BNUR 2003 Biomedical Chemistry & Lab Diagnostics Lecture Notes PDF

2023-09-12 Course Overview BNUR 2003 Biomedical Chemistry & Lab Diagnostics Unit 1- Introduction to biomedical chemistry 1-1 Cell structure and function 1-2 Patient care cycle Professor Ryan Calhoun (Until October 9) Jenna Graham (After October 9) Evaluation 2 Tests Case history assignment* Fina...

2023-09-12 Course Overview BNUR 2003 Biomedical Chemistry & Lab Diagnostics Unit 1- Introduction to biomedical chemistry 1-1 Cell structure and function 1-2 Patient care cycle Professor Ryan Calhoun (Until October 9) Jenna Graham (After October 9) Evaluation 2 Tests Case history assignment* Final group presentation* Final Exam 2x15% 20% 20% 30% *Assignment details, rubric, etc… will be given after groups are formed in Week 2 1 2 Learning Objectives- Cell structure and function The Cell – Introduction Part 1- Organelles 1. Describe the structure and function of the nucleus. 2. Name the organelles of an animal cell and describe their functions. Nucleus Part II- Membrane 1. List the components of the plasma membrane and describe their functions. 2. Explain the various transport mechanisms in biological systems. Cytoplasm = Cytosol + Organelles Plasma membrane 3 4 1 2023-09-12 The Nucleus The Nucleus The nucleus is the control center for cellular operations. -contains (most of) the information needed to synthesize proteins The nuclear envelope is composed of __ membranes.The outside membrane is continuous with the rough endoplasmic reticulum (RER). Functions of the nucleus: • Store genes on chromosomes • Organize genes into chromosomes to allow cell division • Transport via pores • Produce __________ • Produce __________ • Organize uncoiling of DNA for replication 5 MRNA 6 ribosomes mRNA- translates into protein Organelles- Ribosomes Organelles- Ribosomes Ribosomes coordinate the interaction of ______ and _____ to build polypeptides during protein synthesis. Composed of two subunits, the small ribosomal subunit and the large ribosomal subunit. Free Ribosomes: Scattered through cytoplasm. Manufacture proteins for the cell Free ribosomes (fixed) 7 I Fixed Ribosomes: Attached to the ER. Manufacture proteins that enter the endoplasmic reticulum destined for secretion 8 +RNAs MRNAs docking space to create polypeptides 2 2023-09-12 Organelles- Endoplasmic reticulum Organelles- Endoplasmic reticulum 1 2 smooth endoPlaSMIC reticul ROUGH Free ribosomes ER ↳ fixed (fixed) 3 free riDOSOMeS 9 10 Organelles- Endoplasmic reticulum Organelles- Golgi apparatus The endoplasmic reticulum is a network of intracellular membranes connected to the ______________ that synthesize, store, transport, and detoxify molecules. There are two categories of ER: Smooth ER: No ribosomes attached. Synthesizes lipids and carbohydrates. Free ribosomes Rough ER: Ribosomes attached. After synthesis by the ribosomes, proteins are imported into the lumen of the ER for modification and packaging into vesicles. 11 nuclear envelope (fixed) 12 3 2023-09-12 Organelles- Golgi apparatus Organelles- Lysosomes The Golgi apparatus is made up of flattened disks and membraneenclosed vesicles. It modifies and packages secretions that are destined for _________ and packages enzymes into vesicles.The Golgi apparatus is involved in lysosome and peroxisome synthesis. Free ribosomes (fixed) 13 exocytosis Organelles- Lysosomes 14 Organelles- Peroxisomes Lysosomes are membrane-bound vesicles containing digestive Enzymes (include proteases [proteins], nucleases [nucleic acids] glycosidases [CHO] and lipases [lipids]). Needed for ______________ of excess, damaged, and foreign substances. Free ribosomes (fixed) 15 digestion in 16 creating macro-molecule 4 2023-09-12 Organelles- Peroxisomes Organelles- Mitochondria Peroxisomes are also membrane-bound vesicles.They are roughly spherical and encase oxidative enzymes.They break down fatty acids and organic compounds. In doing so, they produce toxic _____________, but break it down to oxygen and water. Free ribosomes (fixed) 17 hydrogen peroxide Organelles- Mitochondria 18 Organelles- Mitochondria Mitochondria are the powerhouse of the cell. They manufacture most of the cell’s ______ in the citric acid cycle and electron transport chain. Free ribosomes (fixed) 19 ATP ↳ /energy/power cell) 20 maintain homostatic balance 5 2023-09-12 Organelles- Mitochondria Plasma Membrane The cytoskeleton is composed of structural proteins that provide strength and structure to the