Introduction to Clinical Chemistry PDF

Introduction to Clinical chemistry  CLINICAL CHEMISTRY Clinical biochemistry, chemical pathology and clinical chemistry are synonyms Clinical: describes the practical observation and treatment of a patient Biochemistry: is the chemistry of life. It seeks to describe the structur...

Introduction to Clinical chemistry  CLINICAL CHEMISTRY Clinical biochemistry, chemical pathology and clinical chemistry are synonyms Clinical: describes the practical observation and treatment of a patient Biochemistry: is the chemistry of life. It seeks to describe the structure, organization and functions of living matter in molecular terms.  Clinical chemistry: is the branch of laboratory medicine in which biochemical methods are applied to the study of disease. Clinical chemistry: the function of the clinical lab is to perform qualitative and quantitative analysis on body fluids such as blood, urine, and spinal fluid, as well as feces, tissue, calculi, and other material.  Clinical biochemistry: The clinical biochemistry measures change in biochemical compounds as an indicator of health status or disease processes. Clinical biochemical tests comprise over one third of all hospital laboratory investigations. A centralfunction of the clinical chemistry laboratory isto provide biochemical information for the management of patients.  Biochemical results are useful for diagnosis and treatment of disease.  the tests must be performed accurately.  validated analytical methods and good instrumentation should be available with understanding of chemical reactions involved in each test.  A working knowledge of clinical chemistry and biochemistry is essential for the technologist to effectively communicate and interact with other health professionals  The technologist must have a comprehensive background in the terminology and abbreviations used in clinical lab. The technologist must have the necessary background to understand the basis of lab tests that are used to measure physiological parameters. The technologist must be aware of the influence of disease states and drug therapy on the results of lab diagnostic tests.  This course provides the basic information in lab medicine that is necessary for the technologist.  CLINICAL CHEMISTRY How biochemical tests are used Biochemical tests are used extensively in medicine. Biochemical tests are used in diagnosis, prognosis, monitoring and screening.  Use of biochemical tests The results of the biochemical tests are useful to the clinician in: 8  Diagnosis is the art or act of distinguishing one disease from another.  Medical diagnosis is based on the patient’s history combined with the findings on examination. It is usually possible to make a differential diagnosis (is the term used when making a correct decision between diseases presenting a similar clinical picture).  Biochemical and other investigations may then be used to distinguish between them  Investigations may be selected to help either confirm or disprove a diagnosis.  Biochemical tests are important for confirmation or rejection of clinical diagnosis Prognosis: a medical term indicates the doctor's prediction of how a patient's disease will progress, and whether there is chance of recovery, based on knowledge of the course of the disease in other patients together with the general health, age and sex of the patient. Tests used primarily for diagnosis may also provide prognostic information for example, serial measurements of plasma creatinine concentration in progressive renal disease are used to indicate when dialysis may be required. Monitoring (sequential recording, keep watch over): To do this, there should be a suitable analyte, for instance, glucose in patients with diabetes mellitus. To follow the course of an illness and to monitor the effects of treatment. Examples: Glycated haemoglobin in patients with Diabetes mellitus. Biochemical tests may also be used to detect complications of treatment, such as hypokalemia during treatment of diuretics. Screening (examine for the presence or absence of a disease) Biochemical tests are used to determine Subclinical diseases: An illness that stays below the surface of clinical detection, which has no recognizable clinical findings.  Is designed to detect individuals affected with a condition before it is apparent clinically. The best known example is the mass screening of all new born babies for phenylketonuria (PKU), which is carried out in many countries, and congenital hypothyroidism. (Analysis of newborn blood sample for thyroid stimulating hormone (TSH) during 1st week of life.  Qualitative and quantitative analysis  The clinical chemistry tests can be divided into three main category  Core biochemistry Core analysis: are the commonly requested tests which are of value in many patients, carried out in every biochemistry laboratory.  Specialized tests Not every lab is equipped to carry out all possible biochemistry requests, may be referred to larger laboratories (DNA analysis).  