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CONTENTS
RENAL PHYSIOLOGY
Cc
INTRODUCTION TO URINALYSIS
KIDNEY
● Major organ of the excretory system ○ One pair (1) of kidneys ○ Bean-shaped organ located around the third and fourth lumbar area of your vertebrae ○ Major function is to dispose waste products of metabolism ○ Regulates plasma-water volume of the body for water reabsorption and excretion of waste. FUNCTIONS OF THE KIDNEY
- Removal of waste products
- Regulation of plasma, water volume
- Maintain normal acid-base balance
- Regulation of blood pressure
- Endocrine function
NEPHRONS 1 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID
- August 27 ,
- RENAL PHYSIOLOGY
- INTRODUCTION TO URINALYSIS
- URINE FORMATION
- GLOMERULAR FILTRATION
- TUBULAR REABSORPTION
- TUBULAR SECRETION
- RENAL TUBULAR SECRETION
- RENAL FUNCTION TESTS
- GLOMERULAR FILTRATION RATE (GFR)
- BETA-2-MICROGLOBULIN
- RADIONUCLEOTIDES
- ROUTINE URINALYSIS
- TYPES OF COMMON URINE SPECIMENS
- CHANGES IN UNPRESERVED URINE
- PHYSICAL EXAMINATION OF URINE
- URINE VOLUME
- URINE COLOR
- URINE CLARITY
- SPECIFIC GRAVITY
- SPECIFIC GRAVITY TEST
- ODOR
- RECALL
- CHEMICAL EXAMINATION OF URINE
- RECALL QUESTIONS
- CHEMICAL EXAMINATION OF URINE
- REAGENT STRIPS
- PHENYLKETONURIA
- MELANURIA
- BRANCHED CHAIN AMINO ACIDURIA
- DIAPER SYNDROMES
- MICROSCOPIC EXAMINATION OF URINE
- MANUAL MICROSCOPIC EXAMINATION
- SPECIMEN PREPARATION
- Examine fresh or adequately
- preserved specimens (10-15mL)
- CENTRIFUGATION
- SEDIMENT PREPARATION
- EXAMINATION OF SEDIMENT
- REPORTING - CORRELATE RESULTS - URINALYSIS MICROSCOPIC TECHNIQUES - TWO TYPES OF URINARY SEDIMENTS - REPORTING - STAINS AND SOLUTIONS - CASTS - RED BLOOD CELLS - WHITE BLOOD CELLS - EPITHELIAL CELLS - BACTERIA - YEAST - PARASITES - MUCUS THREADS - CRYSTALS - RECALL QUESTIONS - AUTOMATION IN URINALYSIS LABORATORY - SYSMEX UX-2000 FULLY AUTOMATED - INTEGRATED URINE ANALYZER - Specifications - SUMMARY OF RENAL DISEASES - LABORATORY SAFETY - FIRE - SPECIFIC SAFETY MEASURES URINALYSIS - GUIDELINES ON SPECIMEN DISPOSAL (AUBF)
August 27 , 2022 ● Controls the ability of the kidney to clear waste products and maintain the body’s water and electrolyte balance. ○ Each kidney is composed of about 1 to 1.5 millions of nephrons which are capable of urine formation. ● Types of nephrons ■ Cortical nephrons - are located in the cortex of the kidney and remove waste products and reabsorb nutrients ○ Main function is removal of waste products as well as reabsorption of nutrients ○ 80-85% of nephrons are cortical in nature. ■ Juxtamedullary nephrons - extend into the medulla of the kidney and concentrate in the urine ○ Responsible for reabsorption of water ○ Urine that is concentrated would only mean that it has a high amount of solutes and less amount of water.
URINE FORMATION
● Renal blood flow ○ Renal artery → afferent arteriole → efferent arteriole → proximal convoluted tubule capillaries → vasa recta/loop of Henle → distal convoluted tubule capillaries → renal vein ● Normal renal blood flow is approximately 1200 mL/min ● Normal plasma flow is approximately 600 to 700 mL/min ○ Half of the renal blood flow, since plasma is half of the components of whole blood ○ Has a longer Loop of Henle which is in-charge of concentrating the urine.
URINE FORMATION 3 - STEPS
GLOMERULAR FILTRATION
● Glomerular Tuft – filtering apparatus featuring eight (8) lobes of capillaries that are responsible for filtration (glomerular) ● From renal artery → afferent arteriole : Non-selective filtration will occur as long as the substance is less than 70,000 kD, it can pass through. (amino acids, glucose, electrolytes, water) ● 25% of total cardiac output can enter per minute - 1200mL ○ 25% of total cardiac output = 25% of total blood volume ● Total blood volume of an average healthy individual: 5-6 Liters ● Product of filtration: Ultrafiltrate ● Ultrafiltrate: expected specific gravity is 1. ● NOTE: Specific gravity may predetermine if the urine is hyposthenuric, isosthenuric or hypersthenuric. ○ After filtration, there are only about 120 to 130 mL of ultrafiltrate that will transverse in the proximal convoluted tubule where tubular reabsorption will occur afterwards. ○ Substances that are reabsorbed are the essential ones which include: ■ 100% of Glucose (major threshold substance) ● NOTE : Urine should always, AT ALL TIMES, be negative for albumin or protein. ● Healthy urine is negative for protein and glucose as proteins are not allowed to pass through the glomerulus (molecular size) and glucose is reabsorbed fully at 100%. ● it’s not normal that urine turns positive in the presence of protein, which may mean glomerular; it should not also turn positive for glucose, since your not diabetic” ● In cases of DM, wherein the glucose level is high, there may be presence of glucose in the urine because the proximal convoluted tubule (PCT) has a threshold for glucose (160- mg/dL) TUBULAR REABSORPTION ● Amino acids, electrolytes may also be reabsorbed in the PCT ● After passing through the PCT, the ultrafiltrate will now transverse to the descending loop of Henle (limb of Henle) ○ Responsible for holding or reabsorbing water ○ 80% of water (the majority of water) are being reabsorbed here ● Ascending loop of Henle (limb of Henle) ○ Reabsorption of salt (no water reabsorption) ○ “ASINding” for salt ○ This is the only part of the nephron where it is impermeable to water ○ This is also where casts and crystals may start to form since it promotes precipitation of chemicals and proteins because of being free from water TUBULAR SECRETION ● Not all substances are being filtered by the glomerulus; has requirements: ○ Low molecular weight – anything that are big in size, heavy, cannot be eliminated ■ Elimination of drugs through the kidney by tubular secretion ■ In the peritubular portion of the kidney, substances bound to protein will dissociate, and the former will be secreted in the tubules and later on eliminated ● Last part of product elimination ● Urine will be the final product, and will be stored in a bag-like structure called the urinary bladder. When the bladder is full, it sends signals to the brain for the need to urinate
GLOMERULAR FILTRATION