cell. There are three main types of cytoskeletal filaments: • Actin filaments (microfilaments) • Intermediate filaments • Microtubules • • 21 microtubules 22 Plasma Membrane Physical isolation of the cytoplasm and extracellular fluid Regulation and exchange with the environment All PMs have a lipid bilayer The other proteins determine cell function 3 macromolecular components of the plasma membrane: lipids, carbohydrates, proteins hydrophobic/hypo Plasma Membrane- Signal transduction Membrane proteins are responsible for most of the functions of the PM. - out inside membrane Classification: • Integral vs. Peripheral (Exterior or Interior) • Transmembrane proteins Structure of transmembrane proteins: • Hydrophilic regions vs. hydrophobic regions 23 • • ____________ combine to form several other organelles: • Cilia and flagella • Centrioles ↳ Oute fluid Two main functions • Transport – e.g., channels • Communication – e.g., receptors The Plasma Membrane separates the cytoplasm from the extracellular fluid. It has the following functions: ↳ The receipt of information signals by a cell is complex. Cells have an extraordinary array of transmembrane proteins called receptors. Receptors act as the antennae of cells. Second messengers (phosphorylated proteins) Single Transducers RNA Growth Factor stay in phospholipid Growth Factor Receptor by layer DNA Cytoplasmic Receptor Region (a protein Kinase) m-RNA Proteins 24 6 2023-09-12 Cellular Transport Cellular Transport- Diffusion The plasma membrane is a barrier between the cytoplasm and extracellular fluid. BUT, __________ must get in and waste out. Fortunately, it is selectively permeable. Over time, continuously moving particles will randomly mix. Material moves from areas of high concentration to areas low concentration. -> small molecules that fit Passive transport: No energy required. Active transport: Requires energy in the form of ATP. through ↳ Simple Diffusion Influenced by distance, molecular size, temperature, gradient size. of protiens Major categories of cellular transport: - > 1. Diffusion (simple and facilitated) in or out 2. Active transport (primary and secondary) 3. Endocytosis and exocytosis of the cell presence allowing 25 nutrients 26 Cellular Transport- Diffusion Cellular Transport- Osmosis Facilitated diffusion uses membrane ___________________to allow larger and charged molecules to cross the cell membrane. Osmosis is the net diffusion of water over a membrane The speed of facilitated diffusion is limited by the number of channel proteins. 27 protein channels 28 7 2023-09-12 Cellular Transport-Active transport Cellular Transport-Active transport Certain substances must be pumped across the PM. During active transport energy is used to accomplish transport. jogas In primary active transport energy is used at the membrane protein itself to induce a conformational change that results in transport. In secondary active transport energy is used to establish a concentration gradient. Next the energy of the concentration gradient is used to transport another substance. Of WA sodium ↳ pre-established concentration gradient 29 30 Cellular Transport-Active transport Cellular Transport- Endocytosis and exocytosis In endocytosis and exocytosis, materials that are too large to pass directly through the cell membrane are transported into or out of the cell. Endocytosis Pinocytosis Phagocytosis glUCOst> Well cellS Exocytosis aS https://medicoapps.org/glucose-transporters-2/ 31 32 8 2023-09-12 Learning Objectives BNUR 2003 Biomedical Chemistry & Lab Diagnostics 1. Understand how clinical chemistry relates to the patient care cycle and describe what differential, provisional, and definitive diagnoses are. 2. Describe the three major steps involved in quality assurance in clinical chemistry labs. 3. Describe, in detail, the analytical phase of QA, differentiate between accuracy and precision and explain how laboratories evaluate them during the analytical phase of QA. Unit 1- Introduction to biomedical chemistry 1-1 Cell structure and function 1-2 Patient care cycle 4. Describe what a reference interval is and how a reference interval is established. Calculate and interpret various statistics of diagnostic relevance (true negatives, false positives, sensitivity, specificity, etc.) 1 2 Department of Laboratory Medicine Clinical Chemistry The primary goal of the laboratory is to do the right test as accurately as possible on the correct patient in an appropriate