analyses. A quick review of specimen requirements for chemistry analysis, covered previously in the phlebotomy section, follows: 1. Specimens must be properly identifi ed according to standard procedures followed by the facility. 2. Specimens containing an anticoagulant must be mixed properly to avoid clots. 3. Hemolysis (the breakdown of red blood cells) may release interfering substances.

4. Lipema, a high level of fatty materials in the serum or plasma, must be noted for proper interpretation of results, or the specimen should be recollected. 5. Specimens must not leak contents onto the outer portion of the tube. Certain specimens may require that they be sealed in leak-proof containers. 6. Timely delivery to the laboratory and proper storage of specimens that will not be analyzed until later is of great importance. 7. Short draws are those in which the tube was not completely fi lled and therefore contains less volume than designed for a given amount of anticoagulant. This will result in an improper dilution ratio and will affect results. 8. Patients being administered IV solutions or who have other implanted devices will require care in the collection of blood. Blood should never be drawn from above an active IV site. Many safety devices are provided for handling samples, as this process involves the greatest risk when performing clinical chemistry exams. Most of the chemicals utilized in chemistry procedures are quite safe today. However, in the past, many toxic chemicals were used that were carcinogenic (cancer-causing) or could cause serious tissue damage, such as caustics that might burn the skin or the mucous membranes. Uncapping tubes (removing the specialized plastic and rubber tops) presents an opportunity for exposure to biohazards. By using a safety shield, this danger is minimized. Some instruments pierce the cap of the tube and aspirate the sample without the medical laboratory worker being required to uncap the tube. Other considerations in the chemistry laboratory are the collection of timed specimens that either are to be collected at a specifi c time of day or in a timed sequence to determine a patients response to medication or food. Fasting specimens are required for some procedures to obtain a baseline or to avoid lipemia (fats in the blood that cause turbidity). Other reasons for requiring samples from fasting patients are to avoid foods containing certain chemicals that may elevate or decrease the values of some laboratory tests. An example of this would be testing for blood glucose level, which may be elevated if a blood sample is taken within an hour of a meal that contains carbohydrates and glucose. But even if due diligence and care are exercised for preanalytical factors and the condition of supplies and operations of equipment are adequate, random errors (those whose cause is undetermined) will occur on a statistical basis. Random errors affect precision (reproducibility) and may occur due to a variety of factors, including technique and environmental conditions under which the procedures are performed. As one might recall from the chapter on quality control, roughly 5% of results will be outside an acceptable range. Serum and Plasma as Clinical Specimens What is the difference between serum and plasma? A blood tube that contains no anticoagulant may have clot enhancers as well as barrier materials to separate blood cells and platelets from the liquid portion of the blood. When the blood is allowed to clot and is then centrifuged, the resulting liquid portion of the blood that overlays the cells is called serum. The barrier gel will move to an area between the cells and the serum, allowing easy separation. Blood collected in a tube containing an anticoagulant will not clot if the blood and anticoagulant are in the proper ratio to each other. When an anticoagulated sample is centrifuged, plasma results when the cells and liquid portion are separated. The plasma contains coagulation factors that are absent in serum, which is obtained after the blood has clotted, a process that uses up most of the coagulation factors. Clnical Chemistry Procedures The following representative laboratory procedures for chemistry determinations demonstrate a variety of manual procedures for several different analytes and for each of the fi ve major departments of the laboratory. For some chemistry procedures, the results will correlate with and confi rm the results from other departments. The manual chemistry procedures presented here can be performed with a minimum of equipment and supplies. Basic knowledge of each of these procedures will be valuable to the student of medical laboratory technology as a preparation for working at a clinical site. Even though most clinical sites will perform most of their procedures on automated equipment, there are strong correlations between the manual and automated processes. Some instructors may wish to have students perform some of these procedures as an introduction to the performance of more comprehensive laboratory procedures in other courses. Programs are often designed where both theory and practice may be taught in specifi c courses related to each of the major laboratory areas.

