Clinical Microscopy PDF - 2022

CLINICAL MICROSCOPY August 27, 2022 CORRELATE RESULTS 22 CONTENTS...

CLINICAL MICROSCOPY August 27, 2022 CORRELATE RESULTS 22 CONTENTS URINALYSIS MICROSCOPIC TECHNIQUES 22 TWO TYPES OF URINARY SEDIMENTS 23 RENAL PHYSIOLOGY 2 REPORTING 23 INTRODUCTION TO URINALYSIS 2 STAINS AND SOLUTIONS 23 URINE FORMATION 2 CASTS 23 GLOMERULAR FILTRATION 3 RED BLOOD CELLS 24 TUBULAR REABSORPTION 3 WHITE BLOOD CELLS 24 TUBULAR SECRETION 3 EPITHELIAL CELLS 25 RENAL TUBULAR SECRETION 3 BACTERIA 26 RENAL FUNCTION TESTS 3 YEAST 26 GLOMERULAR FILTRATION RATE (GFR) 3 PARASITES 26 BETA-2-MICROGLOBULIN 4 MUCUS THREADS 26 RADIONUCLEOTIDES 4 CRYSTALS 27 ROUTINE URINALYSIS 5 RECALL QUESTIONS 29 TYPES OF COMMON URINE SPECIMENS 5 AUTOMATION IN URINALYSIS LABORATORY 29 CHANGES IN UNPRESERVED URINE 6 SYSMEX UX-2000 FULLY AUTOMATED 29 PHYSICAL EXAMINATION OF URINE 7 INTEGRATED URINE ANALYZER 29 URINE VOLUME 7 Specifications 29 URINE COLOR 7 SUMMARY OF RENAL DISEASES 30 URINE CLARITY 8 SPECIFIC GRAVITY 8 LABORATORY SAFETY 30 SPECIFIC GRAVITY TEST 8 FIRE 30 ODOR 9 SPECIFIC SAFETY MEASURES URINALYSIS 31 RECALL 9 GUIDELINES ON SPECIMEN DISPOSAL (AUBF) 31 CHEMICAL EXAMINATION OF URINE 10 REAGENT STRIPS 10 RENAL PHYSIOLOGY RECALL QUESTIONS 13 Cc CHEMICAL EXAMINATION OF URINE 14 INTRODUCTION TO URINALYSIS REAGENT STRIPS 14 KIDNEY PHENYLKETONURIA 21 Major organ of the excretory system MELANURIA 21 ○ One pair (1) of kidneys BRANCHED CHAIN AMINO ACIDURIA 22 ○ Bean-shaped organ located around the third and fourth lumbar area of your vertebrae DIAPER SYNDROMES 22 ○ Major function is to dispose waste products of metabolism ○ Regulates plasma-water volume of the body for water MICROSCOPIC EXAMINATION OF URINE 22 reabsorption and excretion of waste. MANUAL MICROSCOPIC EXAMINATION 22 SPECIMEN PREPARATION 22 FUNCTIONS OF THE KIDNEY Examine fresh or adequately 22 1. Removal of waste products preserved specimens (10-15mL) 22 2. Regulation of plasma, water volume 3. Maintain normal acid-base balance CENTRIFUGATION 22 4. Regulation of blood pressure SEDIMENT PREPARATION 22 5. Endocrine function EXAMINATION OF SEDIMENT 22 REPORTING 22 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 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 Controls the ability of the kidney to clear waste products and NOTE: Specific gravity may predetermine if the urine is maintain the body’s water and electrolyte balance. hyposthenuric, isosthenuric or hypersthenuric. ○ Each kidney is composed of about 1 to 1.5 millions of ○ After filtration, there are only about 120 to 130 mL of nephrons which are capable of urine formation. ultrafiltrate that will transverse in the proximal convoluted Types of nephrons tubule where tubular reabsorption will occur afterwards. Cortical nephrons - are located in the cortex of the ○ Substances that are reabsorbed are the essential ones kidney and remove waste products and reabsorb which include: nutrients 100% of Glucose (major threshold substance) ○ Main function is removal of waste products as well as NOTE: Urine should always, AT ALL TIMES, be negative reabsorption of nutrients for albumin or protein. ○ 80-85% of nephrons are cortical in nature. Healthy urine is negative for protein and glucose as Juxtamedullary nephrons - extend into the medulla of proteins are not allowed to pass through the glomerulus the kidney and concentrate in the urine (molecular size) and glucose is reabsorbed fully at 100%. ○ Responsible for reabsorption of water it’s not normal that urine turns positive in the presence of ○ Urine that is concentrated would only mean that it has a protein, which may mean glomerular; it should not also turn high amount of solutes and less amount of water. positive for glucose, since your not diabetic” In cases of DM, wherein the glucose level is high, there may URINE FORMATION be presence of glucose in the urine because the proximal Renal blood flow convoluted tubule (PCT) has a threshold for glucose (160-180 ○ Renal artery → afferent arteriole → efferent arteriole → mg/dL) proximal convoluted tubule capillaries → vasa recta/loop of Henle → distal convoluted tubule capillaries → renal TUBULAR REABSORPTION vein Amino acids, electrolytes may also be reabsorbed in the Normal renal blood flow is approximately 1200 mL/min PCT Normal plasma flow is approximately 600 to 700 mL/min After passing through the PCT, the ultrafiltrate will now ○ Half of the renal blood flow, since plasma is half of the transverse to the descending loop of Henle (limb of Henle) components of whole blood ○ Responsible for holding or reabsorbing water ○ Has a longer Loop of Henle which is in-charge of ○ 80% of water (the majority of water) are being concentrating the urine. reabsorbed here Ascending loop of Henle (limb of Henle) URINE FORMATION 3-STEPS ○ 