Examination of Urine (From Manual of Laboratory Medicine Book) .pdf

Urine examination is one of the most important and basic investigations in laboratory medicine. The book explained the complete urine examination step by step, including physical, chemical, and microscopic analysis.

1. ”
9. URINE EXAMINATION

Man has been curous about urine rom the
aries times. The Babylonians and Sumerians
studied urine and attempted to relate ts
‘appearance to various human ailments. Early
Hindu Iorature desenbes "honey uno” as ono,
‘which atracted ant, Prive analyst included!
tasting of urine. The early 190 century marks
the sum of Scenic. meihods urine
examination. In 1827, Richard Bight studied
tine at Guys In 1908,

Benedict described soon for quanttaton of
glucose in une. In 1941, Dr. Waller Compton
Created cito, for the measurement of
Feducing sugars. This est was the forerunner of
the wide range of convenient urine tests to be
Used allover he word.

Urine testing provides rapid, valuable and
relate information ino the halt status ofthe
Patent Uno is a valuable index to many
Formal and pathologic mechanisms. Its. à
Complicated aqueous solution of various organic
and inorganic substances. Those substances
are products trom the body metabolsm (ether
‘normal of abnormal) or products derived directly
from foods

Many urine characteristics and components aro
Unstable. The urines also an excellent cuture
modum. Therefore, al specimens should bo
examined within 30 min ol collection or samples
Should be rfigeraied. The delay in tesüng may
result in gross changes, which affect the test
resuls. Bactoal_acton aflecs pH. glucose,
ketones and RECS. Hydrayis and oxidation
afect birubin. Delay and ‘exposure to light
Foul in photo-degradaton of uoblinogon to
robin and volaisation of acaone, I shoud be
noted that sodimente are ‘unstable even at
Feference temperature he urine saka,
Major sources of error are:

‘Bactral or chemical contamination
Contamination with menstrual blood.
Contamination with vaginal and uretral
discharges.

Inadequate mixing before examination.
Wironginadequate preservative
‘TYPES OF URINE SPECIMEN

First morning specimen
In provides concentrated urine as the bladder
Inadbated it the whole night. is best or nite,

protein, good for microscopic examination and
Culture and sensitivity. The casts may have
Seterorated and bacteria” may afect te
‘lucose reading.

Random specimen

Its the most common type and most convenient
‘sample. It is good for observing physical
Characteristics, "chemical analysis and
‘entiation of cass, crysals and cl
‘Second-voided

‘The fest moming specimen is discarded and
‘second specimen is collecte! Formod elements
amas act

Postprandial
Its collected ater meat (usualy after hours). I
ls good for glucose and protein estimation. Unne
‘sugar testing now has limitod diagnostic or
Prognostic value.

‘Timed specimen
ls a combination of all voiding over a length of
time. Two-hour specimen is good. for
roblinogen and 24-hour specimen 1 good for
uantlaive ufnary components. estimation.
‘Timed urine specimens are colleciad in dynamic
function tests. (seo WATER. DEPRIVATION
TEST on page 364).

Foley catheter

Disinfect a porton ofthe catheter with alcoho.
Punelunng the tubing direciy wih a storie
Syringe and needle and aspirate the urine. Place
‘Fine in a serie container, & shoud never be
colectas rom drainage bag

‘Apart trom these procedures, urine specimen
can also be collected by suprapubic aspiration
‘nd cystoscopy

PHYSICAL EXAMINATION

Volume
‘Measuring volume of urine in a calbrated
‘finder is a very messy procedure, therefore is
‘ot recommended. Beter methods to weigh Ihe
ine with the contanar and container wiheut
Urine. Ding the net weight of urine. wih
‘specie gay gives te volume,
vespa wine

Normal 24 hours volume depends upon age,
fad intake and weather. In an adult le 800:

2. presenti phonytketonuria,
Colour
Normal colour of urine is pale yellow because of

ih ingoston of certain foods, 1004 dyos.
‘medications (Table 9.1). There are

that can impart colour to urine. This possibilty
shoud be excluded before interpreting the
Colour change,

‘Appearance

Freshly voided urine is lea. It may become
‘cloudy’ on standing because of amorphous
phosphates, rates, oxalaes, pus, bacon, fal
‘and cyte.