However, in some medical laboratory programs, the practical portion of your education may be deferred until you are assigned to a clinical site such as a hospital laboratory. BASIC CLINICAL CHEMISTRY PROCEDURES MANUAL CHEMISTRY PROCEDURE #1 Total Protein Principles Abnormal total protein values are most often related to liver disease. Although proteins are found in all body fl uids to varying degrees, serum is the best fl uid for determining the level of protein. Plasma has an abundance of fi brinogen that will affect the results of a total protein determination. Total protein determinations test simultaneously for both albumin and globulin through a dye-binding procedure. Albumin, which comprises more than one-half of the total protein value, is often measured in a second and separate test. The difference between the total protein and the albumin is roughly equal to the globulin value. Albumin is important in maintaining fl uid balance within the body, while globulin includes antibodies, blood coagulation proteins, and proteins that transport elements such as iron throughout the body. The ratio among these major proteins will be altered in diseases that affect the functions of the liver, where albumin is synthesized. In this procedure, the student will perform a serum total protein determination by using a simple dye that colors the protein molecules. The intensity of the color is proportional to the level of protein in the sample. This type of reaction is called an endpoint colorimetric determination (Figure 14-1). The color development is proportional to the absorbance level. The Stanbio Total Protein Method is available as a simple test kit that will enable the student to perform a totally manual method for protein. The kit contains both the standards and the reagent for the quantitative colorimetric determination of total protein in serum or plasma. In addition to the reagents provided in the kit, a spectrophotometer capable of absorbance readings at 500 nanometers (nm) is needed. Protein Calculation (Manual Method) Protein (g/dL) = Abs of unknown (Au) 10 (concentration of the Abs of standard (As) standard) (mg/dL) Quality Control Two to three levels of control material with known ranges of protein levels determined by this procedure are analyzed each day of testing in a medical laboratory. The total protein value of each specimen can be estimated by using a refractometer where the specifi c gravity is determined on the appropriate scale (a separate scale is observable for urine samples). Linearity When performed as directed this method provides for linearity from 0 to 10 g/dL. Reporting of Results Report your fi ndings from the Total Protein procedure in the form supplied by your instructor. Results of the serum total protein are measured in grams per deciliter (g/dL). However, the protein values for some body fl uids, including cerebrospinal fl uid and urine, are measured in mg/dL. MANUAL CHEMISTRY PROCEDURE #2 Glucose Principles Glucose is the major carbohydrate found in the blood and is used for energy for the organs and tissues of the body. The glucose determination is chiefl y used to determine if a person is diabetic or, if the patient has already been diagnosed as diabetic, if he or she is compliant in taking insulin and if the dosage is correct. The test may also determine if a patient is hypoglycemic, a condition in which a low blood glucose is found. The Stanbio Enzymatic Glucose procedure is specifi c for glucose and may be used for manual as well as automated systems. It uses an enzymatic procedure for the quantitative enzymatic-colorimetric determination of glucose in

serum, plasma, or cerebrospinal fl uid. The reagent for this reaction provides for an enzymatic determination, and is the most common method for determining glucose values. Reagents including enzymatic glucose reagent may require reconstitution with deionized water. The water and a glucose standard (100 mg/dL) are provided with the kit. Other materials required but not provided are control samples to insure reproducibility and validity of the results. Although this reagent may be used on automated and semi-automated systems, the procedure outlined here is that of a manual procedure. The process of specimen collection and preparation allows for either serum or plasma determinations. If serum is to be used, it must be removed from the clot within 30 minutes of collection to prevent glycolysis (blood glucose will drop signifi cantly after 30 minutes). Plasma obtained by use of a tube with an anticoagulant containing fl uoride is recommended, but any of the common anticoagulants may be used if the plasma is separated from the blood cells promptly after centrifugation. This procedure can also be used for the determination of glucose levels in cerebrospinal fl uid (CSF). No special preparation is required unless the CSF sample contains blood; in such instance, the sample must be centrifuged and removed from the blood, or glycolysis will occur as it does in blood samples. Only minimal limitations are incurred when using this procedure where a specifi c enzyme reacts with glucose. Both serum and plasma samples are processed in the same manner, and the samples, when separated from blood cells, are stable for up to 40 hours if stored in a refrigerator at 2C8C. Excessive levels of ascorbic acid are the chief interfering substance and may result in falsely low glucose values. Analysis parameters are provided by the manufacturer for procedures using autoanalyzers as well as for adaptations from these parameters than can be used for manual procedures. Manual methods may require the same parameters, but some of them might require manual steps such as manually measuring the samples and timing them. These tasks might otherwise be handled by automated equipment. These procedures will be found on package inserts accompanying the test kits and reagents. Most basic procedures performed in a classroom laboratory, however, will be manual methods. The Stanbio enzymatic glucose procedure utilizes a quantitative enzymatic determination of glucose in plasma, serum, or cerebrospinal fl uid. Glucose Calculation (Manual Method) Glucose (mg/dL) = Abs of unknown (Au) 10 (concentration of the Abs of standard (As) standard) (mg/dL) Quality Control Two to three levels of control material with known ranges of glucose levels determined by this procedure should be analyzed each day of testing in a medical laboratory. Linearity When performed as directed this method provides for linearity from 0 to 500 g/dL. Reporting of Results Report your fi ndings from the Glucose procedure in the form supplied by your instructor. Results of serum or plasma glucose are reported in milligrams per deciliter (mg/dL). The glucose determinations for some body fl uids, including cerebrospinal fl uid (CSF), is always lower than the values found in the blood, but are performed in the same manner as for serum or plasma. The value for CSF fl uid is normally only about 60% 70% of that of the blood. MANUAL CHEMISTRY PROCEDURE #3 Total Cholesterol and High-Density Lipoprotein (HDL) Principles The presence of excess lipids (of which cholesterol is but one type) in the blood has been recognized as a risk factor for cardiovascular disease leading to stroke or heart attack for years. A number of procedures are available for measuring cholesterol. In the manual procedure presented here, total cholesterol is measured fi rst. Low-density lipoproteins (LDL) are then removed from the sample with a precipitating reagent, leaving only the high-density lipoproteins (HDL), and a repeat procedure for cholesterol is performed. The difference between the total cholesterol and the HDL value is the LDL value. The Stanbio Cholesterol and HDL Test Kits are intended for the quantitative, colorimetric determination of cholesterol in serum or plasma. Standard laboratory tests for measuring HDL are not usually performed in the clinical laboratory.

