Lecture 27 Coursework Case Study PDF

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Coursework Case Study...

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Workshop 1 ●

ABX Pentra 400 ○ Performs a wide range of tests from the pre-entered menu using commercial kits (up to 55 on board tests) ○ Analysis method types: ■ Spectrophotometry: colorimetry and turbidmetry ■ Potentiometry: direct (serum or plasma) and indirect (urine) ○ Optional ISE molecule (Na+, K+, Cl-) ○ Maximum throughput: 420 test/h ○ Sample specimen ■ Serum, plasma, urine CSF, whole blood, homogeneous fluids ○ Samples volume: 2 to 380 ul ○ Automatic sample dilution: from ½ to 1/22,500 ○ Optical system: ■ Tungsten-halogen lamp ■ Measurement principle ● Bi-chromatic light absorbance ● Choice of 15 wavelength (340-700nm) ● Wavelength selection with holographic grating technology ■ Sensor - photodiode array

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Holographic grating technology ○ Spectral unit offering 15 different wavelengths ○ Optimised reading by continuous sweep ○ Space-saving design with maximum efficacy provided by the tungsten lamp

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Pentra 400 system components ○ A bench-top analyser with an integrated data station ■ Separate keyboard and printer ○ B floor standing waste ○ C water ○ D cooling unit

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Integrated ISE module ○ 180 tests/hour ○ Simultaneous analysis of sodium, potassium and chloride ○ Applications on serum (most likely to use), plasma (can mess up probes) and urine

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Pentra 400 reagent cassettes and racks ○ Most reagents supplied in cassettes (1 per assay) ■ Bar coded with reagent details ■ Composed of 1 or 2 compartments to separate reagents

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■ Uniquely identified with a number ■ All ready to use 5 reagents - have to be put in separate veils and sits in the cassette area which reduces sources of error and time ■ CK MB, LDH, CO2, magnesium, prealbumin ■ Diluents, cleaners, and third party reagents must be transferred to 4, 10 or 15 mL bottles and loaded into reagent racks

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Cuvette segments and loading cuvettes

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Sample and tube requirements ○ 2 types of sample racks ■ Holds up to five 16mm tubes and five 13 mm tubes ■ Holds 10 samples cups ○ Up to 6 racks can be loaded at one time ○ A cover is fitted over the sample racks ○ Dead volume for sample cups 100ul, primary tubes 1000ul

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This machine tends to be used in clinics and in vet surgeries rather than large labs

Alanine transaminase (ALT) ○ As a liver specific enzyme ALT is only significantly elevated in hepatobiliary diseases

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Increase AST levels, however, can occur in connection with damages oh heart or skeletal muscle as well as of liver parenchyma Paralle measurement of ALT and AST is therefore applied to distinguish liver from heart or skeletal muscle damages The AST/ALT ratio is used for differential diagnosis in liver diseases While ratios 1 are associated with severe, often chronic liver diseases

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ABX Pentra ALT CP method ○ Quantitative in-vitro determination in serum or plasma ○ Optimised UV-test according to IFCC modified method without pyridoxal phosphate ○ Sample volume: 20ul/test ○ Measuring range: 4U/L 600 U/L, with automatic post-dilution: 1800 U/L

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Aspartate transaminase (AST) - ABX Pentra AST CP ○ Human seruma and plasma based on a UV test using L-aspartate and 2oxoglutarate ○ Optimised UV-test according to IFCC ○ MDH = Malate Dehydrogenase ○ Sample volume: 20ul/test ○ Measuring range from 4.0 U/L to 600.0 U/L, extended to 1800 U/L wth the automatic post-dilution

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Alkaline phosphatase (ALP) - ABX Pentra ALP CP ○ Measurements of alkaline phosphatase or its isoenzymes are used in the diagnosis and treatment of liver, bone, parathyroid and intestinal diseases ○ Human serum adn plasma based ona kinetic photometric test using pNitrophenylphosphate ○ Sample volume: 4ul/test ○ Measuring range from 8.0U/L to 1596.2 U/L, extended up to 6000.0 U/L with the automatic post-dilution