time frame in order to affect a positive patient outcome. Clinical Chemistry is a branch of laboratory medicine that examines chemical or biochemical information about the health status of a patient. We seek to understand the relationships between: • Patients’ clinical signs and symptoms • Underlying biochemical/physiological phenomena • Laboratory results Histology 3 4 chem/bio signs 1 2023-09-12 The Patient Care Cycle The Patient Care Cycle Step 1 Individual becomes aware of a problem by assessing their state of health (symptoms). Step 4 Laboratory tests are performed. Step 2 Individual seeks medical care and patient information is obtained (patient interview). Step 5 Laboratory tests are interpreted. Step 6 Therapy implemented and evaluated. Step 3 Clinician seeks objective data to establish a diagnosis (tests). In 20% of patients, there is an uncertainty in a diagnosis established from signs & symptoms alone 5 signs Objective symptoms subjective : 6 : Types of Diagnosis Quality Assurance (QA) Q: Are laboratory tests always “correct”? Differential Diagnosis: list of most probable disease entities that are consistent with observed signs and symptoms. Many conditions could be consistent with the S&S A: Provisional Diagnosis:The most likely of possible diagnoses. Definitive Diagnosis: Established by confirmatory data such as laboratory tests. 7 (signs symptoms observed) - 8 2 2023-09-12 Quality Assurance (QA) QA: Analytical Phase Labs must carefully monitor the quality of their results. This occurs in three phases: intragenerate results generate Pre-analytical phase • Assess controllable _________ that influence test results • e.g., patient preparation, specimen collection, interferences -> ↳ The laboratory must: • use appropriate equipment and methods that can attain goals of accuracy and reproducibility • implement an internal ____________ system for each test that monitors the reproducibility and accuracy of test results being reported instrument failure bad site Analytical phase • Assess accuracy and precision due to processing of the actual test , ↳ result has to be interped Post-analytical phase • Compare test results to external data after the test is complete. ↳"ravest" - miscommunication/data error 9 variables 10 quality control any difference difference QA:Analytical Phase no where near to be an a biologic error Repeated tests on a laboratory standard of known glucose serum Accuracy 7 Precision Precision The degree to which repeated measures under the same conditions return the same result hoped Instead of bullege Accuracy The closeness of a result to the true value is Test 1: 3.7 mmol/L Test 2: 3.6 mmol/L Test 3: 3.8 mmol/L Test 4: 3.7 mmol/L Test 1: 2.0 mmol/L Test 2: 2.1 mmol/L Test 3: 2.0 mmol/L Test 4: 2.0 mmol/L Test 1: 4.0 mmol/L Test 2: 3.3 mmol/L Test 3: 4.0 mmol/L Test 4: 3.4 mmol/L Test 1: 0.2 mmol/L Test 2: 8.3 mmol/L Test 3: 5.0 mmol/L Test 4: 1.7 mmol/L Laboratory Standard = 3.7 mmol/L 11 12 3 2023-09-12 QA:Analytical Phase – Precision QA: Analytical Phase – Accuracy Accuracy is established when a new method is introduced for the first time Three ways to establish accuracy: Example: 1. Compare a patient’s test result using the new method to a result of same sample using a different, well established method. 2. Perform the new method on a series of ___________________ (e.g., linearity check samples). 3. External quality control programs new method Step 1: Establish precision of the new method by performing the new method on two commercial control samples (normal and abnormal) to determine mean values and standard deviation. Step 2: Run a patient’s sample with commercial controls. Step 3: Determine whether the patient’s results are reportable by using both normal and abnormal results 13 reference samples on The precision of a method is determined as part of internal quality assurance programs.These programs are critical in determining whether patient results are ________________. already pre-existing 14 reportable data-reference sample QA:Analytical Phase – Precision QA:Analytical Phase – Precision Step 1: Establish precision Step 1: Establish precision Control Values over 1 month 2.0 2.2 2.4 2.6 3.0 2.9 3.1 2.2 2.5 2.6 2.7 2.7 2.3 2.9 2.5 2.8 2.6 2.3 2.4 2.7 2.7 2.0 2.6 2.3 3.5 3.3 3.0 3.2 2.6 2.4 Standard Deviation (S.D.) = 0.37 Mean +/- 1 S.D. is the range between 2.26 and 3.00 The standard deviation is a useful statistic. Statistically speaking, approximately 66% of points fall within