MACROSCOPIC SCREENING

MACROSCOPIC SCREENING AND MICROSCOPIC CORRELATIONS

Screening Test (Parameters) Significance
Color Blood
Clarity Hematuria vs Hemoglobinuria/Myoglobinuria
Confirm pathologic or non-pathologic cause of turbidity
Blood RBCs, RBC casts
Protein Cast, cells
Nitrite Bacteria, WBCs
Leukocyte Esterase WBCs, WBC casts, bacteria
Glucose Yeast

 SPECIMEN PREPARATION
o Fresh and adequately preserved
o Formed elements (RBCs, WBCs, Hyaline Casts) disintegrate in Dilute Alk. Urine
o Refrigeration = Ppt. of Amorphous Urates and Phosphates
o Warming to 37⁰C = dissolve crystals
o Midstream Clean-Catch, 1st morning Spx. – minimizes external contamination
o Dilute random spx.= F-
 SPECIMEN VOLUME
o 10 and 15 mL (centrifuged in a conical tube)
o 12-mL – frequently used/Ave. – multiparameter reagent strips are easily immersed.
 CENTRIFUGATION
o 5 mins. @ 400 RCF (relative centrifugal force) = prod. an optimum amt. of sediment and least
amt. of damage elements.
 RCF is used over revolutions per minute (RPM)
To convert RPM to RCF:
RCF = 1.118 x 10⁻⁵ x radius in centimeters x RPM²
 SEDIMENT PREPARATION
o 0.5 mL, 1.0 mL – frequently used – amount after decantation
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑈𝑟𝑖𝑛𝑒 𝐶𝑒𝑛𝑡𝑟𝑖𝑓𝑢𝑔𝑒𝑑
o = Concentration Factor
𝑆𝑒𝑑𝑖𝑚𝑒𝑛𝑡 𝑉𝑜𝑙𝑢𝑚𝑒

Sediment Concentration Factor – relates to the probability of detecting elements
present in low quantities
 Urine should be aspirated- to maintain uniform sediment conc. factor
 VOLUME OF SEDIMENT EXAMINED
o Glass-slide method volume = 20 microliter (0.02 mL) covered by 22x22 cover slip
 COMMERCIAL SYSTEMS
o KOVA, Urisystem, Count-10, Quick-Prep Urinalysis System, CenSlide 2000 Urinalysis System,
R/S Workstations
o CenSlide and R/S Workstations (Closed Systems)
 CenSlide – permits direct reading of the urine sediment
 R/S Workstations – consist of a glass flow cell into which urine sediment is pumped,
microscopically examined, and then flushed from the system
  EXAMINING THE SEDIMENT
o Microscopic Exam: Min. of 10 LPFs(10x), 10 HPFs(40x)
o Sediments should be examined under reduced light when using bright-field microscopy
 REPORTING THE MICROSCOPIC EXAMINATION
o Casts – Ave. number per LPF in 10 fields
o RBCs and WBCs – Ave. number per 10 HPF
o Epithelial cells, crystals, and other elements – Semi quantitative terms (Rare, few, moderate, and
many)
o LPO- Gen. Evaluation, Squamous cells, Representation & ct. of casts
o HPO- Identification, for RTE cells, WBCs, RBCs, Bacteria, Transitional ECs

ADDIS CT.
 Dev. by Thomas Addis, 1962
 1st procedure to standardize the quantitation of formed elements in the urine
microscopic analysis
 Used Hemocytometer to ct. the # of RBCs, WBCs, Casts, Epithelial cells in
12hr. spx.
 Used to monitor the diagnosed cases of Renal dse.
 NVs: 0-500,000 RBCs
0-1,800,000 WBCs
0-5000 Hyaline casts

