CM-2024-MTAP-REVIEW-CLINICAL MICROSCOPY PDF
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2024
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This document is a review for Medical Technology students on clinical microscopy, specifically renal function, glomerular filtration, tubular reabsorption, and secretion, complete with notes and some formulas.
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CLINICAL MICROSCOPY MTAP REVIEW II. GLOMERULAR FILTRATION GLOMERULUS...
CLINICAL MICROSCOPY MTAP REVIEW II. GLOMERULAR FILTRATION GLOMERULUS Resembles asieve NOTES T Found within the Bowman’s capsule Coil of approx. 8 capillary lobes (capillary tuft) Non-selective filter of plasma substances with molecular weight of RENAL FUNCTION _________________ M NEPHRON Basic structural & functional unit of the kidney R LOMERULAR G FILTRATE: > SG: 1.010 > NEGATIVE FOR 4 ALBUMIN LBUMINis 69k Da only. Why will it turn outnegative A 2 in the filtrate? 0 2 III. TUBULAR REABSORPTION O 1st function to be affected in renal disease PROXIMAL CONVOLUTED TUBULE ○ Major site(65%) of reabsorptionof plasma substances ote: PCT, LH, DCT & CD alter urine concentration.The ascending LH is highly impermeable to N C water RENAL FUNCTIONS U I. Renal Blood Flow II. Glomerular Filtration III. Tubular Reabsorption IV. Tubular Secretion.J Note: _______________- regulates SODIUM REABSORPTION _ I. RENAL BLOOD FLOW _______________________________- regulates WATER REABSORPTION in the L RENAL BLOOD FLOW:________________ DCT and CD RENAL PLASMA FLOW: 600-700 ml/min Renal threshold for glucose-______________________ 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 1 RENIN-ANGIOTENSIN-ALDOSTERONE SYSTEM (RAAS) - Activated only withLOW BLOOD PRESSURE T M RAAS EFFECTS Release of Aldosterone & ADH (increase Na & H2O reabsorption) RENAL FUNCTION TESTS R Vasoconstriction (increase BP) I. GLOMERULAR FILTRATION- ______________tests (usedto evaluate glomerular Corrects renal blood flow filtration) TUBULAR REABSORPTION Creatinine Clearance Formula: 4 ACTIVE TRANSPORT PASSIVE TRANSPORT here: W Ccr= Creatinine Clearance 2 U= Urine Creatinine (mg/dl) SUBSTANCE LOCATION SUBSTANCE LOCATION V= Urine volume (ml/min) P= Plasma creatinine 0 A= Body surface area lucose, amino G PCT Water CT, Descending P Creatinine clearance is a measure of the completeness of a __________ urine collection. acids, salts LH, CD SAMPLE PROBLEM: 2 Given the ff. data, compute for creatinine clearance: Chloride Ascending LH Urea PCT, Ascending LH Urine creatinine = 120mg/dL Plasma creatinine = 1 mg/dl O Urine volume in 24 hours = 1440 mL Sodium PCTand DCT Sodium Ascending LH Patient average body surface area: > Adults = 1.73m2 > Children = 0.17m2 C IV. TUBULAR SECRETION 2 MAJOR FUNCTIONS CALCULATED GLOMERULAR FILTRATION ESTIMATE USING FORMULA > ○ Regulation of the acid-base balance in the body through secretion of H+ DEVELOPED BY COCKGROFT AND GAULT U ions ○ Elimination of waste products not filtered by the glomerulus Renal Tubular Acidosis Inability to produce an _________ urine (hydrogen ions are not excreted in the urine) ote: Results are multiplied by 0.85 for female patients N VARIABLES: ___________________________________.J > MODIFICATION OF DIET IN RENAL DISEASE (MDRD) SYSTEM FORMULA −0.999 −0.176 𝐺𝐹𝑅 = 170𝑥 𝑆𝑒𝑟𝑢𝑚𝐶 𝑟𝑒𝑎 𝑥𝑎𝑔𝑒 𝑥0. 822(𝑖𝑓𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑖 𝑠𝑓 𝑒𝑚𝑎𝑙𝑒) L −0.170 +0.318 1. 