Less commonly asked tests for.  An urgent test in The emergency lab All clinical chemistry labs provide facilities for urgent tests. Only a small number of test types are available from the emergency lab. These tests are processed rapidly. An urgent test is designed as test on which the clinician is likely to take immediate action. When an immediate treatment will depend on the result.  The clinical chemistry tests 1. Core biochemical tests Sodium, potassium, chloride Urea and creatinine Calcium and phosphate Total protein and albumin Bilirubin and alkaline phosphatase ALT and AST -glutamyl transferase (GGT) Creatinine kinase Glucose Amylase Lipids  The clinical chemistry tests 2. Specialized tests 3. Emergency tests Hormones Specific Urea electrolytes proteins Trace elements Blood gases Glucose Vitamins Drugs Lipoproteins electrophoresis Cardiac PC Troponins enzymes R  Tests performed away from the laboratory: Point of care testing (POC) Instruments are available that can perform certain test at different locations such as at the bedside or in a clinical care units. Blood glucose (glucocheck) Urine analysis Occult blood Blood gases Electrolytes, urea, creatinine Cardiac markers (Troponin I and T, CK-MB) POC tests are always more expensive than the same tests performed in the central laboratory.  Tests performed away from the laboratory: Point of care testing (POC) Why do we need point of care testing? 1. Tests are of urgent importance and results will affect the immediate management of the patient such as: Blood gases ( Operated by respiratory therapists) Electrolytes Glucose 2. tests are so common, simple and cheap that it is more economical to perform them at the point of care such as:  Blood glucose  Urine analysis  Qualitative pregnancy test in urine. HCG (Analysis of human chorionic gonadotropin hormone in urine) Tests performed away from the laboratory: Point of care testing (POC) The most common is the detection of glucose conc., in a finger stab sample in the clinic or at home by pocket-sized instruments. It is important for diabetic patients to monitor their blood glucose on a regular basis. Advantages: Time saving , convenience to both patient and clinician. Provides information so that therapeutic intervention may be initiated immediately. Disadvantage: The selected test or tests should be able to provide the type of information required Reasons for ordering lab tests: To make a definitive diagnosis or to confirm a clinically suspected diagnosis. To rule in (or out) a differential diagnosis list. To screen for hidden disease. To determine the severity of disease (usually an acute disease). To determine the stage of disease (usually a chronic disease). To assist in determining therapy or the effect of therapy. To prepare the patient for a routine admission or operation.  Steps in obtaining a laboratory test: 1. Written order is placed 2.Specimen is collected and properly labeled 3. Specimen and order are transported to the lab. 4.The specimen is accessioned in the lab. 5.The specimen is processed 6.The specimen is analyzed 7. The results are reviewed and verified by an MT 8. The results are released to the patient record.  Specimen collection  In order to carry out biochemical analyses. It is necessary that the laboratory: be provided with both the correct specimen for the requested test all information which will ensure that the right test is carried out the result returned to the requesting clinician with the minimum of delay.  As much detail as possible should he included on the request form to help both laboratory staff and the clinician in the interpretation of results.  This information can be very valuable when assessing a patient's progress over a period  Sample collection and processing Specimens used for biochemical analysis: 1. Venous blood, arterial blood, capillary blood 2. Urine 3. Stool 4. CSF 5. Amniotic fluids 6. Aspirates, Paracentesis fluid: pleural, pericardial and ascitic fluid, joint (synovial) fluid 7. Calculi (stones)  Specimen collection Collection of blood Blood for analysis may be obtained from veins, arteries or capillaries. Venous blood is usually the specimen of choice. Skin puncture: if only a small volume of blood is required for a blood test (e.g., a blood glucose test), A skin puncture may be used to obtain the sample in an infant younger than 1 year, the lateral site surface of the foot should be used for skin puncture. Acceptable sites for skin puncture to collect blood from infant’s foot  Blood Sample Whole blood, plasma or serum can be used for testing. Whole blood: If whole blood is desired for testing, an anticoagulant must be added to the specimen during the collection procedure. Whole blood is rarely required for clinical chemistry tests; only for blood gas, ammonia, and some trace element determinations. Plasma Plasma is the fluid fraction of blood. If plasma is desired for testing, an anticoagulant