one standard deviation of the mean. E.g. 2.63 - 0.37 = 2.26 2.63 + 0.37 = 3.00 15 Control Values over 1 month Mean Value = 2.63 2.0 2.2 2.4 2.6 3.0 2.9 3.1 2.2 2.5 2.6 2.7 2.7 2.3 2.9 2.5 2.8 2.6 2.3 2.4 2.7 2.7 2.0 2.6 2.3 3.5 3.3 3.0 3.2 2.6 2.4 Mean Value = 2.63 Standard Deviation (S.D.) = 0.37 Mean +/- 2 S.D. is the range between 1.89 and 3.37 Approximately 95% of points fall within two standard deviations of the mean. E.g. 2.63 – 2(0.37) = 1.89 2.63 + 2(0.37) = 3.37 "I standard deviation +116 4 2023-09-12 QA:Analytical Phase – Precision QA:Analytical Phase – Precision Step 2: Run a patient sample Step 2 – Levey-Jennings charts Run the quality control samples (normal + abnormal) with patient samples.The frequency that this is done depends on instrument, technologist, and laboratory policy. ‘3.37’ "2sd" "2sd" ‘3.00’ "1sd" "1sd" ‘2.63’ mean mean ‘2.26’ -1sd -1sd ‘1.89’ -2sd At the minimum once per 8 hour shift. QC data (normal + abnormal) are plotted on a Levey-Jennings chart*, which is based on the data from Step 1. Day -2sd 1 2 34 5 6 7 Day 1 23 4 5 67 normal abnormal Mean Value = 2.63 +/- 2 S.D. =1.89 to 3.37 *The LJ chart does not include patient samples normal (Abnormal would have a different mean value) range 17 18 QA:Analytical Phase – Precision QA:Analytical Phase – Precision Levey-Jennings chart over the course of one month Step 3: Decide whether to report patient’s result • in We can use a series of rules known as the Westgard Rules to decide whether or not a patient’s result should be reported (i.e., is reliable). Decreasing trend at end of August 19 arifting - pt results may be bias 20 5 2023-09-12 QA:Analytical Phase – Precision QA:Analytical Phase – Precision Step 3: Westgard Rules Step 3: Westgard Rules Rule 2: If one control is within 2SD and the other is no more than 3 SD from the mean, accept the run and report the patient results. BUT, check next controls carefully. Rule 1: If both controls are within 2SD, accept the run and report patient result +3 sd +3 sd +2 sd +1 sd +2 sd +1 sd Mean -1 sd -2 sd -3 sd Mean -1 sd -2 sd -3 sd 21 both within & SD- if that is continue then +3 sd +3 sd +2 sd +1 sd +2 sd +1 sd Mean -1 sd -2 sd -3 sd Mean -1 sd -2 sd -3 sd 22 with run QA:Analytical Phase – Precision QA:Analytical Phase – Precision Step 3: Westgard Rules Step 3: Westgard Rules Rule 4: If both controls are outside 2SD, reject the run and do not report the patient’s result. Rule 3: If either control is outside of 3SD, reject the run and do not report the patient’s results +3 sd +2 sd +3 sd +3 sd +1 sd +2 sd +1 sd +2 sd +1 sd Mean -1 sd -2 sd -3 sd Mean -1 sd -2 sd -3 sd Mean -1 sd -2 sd -3 sd 23 +3 sd +2 sd +1 sd Mean -1 sd -2 sd -3 sd 24 6 2023-09-12 QA:Analytical Phase – Precision QA:Analytical Phase – Precision Step 3: Westgard Rules Step 3: Westgard Rules Rule 5: If four quality control results in a row, for either the normal or abnormal control, fall on the same side of the mean above or below 1SD, reject the run. Rule 6: If ten consecutive results for either quality control sample fall on the same side of the mean, reject the run. +3 sd +2 sd +1 sd +3 sd +2 sd +3 sd +2 sd +1 sd +1 sd +2 sd +1 sd Mean -1 sd Mean -1 sd -2 sd -3 sd -2 sd -3 sd Mean -1 sd -2 sd -3 sd Mean -1 sd -2 sd -3 sd 25 +3 sd 26 reject things QA:Analytical Phase – Precision QA: Post-Analytical Phase Levey-Jennings chart over the course of one month Two examples of post-analytic QA: at 2 SD 1.Test patterns/grouping: Multiple markers respond to disease at the same time. • e.g., renal failure is accompanied by both elevated creatinine and urea nitrogen 2. Delta Values: Compare test result to previous test results for the same patient. ↳ difference en the 2 below 2 SD 27 28 7 2023-09-12 Interpreting a patient’s result We have one large question remaining “Does the patient’s test result indicate whether the patient can be diagnosed as likely having the disease or not?” Let’s try… Q: Bob has a glucose level of 4.0 mmol/L. Is Bob’s glucose level normal? unknown Q: Chandra has a glucose level of 3.7 mmol/L and the population average is 4.9 mmol/L. Is Chandra’s glucose level normal? unknown Q: Gideon has a glucose level of 7.0 mmol/L and 95% of the population has glucose levels between 3.6 and 6.1 mmol/L. Is Gideon’s blood glucose level normal? of no-indictive a 29 30 Interpreting a patient’s result Interpreting a patient’s result problem We need to understand sources of variation: • 31 We are usually interested in the effect of disease state on test results, but variation could also be due to: • Analytical variation:Variation that is inherent in the test method. • Biological variation: Inter-individual variation can include age, sex, race, diet. Within individual variation can include time of day, social environment, diet, etc… • The most common method of establishing whether a patient’s result should be considered “normal” is the reference interval. • Reference intervals are constructed from test populations that must consist of more than ____ individuals, be well described (age, sex, etc.) and be measured with clearly stated techniques. 