1880( 𝑖𝑓𝑝𝑎𝑡𝑖𝑒𝑛𝑡𝑖 𝑠𝑏 𝑥 𝑙𝑎𝑐𝑘)𝑥𝐵 𝑈𝑁 𝑥𝑆𝑒𝑟𝑢𝑚𝐴𝑙𝑏𝑢𝑚𝑖𝑛 ARIABLES: Ethnicity, BUN, Serum Albumin V 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 2 II. UBULAR REABSORPTION- _________________ tests (usedto evaluate tubular T 3. Second morning/ Fasting nd voided urine after a period of fasting; for glucose 2 determination reabsorption) T 4. 2 hours postprandial For diabetic screening/monitoring BSOLETE O 1. Fishberg test TESTS - Patient is deprived of fluid for 24 hours 5. Glucose tolerance Optional with blood samples in glucose tolerance test M - Urine SG is the measured (SG should be ≥ 1.026) 2. Mosenthal test- compares day and night urine interms of volume and SG 6. Fractional specimen At least 2 voided collection OMMONLY C . Specific gravity- influenced by thenumber & densityof particles 3 7. ________________ For routine screening and bacterial culture (OPD) USED TESTS in a soln 4. Osmolarity- influenced by thenumberof particlesin a solution R 8. ________________ For bacterial culture 9. ________________ ladder urine for anaerobic bacterial culture and urine B III. UBULAR SECRETION AND RENAL BLOOD FLOW T cytology 4 1. PAH (p-aminohippuric acid) test (aka: Diodrast test) 2. PSP (phenolsulfonphthalein) test 10. Pediatric specimen se of soft, clear plastic bag with adhesive U Sterile spx obtained by catheterization/suprapubic 2 INTRODUCTION TO URINALYSIS aspiration URINE COMPOSITION 0 - 95-97% - Water 11. Three- glass technique For prostatic infection 1. First portion of urine voided - 3-5% - Solids - 60 grams total in 24 hours 2. Middle portion of urine voided 2 3. Urine after prostatic massage Examine the 1st and 3rd specimen O microscopically, then compare the no. of WBC TOTAL SOLIDS and bacteria tamey-Mears test for S Prostatic infection -if the no. of WBC and Prostatitis - 4 GLASS bacteria in the3rd specimen is 10X GREATER 5 grams(Organic 3 __________-major _ METHOD than that of the 1st (3rd > 1st) substances) Others: Uric acid, hippuric acid, creatinine, carbohydrates, C 2nd spx = ______________(for bladder and pigments, fatty acids, mucin, enzymes, hormones kidney infection) If + for WBC and bacteria, the results from the 3rd specimen is considered 5 grams(Inorganic 2 HLORIDE(major) > Sodium > Potassium C invalid U substances) Principal salt: NaCl Others: Sulfate, Phosphate, Ammonium, Magnesium, Calcium 2. Timed specimen (For 1 fternoon (2-4pm) - _____________________ A quantitative testing) 4 hours - _____________________________ 12 hours - _____________________________.J TYPES OF URINE SPECIMENS 24 hours - _____________________________ .Random/ Occasional/ 1 For routine and qualitative UA Single 2. First morning ___________________________________ _ L Most conc. / Most acidic ; For evaluation of orthostatic proteinuria 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 3 DRUG SPECIMEN COLLECTION NOTE: ______________- LEAST AFFECTED PARAMETER HAIN OF CUSTODY- process that provides documentationof proper sample identification C from the time of collection to the receipt of laboratory results T URINE PRESERVATIVES Required urine volume- ________________(60 ml :container capacity) > PRESERVATIVE FUNCTION > Temperature (within 4 mins)-_____________________ M > ___________________is added to the toilet water reservoir to prevent specimen adulteration. REFRIGERATION est