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 GLOMERULAR FILTRATION Urine will be the final product, and will be stored in a bag-like Glomerular Tuft – filtering apparatus featuring eight (8) lobes structure called the urinary bladder. When the bladder is full, it of capillaries that are responsible for filtration (glomerular) sends signals to the brain for the need to urinate From renal artery → afferent arteriole : Non-selective filtration will occur as long as the substance is less than 70,000 kD, it GLOMERULAR FILTRATION can pass through. (amino acids, glucose, electrolytes, water) Capillaries drop off particles in the blood the body needs to 25% of total cardiac output can enter per minute - 1200mL get rid of ○ 25% of total cardiac output = 25% of total blood volume ○ There are about 8 tufts in the glomerulus Total blood volume of an average healthy individual: 5-6 Liters Product of filtration: Ultrafiltrate Nonselective filtration of plasma substances with MWs less Ultrafiltrate: expected specific gravity is 1.010 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 ○ How come albumin is not allowed to pass although it is hydrogen molecules, producing bicarbonate that can be less than 70,000 kD: the net electric charge (albumin secreted back into the blood being negatively charged and the membrane is the The kidneys are important in maintaining acid-base balance same, thus, it will repel) particularly the tubular secretion function ○ Defect in the glomerulus allow albumin to pass through Driven by blood pressure (filtration pressure) RENAL FUNCTION TESTS ○ Since it is nonselective, filtration is affected by several We can consider urinalysis to be the first step/initial test to gradients (size/pressure) assess the kidneys Produces 180 L of fluid every 24 hours ○ If the urinalysis results are significant or has flags, then we Particles filter into the glomerulus capsule (mostly water) can perform more specific tests to find out what the problem is ○ Composition is very close to tissue fluid, with a specific gravity of 1.010 GLOMERULAR FILTRATION RATE (GFR) Normal value: approximately 120mL/min TUBULAR REABSORPTION Classic test: Creatinine clearance test (usually 24 hours) Occurs mostly in the proximal tubule (PCT) ○ Sex-specific: males (higher) and females (lower) have Transports items out of the tubular fluid (urine) back into the different creatinine values blood (peritubular capillary) ○ What is the earliest or first glomerular filtration test in ○ What the body still needs (what are reabsorbed) assessing GFR? Glucose, amino acids, water, urea (about 50% of it), Urea test is the first glomerular filtration test however it is not performed because of its flaws. and uric acid The requirements in choosing markers for GFR 65% of filtrate reabsorbed here 1. Substance must freely pass the glomerulus Reabsorption of sodium and potassium 2. It must be not reabsorbed nor secreted **Renal tubular concentration ○ Urea are also reabsorbed by the body so ○ Takes place in the ascending and descending loops of determining the amount of urea in the blood and Henle urine might be a problem since we won’t know if it ○ The expected specific gravity in concentrated urine will be has increased or decreased due to being reabsorbed. high ○ Creatinine on the other hand is not reabsorbed so that is why Urea Test is not being used TUBULAR SECRETION Inulin clearance test is considered as the gold standard for Late filtering process this glomerular filtration rate. Inulin is totally and completely ○ There are some substances that are considered as wastes excreted. It is an exogenous compound that is administered but are unable to be filtered by the glomerulus due to their to the patient at a constant rate. In 24 hours, that inulin must size or due to being plasma-bound be completely excreted. If not 100% excreted, the glomerulus cannot do its function. Although it is an old standard, it is not Transports items out of the blood (peritubular capillaries) into practical so that is why we do other testing and one of which the renal tubules is Cystatin C ○ Processing of potassium, water, and some uric acid and GFR can be categorized into exogenous and endogenous urea ○ If substance is normally produced by our body, these ○ Removes non filtered waste products from the blood to methods are considered endogenous the filtrate and maintains the acid-base balance in the ○ Inulin is exogenous because it is a foreign substance introduced by our body Formula: C=UV/P ○ Substances such as medication are bound to plasma ○ U = Concentration of creatinine in urine carrier proteins and are too large to be filtered ○ P = concentration of creatinine in plasma ○ V = volume of urine in ml per minute RENAL TUBULAR SECRETION ○ We assume that the patient has a normal body surface