304BMS Clinical Biochemistry Exam Revision PDF

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304BMS Clinical Biochemistry Exam RevisionSECTION AEmma Clinical Biochemistry Laboratory  Samples and Pre-analytical errors  Clinical interpretation  Diagnostic enzymology – liver enzymes and cardiac enzymes  Laboratory Quality Insurance  Point of Care  Antenatal and Newborn Screening  Works...

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304BMS Clinical Biochemistry Exam Revision SECTION A Emma           

Clinical Biochemistry Laboratory Samples and Pre-analytical errors Clinical interpretation Diagnostic enzymology – liver enzymes and cardiac enzymes Laboratory Quality Insurance Point of Care Antenatal and Newborn Screening Workshop 2 - Quality Assurance and Automation (EF) Workshop 3 - Liver and Cardiac Case Studies (EF) Workshop 6 - Point of Care (EF) Workshop 7 - Neonatal Screening (EF)

Lecture 1 Quality Assurance and Result Interpretation Pathology      

Clinical Biochemistry Haemotology and Coagulation Immunology Microbiology and virology Histopathology Cytology Molecular

Laboratory Testing Laboratory medicine aids the clinician not only in diagnosis but also in: Screening – Generic testing  Antenatal screening  Newborn screening  Cancer screening Prognosis – outcome of what will happen to the patient. Tumour marker monitoring shows returning of cancer. (Recovery).  Monitor changes in disease  Reoccurrence of disease  Likely outcome for patient Treatment – sensitivity and molecular testing. Narrow effective range: Digoxin and Warfarin  What treatment?  Therapeutic drug monitoring  Is treatment working? Testing is required for the diagnosis of diseases or differentiation of diseases that present in a similar way

1. Over-investigation can harm patient -

Discomfort and anxiety Repeated venesection may cause or aggravate anaemia May delay treatment/discharge Wastes resources

2. Errors associated with lab testing -

An inappropriate test is ordered

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An appropriate test is not ordered A test result is misapplied Result delay Result is inaccurate

Typical Biochemistry Laboratory     

Highly automated Computerised Open 24 hours Repertoire > 100 tests Biochemical tests are increasingly being performed outside of the lab – point of care testing

Patient request form      

Patient details Sample type What tests are requested? Who is requesting them? Clinical details E-requests – saves time and reduces errors

Blood Samples    

Venous sample Arterial sample Capillary sample: different to venous blood - Finger prick testing and scalp blood sample CSF (Cerebral spinal fluid): protein and glucose - bleeding on brain not evident on CT scan

Sample Analysis Consists of serum, plasma, whole blood, red cells or white cells Plasma: anticoagulant is used Serum: blood is allowed to clot before spinning  Check for haemolysis  Lyse and rupture red cells  Contents are released so results are elevated  Red top: plain tube for general chemistry  Yellow top: contains separator gel for general chemistry  Green top: contains lithium heparin anticoagulant  Purple top: EDTA anticoagulant for whole blood, red cell and lipoproteins  Pink top: EDTA anticoagulant for blood bank  Blue top: Sodium citrate anticoagulant for clotting tests  Grey top: fluoride oxalate anticoagulant for glucose, lactate and alcohol.

Timing of Samples    

For drug estimations, time since last dose is required Fasting may be required to measure glucose and lipids Some analytes show dirunal variation – changes throughout the day. To measure sample must be taken at certain times Sex hormones vary during menstrual cycle

Specimen reception 1. Samples sorted and checked against request forms

2. Patient identification details and requested tests entered into laboratory information system 3. Given unique barcode 4. Specimen is centrifuged, separated and stored or sent for analysis

Sample integrity Generally, serum/plasma should be separated from blood cells as soon as possible by centrifugation and stored in a fridge For longer term storage or for unstable analytes are frozen (-20 oC)

Collection Tubes Order of which tubes are drawn is extremely important Once the needle comes into contact with the tube stopper, additives can be transferred from one bottle to the next, even a small amount can drastically alter results. For example, the purple EDTA is high in potassium, which if transferred to another bottle used for testing potassium, can elevate levels to become normal, even if the patient suffers from low potassium levels leading to an incorrect diagnosis The site of draw and sufficient sample must also be considered. Order: Sterile  Light blue  Red  SST  PST  Green  Lavender  Grey

