M4U1: SEDIMENT PREPARATION AND EXAMINATION Flashcards
This is a specialized centrifuge procedure used to produce a monolayer of the cellular constituents in various body fluids on a microscopic slide
cytocentrifugation
a chemical reaction used to identify the presence of iron in body fluids
Rous Test/Prussian blue staining
a glycoprotein that is secreted by the renal tubular cells of the thick ascending loop of Henle and distal convoluted tubules
Uromodulin/Tamm-Horsfall protein
This is the cellular pellet remaining after urine centrifugation
urinary sediment
A normal urinary sediment should be free of ___, contain ___
crystals
less cells and very low concentration of proteins
This refers to the ability of a substance to refract light in two directions
Birefringent/Double refractile
Type of microscopic illumination in which a lamp condenser located above the light source focuses the image of the light source onto the front focal plane of the condenser
Kohler illumination
Term describing objective lenses that remain in focus when the user switches from one objective to another of a different magnification
Parfocal
A traditional method developed to standardize the quantitation of formed elements in the urine
Addis count
Addis count uses a ___ to count the number of RBC, WBC, casts and epithelial cells present in a ___
hemocytometer
12-hour specimen
The ___ contribute formed elements of urine
blood, kidney, lower genitourinary tract and external contamination
These are to ensure the accuracy and precision of the urine microscopic examination
appropriate microscopy techniques
protocols on the standardization of sediment preparation should be implemented
What is the ideal specimen for sediment preparation ?
freshly collected or adequately preserved first morning specimen (midstream clean catch)
Formed elements such as RBC, WBC, and hyaline casts disintegrate rapidly in ___ urine
dilute, alkaline
How much specimen volume is used for sediment preparation ?
12 ml or volume ranging from 10-15 ml
If obtaining a 12 ml specimen is not possible for pediatric patients, the volume can be reduced to ___
6 ml
What must be done if the volume for sediment preparation is below 12 ml ?
noted on the report form to allow necessary corrections
What is the centrifuge speed used for sediment preparation and why ?
400-450 x g for 5 minutes
allow optimal sediment concentration without damaging fragile formed elements such as cellular casts
Why is it important that the centrifuge brake is not used ? and how does it affect the results ?
it will cause the sediment to resuspend, resulting in erroneously decreased numbers of formed elements in the concentrated sediment
What sediment concentration is desirable in manual techniques ?
24:1 or 12:1
This is done to maintain a uniform sediment concentration factor when decanting supernatant urine
should be aspirated off rather than poured off, unless otherwise specified by the method in use
Other than using a pipetter what else can be done for resuspension ?
gently flicking the bottom of the tube
Why is thorough resuspension of urinary sediment essential ?
provide equal distribution of elements in the microscopic examination field
What can too vigorous agitation of the sediment do ?
ma cause fragile and brittle formed elements, such as RBS casts and waxy casts, to break into pieces
When using the conventional glass slide method, the reccomended volume is ___
20 ul
Why must you ensure that the specimen does not flow outside of the coverslip ?
may result in the loss of heavier elements such as casts
A minimum of __ fields under both low and high-power objectives is examined
10
What is the purpose of examining the the slide under low power first ?
detect casts
ascertain the general composition of the sediment
The magnification is switched to high power field to ____ if the conventional glass-slide method is being used
delineate the structures seen
What happens to RBC in hypertonic urine ?
become smaller
How does an RBC look like when viewed from above and from the side ?
above: disk with central pallor
side: hourglass
What happens with RBC crenates ?
lose their biconcave shape
become spheres covered with spicules or crenation’s
What are the conditions that need to happen for an RBC to become a ghost cell ?
hypotonic urine
release their hemoglobin
What are the characteristics of a ghost cell ?
colourless
empty
intact membrane
no hemoglobin
What type of microscopy is best used to observe ghost cells ?
phase contrast
interference contrast
___ urine promotes RBC lysis and disintegration, resulting in ghost cells and RBC remnants
alkaline
What formed element is associated with acanthocytes or sickle cells in increased numbers ?