In the tubules they disassociate from the carrier protein and area so we multiply it by 1 then secreted into the filtrate ○ Usual problem here is the volume is given in Liters per 24 hours so you need to convert Liters per 24 hours to Small hydrogen molecules are easily filtered and must be ml/minute returned to the blood. In the filtrate, they combine with ○ Formulas may use serum creatinine and combinations of phosphate ions or ammonia secreted by the renal tubular age, sex, ethnicity, blood urea nitrogen (BUN) and serum cells and are secreted back to the blood albumin Small bicarbonate ions needed for the acid-base buffering Measurements of serum marker system are also easily filtered. They combine with the ○ Cystatin C is a small molecule produced at a constant rate by all nucleated cells 3 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 ○ It is completely filtered, reabsorbed and then broken down *effective renal plasma flow : 600-700 ml/min by the renal tubular cells. Serum level remains constant average blood flow: 1200ml/min unless the GFR decreases causing the serum level to rise. Initially, once the PAH Test is introduced to the body, it ○ *may not be applicable pediatrics, diabetics, geriatrics, must not pass the glomerulus. It must be excreted and critically sick patients wherein the kidneys have through secretion in the peritubular capillaries problems ○ **independent of muscle mass REGULATION OF BLOOD PRESSURE 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 If the individual has low blood pressure, then it must be 125I-iothalamate corrected. In that case, once blood pressure decreases, **Plasma clearance of the radioactive materials then there will be secretion of renin. This will react to a Used to monitor the viability or success of kidney transplant blood-borne substance, your angiotensinogen which is Radioactive products will be introduced and if the products inactive. The angiotensinogen will be activated in the are not recovered, the kidney transplant is not successful. presence of renin, converting it to Angiotensin 1. In the Renal tubular reabsorption lungs, the ACE or angiotensin converting enzyme will ○ Primary tests are serum and urine osmolality convert angiotensin 1 to angiotensin 2. serum osmolality: 275-300 mOsm urine osmolality: Angiotensin 2 will dilate afferent arterioles by 50-1400 mOsm vasodilation and this will make the afferent arterioles ○ The free water clearance test measures the ability of the receive more blood kidney to respond to the body hydration Efferent arterioles will be constricted, making the Fish bird method is an old method wherein the patient blood pressure high will be challenged to not take fluid for 24 hours and Aldosterone will become activated and it will promote collect the urine after it. If the urine has low specific reabsorption of salt, specifically sodium, making the gravity, it is not normal and it means that the urine has blood pressure high. no concentrating ability This process is a negative feedback mechanism If the body is dehydrated, the kidney will reabsorb water and result in low urine output and high specific ROUTINE URINALYSIS gravity which means that the tubules has no capability to reabsorption of water Tubular secretion tests URINALYSIS ○ 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 Routine analysis comprises of: cleared as blood passes through peritubular capillaries ○ 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 ○ Chemical examination Note: When a routine urinalysis and a culture are both ○ Microscopic examination ordered, perform the culture first It is important because it aids in the diagnosis of disease, not This is done to patients who cannot void voluntarily limited to kidney diseases like diabetes. It can be used as bacterial culture if the sample is not It is also used as a screen for asymptomatic populated obtained from the polycat bag. undetected disorders. ○ If sample is collected in the bag and the urine was These tests can screen symptomatic or asymptomatic standing for 2 hours, there will be an increase of bacteria, patients which may produce false positive results It can monitor progress of disease and effectiveness of Culture must be prioritized to prevent further contamination therapy It is rapid, reliable, accurate (if well trained), collection of 24 HOUR / 12 HOUR (TIMED) sample is not invasive, safe 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 TYPES OF COMMON URINE SPECIMENS and prior to the start of the time, there is already 2ml of Random urine so we have to discard it first First Morning Collects all urine during the designated period Clean-Catch Voids and collects urine at the end of the period Catheterized Specimens can provide quantitative results 24Hour (Timed) Usedsed to measure hcg titer