Total Cholesterol and HDL Procedure Tube 1 Reagent blank Tube 2 Total cholesterol standard (1 mL reagent and 10 L standard200 mg/dL) Tube 3 Total cholesterol unknown (1 mL reagent and 10 L serum) Tube 4 HDL cholesterol standard (1 mL reagent and 25 L standard50 mg/dL) Tube 5 HDL cholesterol unknown (1 mL total cholesterol reagent and 25 L supernatant from the serum preparation precipitate)

Total Cholesterol Calculation (Manual Method) Total Cholesterol (mg/dL) = Abs of unknown (Au) 200 (concentration of Abs of standard (As) the standard) (mg/dL) HDL Calculation (Manual Method) HDL (mg/dL) = Abs of unknown (Au) 50 (concentration of the Abs of standard (As) standard) (mg/dL) Quality Control Two levels of control material with known cholesterol levels determined by this procedure should be analyzed each day of testing in a medical laboratory. Linearity When performed as directed, the method is linear from 0 to 200 mg/dL. Reporting of Results Report your fi ndings from the Total Cholesterol and High-Density Lipoprotein (HDL) procedure in the form supplied by your instructor. Results of total cholesterol and HDL are reported in milligrams per deciliter (mg/dL). MANUAL CHEMISTRY PROCEDURE #4 AST/GOT (Manual) Chemistry Procedure Principles This test is for the quantitative determination of serum aspartate aminotransferase (AST), needed in the diagnosis and treatment of certain types of liver and heart disease. Organ cells deteriorate under certain conditions (e.g., infection, diminished blood fl ow). The death of these cells releases enzymes that are richer in some organs than others. The physician can rule out certain ailments or suspect others based on this test. Usually, a battery of enzymes tests are run and the results compared for signifi cance. A working reagent is prepared by reconstituting a substrate upon which the enzyme AST will react. The working reagent is prepared by pouring the contents of a small AST additive (substrate) bottle into a larger AST reagent (coenzyme) bottle. Replace the cap and mix well by gentle inversion (do not shake vigorously). For ease in transferring the working reagent, some kits provide a fl ip-top cap to replace the screw cap. The absorbance of freshly prepared working reagent should be at least 1.200 when measured at 340 nm in a spectrophotometer with a 1-cm light path. The reagent should be combined with reagent grade water or the provided diluent and mixed well