● Capillaries drop off particles in the blood the body needs to get rid of ○ There are about 8 tufts in the glomerulus ● Nonselective filtration of plasma substances with MWs less than 70,000 kD (MW of albumin is 67,000) 2 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 ○ It is completely filtered, reabsorbed and then broken down by the renal tubular cells. Serum level remains constant unless the GFR decreases causing the serum level to rise. ○ *may not be applicable pediatrics, diabetics, geriatrics, and critically sick patients wherein the kidneys have problems ○ **independent of muscle mass
BETA- 2 - MICROGLOBULIN
● Dissociates from HLA at a constant rate and is rapidly removed by filtration ○ ↑ plasma Beta-2-microglobulin: ↓ glomerular filtration rate ○ * Not for patient with immunologic disorders and malignancy ○ It is an endogenous process ○ They may have high levels of this microglobulin. This is not associated with GFR but with oveflow type. ■ This means our body is producing more than expected and the kidneys cannot cope with it. ● It is a microglobulin so it is expected that it will pass through in your glomerulus, and therefore, in the blood it must not be elevated. In the event there is elevation, GFR is not normal.
RADIONUCLEOTIDES
● Exogenous process ● 125I-iothalamate ● **Plasma clearance of the radioactive materials ● Used to monitor the viability or success of kidney transplant ● Radioactive products will be introduced and if the products are not recovered, the kidney transplant is not successful. ● Renal tubular reabsorption ○ Primary tests are serum and urine osmolality ■ serum osmolality: 275-300 mOsm urine osmolality: 50-1400 mOsm ○ The free water clearance test measures the ability of the kidney to respond to the body hydration ■ Fish bird method is an old method wherein the patient will be challenged to not take fluid for 24 hours and collect the urine after it. If the urine has low specific gravity, it is not normal and it means that the urine has no concentrating ability ● If the body is dehydrated, the kidney will reabsorb water and result in low urine output and high specific gravity which means that the tubules has no capability to reabsorption of water ● Tubular secretion tests ○ Titratable acidity detects the inability of the proximal convoluted tubule to secrete hydrogen molecules ○ Urinary ammonia detects the inability to produce ammonia in the proximal and distal convoluted tubules ○ This is the one that regulates our acid base balance and we are doing PAH Testing or para aminohippuric acid testing to test this tubular secretion test ○ The simplest test that will determine our kidney’s ability to concentrate urine is Specific Gravity Testing ● Renal Blood Flow Test ○ PAH Test — Measures the amount of blood flowing through the kidney ■ exogenous procedure ■ loosely bind to plasma protein ■ cleared as blood passes through peritubular capillaries ■ *effective renal plasma flow : 600-700 ml/min ■ average blood flow: 1200ml/min ■ Initially, once the PAH Test is introduced to the body, it must not pass the glomerulus. It must be excreted through secretion in the peritubular capillaries
REGULATION OF BLOOD PRESSURE
■ If the individual has low blood pressure, then it must be corrected. In that case, once blood pressure decreases, then there will be secretion of renin. This will react to a blood-borne substance, your angiotensinogen which is inactive. The angiotensinogen will be activated in the presence of renin, converting it to Angiotensin 1. In the lungs, the ACE or angiotensin converting enzyme will convert angiotensin 1 to angiotensin 2. ● Angiotensin 2 will dilate afferent arterioles by vasodilation and this will make the afferent arterioles receive more blood ● Efferent arterioles will be constricted, making the blood pressure high ● Aldosterone will become activated and it will promote reabsorption of salt, specifically sodium, making the blood pressure high. ● This process is a negative feedback mechanism
ROUTINE URINALYSIS
URINALYSIS
● Routine analysis comprises of: ○ Physical examination 4 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 ○ Chemical examination ○ Microscopic examination ● It is important because it aids in the diagnosis of disease, not limited to kidney diseases like diabetes. ● It is also used as a screen for asymptomatic populated undetected disorders. ● These tests can screen symptomatic or asymptomatic patients ● It can monitor progress of disease and effectiveness of therapy ● It is rapid, reliable, accurate (if well trained), collection of sample is not invasive, safe
TYPES OF COMMON URINE SPECIMENS
● Random ● First Morning ● Clean-Catch ● Catheterized ● 24Hour (Timed) ● Drug Screening ● Three-glass collection ● Suprapubic Aspiration ● Pediatric Specimen RANDOM ● Routine screening ● May require confirmatory testing based on diet and exercise ● You can collect the sample anytime of the day without specific instruction. ● May confirm obvious pathology ○ Expected to be diluted but the same substance are elevated. FIRST MORNING ● Collected immediately on arising ● Routine screening/confirmatory testing ● Orthostatic proteinuria ○ If protein is traced to 1+ in the late afternoon, collection must be repeated and sample must be first morning ■ If the sample is positive, then it is true proteinuria ■ If negative, it is orthostatic ● Pregnancy tests CLEAN-CATCH ● Requires patient to cleanse the genital area ● Void first into the toilet, then collect specimen and finish voiding into the toilet ● Bacterial cultures ● The process is the same with midstream catch but additional cleansing with soap and water of the genitalia must be done ● Bacteriuria will be reported if the colony forming units are greater than 100,000. ○ If the specimen is suprapubic aspirated urine, the 100, cfu will not be applicable because the aspirated urine sample is sterile. So if the colonies are less than 100,000, it should still be considered as significant CATHETERIZED ● Collected from a catheter passed into the bladder ● Bacterial Cultures ● Note: When a routine urinalysis and a culture are both ordered, perform the culture first ● This is done to patients who cannot void voluntarily ● It can be used as bacterial culture if the sample is not obtained from the polycat