Drug Screening Container must hold up to 3L, sometimes colored container Three-glass collection because some substances are photosensitive Suprapubic Aspiration In history, 12 hours timed urine is used in addis count which Pediatric Specimen will quantify the following: ○ WBC RANDOM ○ RBC Routine screening ○ Epithelial cells May require confirmatory testing based on diet and exercise ○ Casts You can collect the sample anytime of the day without specific instruction. DRUG SCREENING May confirm obvious pathology Strictly follow chain-of-custody (COC) form requirements ○ Expected to be diluted but the same substance are ○ The sample must be safe and secured from collection up elevated. to testing No tampering, substitution, spiking, and or dilution FIRST MORNING Temperature: 32.5-37.7 (within 4 minutes) The label itself is capable of measuring temperature Collected immediately on arising Sample is collected while being witnessed by an accredited Routine screening/confirmatory testing drug analyst Orthostatic proteinuria In the US, the container is capable of measuring the ○ If protein is traced to 1+ in the late afternoon, collection temperature must be repeated and sample must be first morning ○ The small portion below the label is colorless at room If the sample is positive, then it is true proteinuria temperature If negative, it is orthostatic ○ Once urine is added, the small portion turns brown, which Pregnancy tests means it is warm. If there is no change in color, the urine is cold and the CLEAN-CATCH incident must be report Requires patient to cleanse the genital area ○ If the patient cannot void urine, tell them to drink at least Void first into the toilet, then collect specimen and finish 500 mL of water. voiding into the toilet Bacterial cultures 3-GLASS COLLECTION The process is the same with midstream catch but additional Used to diagnose prostatic infection, to differentiate prostatitis cleansing with soap and water of the genitalia must be done from genitourinary tract infection (astitis/urethritis) Bacteriuria will be reported if the colony forming units are For male patients greater than 100,000. Used in the diagnosis of prostatic infection ○ If the specimen is suprapubic aspirated urine, the 100,000 1st vial - first fraction of urine cfu will not be applicable because the aspirated urine 2nd vial - midstream part of urine sample is sterile. So if the colonies are less than 100,000, 3rd vial - prostatic fluid with remaining urine volume (the it should still be considered as significant urologist massage the prostate) ○ Digital Prostate Massage - The index and middle finger is CATHETERIZED inserted in the anal region of the male and massage the Collected from a catheter passed into the bladder prostate (right plump) Bacterial Cultures 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 ○ Inorganic - major component is chloride SPA (SUPRAPUBIC ASPIRATION) Anaerobic urine culture CHEMICAL PRESERVATIVES (REFRIGERATOR/LOSING ○ Direct puncturing of the bladder DRY ICE) Bladder cytology/cytologic examination Chemical Advantage Disadvantage Critical quantitative bacteriology Preservatives Thymol Glucose, Acid precipitation PEDIATRIC SAMPLE sediments protein Urine bag “Wee Bag” or pediatric bag Boric acid Formed elements Acidic pH of urine, ○ The plastic container has an adhesive that is attached to For preserving interferes with the genital of the infant to collect urine sample urine formalin drugs and ○ Prone to contamination culture hormones testing Formalin Excellent/Unebeat Reducing agent PRESERVATION OF URINE SPECIMENS ble preservative for (Formalin will Test specimens within 2 hours of collection formed elements cause a false Refrigerate specimens that cannot be tested within 2 hours (Ex: RBC, WBC, increase if used in casts) reduction tests, CHANGES IN UNPRESERVED URINE such as Fehling’s False increase tests, as it will ○ Sp. Gravity participate in the ○ pH reduction ) ○ Urobilin Toluene Not interfere with Floats on surface ○ Nitrite chemical tests of urine ○ Bacteria Cannot prevent ○ Odor bacterial growth False Decrease Sodium fluoride Prevent glycolysis, Inhibit enzyme ○ Glucose Quantitative action (Glucose Decreases because of glycolysis and some of bacteria glycolysis, oxidase test cannot utilized glucose as energy source Drug analyses be performed as ○ Clarity the enzymatic Decreases because some of the crystals will form and reaction will be bacteria cells will increase which will result to a hazy inhibited, use result Orthotolidine test ○ Urobilinogen instead or an Oxidized to urobilin, therefore decreases in alternative concentration preservative such ○ Bilirubin as sodium Oxidized to other compound benzoate) ○ Ketone bodies Phenol Bactericidal Change in urine Evaporate/Volatilized color ○ Trichomonas BD Urine