choice for _______________ & B _______________ Prevents bacterial growth up to 24 hours SPECIMEN INTEGRITY Following collection, urine specimens should be delivered to the laboratory promptly ORMALIN F ediment preservative (cells & casts) S and tested within ____________. (FORMALDEHYDE) ADDIS COUNT CHANGES IN UNPRESERVED URINE R SACCOMANO FIXATIVE 0% Ethanol + 2 % Carbowax 5 INCREASED “p(a)BaON” CAUSE Used forCytology study 50 ml urine 1. pH Urea --- (Urease) ---> Ammonia BORIC ACID acteriostatic B 4 Keeps acidic pH (6.0) 2. Bacteria Multiplication For culture transport 2 3. Urea --- (Urease) ---> Ammonia PHENOL auses odor change C Add 1 drop per oz of spx 0 4. Due to bacterial contamination SODIUM FLUORIDE ntiglycolytic A Inhibits rgt strip test for glucose, blood & DARKENED/MODIFIED CAUSE leukocytes 2 Substitute w/ Na benzoate for rgt strip testing 5. Color Oxidation or reduction of metabolites DECREASED CAUSE O TOLUENE THYMOL Floats on urine surface reserves glucose & sediments P Interferes w/ acid ppt test for protein 6. Clarity Bacterial contam , precipitation of amorphous material C HERRY RED/ YELLOW TOP C reservative: Sodium propionate P 7. Glucose Glycolysis TUBE Conical bottom U YELLOW PLAIN UA TUBE sed on automated instruments U 8. Ketones ______________(if left uncapped) Round or conical bottom 9. Bilirubin (CB) Light exposure GRAY C & S TUBE reserves bacteria P Preservative: Boric acid.J Do not use on routine UA 10. Urobilinogen Oxidation to urobilin URINE COLLECTION KITS ontains collection cup, C & S preservative C 11. RBC/WBC Casts Disintegrate in alkaline urine tube or UA tube 12. Trichomonas ecomes immobile or die ; possible misidentification B OMMERCIAL C ossible substitute if refrigeration is not P L as WBCs PRESERVATIVE TABLETS possible Check inserts for possible test interferences 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 4 Pale yellow Polyuria, dilute random spx URINE VOLUME Dark yellow oncentrated spx , Strenuous exercise, first morning spx, C T ormal range (24 hours) : _____________________________________ N Carotene Average (24 hours) : _________________________________________ Night urine output : __________________________________________ Night:Day ratio : ____________________________________________ Amber Dehydration, Feverm Burns M Container capacity (UA) : _____________________________________ Required for routine UA : _____________________________________ range O Bilirubin (yellow foam) > “Tea-colored > Phenazopyridine: Orange & viscous urine w/ orange foam URINE VOLUME urine” Others: Acriflavin, Nitrofurantoin, Phenindione DEFINITION CAUSES ellow-green, Y Bilirubin –(oxidized)--> Biliverdin R Yellow brown POLYURIA Increased urine volume Increased fluid intake, Diuretics, > (>2000 ml/ 24 hours in Nervousness Green Pseudomonas infection adults) > Diabetes Mellitus - ________ > Diabetes Insipidus - _______ 4 Blue-green Indican,Amitriptyline, Methocarbamol, Clorets, Methyleneblue, Phenol.Chlorophyll OLIGURIA ecreased urine volume D Dehydration > 2 ( Renal diseases > renal calculi or tumor Pink,Red RBCs (Cloudy/Smoky Red): Hematuria > > Hemoglobin (Clear Red):Intravascular hemolysis 0 > Myoglobin (Clear red/reddish-brown/cola colored): ANURIA omplete cessation of C Complete obstruction (stones, > Rhabdomyolysis urine flow ( Others: Beets, Menstrual contam, Rifampin (Anti-TB