must be added to the specimen during the collection procedure. Serum Serum is the supernatant fluid which forms when blood clots. Serum vs. Plasma Blood Sample If blood is collected into a plain tube and allowed to clot after centrifugation a serum specimen is obtained. Serum from coagulated blood is the specimen of choice for many assay systems, but plasma obtained with an appropriate anticoagulant may be an equally valid specimen. The use of plasma accelerates analysis in medical emergencies and when the analyte is unstable (no time is wasted waiting for the specimen to clot. Because serum requires a wait of 15 to 30 min for coagulation. The formation of fibrin clots or fragments when plasma is stored and the subsequent risk of blockage sample probes of automated analytical instruments is a disadvantage. Plasma is also not suitable for electrophoresis analysis, because the presence of fibrinogen can confuse interpretation of electrophoresis patterns. Preparation of serum sample: Safe Re-cap Methods Blood specimen tubes for specific biochemical tests Serum separator tube (SST) contains a gel at the bottom to separate serum, sodium citrate tube, the blood and anticoagulant ratio should be precisely known Since the tube is used for coagulation study.  Urine collection The type of urine to be collected is determined by the tests to be performed. A clean, early-morning, fasting specimen is generally the most concentrated specimen and thus is preferred for microscopic examinations. It is satisfactory in most cases to use specimen collected with careful attention to cleansing and to keeping the urine cool. Collection is preferred to be fresh, no need for preservatives. But 24 hour urine sample, must introduce a preservative. The most common preservatives are freeze, glacial acetic acid, nitric acid. Preservatives have different roles but are usually added to reduce bacterial action or chemical decomposition or to solublize constituents that might otherwise precipitate out of solution. Small aliquots of faeces are frequently analyzed to detect the presence of “hidden” or so called occult blood, which is recognized as one of the most effective evidences to the presence of bleeding ulcer or a malignant disease in the GIT. Collection of spinal fluid Spinal fluid is normally obtained from the lumbar region. Spinal fluid is examined when there is a question as to the presence of meningitis. Collection of other fluids and tissues for analysis Synovial fluid aspiration: synovial fluid is withdrawn from joints to aid characterization of the type of arthritis and to differentiate non- inflammatory from inflammatory fluids. Dangerous specimens All specimens from patients should be considered dangerous. A similar label attached to the request form !!!!. Hepatitis B and HIV are of most concern of the laboratory staff.!!!!! All specimens should always be treated both by clinicians and biochemistry staff as potentially hazardous.  Sources of error in laboratory results: Pre-analytical error Analytical error Post analytical error SAMPLING ERRORS There are a number of potential errors which may contribute to the success or failure of the laboratory to provide the correct answers to the clinician questions. 1. pre-analytical Errors: Collection:  Was the right tube used?  Was vein puncture performed correctly?  Was the specimen properly stored? Blood Sampling technique: Difficulty in obtaining a blood specimen may lead to haemolysis with consequent release of potassium and other red cell constituents (LDH). Results of these tests will be falsely elevated.  Prolonged stasis during vein puncture: Plasma water diffuses into the interstitial space and the serum or plasma sample obtained will be concentrated. Proteins and protein-bound components of plasma such as calcium or potassium will be falsely elevated. Insufficient specimen: analysis requires certain volume of specimen to enable the test to be carried out PT & PTT. Error in timing: The biggest source of error in the measurement of any analyte in a 24-hour urine specimen is in the collection of an accurately timed volume of urine. Specimens requiring special handling: should be placed immediately on Ice: Ammonia, Acid phosphatase. Incorrect specimen container: for each blood sample, the correct container with the proper anticoagulant should be used. Samples for glucose should be collected into a special container containing fluoride which inhibits glycolysis. If a sample is collected into the wrong container, it should never be decanted into another type of tube. For example, blood which has been exposed to EDTA (an anti-coagulant used in sample containers reduced calcium concentration approaching zero. Inappropriate sampling site: Blood samples should not be taken 'downstream‘ from an intravenous drip. e.g Blood sample for glucose taken from the same arm into which 5%, glucose is being infused. Incorrect specimen storage: A blood sample stored long time before analysis show falsely high potassium, phosphate and RBC enzymes as lactate dehydrogenase 2. Identification: was the blood collected from the correct patient? Was the blood correctly labeled? Patient name, ID, Date, time of collection, phlebotomist. Specimen identification: one of the commonest sources of erroneous lab results is misidentified specimen. Blood bank has stricter requirements for specimen identification.  