AUB F LEC 5 Trans 5 PDF

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OUTLINEI. Roles of Microscopic Examination of Urine II. Manner of Reporting III. Sources of Errors in Urine Handling IV. Addis Count V. Microscopic Techniques VI. Sediment Constituents A. Cells B. Casts C. Crystals VII. Normal Findings Acid Urine VIII. Abnormal Metabolism Acid Urine IX. Crystals of ...

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OLFU RMT 2023

I. II. III. IV. V. VI.

VII. VIII. IX. X. XI.

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Microscopic Examination of Urine ANALYSIS OF URINE AND BODY FLUIDS Instructor: Prof. Maricho P. Barnachea, RMT, MPHc Date: November 1, 2021

OUTLINE Roles of Microscopic Examination of Urine Manner of Reporting Sources of Errors in Urine Handling Addis Count Microscopic Techniques Sediment Constituents A. Cells B. Casts C. Crystals Normal Findings Acid Urine Abnormal Metabolism Acid Urine Crystals of Iatrogenic Origin Normal Metabolism Alkaline Urine Urine Sediment Artifacts

I. ROLES OF MICROSCOPIC EXAMINATION OF URINE Determine the correctness of diagnosis of a renal system infection and disease Refrigerated urine preserves almost all cellular elements, crystals, and casts very well Fresh and concentrated urine provides more accurate results Proper collection avoids extra debris of the urethral meatus, vaginal secretions Proper use of subdued light in LPO before HPO: for accurate results Examine the urine specimen within 1 to 2 hours prior to collection II. MANNER OF REPORTING Examine the urine specimen within 1 to 2 hours prior to collection RBC and WBC – Average number /HPF Casts – Average number /LPF Epithelial cells and crystals: occasional, rare or few, moderate and abundant, too many to count or too numerous to count, plenty III. SOURCES OF ERRORS IN HANDLING URINE Careless transfer of sediment (contamination) Too much light (retractile bodies cannot be seen) Using the HPO only Specimens up only on long standing (false elements are seen) Dirty equipment Scratches on slides

Figure 3.0 Urine Sediment Preparation URINE SEDIMENT PREPARATION 10 – 15 mL of urine (average of 12mL) ↓ Centrifuge at 400 RCF for 5 mins ↓ Decant urine (0.5 or 1.0 mL remains) ↓ Transfer 20 uL sediment to glass slide ↓ Coverslip ↓ Examine microscopically ↓ 10 LPF and 10 HPF under reduced light

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LEC 5 TRANS 5

2021 – 2022 1st Semester AUBF 311 LEC

IV. ADDIS COUNT Quantitative measurement of formed elements of urine using hemacytometer Specimen of choice: 12-hr urine Preservatives: Formalin Normal count:  RBC: 0 – 500,000 / 12-hr urine  WBC: 0 – 1,200,000 / 12-hr urine  Hyaline cast: 0 – 5,000 / 12-hr urine Abnormalities in the physical and chemical portions of the urinalysis play a primary role in the decision to perform a microscopic analysis, thus the use of the term macroscopic screening, also referred to as chemical sieving V. MICROSCOPIC TECHNIQUES

A. Bright Field Microscopy  Used for routine urinalysis  Most commonly used technique B. Phase-contrast Microscopy  Enhances visualization of elements with low refractive indices (hyaline cast) C. Polarizing Microscopy  Identification of cholesterol in oval fat bodies, fatty casts, and crystals D. Dark-Field Microscopy  Identification of T. pallidum (C.A of syphilis) E. Fluorescence Microscopy  Visualization of fluorescent microorganisms or those stained with fluorescent dyes F. Interference Microscopy 1. Nomarski microscopy or Differential interference contrast 2. Hoffman contrast  Bright field microscopy can be adapted Table 5.0 Macroscopic Screening Correlations SCREENING TEST SIGNIFICANCE Color Blood Clarity Hematuria versus hemoglobinuria/ myoglobinuria Confirm pathologic or nonpathologic cause of turbidity Blood RBCs/RBC casts Protein Casts/cells Nitrite Bacteria/ WBCs Leukocyte esterase WBCs/WBC casts/ bacteria Glucose Yeast Table 5.1 Routine Urinalysis Correlations Microscopic Physical Chemical Elements RBCs Turbidity + Blood Red color WBCs Turbidity + Protein + Nitrite + Leukocytes Epithelial cells Turbidity Casts + Protein Bacteria Turbidity pH + Nitrite + Leukocytes Crystals Turbidity pH Color