bag. ○ If sample is collected in the bag and the urine was standing for 2 hours, there will be an increase of bacteria, which may produce false positive results ● Culture must be prioritized to prevent further contamination 24 HOUR / 12 HOUR (TIMED) ● Patients void into the toilet and then begins timing ○ Empty the bladder first by discarding the urine in the first hour since the nephrons are continuously making urine and prior to the start of the time, there is already 2ml of urine so we have to discard it first ● Collects all urine during the designated period ● Voids and collects urine at the end of the period ● Specimens can provide quantitative results ● Usedsed to measure hcg titer ● Container must hold up to 3L, sometimes colored container because some substances are photosensitive ● In history, 12 hours timed urine is used in addis count which will quantify the following: ○ WBC ○ RBC ○ Epithelial cells ○ Casts DRUG SCREENING ● Strictly follow chain-of-custody (COC) form requirements ○ The sample must be safe and secured from collection up to testing ● No tampering, substitution, spiking, and or dilution ● Temperature: 32.5-37.7 (within 4 minutes) ● The label itself is capable of measuring temperature ● Sample is collected while being witnessed by an accredited drug analyst ● In the US, the container is capable of measuring the temperature ○ The small portion below the label is colorless at room temperature ○ Once urine is added, the small portion turns brown, which means it is warm. ■ If there is no change in color, the urine is cold and the incident must be report ○ If the patient cannot void urine, tell them to drink at least 500 mL of water. 3-GLASS COLLECTION ● Used to diagnose prostatic infection, to differentiate prostatitis from genitourinary tract infection (astitis/urethritis) ● For male patients ● Used in the diagnosis of prostatic infection ● 1st vial - first fraction of urine ● 2nd vial - midstream part of urine ● 3rd vial - prostatic fluid with remaining urine volume (the urologist massage the prostate) ○ Digital Prostate Massage - The index and middle finger is inserted in the anal region of the male and massage the prostate (right plump) 5 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 ○ Usually observed when there is a complete obstruction (renal stones, toxic agent) ● Nocturia: Increased urine output at night ○ Exceeding 500mL/night is already considered nocturia ○ Physiologically observed among pregnant women ○ Observed among elder male patients with enlarged prostate ○ Babies do not suffer from nocturia (it is their diet) ● Polyuria: Increased urine output greater than 2.5L/day ○ Diabetes mellitus: increased urine output to excrete excess in urine glucose (way of kidney to eliminate glucose) ○ Diabetes insipidus: increased urine output caused by lack of dysfunction/absence of antidiuretic hormone (ADH
- hormone needed to concentrate urine in nephrons) ○ Results in polydipsia (excessive thirst) since body is losing too much water ● Diabetes Mellitus ○ Polydipsia (3Ps) ○ Polyuria ○ Polyphagia (excessive eating) ○ High Sp. Grav. ○ Low insulin/ No insulin ○ Hyperglycemia ○ Glucosuria ● Diabetes insipidus ○ Polydipsia ○ Polyuria (2Ps unlike DM with 3Ps) ○ Low sp. Gravity (as low as 1.003) ○ Decreased/ function ADH ○ No hyperglycemia ○ No glucosuria
URINE COLOR
● Normal urine is yellow. ○ Normal urine may range from straw to amber ○ Pigments responsible for yellow color: urochrome, urobilin, and uroerythrin ● Shades of yellow are based on fluid consumption and vary from pale yellow (dilute) to dark yellow (concentrated) URINE COLOR Color Cause Correlation Orange Bilirubin (B2 form) ● Bilirubin 1 is bound to albumin and cannot pass through the glomerulus Produces yellow foam when shaken, abnormal liver function Pyridium ● Interferes with reagent strip - all colors are errors, so every test must be done manually Produces thick orange pigment that can interfere with reagent strip tests Red ● Most commonly observed RBCs Cloudy red urine,^ or positivesmoky abnormal color of urine ● Abnormal pathologic ○ bleeding ● Abnormal non-pathologic ○ medication or consumption of red beans ● Speckled reaction pad in the reagent strip tests for blood, microscopic RBCs Non-hemolyzed blood Hemoglobin Clear urine, positive test for blood Hemolyzed blood Clear red Myoglobin ● Excessive myoglobin is toxic to the kidney and can be lethal or fatal (ex. hazing) Clear urine, positive test for blood, need further testing Intravascular hemolysis - plasma will appear color pink or red Myoglobinuria - plasma will appear clear yellow Clear red Porphyrins Negative tests for blood, needs further testing Described as port wine red or purplish red color (observed in cases of porphyrinuria, or defects with hemoglobin synthesis) Black Oxidized RBCs, denatured Hgb Clear urine, positive test for blood Melanin Clear urine, darkens on standing Methemoglobin From red to brown in use of acid hematin test (acidification) From dark brown to black upon exposure to light or air due to melanin (melanogen converted to melanin upon exposure to light) Homogentisic acid In cases of alkaptonuria, dark brown will turn black upon standing or upon alkalinization ● Other tests to differentiate hemoglobin from myoglobin – the use of ammonium sulfate, which precipitates hemoglobin, but not myoglobin, then repeat test. If the test is negative, it is hemoglobin. 7 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022
URINE CLARITY
● Terminology: Clear, hazy, cloudy, turbid, milky ● Freshly voided normal urine is clear ● Refrigerated normal urine ○ White Turbidity in urine with an alkaline pH from amorphous phosphates and carbonates ○ Pink turbidity in urine with an acid pH from amorphous urates ■ Because of urates bind to the pigment uroerythrin, and forms pink sediments when accumulated ● Adjust transparency based on microscopic findings NON-PATHOLOGIC TURBIDITY ● Squamous epithelial cells ○ Poor collection, not midstream, uncircumcised ● Mucus ● Amorphous phosphates, carbonates, and urates ● Semen ● Feces ● Radiographic Contrast Media ● Powder and Creams ○ e.g. gloves powder (gloves powder not recommended to use in laboratory) PATHOLOGIC TURBIDITY ● Red Blood Cell (RBC’s) ○ About 500 cells/cumm can cause haziness ● White Blood Cell (WBC’s) ○ About 200 cells/cumm can cause haziness ● Yeast ● Urothelial and Renal Tubular Epithelial Cells ● Abnormal Crystals ● Lipids (Milky) ○ Filarial worms ○ Long bone fracture ● Bacteria ○ Causes uniform turbidity ○ If bacteria is high, sample may remain turbid even after centrifugation ○ Spinning can reduce bacteria but can not achieve clarity ○ Adsorbent can be used to remove bacteria
