Urine Under MicroscopeDr. Abrar Ali KatparDepartment of Nephrology

King Khalid Hospital-Hail

Kingdom of Saudi Arabia.

Introduction

Urinalysis is perhaps the most common investigation performed in clinical

practice it is noninvasive inexpensive and easy to perform test, which

provides wealth of useful information especially on patients with renal

disease. It not only assists in the diagnosis of renal disease but can also

provide information about the severity and outlook of the disease.

In order to obtain maximum information from this elementary investigation,

the physician looking after the patients should preferably do itself. Because

he alone will ultimately be in better position to correlate the findings with

the disease and fully grasp its implications.

Urine Collection

For microscopy, urine should be collected in an open-mouth, clean but not

necessarily a sterile container. Although the first morning specimen is most

concentrated and acidic due to overnight fast, the second urine of the

morning is preferable for microscopy, as it too is concentrated and acidic but

without overnight stay in the bladder which causes lysis of the cells and cast.

For reliable urine microscopy and culture, carefully collected Midstream Urine without contamination from the genitalia is essential. For this, the patient must be instructed about the correct procedure. The patient should be explained in plain simple language that before collecting a specimen of his/her urine, hands and external genitalia should be gently washed with water or saline: disinfectants should not be used. Also that overzealous cleaning is harmful, because it can possibly cause abrasions of the periuretheral area and result in bleeding; thus contaminating the specimen. The patient should also be explained that it would be ideal for him/her to have a moderately full bladder. Furthermore to avoid contamination men should be advised to retract their foreskin (if Present) and women should hold their labia apart. Once they start voiding, the first 200ml of urine should be discarded, and the next 100-200 ml of urine should be collected in container by moving it in and out of free-flowing urine stream.

If a proper mid-stream specimen cannot be collected as in the case of infants,

or young children, or aged persone because of there physical shortcoming or

mental handicap, the requisite urine specimen can be obtained either by an

open-ended catheter or suprapubic aspiration.

STORAGE OF URINE

To avoid any possible alterations in physical or chemical feature, urine should

ideally be analyzed with in an hour of Voiding. With passage of time the cells

in the specimen tend to lyse and cast disintegrate especially in urine with low

specific gravity and or alkaline pH. Moreover, with prolong stay at room

temperature, bacteria have tendency to multiply obscuring the very cells and

casts. Several means of preservations of cells with chemical reagents (0.5%

Glutaryldehyde, Boric Acid, Formalin) and refrigeration at 4 degrees

centigrade have been proposed. But these tends to interfere with chemical

reaction. On the other hand refrigeration of urine causes crystals to

precipitate, obscuring cells and cast.

Starting

URINE MICROSCOPY

To obtain maximum possible information it is important to examine the urine

using a phase-contrast microscope with the added facility of polarized light.

Colorless, or unstained objects, have little effect on the amplitude of light

waves; hance observing such objects under bright field is not very helpful.

With phase-contrast microscopy, light passing through a relatively thick or

dense part of a cell, such as the nucleus, is retarded compared with light

passing through media only, or a thin part of the specimen. The degree of

retardation, and the extent of the phase-shift that result, is proportional to

the thickness of the object.

The the phase-contrast microscope thus exploit the interference effects that

takes place when the two types of light recombine. If the waves complement

each other, the light is brighter, while if they are 180 degrees out of phase,

one cancels other, and the image becomes dark. To sum up phase-contrast

microscopy enhances the ability to see and identify cells and cast, which are

not visible with bright-light microscopy. Polarized light is particularly useful in

identifying free fat, cholesterol and crystals of calcium oxalate, uric acid.

To quantify formed elements in the urine, most authors recomned

centrifugation of a fixed volume of urine, transferring a drop of urine from

the deposit to a glass slide and then counting the cells under high power. With

this semi-quantitative method, cells are described as number or a range of

cells observed per high power field(HPF).

This method has been found open to error at almost every step. The number

of cells/HPF depend largely upon various factors namely:

a- The quantity of urine centrifuged

b- The type and speed of centrifuged

c- The duration of centrifugation

d- The method of discarding the supernatant and

e- The amount of urine in which the sediment is re-suspended.

The volume of urine under the cover slip also varies and depends upon the

size of the drop of urine, weight of the coverslip and viscosity of the urine.