dysmorphic or distorted erythrocytes
Abnormal urine concentration affects RBC appearance, and small numbers of ____ cells are found with non-glomerular hematuria
dysmorphic
Hematuria accompanied by red blood cell casts, glomerular or tubular bleeding may be indicated, such as in cases of ___
glomerulonephritis
pyelonephritis
cystitis
presence of calculi
When an increased number of RBC’s is present without casts or proteinuria, what may be the cause of the bleeding ?
there is bleeding below the kidney
caused by contamination (menstrual blood, hemorrhoid)
Non-renal disorders such as ____ may also be attributed to the occurrence of hematuria
hypertension smoking appendicitis tumors trauma drugs (anticoagulant drugs and drugs inducing a toxic reaction like sulfonamides)
What may be the reason to a specimen having a positive chemical test for blood but microscopic examinations reveals no RBCs ?
lysis might have occurred especially if the sample is hypotonic or alkaline
lysis have already occurred within the urinary tract before collection
What may cause a false positive blood chemical test ?
myoglobin
microbial peroxidases
strong oxidizing agents
What may be the reason to a specimen having a negative chemical test but RBCs are present microscopically ?
ascorbic acid interference
possible that formed elements observed are not RBCs but look-alike components
What are formed elements that may look-alike RBC ?
yeast
oil droplets
bubbles
monohydrate calcium oxalate crystals
What are the solutions to confirm identity of RBC from look-alikes ?
staining
polarizing microscopy
What is the use of sternheimer-malbin stain ?
characteristically colours RBC
neither yeast nor calcium oxalate crystals stain
How to differentiate calcium oxalate crystals from RBC ?
polarizing microscopy - calcium oxalate crystals polarize light while RBC doesn’t
sternheimer-malbin stain - doesnt stain while RBC does
add 2% acetic acid - lyse RBC but doesn’t eliminate calcium oxalate crystals
How to differentiate yeasts from RBC ?
sternheimer-malbin stain - doesnt stain while RBC does
add 2% acetic acid - lyse RBC but doesn’t eliminate yeasts
tends to be spherical or ovoid rater than biconcave, often exhibits budding
How to differentiate droplets of oil from RBC ?
Bubbles and fat droplets:
variation in size
uniformity in appearance
high refractility
How to distinguish WBCs from crenated RBCs in a hypertonic urine specimen ?
acetic acid or toluidine blue stain
make it easier to see the nuclei of WBC
What WBC is most often observed in urine and why ?
neutrophils because they predominate in the peripheral blood
What is the reason for naming neutrophils in dilute, hypotonic, alkaline urine “glitter cells” ?
Brownian movement of the refractive cytoplasmic granules
This refers to the uncontrolled and fat movement of granules within the cytoplasm as a result of collision forces
Brownian movement
What are the degenerative changes of neutrophils as they age ?
lobed nuclei fuse, resemble a mononuclear cell
formation of blebs
blebs can detach and become free floating in urine
blebs may develop and remain within the cell, pushing the cytoplasm to one side and giving rise to large pale areas intracellularly
development of myelin forms, from the breakdown of the cell membrane
additional vacuolization
rupturing
pseudopod formation
What stains are used for eosinophils ?
Hansel strain is the primary stain
Wright stain can be alternatively used
What conditions allow neutrophils to lyse rapidly ?
room temp
dilute, hypotonic, alkaline urine
50% in 2-3 hours
These mononuclear cells are large cells and may appear vacuolated or contain inclusions
monocytes
macrophages
histiocytes
When are macrophages called histiocytes ?>
reside in interstitial tissue
What are the characteristics of enlarged macrophages ?
irregular, kidney-shaped nuclei
abundant cytoplasm
This refers to the increased pus excretion in urine
Pyuria
This refers to the increase in urinary WBCs
Leukocyturia
When would trichomonads and mycoses appear in urine ?
women: contaminants from vaginal secretions
men: UTI
These formed elements predominate in renal transplant rejection
lymphocytes
____ is a good predictor of acute interstitial nephritis associated with drug hypersensitivity, particularly to penicillin and its derivatives
eosinophiluria
Urinary eosinophils are primarily associated with ___
drug induced interstitial nephritis
In cases of acute allograft rejection, the presence of large numbers of ____ in a kidney biopsy specimen is considered a poor prognostic indicator
eosinophils
What would consist of the physical and chemical examination of urine with increased WBC ?
cloudy
gray-white sediment button
depending on extent of infection, strong foul odor
increased specific gravity
increased pH
How much WBC is usually detected in LE screening test ?