drug) > Toxic agents 2 > Decreased renal blood flow urplish red, P Porphyrins Portwine NOCTURIA xcretion of more than E O > __________________ 500 ml urine at NIGHT (SG: > Homogentisic acid (alkaline urine): Alkaptonuria > Melanin (upon air exposure) C >Others: Phenol derivatives, Argyrol, Methyldopa/Levodopa, URINE COLOR Metronidazole (Flagyl) Rough indicator of thedegree of hydration U URINE CLARITY DETERMINATION Note: Thoroughly mix the specimen Examine the spx under a good light Examine the specimen while holding source in front of a light source Look down through the container.J View through a against a _____________ ______________________ LABORATORY CORRELATIONS OF URINE COLOR COLOR POSSIBLE CAUSE L Colorless Recent fluid consumption 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 5 URINE ODOR > CHEMICAL EXAMINATION OF URINE REAGENT STRIP TECHNIQUE ODOR CAUSE READING URINE PRINCIPLE POSITIVE COLOR T TIME PARAMETER Aromatic Normal 30 secs Glucose ouble sequential D Green to brown M enzyme reaction _____________ Acute tubular necrosis(acute renal failure) 30 secs Bilirubin Diazo reaction Tan or pink to violet Foul, ammoniacal UTI 40 secs Ketones Na nitroprusside rxn Purple R Fruity, sweet Ketones (DM, starvation, vomiting) 45 secs S.G Ka change of a p Blue to yellow polyelectrolyte aramelized sugar, curry, C Maple Syrup Urine Disease (MSUD) maple syrup 60 secs Protein rotein (sorensen’s) P Blue 4 error of indicators Mousy, Musty Phenylketonuria (PKU) 60 secs pH ouble indicator D Orange to blue 2 Rancid butter Tyrosinemia system 0 Sweaty feet, acrid Isovaleric acidemia, glutaric acidemia 60 secs Blood seudoperoxidase P niform green/blue U activity of Hgb (Hgb or Mb) speckled/spotted Cabbage, Hops Methionine malabsorption (Oasthouse syndrome) 2 (intact RBCs) Bleach contamination O 60 secs Urobilinogen Erlich’s rxn Red Sulfur Cystine disorder 60 secs Nitrite Greiss rxn Uniform pink Rotting fish Trimethylaminuria C 120 secs Leukocytes Leukocyte esterase Purple Pungent Ingestion of onions, garlic & asparagus OTE: AUTOMATED REAGENT STRIP READERS N U Principle: _________________________________ Swimming pool Hawkinsinuria I.SPECIFIC GRAVITY Cat urine 3-hydroxy-3-methylglutaric aciduria > URINOMETRY (URINOMETER/HYDROMETER).J Calibration temperature:20’C Tomcat urine Multiple carboxylase deficiency Calibration: Potassium Sulfate soln (20.29g K2SO4to 1 liter H20) = SG reading should be 1.015 Requires temp correction: -_______ for every 3’C that the specimen isBELOW the calib temp L + ______for every 3’C that the specimen isABOVE the calib temp 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 6 Requires correction for glucose and protein: REAGENT STRIP REACTION FOR SG ○ 1 g/dL of glucose = - 0.004 ○ 1 g/dL of protein = - 0.003 PRINCIPLE pKa change of a polyelectrolyte T Urine volume required:10-15 ml (disadv) Blue (1.000)—---> Green —---->Yellow (1.030) > REFRACTOMETRY (REFRACTOMETER, Rf/TS(Total Solids) meter) Indirect method based onrefractive index (RI) M REAGENTS Bromthymol blue Compensated to temp (15-38’C) No need for temperature correction Requires correction for glucose and protein Calibration ○ Distilled H20 = 1.000 ○ 3% NaCl = 1.015 ± 0.001 R ○ 5% NaCl = 1.022 ± 0.001 ○ 9% Sucrose = 1.034 ± 0.001 SAMPLE PROBLEM : 4 Urine SG reading by urinometer: 1.025 Urine temperature: 26’C Urinometer calibration temp: 20’C 