Urine C&S Volume specific Die and if it becomes no longer motile, it can no longer Collection kit be identified in urinary sediments. Can only be mistaken Yellow UA plus Automated Must be for WBC/greater cells/macrophages tube Urinalysis refrigerated within ○ Casts 2 hours Disintegrated If urine has high pH (alkaline) and has low specific gravity, these casts are easily disintegrated even if 10% formalin is added. Specially the RBC PHYSICAL EXAMINATION OF URINE Bacteria increase = pH increase = nitrite increase = specific gravity increase URINE VOLUME ○ There are bacteria that produces urates and nitrate Normal: 600-2000 mL/day ○ Bacteria is the cause of the elevation of pH, odor, and Oliguria: Decreased output, less than 400 mL/day nitrite ○ Can be physiologic or pathologic ○ Only urobilin is not caused by bacteria, this is produced by Ex1: water restriction for a day = physiologic oxidation of urobilinogen. Urobilinogen is oxidized to Ex2: Renal tumor, cancer, disease = pathologic urobilin Anuria: No urine output Urine is composed of urea, in the presence of urease, will ○ Prefix “a-” means without breakdown urea to ammonia, therefore, ammonia is ○ Not completely walang wala, less than 100mL/day can responsible for the increase in pH and change in odor already be considered anuria ○ Organic - major component is urea 6 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 ○ Usually observed when there is a complete obstruction abnormal color Speckled tests for blood, (renal stones, toxic agent) of urine reaction pad in microscopic RBCs Nocturia: Increased urine output at night Abnormal the reagent strip Non-hemolyzed ○ Exceeding 500mL/night is already considered nocturia pathologic blood ○ Physiologically observed among pregnant women ○ bleeding Clear urine, ○ Observed among elder male patients with enlarged Abnormal positive test for prostate non-pathologic Hemoglobin blood ○ Babies do not suffer from nocturia (it is their diet) ○ medication or Hemolyzed blood Polyuria: Increased urine output greater than 2.5L/day consumption Clear red ○ Diabetes mellitus: increased urine output to excrete of red beans Clear urine, excess in urine glucose (way of kidney to eliminate positive test for glucose) blood, need further ○ Diabetes insipidus: increased urine output caused by Myoglobin testing lack of dysfunction/absence of antidiuretic hormone (ADH Excessive Intravascular - hormone needed to concentrate urine in nephrons) myoglobin is hemolysis - plasma ○ Results in polydipsia (excessive thirst) since body is losing toxic to the will appear color too much water kidney and can pink or red Diabetes Mellitus be lethal or fatal Myoglobinuria - ○ Polydipsia (3Ps) (ex. hazing) plasma will appear ○ Polyuria clear yellow ○ Polyphagia (excessive eating) Clear red ○ High Sp. Grav. Negative tests for ○ Low insulin/ No insulin blood, needs ○ Hyperglycemia further testing ○ Glucosuria Described as port Diabetes insipidus wine red or ○ Polydipsia Porphyrins purplish red color ○ Polyuria (2Ps unlike DM with 3Ps) (observed in cases ○ Low sp. Gravity (as low as 1.003) of porphyrinuria, or ○ Decreased/ function ADH defects with ○ No hyperglycemia hemoglobin ○ No glucosuria synthesis) Oxidized RBCs, Clear urine, URINE COLOR denatured Hgb positive test for Normal urine is yellow. blood ○ Normal urine may range from straw to amber Clear urine, ○ Pigments responsible for yellow color: urochrome, urobilin, Melanin darkens on and uroerythrin standing Shades of yellow are based on fluid consumption and vary From red to brown from pale yellow (dilute) to dark yellow (concentrated) in use of acid hematin test URINE COLOR (acidification) Color Cause Correlation From dark brown Bilirubin (B2 form) to black upon Black Methemoglobin Bilirubin 1 is exposure to light or Produces yellow air due to melanin bound to foam when (melanogen albumin and shaken, abnormal converted to cannot pass liver function melanin upon through the Orange glomerulus exposure to light) Pyridium In cases of Interferes with Produces thick alkaptonuria, dark reagent strip - all orange pigment Homogentisic brown will turn colors are errors, that can interfere acid black upon so every test with reagent strip standing or upon must be done tests alkalinization manually Other tests to differentiate hemoglobin from myoglobin – the Red use of ammonium sulfate, which precipitates hemoglobin, Cloudy or smoky but not myoglobin, then repeat test. If the test is negative, it Most commonly RBCs red urine, positive observed 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 URINE CLARITY ○ Its based on the number of ionized particles, more Terminology: Clear, hazy, cloudy, turbid, milky specific, not affected by particle size Freshly voided normal urine is clear ○ Unlike SG which is a reflection of the dissolved