SPECIFIC GRAVITY
● Screening test for renal tubular reabsorption of essential elements filtered by the glomerulus ● Based on the fact that the glomerular filtrate has a specific gravity of 1.010 (isosthenuria) ● >1.010 hypersthenuria ● <1.010 hyposthenuria ● Correlate with water intake and physical activity ● Comparison of the density of urine to the density of distilled water (1.000) ● Urine contains dissolved substances that produce density by their size and number ● Osmolarity is a better method of measuring solutes ○ Its based on the number of ionized particles, more specific, not affected by particle size ○ Unlike SG which is a reflection of the dissolved substance based on size and amount ○ If high density, high SG
SPECIFIC GRAVITY TEST
REAGENT STRIP
● Most commonly used method ● Primary test for routine urinalysis is the reagent strip test ● Principle is based on the number of hydrogen ions (H+) released from a polyelectrolyte (pKa) is proportional to the number of ions in the urine ● Increased urine concentration = increased H+ released = low pH ● Tye indicator on the strip is bromothymol blue ● Reaction = Yellow-Green (acid) → Green-Blue (alkaline) ● Not applicable if there is thyridium (use refractometer) ● Increment is by 0. REFRACTOMETER ● Principle: The concentration of dissolved particles in a solution determines the velocity and angle of light passing through a solution ● The refractometer uses a prism to direct a wavelength of light that can be read on a scale calibrated with distilled water (1.000) ● Standardizing Solutions: ○ Distilled water: 1. ■ Readily available ○ 5% NaCl: 1.022 +/- 0. ○ 9% Sucrose: 1.034 +/- 0. ● Each line is equivalent/ to increments of 0. OSMOLARITY ● considered more representative of renal concentrating ability than specific gravity because it measures only the number of particles and their size is not relevant ● Measurement is the number of particles into which 1g molecular weight of a substance dissociates ○ Ex. Non ionizing urea (mol. weight [MW] 60) = 1 particle, ionizing NaCl (MW 58.5) = 2 particles ○ Each specific particle is accounted ● Reported in milliosmoles (mOsm) ● Colligative properties measured in the clinical laboratory ○ Freezing point depression ■ One mole of a non ionizing substance will lower the freezing point 1.86 °C ■ Volatile substances such as alcohol can interfere ○ Vapor pressure depression ■ Actual measurement is the dew point of the urine sample ■ Uses micro-sample on filter paper disc. Care must be taken to avoid evaporation ■ No interference from volatile substances CLINICAL SIGNIFICANCE ● Normal serum osmolarity; 275 to 300 mOsm ● Fluid intake influences urine osmolarity 8 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 ● These are basic knowledge that we need to remember ● Tinatanong din sa board exam (e.g. storage temperature, when to run controls, and other important practices in using reagent strips ● Store in tightly closed opaque bottles ○ Strip should always be open specially if not used because it is highly absorbent ● Removed immediately before uses ○ Do not put back used strip as it will contaminate the entire stirp and it can no longer be used ● Do not refrigerate (room temp below 30°C) ○ It will accumulate moisture ● Run positive and negative controls every 24 hours (at least once a day) ○ Controls: ■ Kovatrol I - Pathologic Control ■ Kovatrol III - Normal Control ● Usually in CC control I is normal and control II is pathologic ● Must be run at least once a day ○ Better if you have 3 controls ■ If you have 3 controls, the rule is if 2 out of 3 is in range = valid (pasok pa rin and control), both of these controls must be in control ■ If you are using only two controls ○ You cannot use distilled water as negative control. We are also using a normal urine (Kovatrol I - abnormal urine) ○ usually , these are lyophilized and we just simply reconstitute when we are ready to perform the testing ● Run controls when a new bottle is opened ○ Or we use a new lot number for reagent strip ● Observe expiration date ● Observe discolored reagent pads TECHNIQUE ● Thoroughly mix specimens ● Warm refrigerated specimens ○ If in case the sample is from refrigerator, then you may stand it for few minutes until it reaches room temperature ● Briefly dip reagent strips ● Blot strip while removing from urine ● Observe manufacturer timing instructions ● Relate chemical with physical and microscopic results REAGENT STRIP SOURCES OF ERROR AND CORRELATIONS Test Source of Error Test Correlations pH ● Runover from adjacent strips ● Old specimens + ○ Make urine alkaline ○ Reason: conversion of urea to ammonia ● Nitrites ● Leukocyte Esterase ● Microscopic making pH elevated ○ If the specimen is freshly collected and yet the pH is high, then try to correlate it with bacteriuria ○ Genus: PROTEUS ■ There are some species of bacteria that causes UTI that can really alter the pH of urine from acidic to alkaline ■ Produces rapid urease enzyme ■ It is correlated with the formation of triple phosphate crystal Protein ● Highly buffed alkaline urine + ● Detergents + ● Pyridium + ● Chlorhexidine + ● High specific gravity + ○ Specially if nag run over and you fail to blot, it could be a problem ● Microalbuminuria - ○ Reagent strip will not yield positive result since protein strip test is based on protein error of indicator involving macromolecular weight protein ○ Minsan pag nagpapacheck ng cast pero negative sa protein strip - proceed to perform confirmatory test (sulfosalicylic acid) ○ sulfosalicylic acid will precipitate all proteins both albumin and globulin ○ Reagent strip only detects albumin ○ Situational question: what will be your ● Blood ● Nitrites ● Leukocyte esterase ● Microscopic 10 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 interpretation of your reagent strip for