2 What is the corrected SG? 0 2 SAMPLE PROBLEM : Urine SG reading by refractometer is 1.025 w/ 2g/dL glucose and 2 g/dL protein. Temperature is 37’C. O What is the corrected SG? C ADDITIONAL NOTES: Both refractometer (Rf) and urinometer (U)requirecorrections for glucose and U protein Refractometry reading is lower than the urinometer reading by0.002(Rf < U by 0.002) SPECIFIC GRAVITY DILUTION.J Spx with very high SG readings can be diluted and retested To obtain actual SG,multiply the decimal portion of SG by dilution factor xample: E Urine spx diluted 1:4 has a reading of 1.014. What is the actual SG reading? L 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 7 T M INTERFERENCES alse (+) = High concentration of protein F False (-) = High alkaline urines (>6.5) NOTES Not affected by glucose, protein & radiographic dye III. PROTEIN R Most indicative of renal disease Produces ____________ in urine when shaken > HARMONIC OSCILLATION DENSITOMETRY Least affected parameter in unpreserved urine Based on frequency of soundwave entering a soln changes in proportion to the ALBUMIN- major serum protein found in the urine density of soln Other proteins: 4 Ex: _______________ (International Remote Imaging System) ○ Serum and tubular microglobulins ○ Tamm-Horsfall protein ○ Proteins derived from prostatic and vaginal secretions 2 II. pH Important in theidentification of crystalsanddetermination of unsatisfactory PRE-RENAL (“BEFORE”) OR OVERFLOW PROTEINURIA 0 specimens Caused by conditions that affect the plasmaprior toits reaching the kidney: ○ Intravascular hemolysis - hemoglobin Diabetes mellitus > ○ Muscle injury - myoglobin 2 > Starvation CAUSES OF ACID > High protein diet ○ Severe infection & inflammation - increase APRs URINE > Cranberry juice ○ _________________- proliferation of Ig-producing plasma cells > Emphysema, dehydration, diarrhea, presence of O (Bence-Jones proteins) acid-producing bacteria (ex: E.coli), medications Tests: serum electrophoresis, immunofixation electrophoresis Urine: precipitates at 40-60’C (cloudy) and dissolves at 100’C Renal tubular acidosis > (clear) > Vegetarian diet C CAUSES OF ALKALINE > After meal due to alkaline tide RENAL PROTEINURIA (“true renal disease”) URINE > Vomiting I. Glomerular Proteinuria > Old specimens, hyperventilation, presence of A. Diabetic nephropathy urease-producing bacteria U B. Orthostatic/Cadet/Postural proteinuria- proteinuria when standing due to increased pressure to renal veins REAGENT STRIP REACTION FOR pH (60 secs) Principle:_______________________________________.J Orthostatic proteinuria Clinical proteinuria First morning spx 2 hours after standing L 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 8 II. Tubular Proteinuria- normally filtered albumin can no longer be reabsorbed 2+ 100-200 . Fanconi syndrome A B. Toxic agents/heavy metals 3+ 200-400 T C. Severe viral infections POST RENAL PROTEINURIA (“after”) 4+ Clumps of protein >400 M ower UTI/Inflammation L rostatic fluid/sperms P Menstrual contamination Injury/trauma IV. GLUCOSE (DEXTROSE) Vaginal secretions Most frequently tested in urine RENAL THRESHOLD FOR GLUCOSE: 160-180 mg/dL Other Sugars in Urine REAGENT STRIP REACTION FOR PROTEIN (_____secs) ○ Fructose (Levulose) R ○ Galactose ○ Lactose PRINCIPLE PROTEIN (SORENSEN’S) ERROR OF INDICATORS Indicator + Protein —----> (+) Blue-Green / (-) Yellow ○ Pentose (Yellow) ○ ___________ = NEGATIVE FOR COPPER REDUCTION TEST 4 CLINICAL SIGNIFICANCE OF URINE GLUCOSE 2 Hyperglycemia-associated Renal-associated 0 ___ Blood glucose = ___ Urine glucose ___ Blood glucose = ___ Urine glucose REAGENTS ultistix -Tetrabromphenol blue M Chemstrip - Tetrachlorophenoltetrabromosulfonphthalein 2 Causes: Impaired tubular reabsorption of glucose iabetes mellitus D INTERFERENCES False (+) = high SG Cushing’s syndrome (cortisol) Causes:Fanconi Syndrome NOTES O Indicator isSENSITIVE to_________________ Pheochromocytoma Acromegaly (growth hormone) Hyperthyroidism (T3,T4) Defective tubular reabsorption of glucose and amino acids C PROTEIN DETERMINATION SULFOSALICYLIC ACID (SSA) PRECIPITATION TEST REAGENT STRIP REACTION FOR GLUCOSE (30 secs) ○ A cold precipitation test that reacts equally withall forms of protein U ○ Procedure: PRINCIPLE Double sequential enzyme reaction 3 mL of 3% SSA + 3 mL centrifuged urine -> (+)Cloudiness SSA GRADING Glucose + O2 ------------------------------> Gluconic acid + H2O2 GRADE TURBIDITY PROTEIN RANGE (mg/dL).J Negative No increase in turbidity Oxidized chromogen + H2O Trace Noticeable turbidity 6-30 L 1+ 30-100 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 9 REAGENT STRIP REACTION FOR KETONES (40secs) T PRINCIPLE Sodium nitroprusside reaction (Legal’s test) Acetoacetic acid + Na nitroprusside ---------->(+) PURPLE M (Acetone) (Glycine) REAGENTS ultistix = Glucose oxidase , peroxidase,potassium iodide(blue to M green to brown) Chemstrip = Glucose oxidase , peroxidase,tetramethylbenzidine (yellow to green) INTERFEREN alse (+): Oxidizing agents, detergents F R CES False (-):High levels of ascorbic acid, ketones, high SG,LOW TEMP, improperly preserved specimen REAGENTS Sodium nitroprusside, Glycine NOTES ensitivity: 100 mg/dL S 4 Correlation w/ other tests:Ketones, Protein INTERFERENCES False (-) : improperly preserved spx (volatile) NOTES cetone is detected only when glycine is present A 2 COPPER REDUCTION TESTS 1. Benedict’s test Correlation w/ other test: Glucose ○ Non-specific test for reducing sugars - glucose, galactose, lactose and 0 fructose(SUCROSE is negative!!) ○ Principle: copper reduction ○ Positive result:blue to brick-red 2 ○ Cause of false positive:Reducing substances (ex: ascorbic acid, uric acid) ○ Cause of false negative:Oxidizing substances (ex: detergents) VI. BLOOD 2. Clinitest O V. KETONES HEMATURIA HEMOGLOBINURIA MYOGLOBINURIA Results fromincreased fat metabolismdue to inability to metabolize carbohydrates C Seen in: ________ red urine ________ red urine Clear red (reddish-brown) ○ Type I DM urine ○ Vomiting ○ Starvation Seen in: een in: S een in: S U ○ Malabsorption lomeruloneph G ______________________ ____________________ ritis Transfusion Muscular trauma Renal calculi, reactions Crush syndrome KETONE BODIES tumors Hemolytic anemia Extensive Strenuous Severe burns exertion.J exercise Brown recluse 78% Beta-hydroxybutyric acid -major ketone but NOT DETECTEDIN rgt strip spider bites icroscopic: M 20% Acetoacetic acid(AAA) / Diacetic acid - parent ketone INTACT RBCs L 2% Acetone 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 10 HEMOGLOBIN VS. MYOGLOBIN INTERFERENC alse (+) :Highly pigmented urine, phenazopyridine, indicans F ES False (-): Exposure to light, high conc. of nitrite, ascorbic acid (>25mg/dL) TEST HEMOGLOBIN