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Albumin - ABX Pentra Albumin CP ○ Albumin measurements are used in the diagnosis and treatment of numerous diseases involving primarily the liver or kidneys ○ Intended fo the quantitative in vitro diagnostic determination of albumin in serum or plasma by colorimetry ○ Bromocresol green dye-binding procedure ○ Method is simple and quick unlike electrophoresis or salt fractionation determinations which are not very convenient in laboratories ○ At pH 4.20 in succinate buffer and with a non-ionic surfactant Brij35, the Bromocresol green fixes itself selectively to the albumin of the sample, producing a blue colour which is measured at 628nm ○ The intensity of the colouring is directly proportional to the albumin concentration ○ Sample volume: 2ul/test ○ Measuring range from 4.6g/L to 56.0 g/L, extended up to 1696 112.0g/L with the automatic post-dilution

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Total protein - ABX Pentra Total Protein CP reagent ○ Used in the diagnosis and treatment of a variety of diseases involving the liver, kidney or bone marrow as well as other metabolic or nutritional disorders ○ Intended for the quantitative in vitro diagnostic determination of total protein in serum and plasma by colorimetry ○ End point methodology, simple, rapid and precise ■ Simplified using a single working reagent ■ Increased the stability of the Biuret reagent with the addition of ethylene glycol, tartrate or citrate ■ Methodology based on the formation, in alcaline solution, and in the presence of copper ions, of a characteristic purple coloured complex between the Biuret and two consecutive peptidic connnections ■ Resulting coordination complex obtained absorbs mostly in the blue colour ■ The intensity of the colouring is directly proportional to the protein concentration ■ Sample volume: 2.0uL/test ■ Measuring range from 6.0 g/L to 100.0g/L, extended up to 200.0 g/L with the automatic most dilution

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Case study 1 - Clinical Presentation ○ 47 year old woman ○ Loss of appetite ○ Nausea ○ Flu-like symptoms ○ Dark urine last two days ○ Tenderness in liver (right size under rib cage) ■ Dark urine may show some liver problem ■ Symptoms are consistent with hepatitis - or an acute infection ○ Serum biochemistry ■ Bilirubin 62 umol/L (RR 3-22 umol/l) ■ Aspartate aminotransferase 935U/l (RR 12-48 U/l) ■ Alanine aminotransferase 2700 U/I (RR 3-55) ■ Alkaline phosphatase 411 U/I (RR 80-280) - you would do a test on this which would help you rule out alcohol being the problem here ● These results are consistent with a problem with hepatocytes

which will produce more ALT and AST ○ Possibly hepatocyte disease ○ Causes of this: ■ Hepatitis ● Due to fever ■ Alcohol

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Acute hepatitis B infection ○ Patient can die of acute disease ○ Can get better ○ Usually have chronic inflammatory disease ○ Dont usually get low albumin in acute disease U&Es and eGFR

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Urea - ABX Pentra Urea CP ○ Urea is the nitrogen containing end product of protein catabolism ○ Diagnosis and treatment of certain renal and metabolic diseases ○ Human serum, plasma and urine based on an enzymatic UV test using urease and glutamate dehydrogennase ○ Sample volume: 3.0 uL/test ○ Measuring range from 0.31 to 50.00 mmol/L, with an automatic post-dilution up to 250.00mmol/L - a lot of modern equipment does have automatic features

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Ion selective electrode (ISE) intended for the quantitative determination of electrolytes ○ Electrolytes take part in most of the metabolic functions of the organism

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Sodium, potassium and chloride belong to the most important physiological ions Quantitative determination of electrolytes with ISE module by potentially using ion selective electrode Direct (non diluted serum and plasma) Indirect (diluted urine) Sample volume: 60 uL/test 1, 2 or 3 electrolytes

