4. Transcription and Translation_GNA.pptx.pptx

4. Transcription and Translation_GNA.pptx

1. SOURCES AND CONTROL OF ANALYTICAL VARIABLES MLAB 208 By E. A. Tagoe 2020

2. Course Objectives Upon completion of the course students are expected to; Sources of specimen for most chemical pathological tests explain analytes and analytical variables know analytical phases and sources of analytical variables know importance and control of analytical variables

3. Sources of specimen for laboratory analysis Laboratory investigation uses specimen and in chemical pathology, the body fluids are the source of materials for laboratory analysis. Biofluids include; Serum, Plasma, Whole blood, Tears, Cerebrospinal fluid, Urine Synovial, Semen, Saliva, Mucus, Amniotic fluid, Lymph, etc.

4. Analytes: These are the chenicals or biomolecules detected in samples during laboratory testing ( also known as variables or parameters ). Some of the laboratory test results (analytes) are measured directly whiles other are derived from already measured ones. Eg : Direct measurement: - Creatinine level in serum is used to measure kidney function. Albumin level is used to measure liver function. Sample analytes

5. Creatinine level in serum (measured directly from serum using appropriate instrument) Fasting plasma glucose – (direct estimation) Cholesterol level in serum – (direct estimation) - Haemoglobin level in whole blood – (direct estimation) Estimation of analytes Direct and Indirect estimation: Direct:

6. Importance of analytes - Fasting plasma glucose - how body cells efficiently utilize blood glucose. Free fatty acids in serum - measure of lipolysis - breakdown of triglyceride High density lipoprotein cholesterol ( HDL -c) – blood lipids clearance Low density lipoprotein cholesterol ( LDL -c) – how blood is loaded with lipids - Mention a variable that suggests infection? (Not the presence or materials of the pathogen).

7. Creatinine clearance {estimates glomerular filtration rate ( GFR )} is calculated using factors including gender (male or female), age, body weight and serum level of creatinine - Derived or indirect estimation. LDL -c levels can be calculated from the triglyceride (TG), HDL -c and Cholesterol in the blood - Derived or indirect estimation. N.B : Errors introduced during acquisition of the direct variables will affect the calculated variables. Estimation of analytes Indirect estimation:

8. The Total Testing Process The total laboratory testing process is the entire process from ordering of a test to the interpretation of test result. It starts and ends with the patient, and can be subdivided into three phases; pre-analytical phase analytical phase post-analytical phase

9. Analytical variables Suspected increased variability in clinical testing or diagnostic testing is referred to as ‘‘laboratory error or analytical variability ” Clinical results variability may occur at any phase of sample testing. ie . pre-analytical phase, analytical phase, post-analytical phase

10. T he pre-analytical phase G enerally th e phase is external to the clinical pathology laboratory and error occurs prior to the sample analysis. Pre-analytical error accounts for than 50% of laboratory errors which leads to variation and bias in the test results.

11. Pre-analytical phase and errors Ordering of test or correct test selection A diagnostic test should only be requested when the result could positively alter the management of the patient. The test must be specific and sensitive. ii. Correct dynamic test procedure A number of biochemical or chemical pathological tests are part of a dynamic test of a patient’s physiological functioning. Dynamic testing procedure is developed appropriate for the variable/ analyte . Example is plasma glucose measurement as part of the glucose tolerance test.

12. iii. Correct patient preparation Pre-analytical phase and errors To reduce variability, biochemical tests may benefit from special instruction to, or preparation of, the patient before the samples are taken. This preparation may include: • Dietary restriction Examples are fasting (lipid profile, C-peptide), fluid restriction (urine cortisol) • Drug restriction . A common error is to overlook withholding steroids for at least 8 hours prior to a Synacthen test.

13. Identification of patients is a major concern for laboratories. Most of the errors occur due to improper identification of patient. One method for checking identification is to compare identifiers such as patient’s name, hospital number. Pre-analytical phase and errors iv. Identification of patients/specimen

14. Illegible handwriting

15. Clear instructions and standard procedures must be readily available for sample collection. There are many variables in sample collection that may influence test results. Some considered procedures include; - Blood collection ( venepuncture ):- Avoid use of drip arm and hence dilution by intravenously administered fluids (“drip arm” specimen). Blood that is drawn from a vein that has an intravenous (IV) line may be diluted by the IV fluid. Venous stasis:- A condition of slow blood flow in the veins. This should be minimized, especially for calcium and protein. Tube type: - Appropriate tubes are required for sampling. EDTA can interfere in some enzymatic tests. In some tubes preservatives, additives are needed. Pre-analytical phase and errors v. Correct sample collection

16. Haemolysis :- this should be avoided. This is common and often interferes with analyses. Eg : raises serum potassium (red blood cell potassium) and lowering serum insulin (by increasing proteolysis). Clinically significant changes in AST, chloride, LDH , and sodium. Collection volume : - This should be sufficient per tube to give the correct concentration of additives and sufficient in total to allow for repeat analyses. Stress:- If venepuncture is unusually stressful, or the patient faints, this should be recorded, especially for stress-responsive hormones. These include cortisol, ACTH, GH , prolactin, catecholamines and AVP (arginine vasopressin). v. Correct sample collection Pre-analytical phase and errors