substance Refrigerated normal urine based on size and amount ○ If high density, high SG ○ White Turbidity in urine with an alkaline pH from amorphous phosphates and carbonates SPECIFIC GRAVITY TEST ○ Pink turbidity in urine with an acid pH from amorphous urates REAGENT STRIP Because of urates bind to the pigment uroerythrin, and Most commonly used method forms pink sediments when accumulated Primary test for routine urinalysis is the reagent strip test Adjust transparency based on microscopic findings Principle is based on the number of hydrogen ions (H+) released from a polyelectrolyte (pKa) is proportional to the NON-PATHOLOGIC TURBIDITY number of ions in the urine Squamous epithelial cells Increased urine concentration = increased H+ released = low ○ Poor collection, not midstream, uncircumcised pH Mucus Tye indicator on the strip is bromothymol blue Reaction = Yellow-Green (acid) → Green-Blue (alkaline) Amorphous phosphates, carbonates, and urates Not applicable if there is thyridium (use refractometer) Semen Increment is by 0.5 Feces Radiographic Contrast Media REFRACTOMETER Powder and Creams Principle: The concentration of dissolved particles in a ○ e.g. gloves powder (gloves powder not recommended to solution determines the velocity and angle of light passing use in laboratory) through a solution The refractometer uses a prism to direct a wavelength of light PATHOLOGIC TURBIDITY that can be read on a scale calibrated with distilled water Red Blood Cell (RBC’s) (1.000) ○ About 500 cells/cumm can cause haziness Standardizing Solutions: ○ Distilled water: 1.000 White Blood Cell (WBC’s) Readily available ○ About 200 cells/cumm can cause haziness ○ 5% NaCl: 1.022 +/- 0.001 Yeast ○ 9% Sucrose: 1.034 +/- 0.001 Urothelial and Renal Tubular Epithelial Cells Each line is equivalent/ to increments of 0.01 Abnormal Crystals Lipids (Milky) OSMOLARITY ○ Filarial worms considered more representative of renal concentrating ability ○ Long bone fracture than specific gravity because it measures only the number of Bacteria particles and their size is not relevant Measurement is the number of particles into which 1g ○ Causes uniform turbidity molecular weight of a substance dissociates ○ If bacteria is high, sample may remain turbid even after ○ Ex. Non ionizing urea (mol. weight [MW] 60) = 1 particle, centrifugation ionizing NaCl (MW 58.5) = 2 particles ○ Spinning can reduce bacteria but can not achieve clarity ○ Each specific particle is accounted ○ Adsorbent can be used to remove bacteria Reported in milliosmoles (mOsm) Colligative properties measured in the clinical laboratory ○ Freezing point depression SPECIFIC GRAVITY One mole of a non ionizing substance will lower the Screening test for renal tubular reabsorption of essential freezing point 1.86 °C elements filtered by the glomerulus Volatile substances such as alcohol can interfere Based on the fact that the glomerular filtrate has a specific ○ Vapor pressure depression gravity of 1.010 (isosthenuria) Actual measurement is the dew point of the urine >1.010 hypersthenuria sample darkening of urine after being exposed SPECIMEN PREPARATION to air Examine fresh or adequately Increased urine melanin indicates over proliferation of preserved specimens (10-15mL) melanocytes ↓ Melanin reacts with ferric chloride & produces gray/black precipitate CENTRIFUGATION Sodium nitroprusside test provides additional screening test 5 minutes @ 400 RCF to for melanin & produces red color produce optimum amount of ○ Not only reacts with ketones but also reacts with melanin, Sediment. But in our lab, we only use RPM (1,800-2,000) for addition of glycerine will make it more specific in ketone 10-12 mins. After that, we decant it 90° and resuspend it by bodies. tapping against the palm 21 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 ↓ SEDIMENT PREPARATION 0.5 and 1.0mL of sediments are frequently used ↓ EXAMINATION OF SEDIMENT Minimum of 10 fields under both low and high power, under reduced light (unstained) ↓ REPORTING Casts = average number per lpf RBC and WBC = average number per hpf EC, crystals, etc. = semiquantitative ↓ CORRELATE RESULTS URINALYSIS MICROSCOPIC TECHNIQUES Technique Function Bright-field Microscopy Used for routine urinalysis Dark-field Microscopy Aid is identification motility of spirochetes (Treponema pallidum) Fluorescence Microscopy Visualization of naturally fluorescent microorganisms or by adding stain Polarizing Microscopy Used in identification of crystals , cholesterol in oval fat, and fatty casts Interference-Contrast Three dimensional Microscopy microscopy image ( specimen with little or no contrast when General term for the structures found in the middle viewed (unstained sample) ○ Organized/Formed elements (CREBPAYS) Phase-Contrast Microscopy Visualization of elements