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Creatinine ○ Creatinine measurements are used in the diagnosis and treatment of renal diseases, in monitoring renal dialysis, and as a calculation basis for measuring other urine analytes ○ ABX Pentra Creatinine 120 CP reagent is a diagnostic reagent for quantitative in vitro determine of creatinine in human serum, plasma and urine based on a kinetic method using alkaline picrate (Jaffe method) ○ Improved jaffe chemistry is a kinetic procedure which does not require deproteinisation of the sample and is formulated to reduce the interference in serum proteins ○ At an alkaline pH, creatinine reacts with picrate to form Janovsky complex ○ The rate of increase in absorbance at 510nm due to the formation of creatininepicrate comlex is directly proportional to the creatinine concentration present in the sample ○ Sample volume: 10 uL/test ○ Measuring range from 12.2 umol/L to 1600umol/L ■ Extended up to 4800 umol/L with the automatic dilution

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ABX Pentra enzymatic creatinine CP reagent ○ Multi-step approach ending with a photometric end-point reaction ○ The enzyme creatinine amidohydrolase is used to convert creatinine to creatine ○ Creatine is broken down to sarcosine and urea by creatine amidinohydrolase ○ Further enzyme linked steps with sarcosine oxidase and peroxide yield a coloured chromogen read at 545 nm ○ ESPMT: N-ethyl-N-sulfopropyl-m-toluidine

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Glomerular filtration rate (GFR) ○ The volume of plasma from which a given substance is completely removed from the plasma by glomerular filtration in a unit time ○ An estimation of the efficiency with which substances are cleared from the blood by glomerular filtration and so is a measure of nephron function ○ Determined by measuring the clearance of an exogenous or endogenous substance by the kidneys ○ A substance is only suitable for estimating GFR if: ■ It occurs at a stable concentration in the plasma ■ Physiologically inert ■ Freely filtered at the glomerulus ■ Not secreted, reabsorbed, synthesised, or metabolised by the kidneys ○ In these conditions, the amount filtered at the glomerulus is equal to the amount excreted in the urine

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Creatinine as a marker ○ Creatinine (MW 113) ○ Derived from creatine and phosphocreatine breakdown in muscle ○ Freely filtered at the glomeruls ○ Its plasma concentration is inversely related to GFR ○ It is convenient and cheap to measure ○ But it is convenient and cheap to measure ○ But its concentration in the plasma is affected by age, sex, exercise, muscle mass, certain drugs (e.g. cimetidine, trimethoprim) and nutritional status ○ Neither reabsorbed from, nor secreted into, the tubule from urine in sheep, cattle, cats and dogs, and may be used as an endogenous marker for estimation of GFR in these species

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Creatinine - creatinine clearance ○ As a GFR marker, creatinine can be expressed as its serum conc or its renal clearance

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Measurements of both the daily creatinine output in urine and creatinine concentration in plasma can be used to estimate GFR Its urinary clearance requires a timed urine collection ■ Introducing its own inaccuracies ■ Inconvenient and unpleasant In adults, the intra-individual day-to-day coefficient of variation for repeated measures of creatinine clearance exceeds 25% Additionally, owing to tubular secretion of creatinine, creatinine clearance usually overestimates GFR in adults by 10-40% at clearances about 80mL/min and by a much greater percentage at lower lovers of GFR Creatinine clearance can only provide a crude index of GFR

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Cockcroft and Gault modification of diet in renal disease (MDRD) ○ The mathematical relationship between serum creatinine and GFR can be improved by correcting for the confounding variables that make that relationship nonlinear ○ Many equations have been derived that estimate GFR using serum creatinine corrected for some of all of sex, body size, ethnic origin, and age ○ This is known as estimated GFR (eGFR) and may produce a more reliable estimate of GFR than using serum creatinine aline ○ The Cockcroft and Gault modification of diet in renal disease (MDRD) study equations have been widely used in adults - two further equations ○ Schwartz and Counahan-Barratt are recommended for use in children ○ It has recently been recommended by many healthcare organisations, including the UK department of health, that GFR should be estimated using the MDRD equation in addition to serum creatinine