protein is negative but the sulfosalicylic acid is positive ■ A: presence of protein but not albumin ■ You see clearly in the microscope that there are casts, but when you use a reagent strip it is negative for protein therefore you need to do sulfosalicylic acid (a positive result in sulfosalicylic acid means that other proteins are present in the urine but not albumin) ○ Casts major component or the protein matrix of all types of casts are tamms horsfall protein or uromodulin ■ Uromodulin are low molecular weight proteins produced by renal tubular epithelial cells ■ It would not react in the strip but it will be detected in the sulfosalicylic acid test Glucose ● Oxidizing agents + ● Detergents + ● Increased ascorbic acid - ○ Can cause false negative result for glucose test in reagent strip ○ Ascorbic acid will inhibit enzymatic reaction this is why some reagent ● Ketones ● Proteins strip have their own pad for ascorbic acid ■ The pad is used to at least validate the result in the reagent strip ■ Ex. 200 mg/dL is the plasma glucose, but the strip method is negative, then ascorbic acid is strongly positive ■ In this scenario, you know that the test for glucose is false negative because of the high level of ascorbic acid ○ HOWEVER, if the test is based on COPPER REDUCTION, Ascorbic acid will serve as an oxidizing reagent and will have a false positive ○ False negative: Enzymatic ○ False positive: Copper reduction (Fehling’s, benedict, Folin Wu, etc.) ● Low temperature - ● Old specimens - Ketones ● Red urine + ○ Highly pigmented urine samples can be false positive ○ The reagent used for ketone testing is Sodium nitroprusside ■ The positive result for Sodium nitroprusside test for ketone is red in color ● Sulfhydryl medications + ○ Can make sample highly pigmented ● Levopoda + ● Glucose ○ In cases of uncontrolled diabetes, glucose level is high and patient is prone to have ketoacidosis or Diabetic Ketoacidosis (DKA) 11 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 Specific gravity ● Increased protein + ● Highly alkaline urine - ● Add 0.005 to any urine with a pH of 6. or higher ○ Not normally applied ● Sources of error - favorite question in examinations ● Indicated in the reagent strip if the interfering substance will cause false positive or false negative ● You are expected to know at least the reason behind
RECALL QUESTIONS
Question #1: A negative glucose oxidase test and a positive Clinitest result was noted in a given urine sample, what is the most probable reason for this reaction?
- Urine is positive for sugar except glucose
- Urine may have high ascorbic acid content
- Indicators interfere with the strip A. 1,2,3 are correct B. 1 and 2 are correct C. Only 1 is correct D. Only 2 is correct E. All are not correct Ratio: 1 and 2 are correc t - Clinitest is urine positive except for glucose. Ascorbic acid may act as a reducing agent that gives a positive result to the urine Question #2: The following are interferences for Clinitest, except: A. Lactose B. Ascorbic acid C. Fructose D. Formalin E. Sucrose Ratio: Sucrose - Lactose, ascorbic acid, fructose, and formalin all have the ability to reduce Cupric oxide to cuprous, therefore all are positive for Clintest except for sucrose since it is a non-reducing sugar. Question #3: Which of the following would be affected by allowing a urine specimen to remain at room temperature for 3 hours before urinalysis A. pH B. Protein C. Occult blood D. Specific gravity Ratio: pH - pH would first to be affected compared to other choices, protein may be affected but not as early and as of the same magnitude than pH Question #4: Pathologic causes or urine turbidity: 1. Blood cells 2. Non Squamous epithelial cells 3. Lipid 4. Lymph fluid A. 1, B. 2,
C. 1,2,
D. 1,2,3,
Ratio: 1,2,3,4- All of them can cause pathologic conditions. Blood cells did not specify if it is wbc or rbc, so it can be hematuria or pyuria. Lipiduria can have long bone fracture or hyperlipidemia. Significant non squamous epithelial cells are RTE cells. Lymphatic fluid (chyle) in lymphatic duct obstruction in case of filariasis or elephantiasis Question #5: A tea-colored urine may be caused by: A. Drinking green tea B. Rifampin C. Bilirubin D. Coffee Ratio: C - Confirmation must still be performed via bilirubin tests with Diazo reaction. Question #6: Use of refractometer over a urinometer is preferred due to the fact that the refractometer uses: A. Small urine volume and compensates for temperature B. Small urine volume and compensates for protein C. Large urine volume and compensates for temperature D. Small urine volume and compensates for glucose Ratio: A - Despite using the refractometer, if the specimen is positive or has high level of protein or glucose, correction must still be applied. For every 1 g/dL of glucose, .004 is subtracted and for protein, it is .003. For temperature, no need to correct. Urinometer correction states that for every 3°C higher than the room temperature, .001 is added, and vice-versa. When water cools, it expands, which is why subtraction must be applied to the specific gravity values. It is why in the laboratory, if the sample came from the refrigerator, stand it for a few minutes until the sample is already at room temperature before processing it. Question #7: Which of the following tests is least affected by standing or unpreserved urine? A. Glucose B. Protein C. pH D. Bilirubin Ratio: B - All of these factors are affected, but it is the protein that is LEAST affected. Glucose and pH will increase, and bilirubin is decreased in standing or unpreserved urine. Protein will increase due to the increase of the bacteria, since the cell wall of the bacteria is made up of glycoproteins, peptidoglycan, lipid bilayer. Question #8: Preferred urine sample for pregnancy testing A. Random B. Fasting C. First morning D. Midstream clean-catch 13 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 Ratio: C - First morning urine sample is preferred for pregnancy testing. While fasting is known as the second morning urine. After the first urine, eating and drinking is prohibited until the second urine voiding. Fasting urine is also utilized to monitor or diagnose diabetes. Question #9: A black urine with a ph of 8 should be: A. Tested for melanin and homogentisic acid B. Tested for blood and bilirubin C. Returned to the nurse’s station D. Discarded and the doctor notified Ratio: A - This is in case for melanuria and alkaptonuria. Question #10: Cloudiness in a freshly voided urine could indicate the presence of: A. Protein B. Glucose C. WBC D. Metabolites of vitamins Ratio: C ● Pyuria - clinical term used to indicate the presence of WBC in urine ● Glucosuria - presence of glucose in urine ● Glycosuria - presence of sugar in urine ○ This term is used if sugar is not identified or specified