 CORRELATING RESULTS

ROUTINE URINALYSIS CORRELATIONS

Microscopic Physical Chemical Exceptions
Elements
RBCs Turbidity +Blood Number
Red Color +Protein Hemolysis
WBCs Turbidity +Protein Number
+Nitrite Lysis
+LE
Epithelial Cells Turbidity Number
Casts +Protein Number
Bacteria Turbidity ↑pH Number and Type
+Nitrite
+Leukocytes
Crystals Turbidity pH Number and Type
Color +Bilirubin

SEDIMENT EXAMINATION TECHNIQUES

Sediment Stains
URINE SEDIMENT STAIN CHARACTERISTICS (in KOVA Stain)
Stain Action Function
Sternheimer-Malbin Delineates structure and contrasting Identifies WBCs, epithelial cells,
(Crystal Violet, Safranin O) colors of the nucleus and cytoplasm and casts
-KOVA Stain, Sedi-stain
0.5% Toluidine Blue Enhances nuclear detail Differentiates WBCs and RTE
-metachromatic stain cells
2% Acetic Acid Lyses RBCs and enhances nuclei of Distinguishes RBCs from WBCs,
WBCs yeast, oil droplets, and crystals
Lipid Stains: Oil Red O and Stain triglycerides and neutral fats Identify free fat droplets and lipid-
Sudan III orange-red; containing cells and casts
Do not stain cholesterol
Gram Stain Differentiates gram-positive (blue) Identifies bacterial casts
and gram-negative (red) bacteria
Hansel Stain Methylene Blue and Eosin Y stains Identifies urinary eosinophils
eosinophilic granules
Prussian Blue Stain Stains structures containing iron Identifies yellow-brown granules
of hemosiderin in cells and casts

EXPECTED STAINING REACTIONS OF URINE SEDIMENT CONSTITUENTS

Elements in Urinary Usual Distinguishing Comments
Sediment Color
RBCs Neutral – pink to purple
Acid – pink (unstained)
Alkaline – purple
Nuclei Cytoplasm
WBCs (dark-staining Purple Purple Granules
cells)
Glitter Cells Colorless or light blue Pale blue or gray Some glitter cells exhibit
(Sternheimer-Malbin Brownian movement
positive cells)
Renal Tubular Dark shade of blue-purple Light shade of
Epithelial Cells blue-purple
Bladder Tubular Blue-purple Light purple
Epithelial Cells
Squamous Epithelial Dark shade of Orange- Light purple or blue
Cells purple
Inclusions and Matrix
Hyaline Casts Pale pink/pale purple Very uniform color; slightly
darker than mucous threads
Coarse granular Dark purple granules in
inclusion casts purple matrix
Finely granular Fine dark purple granules
inclusion casts in pale pink or pale purple
matrix
Waxy Casts Pale pink/pale purple Darker than hyaline casts, but
of a pale even color; distinct
broken ends
Fat Inclusion Casts Fat globules unstained in Rare; present if polarized light
a pink matrix indicates double refraction
Red cell inclusion Pink to orange-red Intact cells can be seen in
casts matrix
Blood (hemoglobin) Orange-red No intact cells
casts
Bacteria Motile: Do not stain Motile organisms are not
Nonmotile: purple impaired
Trichomonas vaginalis Light blue-green Motility in unimpaired in
fresh specimens when
recommended volumes of
stain are used; immobile
organisms also identifiable
Mucus Pale pink or pale blue
Background Pale pink or pale purple
Cytodiagnostic Urine Testing – detection of malignancies of the Lower UT (Papanicolaou stain)

URINALYSIS MICROSCOPIC TECHNIQUES

Bright-field microscopy Used for routine urinalysis
Phase-contrast microscopy Enhances visualization of elements with low refractive indices, such as
hyaline casts, mixed cellular casts, mucous threads, and Trichomonas
Polarizing Microscopy Aids in identification of cholesterol in oval fat bodies, fatty casts, and
crystals
Dark-field Often used for unstained specimens; and aids in identification of
Treponema pallidum
Fluorescence Microscopy Allows visualization of naturally fluorescent microorganisms or those
stained by a fluorescent dye including labeled antigens and antibodies
Interference-contrast Produces a three-dimensional microscopy image and layer-by-layer
imaging of a specimen