MYOGLOBIN T NOTES OSITIVE COLOR RESULT: ___________________ P LONDHEIM’S TEST B Precipitated Not precipitated Correlation with other tests: Urobilinogen (Ammonium Sulfate) M ILIRUBIN DETERMINATION B rocedure: P ICTOTEST (TABLET) Urine + 2.8g NH4Sulfate → Filter/Centri ○ More sensitive than strip w/ less interference → Test supernatant for Contains: blood w/ rgt strip ○ P-nitrobenzene-diazonium-p-toluenesulfonate ○ SSA R ○ Sodium carbonate ○ Boric acid REAGENT STRIP REACTION FOR BLOOD (60 secs) POSITIVE RESULT: __________________ 4 PRINCIPLE Pseudoperoxidase activity of Hemoglobin VIII. UROBILINOGEN H2O2 + Chromogen - - - - - - - - - - - - - - - > Oxidized chromogen + H2O Bile pigment that result from hemoglobin degradation 2 (Yellow) (Green to Blue) Specimen:________________________ Normal value for UBG:(+) HERRY RED C C VII. BILIRUBIN Conjugated bilirubin (CB)- water soluble Early indication of liver disease REAGENTS ultistix = p-dimethylaminobenzaldehyde (PDAB or Ehrlich M U Tea-colored / amber urine w/ _______________ reagent) Clinical significance - liver disorders: Chemstrip = 4-methoxybenzene-diazonium-tetrafluoroborate ○ Hepatitis, cirrhosis (specific for UBG) ○ Biliary obstruction (gallstones, carcinoma).J REAGENT STRIP REACTION FOR BILIRUBIN (30secs) INTERFERENCES alse (+) : Ehrlich reactive compounds F False (-) : Old spx, preservation in formalin, high conc. of nitrite PRINCIPLE DIAZO REACTION Bilirubin diglucuronide (CB) + Diazonium salt ----------> Azodye NOTES Correlations with other tests: Bilirubin L REAGENTS ultistix =2,4-dicholoroaniline diazonium salt M Chemstrip = 2,6-dichlorobenzene diazonium salt 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 11 ROBILINOGEN DETERMINATION U X. LEUKOCYTES > WATSON-SCHWARTZ TEST Differentiate urobilinogen (UBG), porphobilinogen (PBG) and other Ehrlich-reactive REAGENT STRIP REACTION FOR LEUKOCYTES (120secs) T compounds(ERC) Uses extraction with organic solvents: ______________________ PRINCIPLE EUKOCYTE ESTERASE REACTION L Indoxylcarbonic acid ester ----> Indoxyl + Acid indoxyl + Diazo salt --->(+) Purple M REAGENTS ultistix = Derivatized pyrrole amino acid ester, Diazonium salt M Chemstrp = Indoxylcarbonicacid ester, Diazonium salt R INTERFERENCES alse (+): Formalin F False (-): High conc. protein, ascorbic acid, gentamicin, cephalosporins, tetracyclines, inaccurate timing > HOESCH TEST (Inverse Ehrlich reaction) 4 Rapid screening test forporphobilinogen(≥2 mg/dL) NOTES Strip can detect lysed WBCs Procedure: 2 gtts urine + 2 mL Hoesch reagent (Ehrlich rgt in 6M or 6N HCl) 2 (+)RED at the top of the mixture XI. ASCORBIC ACID CausesFALSE NEGATIVEreactions on: “BBLNG” B- 0 IX. NITRITE B- Rapid screening test for UTI or bacteriuria L- N- 2 REAGENT STRIP REACTION FOR NITRITE (60 secs) G- 11th reagent pad:Ascorbic acid (≥5 mg/dL)+ Phosphomolybdate → (+) Molybdenum PRINCIPLE O _______________________ P-arsanilic acid + Nitrite -----> Diazonium salt blue > MICROSCOPIC EXAMINATION OF URINE Diazonium salt +Tetrahydrobenzoquinolin----->(+) Uniform pink C MICROSCOPIC TECHNIQUES TECHNIQUE DESCRIPTION U REAGENTS ultistix =p-arsanilic acid, tetrahydrobenzo(h)quinolin-3-ol M Chemstrip = Sulfanilamide, hydroxytetrahydro RIGHT FIELD B For routine UA benzoquinoline MICROSCOPY.J INTERFERENCES alse (-): large quantities of bacteria converting nitrite to F HASE CONTRAST P