before obtaining an initial reading from a spectrophotometer. Storage of the reagent is possible for up to 10 days when stored at 2C to 8C, and for 1 day when stored at 20C to 25C (room temperature), providing that the reagent has not expired. The expiry date is provided on the box in which the reagent was received. The reagent as packaged will be clear and colorless to straw-colored. The additive as packaged will also be clear and colorless. After mixing the two reagents, the working reagent should also be clear and colorless. Discard the working reagent if it is turbid, as this may suggest contamination has occurred. The preferred specimen is fresh and unhemolyzed serum. The samples should be collected in the usual manner for any other clinical test requiring serum. Separate serum and plasma from red blood cells promptly to minimize hemolysis, although slight hemolysis will not signifi cantly affect results. However, if erythrocytes remain in the sample and become hemolyzed, they contain approximately 10 times more intracellular AST than normal serum. If plasma must be used, the recommended anticoagulants are oxalate, citrate, EDTA, and heparin. In addition to hemolysis, other interfering substances such as certain drugs (including illicit drugs) may affect AST determinations. Interferences with the AST procedure commonly occur when triglyceride levels exceed 600 mg/dL. Bilirubin values above 14 mg/dL, a common occurrence in jaundiced persons and those with severe liver damage, may also interfere with this method. Most kits for AST determination are suitable for manual methods as well as semiautomated and automated instruments. The instrument settings are provided through a package insert. The AST procedure is based on a kinetic rate change over a period of time (Figure 14-3). Quality Control Quality control is important in all departments of a clinical laboratory. Fresh commercial control sera prepared each day may require reconstituting with deionized water or may come as a liquid and is recommended with each assay batch to monitor procedural para-meters. Use two controls containing normal and abnormal levels of the enzyme if possible. If r esults fall outside the acceptable activity limits, check procedural parameters (i.e., photometer, cuvette, pipettes, tubes, and temperature). If problems persist, call your technical representative. Please note that some control sera may show increases of AST activity if activated with pyridoxyl phosphate. Reporting of Results Report your fi ndings from the AST/GOT procedure in the form supplied by your instructor. Activity is expressed in units per liter of specimen (U/L). One unit is defi ned as the amount of enzyme that catalyzes the conversion of one micromole of substrate per minute under specifi ed assay conditions. Data used in factor calculations are found on the assay sheet provided by the manufacturer. These data are a factor from the manufacturer of the substrate and are derived from the concentration of the substrate. The results of the test(s) are calculated by multiplying the absorbance change for one minute by the calculated factor. AST/GOT Rate Reaction Calculation U/L = Delta change per minute Derived Factor = Value of Unknown in IU/L CHEMISTRY PROCEDURE #5 Electrolytes Principles Electrolytes may be referred to as a single entity because four of seven components are interrelated, and are balanced between the four basic analytes. The tests for electrolytes refer to positively and negatively charged ions. The positively charged ions, called cations, are sodium, potassium, calcium, and magnesium; sodium (Na+) and potassium (K+) are tested for routinely in a set of electrolytes. Negatively charged ions, called anions, include chloride (Cl), bicarbonate (HCO-3), and phosphate; bicarbonate and chloride are the chief electrolytes routinely monitored. Bicarbonate determinations may be referred to as CO2 (carbon dioxide) as most of the CO2 of the body is found in the bicarbonate molecule . In addition, it is simpler to test for

bicarbonate than for CO2, as the later is a gas and testing for it requires a great deal more preparation. Properly balanced electrolytes are vital for maintaining the narrow range of values necessary for storage of fl uids and proper cellular function and excretion (see Table 14-1). Electrolyte imbalances are caused by a variety of medical conditions. Diabetic comas may occur from electrolyte imbalances (a patient rapidly becomes acidotic, which is a life-threatening condition when the bodys fl uids fall outside a slightly alkaline range of 7.35 to 7.45). Vomiting, diarrhea, and administration of certain medications may lead to an electrolyte imbalance; such imbalances must be rectifi ed quickly and thus may require accurate laboratory measurements stat (immediately). Imbalances of electrolytes often become life-threatening and affect all organs and systems of the body. Electrolytes may be measured by small portable instruments at the bedside, providing continual monitoring when necessary. Electrolytes are also included on most routine biomedical panels of chemistry tests used for screening patients and can be performed quickly by automated means. Proper fl ow of electrolytes between intracellular spaces and extracellular spaces must occur to maintain a balance (Figure 14-4). The methodology for measuring electrolytes has advanced greatly over the years. Initially, sodium, potassium and even calcium determinations were performed by fl ame photometry, where burning of ions emitted light of various wavelengths. Chloride was measured by colorimetry or by the formation of silver chloride when a sample was introduced to a silver wire. Carbon dioxide was measured by manometry or colorimetry. Today, most analyzers use ion-selective electrodes that directly measure these components. A number of instruments are designed to measure either plasma, serum, or whole blood, depending on their operating capacity. Some of these small portable instruments are adapted for use in physician offi ce laboratories and for point-of-care testing in the home or the clinic (Figure 14-5). Reporting of Results Report your fi ndings from the Electrolytes procedure in the form supplied by your instructor. The four major electrolytes most often tested for are reported as follows: Electrolyte Normal Values