Biological factors affecting the interpretation of results Sex of the patient. Age of the patient (ALK). Effect of diet (glucose lipids). Time when sample was taken (Cortisol). Posture of the patient (supine vs. sitting or standing), Effects of exercise (increase CK and decrease lipids). Medical history. Pregnancy (ALK and urea). Drug history (warfarin). Smoking ( increase ammonia)  Biological factors affecting the interpretation of results Sex of the patient: Blood hemoglobin concentrations are lower in women; thus, the serum bilirubin concentrations are slightly lower. Individual’s typical diet: Vegetarianism: in long-term vegetarians The concentrations of LDL and VLDL are low. The total lipid and phospholipid concentrations are reduced, The concentrations of cholesterol and triglyceride may be only two-thirds of those in persons on a mixed diet. Age of the patient:  In infants, bilirubin rises following birth and peaks about the fifth to seventh day of life (the physiological jaundice).  The blood glucose concentration is low in newborns because of their small glycogen reserves.  The plasma nitrogen concentration decreases following birth as the infant synthesis new protein, and the concentration does not begin to rise until tissue catabolism becomes prominent. Time when sample was taken: Many constituents of body fluids exhibit cyclical variations (Circadian variation); throughout the day: occurring in 24- hour periods. These cyclical variations may be quite large the drawing of specimen must be strictly controlled. The concentration of serum of cortisol may change by 50 % between 08:0 and 16:0. Effects of exercise. The influence of exercise on the composition of body fluids is related to the duration and intensity of the exercise. The stress-response increases the blood glucose which stimulates insulin secretion. Plasma pyruvate and lactate are increased with increased metabolic activity of skeletal muscle. Posture of the patient. The blood volume of an adult in an upright position is typically less than that of an adult in lying down position. total protein and albumin decreased in the supine patient.  Fluid reduction in plasma is associated with a comparable increase in the plasma protein concentration. The concentrations of all proteins, including enzymes and protein hormones, and of such compounds as drugs, calcium and billirubin, which circulates partly bound to protein, are also affected. Drug history. Drugs may affects biochemical test results. It may be dependent on their side effects and patient individual response to certain drugs Effects of drugs on the composition of body fluids are likely to be apparent when large doses of a drug are administered. Example: diuretic drugs often cause mild reduction of the plasma potassium concentration; hyponatremia may be observed. Causes of misleading results from discrepancies in specimen collection Precollection Toilet within 30 min: water intake within 2 hours. Smoking Physical activity Drug or dietary supplement within 8 hours. During Collection Time (Diurnal variance) Posture Hemo-concentration from prolonged tourniquet pressure Excessive negative pressure in syringe Incorrect type of tube Capillary versus venous blood Handling of specimen Insufficient of excess anticoagulant Mixing of blood Specimen identification Specimen storage Delay in transit to laboratory  Biochemical test results are usually compared to a reference range considered to represent the normal healthy state of the specific population. Reference ranges Biochemical test results are usually compared to a reference range considered to represent the normal healthy state. Most reference ranges are chosen randomly to include 95% of the values found in healthy volunteers by definition 5%, of the population will have a result out of reference range. Practically, there are no rigid limits to separate the diseased population from the healthy. The further a result is from the limits of the range, the more likely it is to represent pathology.  QUALITY CONTROL Every analysis in the lab. generates a result The validity of that result cannot be taken for granted, however, without some body of supporting evidence. The most convincing evidence is the establishment of a rigorous comprehensive Quality control program that is faithfully followed This the only way to guard against the possible deterioration of an analytic system, to become altered to imprecision of results when they occur, and have confidence in test result when everything is in control  QUALITY CONTROL A comprehensive quality control (QC) program consist of all the means used by a laboratory to ensure the reliability of every assay performed on specimens arriving in the lab. The program includes much more than the analysis of a control serum with every run, and it involves: (1) lab. Director, (2) Technologists, (3) all other personnel who are an integral part of the specimen collecting, and (4) The analytical system.  