URINE SEDIMENT CONSTITUENTS

RBCs

 Smooth, anucleted, biconcave disks, 7µm, Normal: 0-2/HPF

 Ave. reporting: 10 HPFs
 Conc. urine (Hypersthenuric)-CRENATED CELLS~ shrink due to loss H2O, rough appearance
resembles granules of WBCs but smaller (Add Acetic acid or used Supravital stain)
 Diluted urine (Hyposthenuria)- GHOST CELLS~ absorb H2O, swell, lyse rapidly, release Hb,
leaving only the cell membrane
 Most diff. to recognize by students
Reasons:
 Lack of charac. structures
 Vary in size, resembles to other urine sediments
 Freq. confused w/ Yeast cells (exhibit budding), Oil droplets, Air bubbles (highly refractile
when fine adjustment is focused)
 Determine the site of Renal Bleeding
 DYSMORPHIC RBCs- vary in size, w/ cellular protrusions, fragmented
 # & appearance is considered, AbN urine conc. affects RBCs appearance
 Small #s= Nonglomerular hematuria
 fd. after strenuous exercise= glomerular origin
 Acanthocyte with multiple protrusions- closely assoc. w/ glomerular bleeding
 CS:
 Damaged glomerular membrane or vascular injury within genitourinary tract
 Macroscopic hematuria= Cloudy red to brown
o Advanced glomerular damage
o Damage to the vascular integrity of the UT caused by trauma, acute infection or
inflammation, coagulation disorders
 Microscopic hematuria
o Early diagnosis of glomerular disorders (Glomerulonephritis)
o Malignancy of the UT
o Renal calculi
 Presence of hyaline, granular , RBC casts= Strenuous Exercise (non-patho)
  Menstrual contamination

WBCs

 Granular, larger than RBCs, Normal: <5/HPF

 Neutrophils- predominant WBC fd. in urine
 Glitter cells- sparkling appearance of Brownian movement of neutrophil granules, no CS
 Pyuria/Leukocyturia- ↑WBCs
 Bacterial infections: Upper UTI- Pyelonephritis, Lower UTI- Cystitis, Prostatitis, Urethritis
 Nonbacterial disorders: Glomerulonephritis, Lupus, Erythematous, Interstitial nephritis, Tumors
 Inflammation in the genitourinary system
 Graft rejection
 Eosinophils
 >1% is significant
 Assoc. w/ drug-induced interstitial nephritis
 AIN- Acute Interstitial Nephritis
 UTI, renal transplant rejection
 Hansel Stain- preferred, Wright’s stain- can be used
 Mononuclear cells
 Graft rejection
 Lymphocytes (smallest WBC~ resemble RBCs), monocytes, macrophages, histiocytes
= present in small #s
 Primary concern (in the identification of WBCs): Diff. of mononuclear cells and disintegrating
neutrophils from round RTE cells
 WBCs w/ amoeboid motion, difficult to distinguish bec. of irregular shape
 Supravital stain or addition of acetic acid= enhance nuclear detail
 Fewer than 5 WBCs/HPF in normal urine
 ↑ in females

EPITHELIAL CELLS

3 Types: Squamous, Transitional (Urothelial), RTE cells

1. Squamous Cells
 Largest cells, abundant, no CS
 Good ref. for focusing, Disintegrate in not fresh urine
 Resemble casts
 ↑ in female
 Clue Cell- squamous cells covered w/ Gardnerella vaginalis, vaginal infection (coccobacillus)