CHEMICAL EXAMINATION OF URINE
● In case the request is routine urinalysis, we usually run it only with the 10 parameter reagent strip. ○ However, in the Philippine setting, microscopic examination is still done even if all parameters in the reagent strip test are negative. ○ In the States, it is not automatic that microscopic examination is performed. ■ It is only done if chemical testing for nitrite, protein, leukocyte esterase, and blood are positive. ■ If all the chemical tests are negative, then microscopic examination is not anymore performed. ● Note in the remarks that microscopic examination is not indicated = not needed ● Reflex testing : If chemical tests for nitrite and leukocyte esterase are positive, we automatically perform urine culture. ○ We do not need the physician’s request to perform the urine culture ○ If nitrite is positive, it indicates that there is a possible condition such as bacteriuria or bacterial infection. ○ Presence of leukocyte esterase is an indication of infection ■ Indicates general infection (may be due to parasite, yeast, bacteria) ■ To find out the causative agent of this infection, we perform urine culture. ● Chemical testing may be qualitative or quantitative. ○ Qualitative: we are expected to report it as positive or negative; presence or absence of a certain substance ○ Quantitative: we report the amount of the substance being tested in a given urine sample ■ 24-hour urine is the specimen to be submitted
NOTE:
● Chemical testing may be wet (done a long time ago) ○ We utilize test tubes and add the urine samples and reagents ○ Example: ■ Benedict’s test (for sugars) ■ Heat and acetic acid test (for proteins) ○ Performed one at a time ○ Macrovolume of sample is used
REAGENT STRIPS
● Most commonly used ● We perform this by simply dipping the 10 parameter reagent strip in a 10 mL conical tube and in 2 minutes, the reaction and results are available. ● However, we need to observe care and quality control when using reagent strips Care and Quality Control ● Store in tightly closed opaque bottles ● Removed immediately before uses ● Do not refrigerate (room temp below 30°C) ● Run positive and negative controls every 24 hours ● Run controls when a new bottle is opened ● Observe expiration date ● Observe discolored reagent pads Technique ● Thoroughly mix specimens ● Warm refrigerated specimens ● Briefly dip reagent strips ● Blot strip while removing from urine ● Observe manufacturer timing instructions ● Relate chemical with physical and microscopic results pH ● pH ○ The pH of fresh urine does not reach 9. ■ If your reading is like this, suspect that your sample is contaminated or too old. Request another sample. ○ A reading of 9.0 indicates an old specimen that should be recollected; the normal value is 4.5 to 8.0. ■ Acidic to slightly alkaline ■ Random urine pH may range from 4.5 - 8.0. However, if it is a first morning urine, it would be more acidic. It may have a pH of 5.0-6.0. ○ Reagent strip principle: double indicator Clinical significance ● Detection of systemic acid-base disorders ○ For Medical Technologists, this is very important especially for crystal identification. Example, for dumbbell cell crystals, calcium carbonate or calcium oxalate are your options. You check the pH to determine which is which. Another example is a crystal resembling triple phosphate 14 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 ● Specimen needed for quantitative test for protein in urine is 24-hour urine Microalbuminuria ● Microalbumin - used as an index of diabetic nephropathy ○ Also a manifestation of a problem in glomerular filtration ● Requires a different reagent strip capable of testing for only albumin levels below 10 mg/dL ○ We don't use the multistix or chemstrip here ○ Sensitivity: 10 mg/dL ■ If in case lower than 10, it may give a negative result ● Provides early detection of renal disease, particularly in patients with diabetes ● The Multistix PRO 11 reagent strip tests for microalbumin and creatinine, along with all other routine strips, except urobilinogen ○ Except for Micral, there’s not much question about brands in testing particular analyte ● Micral Test Strip ○ Micral Test - Micral is a brand produced by Roche ■ Based on immunochromatography ■ Indicator: ● Gold-labeled antibody ● B-galactosidase ● Chlorophenol red galactoside ■ In doing Micral Testing, we also require to use First Morning urine ■ Conclusion is made if you have ⅔ positive result ● 1st run - negative, 2nd run - negative; if the first 2 run is negative, stop the procedure, it will be interpreted as negative ● If 1st is negative, and 2nd is positive, then we need to run a 3rd test on 3rd day to tell if it is really negative or positive ● Albumin-to-creatinine ratio corrects for hydration in a random sample to provide an estimate of the 24-hour microalbumin level ○ For diagnosis of diabetic nephropathy ALBUMIN EXCRETINON RATE (AER ug/in or mg/24 hrs) Significant values 20-200 ug/min 30-300 mg/24 hrs >3.4 mg/mmol albumin:creatinine ratio ● When you compute for ratio, simply divide concentration of albumin over creatinine ● If it is greater than 3.4, then it is an indication of diabetic nephropathy Bence Jones Proteinuria ● monoclonal immunoglobulin ● low molecular weight (45,000) ○ Cannot be detected by reagent strip ● seen in case of multiple myeloma, lymphoma, soft tissue organ tumor ○ Prerenal type ● Screening Tests