Exceptions Number Hemolysis Number Lysis Number Number Number and type Number and type

Limon, I. – TRANSCRIBER

Microscopic Examination of Urine

OLFU ANALYSIS OF URINE AND BODY FLUIDS Instructor: Prof. Maricho P. Barnachea, RMT, MPHc Table 5.2 Sediment Stain Characteristics Red cell RMT 2023 2021 Stain ActionDate: November 1,Function casts SternheimerMalbin

Toluidine blue

Delineates structure and contrasting colors of the nucleus and cytoplasm Enhances nuclear detail

2% acetic acid

Lyses RBCs and enhances nuclei of WBCs

Lipid Stains: Oil red O and Sudan III

Stains triglycerides and neutral fats orange-red

Gram Stain

Differentiates grampositive and gramnegative bacteria Methylene blue and eosin Y stains eosinophilic granules Stains structures containing iron

Hansel stain

Prussian stain

blue

st

inclusion

Identifies WBCs, epithelial cells and casts

Blood (Hgb) casts Bacteria

Differentiates WBCs and renal tubular epithelial cells (RTE) Distinguishes RBCs from WBCs, yeast, oil droplets, and crystals Identifies free fat droplets and lipidcontaining cells and casts Identifies bacterial casts

Trichomonas vaginalis

Identifies eosinophils

urinary

Identifies yellowbrown granules of hemosiderin in cells and casts

Table 5.3 Expected Staining Reactions of Sediment Constituents Elements in Usual Comments Urinary Sediment Distinguishing Color of Stained Elements RBCs Neutral – pink to purple Acid – pink (unstained) Alkaline – purple Nuclei Cytoplasm WBCs (dark- Purple Purple granules staining cells) Glitter cells Colorless or light Pale blue or gray – (Sternheimer-Malbin blue some glitter cells positive cells) exhibit Brownian movement RTE cells Dark shade of blue- Light shade of bluepurple purple Bladder tubular Blue-purple Light purple epithelial cells Squamous epithelial Dark shade of Light purple or blue ells orange-purple Inclusions and Matrix Hyaline casts Pale pink or pale Very uniform color, purple slightly darker than mucous threads Coarse granular Dark purple inclusions casts granules in purple matrix Finely granular Fine dark purple inclusion casts granules in pale pink or pale purple matrix Waxy casts Pale pink or pale Darker than hyaline purple casts, but of a pale even color; distinct broken ends Fat inclusion casts Fat globules Rare; presence is if unstained in a pink confirmed examination under matrix polarized light indicates double refraction

2021 – 2022 LEC 5 1 Semester AUBF 311can be Pink TRANS to orange-red 5Intact cells LEC seen in the matrix Orange-red Motile: do not stain Non-motile: stain purple Light blue-green

Mucus

No intact cells Motile organisms are not impaired Motility is unimpaired in fresh specimens when recommended volumes of stain are used; immobile organisms are also identified

Pale pink or pale blue Pale pink or pale purple

Background

VI. SEDIMENT CONSTITUTENTS A. Cells 1. RBCs (Hematuria)      

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Normal Value = 0-2 or 0-3/HPF Smooth, non-nucleated, biconcave disks Hypertonic: Crenate/shrink Hypotonic: Swell (hemolyze) or ghost Glomerular membrane damage: dysmorphic with projections Sources of error:  Yeasts  Oil droplets  Air bubbles  Calcium oxalate crystals Remedy: add 2% acetic acid to lyse the RBC

Table 6.0 Summary of Microscopic RBCs Appearance: Non-nucleated biconcave disks Crenated in hypertonic urine Ghost cells in hypotonic urine Dysmorphic with glomerular membrane damage Sources of Yeast cells Identification Oil droplets Error Air bubbles Reporting Average number per 10 hpfs Complete Color urinalysis Reagent strip blood reaction correlations 2. WBCs (Pyuria)  

Normal Value = 0-5 or 0-8/ HPF Larger than RBC

A. Types of WBC a. Neutrophils (most predominant)  Granulated and multilobulated  Hypotonic: swells and undergoes Brownian movement b.   