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Simplified equation ○ The most widely used approach to GFR estimation internationally

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When an accurate GFR necessary ○ Equation-based estimates of GFR should be avoided ○ These includ cancer chemotherapy, or the use of any other drug that is excreted in urine and has a narrow therapeutic margin; the assessment of potential living related kidney donors and the assessment of GFR in patients with musclewasting disorders including spina bifida and paraplegia ○ Reference methods should be used i.e. Cstatin C ○ However, owing to its higher reagent costs compared with creatinine, cystatin C is not widely used in clinical practice

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The international classification of CKD

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eGFR Equations ○ Clinical data from Gaw Case 9 ■ Male age 35 ■ Lion pain ■ Serum creatinine 150 umol/L ■ 24 hour urine 2160 mL ■ Urine creatinine 7.5 mmol/L ■

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Calculate the creatinine clearance, is it normal? ● U x V/P ○ 75 ml/min ■ This is not a normal result for a man of this age ■ It would show that he has significant kidney problems ■ There may have been inaccurate collection of the urine

Case 3 - Clinical Presentation ○ 16 year old male - road traffic accident ○ Both femurs were broken ○ Spleen was ruptured ○ Required surgery and 16 units of blood

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Impaired kidney function - acute kidney injury ○ The urea and creatinine is high ○ Very low urine output ○ Low sodium ○ High phosphate - decreased secretion out of kidneys ○ High potassium ○ Low bicarbonate - implies metabolic acidosis ■ Pre-renal ● Lead by low blood pressure caused by blood loss and lack of filtration pressure

Workshop 2 ●

Workshop Bone Disease, CVD Risk ○

Bone disease ■ Bone profile test ● Calcium ● Phosphate ● Albumin ● Alkaline phosphatase ● Calculate corrected or adjusted calcium to estimate free calcium ■

Follow up tests ● PTH ● Magnesium ● Urine calcium excretion ● 25 hydroxcholecalciferol- vitamin D ● Urine hydroxyproline excretion ● Osteocalcin

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Calcium measurements ○ Diagnosis and treatment of: ■ Parathyroid disease ■ Variety of bone diseases ■ Chronic renal diseases ■ Tetany (intermittent muscular contractions or spasms) ○ Many colourimetric methods ■ Connerty and Briggs described methods using alizarin 3sulphonate and cresophthalein complexone ■ Gindler and King described a method using thymol blue ○ Many subsequent modifications to these methods ○ ABX Pentra calcium AS is based on the metallochromogen Arsenzo III

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ABX Pentra Calcium AS CP ○ Calcium ion react with Arsenazo III at pH 6.75 to form an intense purple coloured chromophore ○ The absorbance of the Ca-Arsenazo III complex is measured bichromatically at 660/700nm ○ The resulting increase in absorbance of the reaction mixture is directly proportional to the calcium concentration int he sample ○ Arsenazo III has a high affinity for calcium ions ○ Shows no interference from other ctions normally present in serum, plasma or urine

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Calcium AS continude ○ Calibrator: ABX Pentra Mutlical ○ Control: ■ ABX Pentra N control ■ ABX Pentra P control ○ Sample volume: 5ul/test ○ Measuring range: 1 to 4.50 mmolL extended up to 13.50 mmol/L with automatic post-dilution ○ Calibration stability: 10 days

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Corrected calcium ○ Measurements of total calcium are affected by changes in plasma albumin concentrations ○ Approx half of the calcium in blood bound to albumin ○ When plasma albumin concentrations increase, total calcium measurements increase, visa versa ○ Concentrations of free unbound, ionised calcium are not affected by changes in albumin

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■ No physiological or pathogloical change For albumin concentrations...