10-25 WBC/microliter
What is the reason for LE test + but no WBC present microscopically ?
WBC lysis
What is the reason for LE test - but with WBC present microscopically ?
lymphocyte is present, they have no leukocyte esterase
Owing to ____, hypotonic urine could cause the leukocyte to be diluted such that it is below the detection limit of the LE reaction
hydration
What is the reason for LE test - with increased WBC found microscopically ?
check if reagent strip is functioning properly
look-a-like elements such as RTE may be present
WBCs in the process of ameboid motion may be difficult to distinguish from epithelial cells because __
of their irregular shape
How to make monocytes and macrophages more identified in the presence of RTE cells ?
suprivital stains on the urine sediment
making a cytocentrifuged preparation followed by Wright’s or Papanicolaou’s stain
addition of acetic acid to enhance nuclear detail
What is the reason for epithelial cells being commonly resulted in urine ?
normal turnover of old cells (sloughing)
epithelial damage caused by inflammatory processes/renal disease
What are the granulations found in squamous epithelial cells referred to as ?
keratohyalin granules
This formed element is the first structure observed when the sediment is examined under low-power magnification
squamous epithelial cells
Where do squamous epithelial cells originate from ?
linings of the vagina and the entire female urethra
lower/distal portion of the male urethra
What pathologic significance does increased squamous epithelial cells mean ?
women: indicate vaginal or perineal contamination
men: uncircumcised, specimen contamination
How can a squamous cell be considered a clue cell ?
covered with Gardnerella coccobacillus
bacteria should cover most of the cell surface and extend beyond the edges of the cell
What is the routine testing for clue cells and what is it used to assess for ?
examine vaginal wet preparation
assess vaginal infection by bacterium Garnerella vaginalis
What forms do transitional epithelium cells appear in ?
spherical
polyhedral
caudate
Why do transitional epithelium cells have several forms ?
caused by the ability to absorb large amounts of water
What are the three principal layers of transitional epithelium in the bladder ?
superficial cells
intermediate cells
basal cells
Where do transitional epithelium cells originate from ?
lining of the renal pelvis, calyces, ureters, and bladder
upper portion of the male urethra except the distal portion
Transitional epithelium cells are usually present in small numbers in normal urine of ____, representing normal cellular sloughing
0-2/hpf
This is the increased number of transitional cells seen singly, in pairs, or in clumps
Syncytia
Syncytia is present following invasive urologic procedures such as
catheterization or other types of instrument procedures (chemotherapy and radiation)
An increase in transitional cells exhibiting abnormal morphology such as vacuoles and irregular nuclei may be indicative of ___
malignancy (transitional cell carcinoma and neoplasia in the genitourinary tact)
viral infection
Urine specimens with increased transitional epithelial cells will appear ___ and is ___ for blood and protein if malignancy is considered
turbid
positive
How to differentiate PCT cells from a granular or fatty cast ?
casts do not have nucleus
How to differentiate DCT cells from WBC’s and spherical transitional epithelium cells ?
observation of the eccentrically placed round nucleus
In observing a collecting duct cell, what must be noted for it to be identified as a fragment ?
at least three cells must be sloughed together with a bordering edge intact
What is the one distinguishing characteristic of a small duct cell ?