Eosinophils Normal value: 1% Associated with drug-induced interstitial nephritis

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Mononuclear cells Lymphocytes, monocytes, macrophages, histiocytes

Table 6.1 Summary of Microscopic WBCs Appearance: Larger than RBCs Granulated, multilobed neutrophils Glitter cells in hypotonic urine Mononuclear cells with abundant cytoplasm

Limon, I. – TRANSCRIBER

Microscopic Examination of Urine

OLFU ANALYSIS OF URINE AND BODY FLUIDS Instructor: Prof. Maricho P. Barnachea, RMT, MPHc Complete Sources of RTE cells RMT 2023 Date: November 1, 2021 UA identification error Reporting Complete urinalysis correlations:

Average number per 10 hpfs Leukocyte esterase Nitrite Specific gravity pH

3. Epithelial Cells  

Not a normal constituent of urine because they are derived from the linings of genitourinary tract Clinically significant unless they are present in large numbers or it abnormal forms

a. squamous epithelial cells  Largest cell with abundant, irregular cytoplasm and abundant nucleus  From the linings of vagina, female urethra, and lower portion of male urethra  Variations: Crew cell  Squamous epithelial cell covered with Gardnella vaginalis  Associated with bacterial vaginosis Table 6.2 Summary of Squamous Epithelial Cells Appearanc Largest cells in the sediment with abundant, e irregular cytoplasm and prominent nuclei Sources of Rarely encountered, folded cells may resemble error casts Reporting Rare, few, moderate or many per lpf Complete Clarity UA correlation s b. Transitional epithelial cells  Also known as urothelial cells  Spherical, polyhedral, or caudate  Centrally located nucleus  Derived from the renal pelvis, ureter, urinary bladder, and upper portion of the male urethra Table 6.3 Summary of Transitional epithelial cells Appearanc Spherical, polyhedral, or caudate with centrally e located nucleus Sources of Spherical forms resemble RTE cells error Reporting Rare, few, moderate or many per lpf Complete Clarity; blood if malignancy-associated UA correlation s c. Renal tubular epithelial cells (RTE)  Most clinically significant epithelial cell  Origin: nephron  Rectangular, polyhedral, cuboidal, or columnar  Eccentric nucleus  RTE cell variation  A. oval fat body  Lipid containing RTE cells  Seen in lipiduria (nephrotic syndrome)  Identified by lipid stains and polarizing microscope  B. bubble cells  RTE cell with nonlipid-filled vacuoles  Seen in tubular necrosis Table 6.4 Summary of RTE cells Appearanc Rectangular; columnar; round, oval, or cuboidal e with an eccentric nucleus possibly bilirubin-stained or hemosiderin-laden Sources of Spherical transitional cells error Granular casts Reporting Average number per 10 hpfs

correlation s

2021 – 2022 LEC 5 1 Semester 311 Leukocyte esterase and nitrite (pyelonephritis) TRANS 5 AUBF LEC Color st

Clarity Protein Bilirubin (hepatitis) Blood

Table 6.5 Summary of Oval fat bodies Appearanc Highly refractile RTE cells e Sources of Confirm with fat stains and polarized microscopy error Reporting Average number per hpf Complete Clarity, blood, protein, free fat droplets/ fatty casts UA correlation s 4. Bacteria  UTI = Bacteria and WBC  Enterobacteriaceae – most common cause of UTI  Staphylococcus – 2nd  Enterococcus – 3rd Table 6.6 Summary of Miscellaneous Structure – Bacteria Appearanc Small spherical and rod-shaped structures e Sources of Amorphous phosphates and urates error Reporting Few, moderate, or many per hpf; the presence of WBCs may be required Complete pH UA Nitrite correlation LE s WBCs 5. Yeasts   

True yeast infection = Yeast + WBC Small refractile oval structures that may or may not bud Candida albicans = seen in DM and Vaginalis moniliasis