with Casts low refractive indices (hyaline Red blood cells cast, Thrichomonas , Epithelial cells Dysmorphic Bacteria RBC/Acanthocytes) Pus cells Animal parasites Yeast cells Spermatozoa GROUPING OF DIFFERENT CELL CASTS AND CRYSTALS General term for crystals (QUICK GUIDE) ○ Unorganized TWO TYPES OF URINARY SEDIMENTS Unorganized ○ due to a change in reaction or concentration (chemical part); ○ examples are crystals and amorphous materials Organized ○ most important (biological part); ○ include casts, rbc, pus cells, epithelial cells, microorganisms, animal parasites, spermatozoa, yeast, and fungi REPORTING Casts 22 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 ○ specify kind per coverslip (total count in 10-20 lpf) In case of renal transplant rejection, there is so much ○ range/lpf exfoliation of renal tubular epithelial cells, then we’ll have RTE, rbc, and pus cells epithelial cell casts. ○ range/hpf Name of the casts depends on what it contains Epithelial cells, microorganism, crystals Formed in the distal convoluted tubule and collecting ducts ○ under hpo (specify kind and report in plus sign): Reported as the number per low-power field rare or occasional (+/-) few (+) CASTS moderate (++) many (+++) Plus system for quantifying urinary sediment: ○ occasional (less than 10) ○ + (11-15) ○ ++ (16-20) ○ +++ (more than 20) Different hospitals may have a different protocol here in the Philippines. Urinalysis reporting is not standardized. STAINS AND SOLUTIONS USEFUL STAINS AND SOLUTIONS IN MICROSCOPIC EXAMINATION OF URINARY SEDIMENTS Stain/Solution Function Sternheimer-Malbin Differentiates pus cells (pink) from glitter cells (pale blue) Stains epithelial cells and casts 0.5% Toluidine Blue Differentiates WBC from RTE 2% acetic acid Differentiates RBC from WBC, yeast Oil Red O, Sudan III, Sudan IV Stains Triglycerides, neutral fats but not cholesterol For clinical microscopy and histopath Gram Stain Differentiate G(+) from G(-) and useful to identify bacterial cast Hansel Stain Stain for urinary eosinophils Prussian Blue Stain iron containing structures ( granular casts if positive for hemosiderin) In what part of the nephrons do casts form or develop? CASTS ○ Ascending loop of henle (impermeable with water) Composed of Tamm-Horsfall protein (aka uromodulin) towards distal convoluted tubule and in the connecting excreted by renal tubular epithelial cells duct If there is a urinary cast, no other part of the excretory system Casts are cylindrical because it assumes the shape of tubule is involved in its formation except tubules, that's why it is ○ Habang lumalaki or nagiging wide yung cast, it is an significant. indication that the tubules are also inflamed When other urinary constituents are present they become ○ RBC Casts- Glomerulonephritis enmeshed in the cast matrix or attached to the matrix. But if it ○ Broad casts- bad prognosis due to inflammation of is a protein matrix alone without enmeshed structure or tubules. constituent, we call that cast as hyaline. End stage renal disease In patients suffering from glomerular bleeding, red cells may ○ Waxy casts- final degenerative form of casts be enmeshed in the protein matrix, then RBC casts will be Granular to waxy formed. Waxy to degeneration (disintegrated) In patients suffering from pyelonephritis, there could be too ○ Epithelial cell cast- Renal transplant and toxic poisoning much WBC. These WBCs can be enmeshed with the protein ○ Hyaline cast- most common and this is the only cast with matrix, and then we’ll have pus casts. normal bodies having a 0-2 higher in casts per low power In case of sepsis (bacteremia) wherein bacteria in the blood is field is considered to be normal enmeshed with protein matrix, then we’ll have bacterial cast. 23 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 Implication: patient may be dehydrated or engaged in Sources of Error strenuous activity Renal tubular epithelial cells, mononuclear lymphocytes and monocytes RED BLOOD CELLS Urinary Correlation Leukocyte esterase, nitrite, pH, and specific gravity Appearance Small, nonnucleated discs Clinical Significance Appear crenated in concentrated urine Urinary tract infection (neutrophils) Appear as larger empty cells in dilute urine (ghost cells) Drug induced interstitial nephritis (eosinophils) Regular (dysmorphic) shapes indicate glomerular bleeding Malignancy (mononuclear cells) Good indicator of infection Sources of Error If positive with nitrite, then it is confirmed as bacterial infection Oil droplets Air bubbles EPITHELIAL CELLS Yeast cells Squamous Cells Clinical Significance Frame of reference; look first for squamous epithelial cells Glomerular membrane damage under the microscope Bleeding in the urinary tract Largest cells in the urine sediment Renal calculi Represent normal sloughing