flat/corner edge on the membrane of the cell
Small duct cells become wider as they approach the ____
renal calyces
____ cells are the most clinically significant of the epithelial cells
RTE
The presence of increased amounts of RTE is indicative of ___
necrosis of the renal tubules (acute ischemic or toxic tubular necrosis) from heavy metals
drug toxicity (aminoglycosides)
____ are found following trauma, shock, or sepsis, and indicate ischemic necrosis and severe tubular injury with basement membrane disruption
collecting duct fragments
____ are often observed in cases pyelonephritis, acute tubular necrosis, kidney transplant, rejection, and salicylate poisoning
large duct cells
RTE cells nucleus is not easily visible in unstained sediment, what is done to make it easier to identify ?
cytocentrifugation followed by Papanicolau’s staining of the urine sediment
RTE can absorb ____ (as a result of liver damage) present in the filtrate and appear deep yellow
bilirubin
RTE can absorb ___ and be converted to hemosiderin
hemoglobin
Following hemoglobinuria, the RTE cells may contain the characteristic ____
yellow-brown hemosiderin granules
Urine specimen with increased RTE would appear ___ and ____ would be positive for chemical tests
turbid
blood
protein
LE
nitrite
___ that absorbed lipids/fats presents in the glomerular filtrate are referred to as oval fat bodies
RTE cells
monocytes
macrophages
Oval fat body presence may indicate +++
glomerular dysfunction
renal tubular cell death
lipiduria caused by nephrotic syndrome, sever tubular necrosis, diabetes mellitus, and trauma that cause release of bone marrow fat from long bones
In cases of acute tubular necrosis, RTE cells containing large, ___ may be seen along with normal renal tubular cells and oval fat bodies
nonlipid-filled vacuoles
Referred to as ___, they appear to represent injured cells in which the endoplasmic reticulum has dilated prior to cell death
bubble cells
Identification of oval fat bodies is confirmed by staining the sediment with ___ or ____ and examining the sediment using ____ microscopy
Sudan III
Oil Red O fat stains
- triglycerides and neutral fats are stained orange-red
polarized
What characteristic is observed when cholesterol is under polarized microscopy ?
maltese cross
Staining increases the overall visibility of elements using bright-field microscopy by ___
changing their refractive index
This is a supravital stain, consisting of crystal violet and safranin, most frequently used in urinalysis
Sternheimer-Malbin stain
What colour does sternheimer-malbin stain epithelial cells, WBCs, RBCs, casts inclusions, and low refractile elements to ?
pink to purple
A metachromatic stain that provides the enhancement of the nuclear details of a cell
0.5% solutions of toluidine blue
This stain can be useful in the differentiation between WBCs and renal tubular epithelial cells and is also used in the examination of cells from other body fluids
0.5% solutions of toluidine blue
This stain with the use of polarizing microscopy, it can be used to confirm the presence of fat
Oil Red O and Sudan III
This stain is used primarily in the microbiology section for the differentiation between gram-positive and gram-negative bacteria
Gram stain
This stain is used to facilitate the visualization of hemosiderin, free floating or embedded in epithelial cells and casts
Prussian blue stain
This is a type of microscopy that produces a magnified image that appears dark against a bright or white background
Brightfield microscopy
This type of microscopy is used to identify and classify birefringent substances that shine brilliantly against a dark background
Polarized microscopy
substances in urinalysis that can polarize light are
uric acid crystals monohydrate calcium oxalate fiber plastic fragments amorphous crystals (urate:strongly, phosphate:weakly) fatty casts cholesterol globules starch granules
substances in urinalysis that do not polarize light are
RBCs casts mucus bacteria cellular debris
Type of microscopy in which variations in the specimen’s refractive index are converted into variations in light intensity or contrast
Phase-contrast microscopy
This type of microscopy is ideal for viewing low-refractile elements (hyaline cast, bacteria, mucus threads) and living cells (trichomonas)
Phase-contrast microscopy
This type of microscopy provides a three-dimensional image of an element, either low or high refractive index, thus enhancing the cellular debris
Interference-contrast microscopy
This type of microscopy is often used for unstained specimens
Dark-field microscopy
Dark-field microscopy is often used to identify the specimen ___
spirochete Treponema pallidum
This type of microscopy is used to detect bacteria and viruses within cells and tissues through a technique called immunofluorescence
Fluorescence microscopy