1. Toluene Sulfonic acid precipitation test ■ highly sensitive ■ Can detect even the smallest amount of Bence Jones Protein 2. Solubility Characteristic/Heat Test ● 40 2 60 degrees Celsius = precipitates ● 80- 100 degrees Celsius = dissolves ● upon cooling 60-40 = re precipitates Heat Test for Bence Jones Protein ● Water bath set-up: bunsen burner (now we use hot plate), water in beaker, thermometer (to determine temperature) ● Sample in water bath ○ 1) Increase temperature; As it reach 40C - > Precipitate ○ 2) increase temperature reaching boiling point -> precipitate will disappear ○ 3) turn off flame which decreases temperature turning back to 60-40C ->reappearance of precipitate ■ If it is below 40 - precipitate will disappear ● Albumin cannot exhibit this phenomenon ○ E.g. once egg is boiled, it cannot go back to its original state ○ Albumin will coagulate 3. Electrophoresis ■ To confirm, electrophoresis needs to be done in order to determine the presence of abnormal protein ■ More reliable procedure Glucose ● Principle: Glucose oxidase test (specific for glucose) ● Double sequential enzymatic reaction ○ We are utilizing 2 enzymes: ■ Glucose oxidase ■ Peroxidase ● The renal threshold for glucose is 160 to 180mg/dL Clinical significance ● Diabetes mellitus ● Gestational diabetes (placental hormones blocking the function of insulin) ○ Hormonal disorders and stress block insulin production and actions ● Renal tubular disorders prevent tubular reabsorption of glucose ○ Situation: plasma glucose of 90 mg/dL (normal), but urine test for glucose is positive (1+). ○ This could mean that there is tubular dysfunction. The specific part of the nephron associated with this is the proximal convoluted tubule. 16 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 ■ Part where 100% glucose is reabsorbed ○ Something related with tubular reabsorption, you need to consider Fanconi Syndrome ● Glucosuria due to hyperglycemia ○ Diabetic in origin ○ Non-diabetic in origin ■ pancreatitis, pancreatic cancer ■ Cushing’s syndrome ■ hyperthyroidism ■ Pheochromocytoma Clinitest ● Principle: Reducing substances including glucose and other sugars can reduce copper sulfate (blue-green) to cuprous oxide (orange-red) ● Based on copper reduction ○ The effect of elevated ascorbic acid - false positive ○ Formalin - false positive ■ may behave as a reducing substance ● May be used to test newborn urine for galactose ○ Reagent strip may not be used since it is specific to glucose only. ■ Because the enzyme that we used is glucose oxidase ○ In enzymatic reaction, it is specific because we are following the lock and key principle. The enzyme cannot be active unless we use a specific substrate. ■ That’s why we use a test for reducing sugar which is the Clinitest ● CLINITEST IS NOT A SPECIFIC TEST FOR GLUCOSE ○ Test for all reducing sugars ○ TRUE OR FALSE: Sucrose will produce positive results with Clinitest. ■ False ; sucrose is non-reducing sugar ● High levels will pass through the reaction and go from blue-green to orange-red to blue-green ○ Pass through reaction or pass through phenomenon happens in the moment of an effervescent tablet mixed with urine and water mixture, reaction will start immediately forming blue, green, orange, red and will revert back to blue. ■ So it will result in looking like a negative result. But the truth is the level of sugar is high. ■ This is the pass-through phenomenon therefore we must observe the reaction carefully or we can modify the testing whenever we suspect that there is a pass-through phenomenon which is the two-drops method. ● Carefully observe the reaction ● Copper reduction (reducing sugars) ○ Clinitest - sensitivity 2% or 200 mg/dl ○ pass through ● In a normal situation, we put 5 drops of urine and 10 drops of distilled water in a test tube. The mixture is there, and after that, we will put a reagent tablet. ○ After putting the reagent tablet, the reaction will start. ■ NEGATIVE: blue ■ 1+: blue-green ■ 2+: yellow green ■ 3+: orange ■ 4+: brick red ● In a pass-through phenomenon, alter the procedure by doing the two-drops method. ○ TWO DROPS METHOD: ■ 2 drops of urine, 10 drops of distilled water, then add the reagent tablet. ■ The moment that the reaction turned GREEN , interpret the result as “glucose is greater than 200mg/dL” Clinistix ● Aka multistick or multistix ● Enzymatic Glucose oxidase (glucose) In a case of: ● Clinistix (-) ● Clinitest (+) It means that it is positive for reducing sugars but not for glucose. ○ Because clinistix is a specific test for glucose (but tested negative) but positive in clinitest which is a test for reducing sugars (lactose, galactose, fructose, pentose, etc.) ■ Bial orcinol test: test for pentoses ● Positive result: green or blue ■ Seliwanoff test: test for fructose ● Positive result: red ● In the presence of enzymatic tests for sugars (glucose in particular), when the DOH inspects, they still look if the laboratory has copper sulfate or Benedict’s reagent. ○ Benedict’s reagent is composed of complex chemicals such as sodium citrate, sodium carbonate, and the pentahydrate of copper(II) sulfate. Because in some cases, this old method should still be done. ● Clinistix ○ Melituria (presence of increased urinary sugar due to ○ inherited disorder) ○ Galactosuria (inability to metabolize galactose to glucose ) ○ Lactosuria (seen during pregnancy & lactation) ○ Fructosuria (associated with parenteral feeding) ○ Pentosuria ( associated with ingestion of large amount of fruits) ○ How will you differentiate different types of protein in a given sample? ELECTROPHORESIS ■ Because proteins are charged substances ○ For carbohydrates, what is the separation method that will help us identify different types of sugar in a given sample? CHROMATOGRAPHY Ketones ● Intermediate metabolites of fat: ○ acetoacetic acid - 20% ■ also known as diacetic acid ■ considered as the “parent compound” ○ acetone- 2% ○ β-hydroxybutyric acid- 78% ■ largest fraction ■ Acetone and β-hydroxybutyric acid are products of oxidation and reduction of diacetic acid. ● Principle: Reaction of acetoacetic acid or acetone (with glycerine) with 17 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 Tests ● Reagent strip diazo reaction ● Ictotest (p-nitrobenzene-diazonium-p-toluenesulfonate) ○ Tablet test or table form of reagent used to detect bilirubin in urine ○ If the specimen in dark-colored and with pyridium, this test is performed ○ There is an asbestos mat or pad where the urine sample is placed ■ On top of the urine, place the tablet ■ Add 