It is not easy to control so many variables; hence this method of semi-

quantitative estimation of cells can be erroneous.

Therefor, with a view to avoid such errors, counting of cells using

uncentrifuged urine and a counting chamber is recommended. A Fuchs-

Rosenthal counting chamber is particularly useful

FORMED ELEMENTS IN THE URINE

CELLS: Red Blood Cells, White Blood Cells, Renal Tubular Epithelial Cells, Transitional Epithelial Cells, Squamous Epithelial Cells, Oval Fat Bodies, Erythrophagocytes.

CASTS: Hyaline, Granular, Red Blood Cells, White Blood Cells, Tubular Cell, Waxy, Broad, Lipid-laden.

CRYSTALS:

Urate, calcium phosphate, Calcium carbonate, Oxalate, Triple Phosphate, Cystine, Cholesterol, Drugs (Sulphadiazine, Acyclovir, Triameterene, Pridoxylate, Primidone).

INFECTING ORGANISIM:

Bacteria, Yeast, Trichomonas, Nematodes.

MISCELLANEOUS: Spermatozoa, Mucus threads, Cylindroids, Fibers, Starch, Hair, Myelin Bodies.

Red blood cells

Red Blood Cells may be found in normal urine. With semi-quantitative methods, up to 1-2

RBC/HPF are considered normal. With phase-contrast microscopy, the upper limit of normal in

un-centrifuged urine is 15000 cells/ml and all are of glomerular origin.

About 30% of cells are lost either with centrifugation or resuspension.

Therefore upper limit of normal in a centrifuged urine is 10,000 cells/ml.

The normal limit with bright light is lower, as some of the cells seen with phase contrast are

either not visible or misinterpreted as debris.

RBC’s are likely to undergo several morphological changes in the urine.

Some of these changes are non specific and due to physiochemical environment. FIG:6

Brich and fairley were

the first to note that

rbc’s cells in the urine of

patients with

glomerulonephritis have

peculiar changes (fig:7-

13).

Other s subsequently

confirmed these

findings.

White blood cells

Like RBC’s, white blood cells (WBC’s) may be found in normal urine.

With the semi-quantitative method of estimation, 1-2 cells/HPF is considered normal.

Using a counting chamber and phase-contrast microscope 2000wbc/hpf is considered to be the upper limit of normal in properly collected midstream urine specimen.

Neutrophils are the most frequent WBC’s in urine, with an average diameter of 10 micrometer they are slightly larger than a RBC.

They have a granular cytoplasm and a lobulated nucleus.

Increased numbers of neutrophils in the urine indicates urinary-tract infection.

When their number increases without

bacteriuria or growth on culture, renal

tuberculosis, renal calculi, renal papillary

necrosis, polycystic kidney disease and

infection with fastidious organism should be

considered.

Lymphocytes are smaller than neutrophils.

Although they can be identified with phase-

contrast microscopy, their identification

usually requires special staining.

They are frequently found in the urine of

renal transplant recipient’s during episodes

of acute cellular rejection.

Eosinophils are slightly larger than

neutrophils and are only identified by

special stains.

They are seen in acute interstitial nephritis

and in patients with athero-embolic disease.

Mucus

Mucus in the urine appears as

ribbon-like threads of variable

width and length.

It is a normal constituent of urine

and of no pathological

significance.

Cylindroids

These are similar to cast with

one of their ends resembling a

mucus thread.

In the past there was some

controversy over their origion.

It is now clear that they are

composed of tamm-horsfall

mucoprotein and are therefore

cast.

They may contain particles like

cast. Fig:

Cast

Cast Are elongated cylindrical structure.

They acquire shape from the tubular lumen (distal tubular and collecting

ducts) in which they are formed. They are composed of Tamm-Horsfall

glycoprotein.

Cast formation is favored by factors which promote aggregation of Tamm-

Horsefall proteins, which include increased urinary concentration of

electrolytes, hydrogen ions, and ultra-filtered proteins.

Iteraction between the protein and hemoglobin, myoglobin, Bence-Jones

proteins or radio contrast media also favor formation.

Different formed elements transported along the nephron are traped in them

producing various types of Cast (Table).