32 108 8 2023-09-12 Interpreting a patient’s result Interpreting a patient’s result The reality is that the diseased and non-diseased populations usually overlap F R E Q U E N C Y • Overlapping populations lead to the possibility of our test having False positives and False negatives. Non-Diseased Diseased 10 100 140 ↓ overlap = better test 140 Test Values 33 34 Interpreting a patient’s result Interpreting a patient’s result Test Parameters • • Some tests are better than others due to varying degrees of overlap between the reference intervals for the healthy and diseased population. We can use test parameters of diagnostic relevance to quantify how good a test is. Sensitivity = × 100 • Proportion of people with disease that test positive for the disease • How good the test is at correctly identifying people with the disease • Calculations only involve people who have the disease (doesn’t tell us if people without disease test positive) • aka – “True Positive Rate” or “TPR” 35 36 true positive rate sensitivity = TP - TP + x 108 F 9 2023-09-12 Interpreting a patient’s result Interpreting a patient’s result Test Parameters Test Parameters Specificity = Predictive Values are the probability of having disease after the tests results are known. × 100 • Proportion of people without disease that test negative for the disease Positive Predictive Value (PPV) = • aka – “Predictive Value Positive Result” or “PVPR” (textbook) • How good the test is at correctly identifying people without the disease • • Proportion of people with a positive test result who have the disease Calculations only involve people who do not have the disease (doesn’t tell us if people with disease test negative) • Sometimes referred to as the ‘post-test probability of disease given a positive test’. • aka – “True Negative Rate” or “TNR” 37 38 Interpreting a patient’s result Interpreting a patient’s result Test Parameters Test Parameters Predictive Values are the probability of having disease after the tests results are known. Negative Predictive Value (NPV) = × 100 False Positive Rate (FPR) = × 100 × 100 • Number of positive test results obtained as a percentage of all people without disease. • aka – “Predictive Value Negative Result” or “PVNR” (textbook) • Proportion of people without disease that test positive for disease. • Proportion of people with a negative test result who do not have disease • Sometimes referred to as the ‘post-test probability of not having disease given a negative test’. 39 40 10 2023-09-12 Interpreting a patient’s result Interpreting a patient’s result Test Parameters Test Parameters Efficiency = × 100 Prevalence = • Number of accurate test results obtained as a percentage of all people tested. • Proportion of population that has disease. • Proportion of patients that receive accurate test results. 41 × 100 42 11 2023-09-12 CHEM 1000 WEEK 1 PROBLEM SET 2 1 2 FN TP FP people with disease that test positive calculations only involve people who have the disease negative test results do not have the disease positive predicted values positive test result who have the disease negative disease 3 test result who do not have the TN TP 100 * TP + FN TN TN + TP TP + x 5500 xp4100 + qu x FN = 100 + x + 100 100 mean calculated 45 : = 91 7 : . - SD given % 500 90 90 + 110 5500 = FP TN TN 100 = x 100 = =350% 15 3 % . 508 x 100 = 14 2 8 3 . . 3 30-15 . 45 8 . 8 30 + 15 8 4 . 4 1 2023-09-12 normal control group abnormal contro group · 6 1 8 9 . . 5 9 8 6 . . · 5 7 . 5 5 MMOl . 8 3 . 8 O L . MMOVL 5 3 7 7 5 1 7 4 . . . . 4 9 7 . . 1 5 5 normal control ISD 2 SD 3 SD = = = 0 2 . 2 3 x x 0 0 abnormal control group MMOl/L 2mM01/L-0 4mMOX/L . 1 . . 2 mmol/L -0 6MMOIIL SD = 0 3 . 2 SD = 3 SD = 2 3 x x . if both controls are within 2SD . accept the run and report 6 MM01/L 0 30 . 0 30 . . . 6 6 MMO1/L 9MMOIL patient findings 7 7 2