PH
DH of the urine is the measure of hydrogen fon
Concentration of the urine. Urine pH has mites
y alone and is useful only when related o
‘other information. I une i eto Stand, is pH
is alered as urea changes to ammona.
Therefore, frosh specimen ts tested for pH
Urine pH isan important screening test in renal
diseases respiratory diseases, certain metabolic
disorders, and some specie therapeutic

regimes,
Normal pH is acidic (6.0 10 6:5) but kidney has
the capabity of changing over a wide range
(46080, mean 60). Urine sample may show
‘one ofthe folowing reactions when tested wit)

‘blue and rod litmus paper:

1. Aid: PH <7.0 (tue mus changes to red).

2. Aline: pH >7.0 (red mus changes to
blue)

3. Neutral pH 7.0 (No change of colour in both
‘he im).

4. Amphoterc (fered): When both the Iimus
‘shows colour change.
Tile Cu ard oa ae

aera
tone
PH of urine Is checked with indicator paper or
‘tips. Stips cany math! red (red stnps) and
blue (ue svp). provide a pH
range of 50-90. Stip is dipped in urine and
touching the edge of container drains of excess
ine. Colour is compared with colour chart
Highly ci urine observed in high proton det,

‘bstructon, vomiting: and metabolic alkalosis.
Alkaline pH ofthe urine is also observed in UTI
win urea producing organisms.

‘Specit Gravity
‘Ths test has significance inthe interpretation of
‘other results. Reference range for unno speciie
gravity is 1010102, In early moming
Specimen it may be 1.020. Is low in kidney

3. diseases, abnormal antfuretic hormone
‘excrion and neubom babies (100251004),
‘and high in dehydration, fever and vomiting
‘Many contrast agents excreted in urine interfere
win comenional speciie gravy
messurements Urino shouldbe collected before
‘2dministraton of contrast medium or at a gap of
{wo or more days alerwards. Contras agents
do not stor. the colormetne metnods. may
‘excoed 1050 i ealesated by urinomelor

Determination
‘Specite gravy can be
termined by urinometer
felraciometer, or by
‘utomatod equipment fa
Urinometer fused for ie
purpose then the urine is à
Slowed to come to room À
temperature and is mid,

wol. Urnomeler tube à

lo,

filed by urine and urinomotor s Road ino 1
Lower meniscus is road on the scale and is
Corrected for temperature as most unnometers.
re calbrated al 20°C. For each change of 3°C,
0007 le added or suhraca. Win sach 1%
Protein in urine the specific gaviy increases by
0.003. wie for each 1% of glucose i increases
by 0004. In “specimens containing these
Substances, specie grauty should be conectas
‘ccordingy. Spectic gravy over 1020
(yperestnenuria occurs in decrease intake of
fads. fever. dehydration, and IV abumin
administration. Specie gaviy less than 1.008
(ypoestrenura) “occur in increased Mu
Intake, hypothermia, alkalosis, progressive renal
flue and sic col anaemia Specie graviy
ed at 1.010 occurs in con renal fase oF
end stage Kidney disease,

CHEMICAL EXAMINATION
PROTEINS

In normal urine protein is undetectable by
rouino method. Is an important indicator ot
renal diseases. It may be used o montor

E

proteoses.
Procedure: Fü 34 ola tubo with urine
and hea upper part of while rotating
the tbe. Tuti wil appear if proteins
© phosphates are present. Add 2-3
rope of acetic aid ri posits
IS due to proteins.

b. Acid precipitation: Many chemical
‘agents “ike suphosaloyic acid. nilo
ais procpiute prleins. Othor
constants of uine may also be
Preciptated th these chemical agents

© Sullosalieyie acid test (Kingsbury
and Clark}; Test is basod on the
Principe that proteins ae denatured and
Procptated by ac.