Automation Labs face huge pressures to produce faster turnover times and reduce errors The goal is to improve patient care Designed to maximise efficiency and minimise errors by intergrating mechanical, electronic and informatic tools. Robot chemist and continuous flow analysers – continually evolving The lab is highly automated - Large random access analysers – urgent sample processing and choose tests - Discrete analysis - Large repertoire of tests - > 400 samples an hour Combination of analysers set up to ensure continuity of service Total Laboratory Automation

Methods     

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Spectrophotometry Fluorimetry Electrochemistry (ISEs) Flame photometry Electropheresis - Agarose - Polyacrylamide - Capillary Immunoassays - Enzyme - Fluorescent - Chemilluminescent - Radiolabels Chromatography - Thin layer - GLC - HPLC

Clinical Biochemistry Process  

A medical report that is delayed or lost, or poorly written can negate all the effort of performing the test correctly A sample that is damaged/altered as a result of improper collection/transport cannot provide a reliable result

What can go wrong? Pre-analytical errors  Test request  Patient preparation  Sample collection  Sample transport  Sample receipt Analytical errors  Working instructions not followed  Reagents  QC out of range  Technical validation  Medical validation Post-analytical errors  Report to wrong location  Transcription error  Sample storage  Record management  Reference ranges

Quality Assurance Quality is about the standards in a research lab. The right result, at the right time, with the right specimen, and the right patient. Assurance are procedures adopted by the lab to make sure results obtained are correct. It ensures that:  Samples are taken, transported and stored in an appropriate manner to avoid pre-analytical errors  Analytical results are “correct” using the same method  Results are correctly matched to the patient from which the sample was taken  Results are delivered to the correct place and in a reasonable time.  Blunders (obvious errors) are rectified by repeating the sample.

Important elements for QA      

Quality control samples External quality assessment scheme Audits Documentation Standard operating procedures (SOPs) Staff training

Analytical Quality Control Analytical performance is monitored by:  Use of internal quality control samples – run at intervals in every batch alongside patient samples, results must be within test limits  Participation in external quality control schemes

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Use of delta checks and looking for clinical consistency

Internal Quality Control Performed during daily routine work Using patient like material Known target value and acceptable range This checks the accuracy of machines Results are plotted on control charts Repeated measurements can show precision of test.

Limits of Internal Quality Control Decision limits are based on Gaussian (normal) distribution of the control material results - A measurement > 3SD’s from the mean is unexpected and can be seen to indicate a problem with the analytical method  likelihood of results falling into that range

Westgard Rules    

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Multi-rule scheme  more complex High error detection with low false rejection Rules are used individually or in combination to evaluate the quality of analytical runs Still small number of results which fall out of range  still effective. - 13s rule violation - 22s rule violation - R4s rule violation - 10x rule violation If the quality control result violates any one of the rules the results are rejected

External Quality Assurance 3-5 samples are sent out monthly to each participant Each lab will analyse the samples, treating as patient samples results are returned to organisers for statistical analysis Each lab is then informed of its performance relative to the others in the scheme and to the consensus result for the sample Anonymity is maintained Poor performers may be reported to the relevant National Quality Assurance Panel These results give assurance that every lab achieves the same result for those using the same methods

Delta Checks Is the comparison of samples to previous samples from the same patient. A significant change could indicate a real pathology or an issue with testing or test sample. Particularly useful in detecting errors in:  Specimen identification  Specimen integrity  Errors in manual data entry  Possible analytical errors Delta checks are not useful for every analyte and are best for stable analytes with little variation and are measured frequently.

Method Validation   

Linearity – range of values between which results regarded as accurate Sensitivity – Ability of test to detect low concentrations Specificity – Ability to measure the analyte of interest

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Interference – Similar analytes, drugs, haemolysis or lipaemic (high lipid or cholesterol)/Icteric (high bilirubin) samples

Result Interpretation Results are quantitative and are expressed as concentrations (amount per volume), but enzymes are expressed as activity Results may vary as a result of analytical variation (precision, sensitivity, specificity of a method) or biological variation (personal biological profile). Must be known how results vary in (a) in health and (b) in disease Must take into account:  Previous results  Medical Details  Biological Variation - Age - Gender - Ethnicity - Time - Pregnancy  Diet  Drugs  Exercise/Stress

Reference Ranges Interpretation of test involves comparing patient results with a reference range This is calculated from a healthy population Mean 2SD – includes 95% of values from population  minimises overlap 5% (1 in 20) healthy results do not fall into the reference range The further a result from the reference range  higher indication of pathology Age or sex related  what is normal for one group may not be normal for another group Each lab establishes and uses their own reference ranges.