2 gtts of distilled water ■ Expect that surrounding the tablet, there is a formation of purple color indicating that the test is positive (+) for bilirubin ● Gmelin Test ○ Historical test ■ No longer used ■ Diazo reaction is used instead ○ HNO3 is used ■ Oxidizes bilirubin, bilicyanin, bilitubrin (???) ○ (+) result = “play of colors” ■ There is visible coloration of green, yellow, red ■ In the specimen, on the junction between urine and HNO3 is where the play of colors is seen ● Protect the sample against light ○ If exposed to light, this will decrease because it will break down or be oxidized to different forms such as biliverdin, bilicyanine, and bilirubin PROCESSING OF BILIRUBIN AND UROBILINOGEN ● Bilirubin metabolism ● Urobilinogen and Urobilin ○ Gives pigment to urine and stool ● Unconjugated bilirubin (B1) ○ Bound to albumin ○ Brought to the liver for conjugation ● In the liver, B2 is formed ● B2 will be released in the small intestine ● B2 will be acted upon by bacteria (normal flora) ○ Upon neutralization or breakdown by bacteria, this will form stercobilinogen and urobilin ● If there is obstruction in the common bile duct, there will be no formation of urobilinogen and stercobilinogen ○ The color of stool will appear as clay-colored ○ Urine will be icteric , not because urobilinogen is high, but because of excessive B ■ Other term is jaundice ● Excess bilirubin will regurgitate in the kidney and be eliminated in the urine as it is water-soluble Urobilinogen ● Some of the conjugated bilirubin is converted to urobilinogen in the intestine ● Then it circulates in the blood t the liver and passes through the kidneys ● A small amount of urobilinogen (1 mg/dL) is found in normal urine ● Principle (depends on the manufacturer) ○ Multistix: Ehrlich’s reaction (reagent: p-dimethylaminobenzaldehyde or PDAB) ■ Multistix is more commonly used ■ Note: 0.5 - 1.0 or 0.5 to 1.0 Ehrlich’s Unit/EU is considered the normal level of urobilinogen, anything beyond that means that there is an increase or rapid 19 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
August 27 , 2022 turnover of your b1, which is observed in case of hemolytic anemia or destruction of your red blood cells or RBCs ○ Chemstrip: Diazo reaction (reagent: diazo compound) Clinical significance ● Early detection of liver disease and hemolytic disorders, constipation ○ impaired liver function (b1 is not converted to b2) ○ hemolytic disorders ○ ( - ) bile duct obstruction (negative or significantly decreased Tests ● Ehrlich's Tube Test ● Watson-Schwartz Differentiation Test ○ Used to differentiate urobilinogen with other Ehrlich’s reactive compound, specifically your porphobilinogen WATSON-SCHWARTZ TEST ● This is a qualitative screening test for the detection and differentiation of urobilinogen and porphobilinogen STEP 1 Urine + Ehrlich reagent + Sodium acetate → Pink or red color → Positive test → Urobilinogen or porphobilinogen is present. STEP 2 Test solution (Above) + Chloroform STEP 3 Pink/red layer + Butanol NOTE: ● If pink color develops in the first step, then continue further (because we are unsure if it’s really the Urobilinogen or the Porphobilinogen that makes the specimen red or pink. ● In the second step, id color develops in aqueous layer, then proceed with butanol test. ● Urobilinogen is soluble to Chloroform and Butanol ● Whereas, Porphobilinogen is insoluble to Chloroform and Butanol ● Remember, in Organic Chemistry, if a substance is soluble to an organic solvent, then it will be extracted. ● Since chloroform is denser than water chloroform will settle at the bottom part of the tube, and since Urobilinogen is soluble to Chloroform, it will combine with the chloroform. The same applies with that of Butanol, however, Urobilinogen will be found in the upper part of the tube since Butanol is lighter than water. ● The reverse applies for Porphobilinogen ● If both layers (aqueous and butanol/chloroform layer) turn out red/pink, you will have another round of extraction. ● So you will get the red portion (top (urobilinogen) and bottom (porphobilinogen) part of each tube). ● If after re-extraction or 2nd extraction both layers are still red/pink, it means that the patient’s urobilinogen levels are extremely high Nitrite ● Detects the presence of reductase-producing bacteria that can convert urinary nitrate to nitrite ● Principle: Griess reaction Clinical significance ● Early detection of Urinary Tract Infection ○ A positive nitrite test should be accompanied by a positive leukocyte esterase test ○ May be used to screen specimens for microbiology testing ■ However, its limitation is that it only detects bacteria, other nitrate reducing bacteria not capable of reducing nitrate may not be able to produce a positive result Leukocyte Esterase ● Detects the presence of granulocytic WBCs, including lysed WBCs ● Principle: ○ Diazo reaction ■ the leukocyte esterase reagent strip reaction should be read 2 minutes after urine exposure Clinical significance ● UTI, including with non-reductase-containing bacteria and parasitic and fungal organisms that would have a negative nitrite tests ● Note: Leukocyte Esterase test is a better indicator of infection than Nitrite ● But for bacteriuria, nitrite is better SUMMARY OF REAGENT STRIP PRINCIPLE Test Principles pH Double-indicator system (methyl red and bromthymol blue) Protein Protein error of indicators Glucose Double sequential enzyme reaction (glucose oxidase) Ketones Sodium nitroprusside reaction Blood Pseudoperoxidase activity of hemoglobin Bilirubin Diazo reaction Urobilinogen Ehrlich’s aldehyde reaction Nitrite Greiss reaction Leukocyte Esterase Leukocyte esterase catalyze the hydrolysis of an acid ester on reagent pad to produce an aromatic compound and acid (also Diazo reaction) Specific Gravity Change in pKa (dissociation constant) of a polyelectrolyte in an alkaline medium
PHENYLKETONURIA
20 THIS REVIEWER IS SOLELY FOR THE USE OF BATCH 2022 STUDENTS. ALL CONTENTS OF THIS FILE SHALL ONLY SERVE AS A GUIDE FOR STUDYING AFOREMENTIONED TOPIC AND THEREFORE, SHALL NOT BE VALID AS EVIDENCE TO JUSTIFY ANY CORRECTIONS IN EXAMINATIONS. NO PART OF THIS MATERIAL SHALL BE REPRODUCED, SOLD, NOR TRANSLATED IN ANY FORM OR BY MEANS, INCLUDING PHOTOCOPYING, SCANNING,