17. Pre-analytical phase and errors v. Correct sample collection Labelling:- When multiple samples are collected during a dynamic test, samples should be labelled with both the clock time and the time elapsed during the test. vi. Correct Sample Handling Many samples need to be transported some distance to the laboratory where the analysis will be done. In this case transport and storage are the major concern. Transportation of samples to the laboratory affect analyses through the following; Time before separation from cells:- (for plasma). For example; ( i ) glucose is well known to decrease during storage of EDTA whole blood. - Centrifugation conditions:- The time must be sufficient to ensure complete sedimentation, the acceleration low enough to avoid haemolysis , and it may be desirable to control the temperature

18. Selecting the Site: Selecting the appropriate site for venipuncture can contribute to a better quality sample. The preferred site is the median cubital vein (superficial vein of the upper limb). This vein is usually the easiest to access and less need to probe to find the vein, which in turn should cause less trauma during the venipuncture. Median cubital vein is the most comfortable vein for patient Pre-analytical phase and errors v. Correct sample collection

19. Pre-analytical phase and errors v. Correct sample collection Tourniquet Application and Time: The tourniquet should be applied approximately three to four inches above the venipuncture site and should be on the arm no longer than one minute. Remove the tourniquet when blood starts to flow into the tube. Prolonged tourniquet time can lead to an increase in various chemistry analytes , including serum protein, potassium and lactic acid due to hemoconcentration of blood at the puncture site.

20. Recommended order of draw Blood Culture Bottles (Aerobic-Anaerobic) Coagulation Tube Serum Tube with or without clot activator, with or without gel separator Heparin Tube with or without gel plasma separator EDTA Glycolytic Inhibitor Pre-analytical phase and errors v. Correct sample collection

21. Recommended order of draw Heparin tube with or without gel plasma separator: Samples collected in these tubes are used for plasma determinations in chemistry. The additive acts as an anticoagulant, and blocks the clotting cascade. Coagulation tube: This tube contains sodium citrate as an anticoagulant and is used for drawing blood for coagulation studies.

22. Recommended order of draw Serum Tube with or without clot activator, with or without gel separate: The wall is coated with microscopic silica particles, which activate the coagulation process. Gel forms a stable barrier between the serum and the blood cells. EDTA Tube: Contains EDTA (the potassium salt, or K2EDTA). Tube is used for full blood counts ( FBC ) and blood films

23. Glycolytic Inhibitor Sterile blood collection tubes for laboratory procedures requiring plasma or whole blood chemistry procedures where glycolytic inhibition of specimen is required. The anti-glycolytic properties prevent the blood cells from using the glucose in the sample. Tests include blood glucose levels Recommended order of draw

24. Pre-analytical phase and errors vi. Correct Sample Handling Special separation requirements :- For example, contamination by the buffy coat must be avoided in plasma for catecholamine determinations as platelets contain high levels. Aliquoting sample:- Volume of aliquots must be sufficient. If the aliquot volume is too small some analysers will report a low result, but without an error message. Temperature and time between separation and analysis :- Cooler is usually better, and frozen usually better than liquid. Exceptions, chilled red cells release potassium, and renin is more stable at room temperature than at 0 o C. Complete thawing :- Adequate mixing and centrifugation of frozen samples before analysis.

25. vii. Correct accept/Reject criteria Pre-analytical phase and errors Acceptability of specimens arriving in the laboratory for requested analyses must be determined. Analyte-specific rejection criteria should be developed for each analyte . Examples; haemolysis exceeding a critical level, insufficient volume, thawed when should be frozen, etc.

26. viii. Laboratory logs Once the sample tube arrives in the laboratory, various logging and monitoring systems are initiated. One should check that patient name and identification number and the test requested on the form match the information on the label of the specimen tube. The specimen should be inspected to confirm adequacy of volume and no problems such as hemolysis. The specimens are then stored appropriately and identification information and arrival time are recorded in master log. Transcriptional errors are more if manual system. Computerization can reduce these errors. T he pre-analytical phase

27. T he a nalytical phase Analytical phase: - this involves the testing of the sample in the laboratory and errors occur during the testing process in the laboratory. The errors are usually attributed to operator or instrument error. Errors in the analytical phase also contribute to inaccurate test results. Pipetting error can be a factor.

28. Incorrect timing of test Eg : Enzymatic reactions are dependent of time. Reaction duration beyond the test time increases reaction product leading to false positive result. ii. Results reported when control results was out of range. Tests are usually run with control. The control test ensures that test reagents and equipment are working. An accepted range of value is expected for the control. iii. improper dilution and pipetting of sample or reagents. Improper dilution and wrong pipetting affect analytes concentration. iv. Reagents stored inappropriately or used after expiration date T he a nalytical phase

29. Post-Analytical Errors occur after the test is completed. In this instance, accurate test results may not cause appropriate patient management. The common errors in this phase are; wrong validation, delayed results, results not reported or reported to wrong patients, incorrect results reported because of post-analytical data entry errors and transcription errors and wrong patient identification. Report not legible, wrong interpretation due to interference of substances not recognized, analytical sensitivity not appropriate, previous value not available for comparison, etc. Post-Analytical

30. Consequences of analytical variables Cost Delay in recovery Time wasting Mismanagement of resources / inefficiency Mistrust / integrity issues