Igor Tamm &

Frank Horsfall

Glycoproteins

Uromodulin

Gene= UMOD

Chromosome16

HYALINE CAST These consist of Tamm-Horsfall

Mucoprotein only.

As they have low refractive

index they easily escape

detection if a bright-field

microscope is used.

They are occasionally present

in normal individuals, but are

increased in renal diseases.

They are also observed in

patients with acute cardiac

failure, fever, those receiving

loop diuretics, and in normal

people after strenuous activity.

GRANULAR CAST

These contain granules that

may be either fine or

coarse.

Granules consist of ultra-

filtered proteins or

degenerated cells.

Their presence in the urine

is always pathological.

Large numbers of coarse

granular casts are

particularly seen in acute

tubular necrosis.

WAXY CAST

Waxy casts are highly

retractile, usually large with

clear cut edges.

Their composition is different

from other cast, and are

resistant to dissolution to

alkaline urine.

They are typically found in

patients with advanced renal

failure.

ERYTHROCYTE CAST

Erythrocyte cast show enormous

Variation in morphology, and

contains variable number of RBC’s.

They indicate glomerular bleeding.

LEUKOCYTE CAST These contain variable number of

neutrophils.

They are commonly found in acute

or chronic pyelonephritis.

They are also seen in proliferative

glomerular lesions like acute post

streptococcal glomerulonephritis,

active mesangiocapillary

glomerulonephritis and lupus

nephritis.

It is at time, difficult to

distinguish leukocytes within the

cast from tubular epithelial cells.

FATTY CAST These contain variable amount

of lipids and are usually seen in

the urine of patients of with

Nephrotic syndrome.

Lipid Droplets Lipid droplets can be seen

in the urine either as free

droplets, within the renal

tubular epithelial cells Fig:

or with in the Cast.

Lipids are usually seen in

the urine of patient with

heavy proteinuria.

How ever they are also

described in patients with

Low-Grade proteinuria.

Oval Fat Bodies Oval Fat bodies are renal

tubular epithelial cells full

of cholesterol esters.

The fat is easily identified

with polarized-light

microscopy when a

‘Maltese Cross’

appearance is seen.

Squamous Epithelial Cells

Thease are large (mean

diameter 55 um) flat cells with

abundant granular cytoplasm

and a small central nucleus.

They are exfoliated from the

bladder and urethra.

Un women with vaginal

discharge, they contaminate the

urine.

Renal Tubular Epithelial Cells Renal tubular epithelial cells,

most commonly found in the urine are from the proximal tubules.

They are round to ovioid cells with an average diameter of 13um and have a single nucleus.

Normally they are not present in the urine.

They may be found in increase in number after exercise and after ingestion of certain drugs.

They are frequently present in case of acute tubular necrosis and acute renal allograft rejection.

Erthryophagocytes

An erythrophagocyte is

renal tubular epithelial cell

whose cytoplasm contains

erythrocytes that are

phagocytosed during their

passage along the nephron.

It may be seen in all form

of the glomerulonephritis

and has the same

significance as erythrocyte

Cast.

Myelin Bodies In Fabry’s disease,

glycosphingolipid laden

epithelial cell of Loop of Henle

and distal convoluted tubules

desquamate and are detectable

in the urine sediment.

Lipids in these bodies

demonstrates “Maltese Cross”

under polarized light.

They can be differentiated from

cholesterol drplets by their

irregular shape. FIG:

Candida Candida is the most frequent

yeast found in the urine.

They appear as pale-green

cells often nucleated, and

with smooth well-defined

walls.

They are commonly seen in

the urine because of

contamination from genitalia.

They can also grow in urinary

tract of patient with: diabetes

mellitus; having indwelling

catheters; on prolong

antibiotic therapy; or

receiving immunosuppression.

Bacteria

Bacteria are frequently seen in

urine sediment.

They do not necessarily indicate

infection and may be the result

of contamination of urine.

Presence of leukocytes with

bacteria increase the probability

of the infection.

Crystals

The urine can contain several types

of crystals.

Uric Acid, Amorphous Urates,

Calcium oxalates, Cystine, Leucine,

Tyrosine, and Cholesterol crystals

are found in acid urine.

Whereas crystals of Calcium

phosphate, Tripple Phosphate and

Amorphus Phosphate are found in

alkaline urine.

Thank you