Table 6.7 Summary of Miscellaneous Structure – Yeast Appearanc Small, oval, refractile structures with buds and/or e mycelia Sources of RBCs error Reporting Rare, few, moderate, or many per HPF; the presence of WBCs may be required Complete Glucose UA LE correlation WBCs s 6. Parasites 

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A. T. vaginalis (Trichomonas vaginalis)  Pear-shaped flagellate with jerky motility  Agent of pingpong disease B. S. haematobium (Schistosoma haematobium)  With terminal spine  Hematuria  Associated with bladder cancer C. E. vermicularis ova (Enterobius vermicularis)  Most common fecal contaminant

Table 6.8 Summary of Miscellaneous Structure – Trichomonas Appearanc Pear-shaped, motile, flagellated e Sources of WBCs, RTE cells error

Limon, I. – TRANSCRIBER

Microscopic Examination of Urine

OLFU ANALYSIS OF URINE AND BODY FLUIDS Instructor: Prof. Maricho P. Barnachea, RMT, MPHc Reporting Rare, few, moderate, or many per hpf Clinical Significance RMT 2023 Date: November 1, 2021 Complete LE UA correlation s

2. Cellular Cast

After sexual intercourse

Table 6.9 Summary of Miscellaneous Structure – Spermatozoa Appearance Taped oval head with long, thin tail Sources of error None Reporting Present, based on laboratory protocol Complete UA Protein correlations 8. Mucus thread 

Major constituent = Tamm Horsfall protein/ Uromodulin

Table 6.10 Summary of Miscellaneous Structure – Mucus Appearance Single or clumped threads with low refractive index Sources of error Hyaline casts Reporting Rare, few, moderate or many per lpf Complete UA correlations None B. CASTS (CYLINDURIA)    

st

Chronic renal disease Congestive heart failure Stress and exercise

WBCs

7. Spermatozoa 

2021 – 2022 LEC 5 1 Semester 311 Glomerulonephritis TRANS 5 AUBF LEC Pyelonephritis

Unique to the kidney Formed within the lumens of the distal convoluted tubules and collecting ducts Major constituent uromodulin or Tamm Horsfall protein  Produced by the RTE cells Performed along the edges of the coverslip with subdued light Hyaline ↓ Cellular ↓ Coarsely granular ↓ Finely granular ↓ Waxy

1. Hyaline Cast A. Prototype Cast  Beginning of all types of casts  NV: 0-2/ LPF  Clinical significance:  Physiologic  Stress  Strenuous exercise  Pathologic  Glomerulonephritis  Pyelonephritis  Congestive heart failure Table 6.11 Summary of Urine Cast – Hyaline Appearance Colorless homogenous matrix Sources of error Mucus, fibers, hair; increased lighting Reporting Average number per lpf Complete UA correlations Protein Blood (exercise) Color (exercise)

A. RBC Cast  Bleeding within the nephron  Clinical significance:  Glomerulonephritis  Strenuous exercise Table 6.11 Summary of Urine Cast – Hyaline Appearance Orange-red color; cast matrix containing RBCs Sources of error RBC clumps Reporting Average number per lpf Complete UA RBCs correlations Blood Protein Clinical Glomerulonephritis Significance Strenuous exercise B. Bacterial Cell Cast  Pyelonephritis Table 6.12 Summary of Urine Cast – Bacterial Appearance Bacili, bound to protein matrix Sources of error Granular casts Reporting Average number per lpf Complete UA correlations WBC cast WBCs LE Nitrite Protein Bacteria Clinical Significance Pyelonephritis C. WBC Cast  Inflammation within the nephron  May be confused with epithelial cells  To differentiate  Phase contrast microscopy  Supravital stain Table 6.13 Summary of Urine Cast – WBC Appearance Cast matrix containing WBCs Sources of error WBC clumps Reporting Average number per lpf Complete UA correlations WBCs Protein LE Clinical Significance Pyelonephritis Acute interstitial nephritis D. Epithelial Cell  Advanced tubular destruction  Renal tubular damage Table 6.14 Summary of Urine Cast – Epithelial Cell Appearance RTE cells attached to protein matrix Sources of error WBC cast Reporting Average number per lpf Complete UA correlations Protein RTE cells Clinical Significance Renal tubular damage 3. Fatty Cast  

Not stained with Sternheimer-Malbin Identification:

Limon, I. – TRANSCRIBER

Microscopic Examination of Urine

2021 – 2022 OLFU ANALYSIS OF URINE AND BODY FLUIDS LEC 5 1 Semester Instructor:  Triglycerides and neutral fats Prof. Maricho P. Barnachea, RMT, MPHc Reporting Average number per lpf 311 RMT 2023 TRANS 5 AUBF Date: November 1, 2021  Oil red O and Sudan 3 LEC Complete UA correlations Protein st

Cholesterol  Polarizing microscope Clinical significance  Nephrotic syndrome 

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Clinical Significance Table 6.15 Summary of Urine Cast – Fatty Appearance Fat droplets and oval fat bodies attached to protein matrix Sources of error Fecal debris Reporting Average number per lpf Complete UA correlations Protein RTE cells Clinical Significance Renal tubular damage

Granular casts WBCs RBCs Waxy casts Extreme urine stasis Renal failure

C. CRYSTALS  Identified on the basis of their microscopic appearance and the pH at which they are present  Factors that influence crystal formation:  The concentration of the solute in the urine  Urine pH  Flow of urine through the tubules

4. Granular Cast  

Granules are derived from the lysosomes of RTE cells during normal metabolism (nonpathologic) Clinical significance  Glomerulonephritis  Pyelonephritis  Stress  Strenuous exercise

Table 6.16 Summary of Urine Cast – Granular Appearance Coarse and fine granules in a cast matrix Sources of error Clumps of small crystals Columnar RTE cells Reporting Average number per lpf Complete UA correlations Protein Cellular casts WBCs RBCs Clinical Significance Glomerulonephritis Pyelonephritis Stress and exercise 5. Waxy Cast   

Final degenerative form of all types of cast Brittle, highly refractile with jagged ends Clinical significance  Stasis of urine flow  Chronic renal failure

Table 6.17 Summary of Urine Cast – Waxy Appearance Highly refractile cast with jagged ends and notches Sources of error Fibers and fecal material Reporting Average number per lpf Complete UA correlations Protein Cellular casts WBCs RBCs Clinical Significance Stasis of urine flow Chronic renal failure 6. Broad Cast    

Also known as Renal failure cast Indicates destruction widening of the tubular walls Any type of cast can be broad Clinical significance  Extreme urine stasis  Renal failure

Table 6.18 Summary of Urine Cast – Broad Appearance Wider than normal cast matrix Sources of error Fecal material, fibers

Table 6.19 Major Characteristics of Normal Urinary Crystals Crystal pH Color Solubility Uric acid Acid Yellow-brown Alkali soluble Amorphous Acid Brick dust or yellow Alkali and urates brown heat Calcium Acid/neutra Colorless Dilute HCl oxalate l (envelopes, oval, oval (alkaline) dumbbell) Amorphous Alkaline White – colorless Dilute phosphates Neutral acetic acid Calcium Alkaline Colorless Dilute carbonate Neutral acetic acid Triple Alkaline Colorless (coffin Dilute phosphate lids) acetic acid Ammonium Alkaline Yellow-brown Acetic acid biurate (thorny apples) with heat Calcium Alkaline Colorless Gas from carbonate (dumbbells) acetic acid Table 6.20 Urine Crystal and their other names Urine Crystal Other Name Calcium Calcium oxalate monooxalate (H2O) hydrate Dumb-bell Calcium Calcium oxalate di-hydrate Oxalate 2 (H2O) Bi-pyramidal envelopes Calcium Calcium hydrogen phosphate phosphate Calcium Tri-calcium phosphate Phosphate Triple Magnesium ammonium phosphates phosphate

Mineral Name Whewellite

Weddelite Brushite Whitlockite Struvite

VII. NORMAL FINDING ACID URINE A. Amorphous Urates  Between pH 5.7 and 7.0  Small, yellow-brown granules,much like sand  Occurs in clumps resembling granular casts  Frequently encountered refrigerated specimen with a pink characteristic (pink-orange)  Often referred to as “brick dust”  Identified by their solubility in alkali or their dissolution when heated to approximately 60°C  Converted to uric acid upon the addition of concentrated acetic acid . B. Acid Urates  Sodium, potassium, and ammonium s...