of old lower genitourinary tract Malignancy cells Folded squamous cells may resemble urinary casts Urinary Correlation Clue cells are squamous epithelial cells covered with A clear red urine with a positive reagent strip RBC and no Gardnerella vaginalis bacteria, indicating a vaginal infection RBCs in the microscopic analysis indicates hemoglobinuria or ○ Clue cells is covered with gram negative short bacilli myoglobinuria. ○ Bubble cells are renal tubular epithelial cells that Don’t be afraid to report RBC in urine kahit negative ang strip absorbed non-lipid substance test for as long as the Red Cell that you will report will not be ○ Oval fat bodies are renal tubular epithelial cells that greater than 10. absorbed lipid or fats Transitional (Urothelial) Cells Found in the renal pelvis, ureters, bladder, and male urethra WHITE BLOOD CELLS ○ This are your bladder cells ○ Donut-like (round) Appearance Normally seen after catheterization procedures Three different forms: Spherical, caudate (pointed) , and Large than RBCs and contain a nucleus polyhedral (can be seen as clusters) Neutrophils have multilobed nuclei and granules ○ Bulging Eosinophils have red granules when stained with Wright or ○ Not flat Hansel stains ○ Eccentric nuclues Glitter cells are neutrophils that have swollen in dilute urine, Spherical cells resemble renal tubular cells, except they have resulting in Brownian movement of the granules in the centrally located nucleus cytoplasm. Sometimes mistaken as RTE in the lab because Increased transitional cells may indicate malignancy glitter cells are the degenerative form of WBC ○ Over proliferation 24 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 Compared to the WBC, you can see the nucleus really good, but you can only appreciate the grand? Unlike in the RTEs you can see the nucleus and the nucleoli Renal Tubular Epithelial Cells and it is called the nucleus is still open? Found in the renal tubes and collecting duct Cell shape varies with location Convoluted tubule cells are rectangular with coarse granules BACTERIA and may be resemble a cast Small spheres (cocci) and rod-shaped organisms Distal convoluted tubule cells are small and round, may Should be accompanied by WBCs resemble spherical transitional cells but have an eccentric Sources or error: Amorphous urates, phosphates, and old nucleus specimens with a high pH ○ The nucleus is open meaning nucleoli can be seen Clinical significance: urinary tract infection Cells from the collecting duct are cuboidal with at least one Reporting: descriptive or semi-quantitative straight edge and are frequently seen in clumps More than two renal tubular epithelial cells per high power field is significant Normal value: 0-2/hpf Clinical significance: ○ Tubular necrosis, often from poisoning or viral infections Absorb filtrate and may be bilirubin stained (liver damage), contain hemosiderin granules or lipids. ○ If it will absorbed lipids then it will become oval fat bodies ○ Non-lipid is bubble cells Oval Fat Bodies Renal Tubular epithelial cells that have absorbed lipids Highly refractile Seen in conjunction with free-floating lipids Confirm by staining with Oil red O, Sudan II or polarized microscopy Clinical significance: Nephrotic syndrome, diabetes YEAST mellitus, and crush injuries Oval structures with buds or mycelia Under polarizing microscope it will appear maltese cross Should be accompanied by WBCs appearance ○ Most common in the lab is the budding yeast ○ Lack of WBC = Candidiasis/yeast infection Associated with acidic urine from patients with diabetes mellitus Sources of error: ○ RBCs ○ To differentiate RBCs from Yeast are the following Shape stain with eosin dissolve with 0.1 NHCl formation Yeast RBC Ovoid & double cell wall Round/biconcave 25 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, RECORDING, RE-POSTING, WITHOUT OBTAINING WRITTEN PERMISSION FROM THE RESOURCE PERSON.  CLINICAL MICROSCOPY August 27, 2022 No central pallor With central pallor ○ May also be confused with WBC cast when mucus Unstained Can be stained threads trapped other structures (refer to the picture Can not be lysed Lysed by 0.1 NHCL below) Budding Rouleauex (pile of point) Clinical significance: ○ Diabetes mellitus ○ Immunocompromised patients ○ Vaginal infections ○ Chemotherapy ○ Antibiotic therapy CRYSTALS Precipitation of urine solutes affected by temperature, solute concentration, and pH + as well as medication Crystals are more abundant in refrigerated urine samples Polarized microscopy aids in their identification Abnormal crystals are found only in acidic or normal urine NORMAL CRYSTALS IN ACIDIC URINE Uric Acid Crystals Appearance ○ Pleomorphic Has many shapes and forms flat-sided rhombic plates, wedges, and rosettes ○ Yellow-brown Sources of error ○ Cystine crystals