Clinical Utility of Tests         

False positive  an abnormal result in a person without the disease False negative  a normal result in a person with the disease Specificity: Ability of test to correctly identify those patients without the disease Ability of test to exclude a true negative Sensitivity: Ability of test to correctly identify patients with the disease Ability of test to detect true positive These are inverse of each other Sensitivity or true positive rate: incidence of positive results with patients with the disease  100% sensitivity if always detects patient with disease Specificity or true negative rate: Incidence of negative results in patients without the disease  100% specificity if always negative for healthy people

Ranges in health and disease     

Ideally a test if both 100% specific and sensitive An overlap leads to false negatives and positives. If a diagnostic cut off is too high, there are no false positives but many false negatives. A test with high specificity is useful for ‘ruling in’ a disease if a person tests positive (Increase specificity to ensure patients with the disease). A diagnostic cut off too low so the number of false positives increases.

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Tests with high sensitivity are useful for ‘ruling out’ a disease if a persons tests negative. (Increase sensitivity to ensure identification of positive results) For example: Distinguishing MI from chest pain Plasma CK is usually raised in MI but may also be raised in patients with chest pain  not specific To identify all MI cases  low cut off for CK (200IU/L). 100% sensitive, 30% specific. To only identify all MI cases  cut off for CK (450IU/L). 100% specific, 75% sensitive. Changing cut offs alters test sensitivity and specificity.

Evidence Based Practice Decisions about cut off values must take into account: - Nature and prevalence of condition - Consequences - Other tests may help to discriminate for diagnosis Evidence based clinical Biochemistry - Test chosen on basis of evidence of utility and on basis of outcome measures - Tests must be evaluated.

Lecture 2 Enzymes, Biomarkers, Liver and Cardiac Disease Biomarkers Defined by NICE as: ‘a characteristic that is objectively measured and evaluated as an indicator of normal biological processes, pathogenic processes, or pharmacological responses to a therapeutic intervention’ They can be used alone or in combination to assess the health or disease state of an individual. Concentration in blood changes in response to a specific disease or clinical condition. A wide variety are used for each biological system (eg. Cardiovascular, liver, kidney or inflammation). This includes: - Enzymes - Structural tissue proteins - Acute phase proteins - C reactive protein - Haptoglobins - Molecular markers such as SNP (single nucleotide polymorphism). - Certain DNA/RNA markers may help in the detection or treatment of cancers and allow for targeted drug treatment/dosage.

Ideal Biomarker These should be: 1. Not present in blood of healthy people 2. Tissue specific 3. Stable 4. Easy to assay 5. Changes in concentration reflect patient’s status 6. Consistent across gender and ethnic groups 7. Cost efficient to follow up 8. Modifiable with treatment

Diagnostic Enzymology Enzymes in the blood may:

1. Have a function - clotting factors or complement  activity is higher in blood than cells 2. Have no function - these can be cell derived enzymes and released due to normal cell turnover Intracellular Enzymes Many enzymes in the cell are linked to metabolic pathways Variation of cells leads to enzymes varying accordingly Normal plasma/serum levels of these enzymes are low By knowing where these enzymes are found  increases can be used to look for tissue damage

Enzymes as Biomarkers Enzymes in exocrine secretions (secretions like amylase from exocrine glands that produce and secrete substances eg. Saliva, sweat and mucous) also present in serum/plasma in low concentrations Healthy cells  steady cell turnover Enzyme activity in plasma/serum is a balance between: 1. Rate of synthesis 2. Rate of release 3. Rate of clearance Increases are seen during cell death or disease

Reasons for Abnormal levels 1. Rate of cell proliferation - E.g. normal growth, tissue repair, malignant disease. 2. Rate of cell turnover - Necrosis or severe damage to cells. Increase depends on rate at which damage occurs and extent of damage. Eg. Liver cell damage or MI. 3. Rate of release - Duct obstruction  enzymes that are normally released into secretions regurgitated into blood as outlet blocked. 4. Rate of clearance - Small peptides cleared through kidneys  impaired glomerular filtration may delay excretion 5. Rate of synthesis - Increased concentrations of enzymes within cells. - Induction by drugs will result in an increased serum level upon normal cell breakdown.