QUALITY CONTROL Responsibility of the director or supervisor 1. Select the most accurate and precise analytical method 2. Adequately train and supervise the activities of lab. Staff 3. Make available printed procedures for each method, with directions, an explanation of the chemical principles, reference values (Normal range). 4. Select good instruments and institute a regular maintenance 5. Institute a good quality control program, provide control sera, inspect control charts. 6. Conduct continuing education sessions with technologists  QUALITY CONTROL The role of the Technologist 1. Fellow assay directions explicitly 2. Use the proper control serum for each run, chart the results, and take appropriate action when the control serum result is beyond the established limts. 3. Always use sound, analytical techniques 4. Be conscientious in instrument maitenance 5. Notify the supervisor immediately when analytical problems develop and when unusual or life-threatening results are obtained on a patient.  Precision & Accuracy The first step in the establishment of a quality control program is to ascertain the limits of uncertainty for each test. Every measurement, every analysis carries with it a degree of uncertainty, a variability in the answer as the test is performed repeatedly. It is essential to determine the precision of each test, which reflects the reproducibility of the test. Precise means "exact, as in performance, execution, or amount. "In physical science it means "repeatable, reliable, getting the same measurement each time." The less the variation, the grater the precision.  Precision & Accuracy Precision must not be confused with Accuracy. Accuracy is the deviation from the true results. Accurate means "capable of providing a correct reading or measurement." In physical science it means 'correct'. A measurement is accurate if it correctly reflects the size of the thing being measured. An analytical method may be precise but inaccurate because of a bias in the test method, e.g. Folin-Wu glucose method. High accuracy, but low precision High precision, but low accuracy  This is a precise pattern, This is a randomlike but not accurate. The pattern, neither precise darts are clustered nor accurate. The darts together but did not hit are not clustered the intended mark. together and are not near the bull's eye.  This pattern is both This is an accurate precise and accurate. The pattern, but not precise. darts are tightly clustered The darts are not and their average clustered, but their position is the center of 'average' position is the the bull's eye. center of the bull's eye.  Precision & Accuracy The degree of precision of a measurement is determined from statistical considerations of the distribution of random error; it is best expressed in terms of Standard Deviation A normal frequency curve (bell- shaped, Gaussian curve) is obtained by plotting the values from multiple analysis of a sample against the Accuracy indicates proximity to the true value, precision to the repeatability or reproducibility of the measurement  Precision & Accuracy Control Specimens: Every assay performed in the Lab. generates a set of numbers (usually a patient’s test results), requested by the physician. Even though the procedures and methods used in the tests is known to be satisfactory, there is no guarantee that every thing is working perfectly on any particular day.  Precision & Accuracy The instrument may be out of calibration, Standards or reagents may be deteriorating, The technologist may have made an error. Therefore, it is necessary to check every assay, every time its done in the Lab. the simplest way is to include a control specimen in the run. Control specimens may be purchased, or use pooled serum, more than one control specimen is used with each run, High, Low and Normal. Use of blind control and external control.  Precision & Accuracy Evaluation of a new methodology Every laboratory seeks to improve its methodology as technology advances, new technology are designed, and new assays are introduced. New method cannot be accepted on faith; it must be rigorously tested and shown to meet the criteria established by the clinical laboratory.  Precision & Accuracy First step in testing the new method is with-run precision (used to test one sample repeatedly) Second step day-to-day precision at normal and abnormal levels over 20 day period. The new method is then compared to the current method or a reference method (40 samples split between the methods) and assayed, The a correlation curve is done to compare. The End