2. Transitional Cells (Urothelial)

 Smaller than squamous, distinct, centrally located nucleiobserved under HPO
 Spherical, polyhedral, caudate (caused of diffs: Ttransitional ECs can absorb large amt. of H2O)
 Cells in singly, in pairs, in clumps (Syncytia)= present in invasive urologic procedures, no CS
 ↑ AbN morphology (vacuoles, irreg. nuclei)= indicative of malignancy, viral infections
 3. Renal Tubular Epithelial Cells
 Resemble casts
 >2/HPF= Tubular injury
 Most clinically significant ECs
 PCT cells (rectangular shape, Columnar/Convoluted cells) are larger than RTE cells
 DCT cells (round, oval) are smaller than PCT cells (eccentrically placed round nucleus)
= mistaken as WBCs & Spherical Transitional ECs
 Collecting duct RTE cells= cuboidal, eccentrically nucleus, @least 1 straight edge,
Grps. Of 2 or more: Renal Fragments
 Indicative of Renal Tubular Necrosis
 CS:
 Condts. producing Tubular Necrosis
o Exposed to heavy metals
o Drug-induces toxicity
o Hb, myoglobin toxicity
o Malignant infiltrations
o Salicylate poison
o Acute allogenic transplant rejection
 Secondary effects of glomerular disorders
 Absorbs bilirubin= Liver damage (w/ viral infections, deep yellow)
 May contain yellow-brown hemosiderin (Prussian blue stain)
 Hb= converts to Hemosiderin
 Single cuboidal= Salicylate poison

OVAL FAT BODIES

 Lipid-containing RTE cells (appear highly refractile)

 Stain w/ Sudan II, Oil Red O fat stain, Polarized microscopy
 Comp. of: Triglycerides, Neutral fats (Orange-red), Cholesterol (no stain)
 Chole. under Polarized light= Maltese cross
 Reported as Ave. # per HPF
 Free-floating fat droplets= confused w/ Starch & Crystal particles
 Lipiduria
 most freq. assoc. w/ damage to the glomerulus caused by Nephrotic Syndrome
 severe tubular necrosis, DM, trauma cases
 Lipid storage dse. = Large fat-laden histiocytes
 Bubble cells (injured cells due to dilated ER)= ATN (Acute Tubular Necrosis) ~large, nonlipid-filled
vacuoles of RTE cells, also seen w/ normal renal tubular necrosis & oval fat bodies

BACTERIA

 Not normally seen in urine, No CS, + Nitrite,

 Significant for UTI, bacteria w/ WBCs
 Correlation: + Urine Culture
 Phase Microscopy
 Motile orgs: Useful in differentiating from Amorphous phosphates & urates
  Enterobacteriaceae- most freq. assoc. w/ UTI
 Staphylococcus, Enterococcus= can caused UTI

YEAST

 Small, refractile oval w/ or w/out bud

 Severe infections appear branched, mycelial forms
 Primarily Candida albicans= present in DM, Immunocompromised pxs., Women w/ vaginal moniliasis
 True yeast infection= presence of WBCs

PARASITES

1. Trichomonas vaginalis
 Most freq. parasite in urine
 Pear-shaped flagellate w/ undulating membrane
 Resemble WBCs, Transitional ECs, RTE cells (when not moving)
 Uses Phase microscopy (enhanced flagella visualization)
 STD w/ vaginal inflammation
 Asymptomatic in males
2. Ova of Schistosoma haematobium
 Assoc. w/ bladder cancer
 Can be a fecal contamination
3. Ova of Enterobius vermicularis (Pinworm)- most common contaminant

SPERMATOZOA

 Oval, slightly tapered heads, long, flagella-like tails

 fd. in female and male urine , No CS
 Urine is toxic to sperms
 + RST for protein due to ↑ amt. of semen

MUCUS

 Protein prod. by glands & ECs of the lower genitourinary tract and RTE cells
 Uromodulin (Tamm-Horsfall Protein)
o major constituent of mucus, Glycoprotein excreted by RTE cells of DCT and upper CDs
 Micro: THREAD-LIKE w/ low refractile index
 Bright-field microscopy, subdued light
 Resemble clumps of mucus w/ Hyaline casts
 ↑ in females, No CS in male or female