What can be determined 1. What tissues have been damaged? - Few enzymes are tissue specific  look for enzyme patterns 2. Has tissue damage occurred and to what extent? - Enzyme levels indicate extent (multiples of reference range). - Enzymes are distributed in different cell compartments  cytoplasmic, mitochondrial and membrane bound Membrane leakage and release of cytoplasmic enzymes only  minor reversible damage All enzymes (cytoplasmic, mitochondrial and membrane bound) are released  complete necrosis

Isoenzymes As few enzymes are tissue specific, isomers present can be measured. Isomers are different molecular forms of the same enzyme with differing combinations of subunits. They have the same catalytic function and found in different tissues. This can give more information about site of damage rather than total activity.

By measuring the isoenzymes, we are able to find out more specific information on the source tissue. This can then be examined and quantified using electrophoresis as they will have differing mobilities.

Measuring enzymes Measured as enzyme activity with International Units (IU)/ litre Standard enzyme assay action of enzyme on a substrate to produce product Colour change measured spectrophotometrically (or fluorimetric or luminescence) a) Visible coloured, (fluorescent or luminescent) product b) Interconversion of NAD(P)+ to NAD(P)H with a change in absorption at 340nm (UV) c) Coupled enzyme reaction  two or more enzymes reactions. Linked by common intermediates. Reagent contains second indicator enzyme. Enzymes can also be used as reagents for measuring substrates eg. GODPAP for glucose measurements

Limitations Lack of specificity to particular tissues Isoenzymes techniques may be expensive and there is a long turn around time for results Time of sample may be important  CK must be taken after a certain period when looking for evidence of an MI. Macroenzymes – CK/amylase. Enzyme joins to an immunoglobulin antibody  interferes with the reticuloendothelial-system clearance mechanism meaning activity increases.

Liver disease The Liver is the largest solid organ in the body Hepatocytes (80% of liver cells) perform the main metabolic functions:  Metabolism  Storage  Metabolism and excretion of drugs  Bile production

Liver Function Tests (LFTs) A single biomarker cannot be used Testing guidelines determine the analytes included in a liver profile panel: 1. Alkaline Phosphatase (ALP) 2. Alanine aminotransferase (ALT) 3. Total bilirubin 4. Serum albumin 5. Total serum protein could also be considered in liver disease. Additional tests include Amino aspartate transaminase (AST), Gamma glutamyl transferase (GGT), Conjugated/unconjugated bilirubin and Lactate dehydrogenate (LDH) Negative results give a low probability of disease. Different results in cholestatic disease (blockage of bile ducts) to hepatic disease  further testing

Albumin Reference range: 36-47g/L Measured by dye binding At pH 4.1, binds to bromcresol green  blue/green complex In liver disease there is an impaired ability to synthesise proteins so albumin levels fall. This reflects long term (chronic) damage (20 day half life in serum so it takes time for levels to fall) Coagulation factors  prolonged prothrombin time.

Bilirubin Formed from the breakdown of haem from haemoglobin.

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Destruction of RBCs in Spleen  form haem and globin chain Two products are formed. Iron is removed and porphyrin ring broken to form bilirubin.

Bilirubin Metabolism Unconjugated bilirubin is not water soluble so bound to albumin and transported. This is taken up by hepatocytes and becomes conjugated to glucuronic acid to become bilirubin glucuronide in the liver. Enzyme UDP-glucuronosyl transferase 1A1 is responsible for catalysing conjugation. Bilirubin glucuronide is conjugated and water soluble so is excreted in bile This is then degraded by bacteria in the colon to urobilinogen Urobilinogen is either oxidised to urobilin and excreted in faeces and stercobilin or a small % is reabsorbed and taken back to the liver only to be re-excreted by the liver or in the urine.

Clinical Jaundice Normal bilirubin range 50μmole/L Serum bilirubin reflects a balance between production and clearance. Increases can be due to: Overproduction of bilirubin Impaired uptake, conjugation, or excretion of bilirub...