nhances visualization of translucent elements (elements with E nitrogen MICROSCOPY low refractive indices, ex: hyaline cast) NOTES (+) nitrite corresponds to 100,000 organisms/mL OLARIZING P Identification of cholesterol in oval fat bodies, fatty casts & MICROSCOPY crystals L DARK-FIELD MICROSCOPY Identification of Treponema pallidum 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 12 Possible sources of ERROR LUORESCENCE F isualization of fluorescent microorganisms of those stained V ○ Yeasts MICROSCOPY by a fluorescent dye ○ Oil droplets T ○ Air bubbles INTERFERENCE- D microscopy-image & layer-by-layer imaging of a specimen 3 ○ Monohydrate CaOx crystals CONTRAST Bright-field microscopes can be adapted Remedy: Add2% Acetic acid, it will lyse the rbcsbut not the others MICROSCOPY M A. Nomarski (Differential) 2. WBCs (Pyuria or Leukocyturia) B. Hoffman NV: 0-5 or 0-8/HPF (Modulation) Increased number indicates presence ofinfection or inflammation Neutrophils (predominant) ○ Granulated and multilobed R ○ In hypotonic urine, neutrophils swells and their STAIN ACTION granules undergo Brownian movement producing a sparkling appearance (glitter cells) TERNHEIMER- MALBIN S Identifies WBC, epithelial cells & casts Eosinophils 4 (Crystal Violet + Safranin O) ○ NV: = 1% (assoc. w/ drug induced ________________) Mononuclear cells (lymphocytes, monocytes, macrophages, histiocytes)- 2 present in small numbers . EPITHELIAL CELLS 3 0 TOLUIDINE BLUE Differentiates WBCs and RTE cells A. Squamous epithelial cell LARGEST CELLw/ abundant, irregular cytoplasm & prominent nucleus 2% ACETIC ACID DistinguishesRBCsfrom WBCs, yeast, oil droplets& crystals 2 From linings of vagina, female urethra & lower portion of male urethra Variation:___________ IPID STAINS L tains triglycerides and neutral fats range red(CANNOT S O ○ Squamous epithelial cell covered with (Oil Red O and Sudan III) STAIN ______________________) Gardnerella vaginalis ○ Associated with bacterial vaginosis GRAM STAIN Differentiates gram positive and gram negative bacteria C ANSEL STAIN H Identifies urinary eosinophils (Eosin Y + Methylene Blue) U PRUSSIAN BLUE Stains structures containing iron (ex: hemosiderin granules) B.Transitional epithelial (Urothelial) cell/ Bladder cell Spherical, polyhedral or caudate with centrally located nucleus Increased TEC may be an indication of malignancy or viral infection ELLS IN URINE C 1. RBCs (Hematuria).J NV: 0-2 or 0-3/HPF Smooth, non-nucleated, biconcave disks Hypertonic urine= crenated shrink Hypotonic urine =swell/hemolyze (GHOST CELL) Glomerular membrane damage= dysmorphic w/ L projections, fragmented 2024 MEDICAL TECHNOLOGY MTAP REVIEW - CLINICAL MICROSCOPY LJUCO2024 13 C. Renal Tubular Epithelial (RTE) cell II. CASTS (_______________) Most clinically significant epithelial cell Unique to the kidney Origin: Nephron Primary located in theDCT and collecting duct T Rectangular, polyhedral, cuboidal or columnar with an eccentric nucleus Major constituent -TAMM HORSFALL PROTEIN(Uromodulin) >2 RTE/hpf indicates _______________________ Performed along the coverslip edges with subdued light ORDER OF CAST DEGENERATION M 1. Hyaline 5. ____________ 2. Cellular cast (RBC, WBC, RTE) 3. Coarse granular 4. Finely granular URINARY CASTS R REPORTING OF E.C. > Rare, Few, Moderate, Many: > Average number per HPF CAST INFO CLINICAL SIGNIFICANCE