Procedure One mi contifuged urine is
Taken in Mo test tubes. To ono tubo 3
m of 3% sulfosalcyic acd is added,
ie the other tube with unne only acts
8 a bank, Both the tubes are alowed
to stand for 10 min. The tides” are
Compared fr Biya ao min
‘svaiable. standards

{Kingsbury "Ca standar)” Normal
tune contains proton up o 75 mg/100
mi and does not produce tur. The
results are reported as taco, or + D +24
roughiy corresponding to. protein
concentration of 20 mg, 30 mg. 50 mg
nd 75 mg/100 respecte, Tu
Produced by albumin à 4 times tat
Produced by gobuins. False postive
Fesulls are obtained with mucous ine
Contrast media, meiaboltes of
tolbutamide, plasma "expanders, IV
abumn and” sufsoxazole. X-Ray
contrast meda (false postive) may
persist "Tor two, ater
ministration. Akalino, highly bufeed
ue gives false negative resul
Improper est technique may give false

Pose or negative resul

2. Colorimetric: A pH 30 tetrabromophenot

‘Bue ls yelow in absence of proton whereas

in presence of protein A becomes green to

N

32

bromophenol Bo) i used. These tots
“sense and wil detec proteins
0291. The resuts, therefor.

‘onfemed wih ubiameine

est ls specii for albumin.
tive. resus “are common in
high. buttered urine and

ant
fn

;
i

4. Iypochiorte. Haemogiobin, gobuins, Bence
Jones proteins give false negative reaction.
improper matching colour Docks, poor
lighting, ete. may give false postive or
gate resus

GLUCOSE AND REDUCING SUGARS

Principal: Monosaccharide hexoses (Table 9.2)
are all reducig sugars producing colour
‘eacton when tested wih Benedict reagent or
with cintest tablets (Ames Division. Mies
Laboratories). Naturally occurring polysaccha-
rides are long-chain camonyáraos composed
uc stunts

Glycogen. tun anima tue 1 iy

branched polysaccharide;

+ Starch, found in plans, is a mure of
amylose (straight chains) and amlepecin
(branched chains).

Tat Common manga sp

Te most common sugar excreted in
urine. The normal acu may excrete up 10 130
mg gucose24 hours. However, Were are a
‘umber of over reducing sugars and reducing
Substances, which can be present in urine
(Table 94), The gucose appears in excess of
formal minute” amount in une in dabetos
malilus, renal gycosura, post gastrectomy.
‘pinophiine excosa eiher rom the adrenals oF
Injected for therapeutic purposes, pancreatits,
hyperthyroidism, Iver damage, ronal tubular
disease, heavy meal and emotional sess.

Benedict's test:

Ad 0.5 mi urine. Bol for another 2 min and cool
{under running tap water. Lok for the colour of
rocio. Interpret the result according to
Table 93. Method is sensive to glucose
concentration as low ae 02%. The test is

[ca
Em
A
= em neater
Enzymatic test

‘This spect for glucose and is now available
on dipsieks. The tests based on the princi
that gluse is converte to guconie acid and
HO, by cote oxidase in presence of oxygen.
‘This 4,0; reacts wih onhotoludne in presence
of peroxidase to produce coloured compounds.
In "mie caso oxdeed erhotoudie (us) +
water. Al reagents are provided on a dipstick
Pad. This testis sensitive to as low as 0.1%
Bucose. No normal usne constituent gives flee
negative or posive result. Presence of bleach
and peroxides (used for cleaning containers)
‘ay give false positive resul. Very high doses
of Vitamin C and homogentsic add may give
falso negative meute. For using ac
precautons given by manufacturers must be
followed. A postive Bonadicts test and negative
‘enzymatic glucose test may indicate presence of

phosphate in iver Iis defeency result in
Sccumuaton of galacose due to metabolic
Bock ti nat a common condition and occur in.
infancy. The infant cannot propery metabolse
lactose or galactose and develop cataracts, ver
domage and possibly mental retardaton. The
final ideniticaton of galactose in urine can be
done by chromatography (see THIN LAYER

5. (CHROMATOGRAPHY on page 40 and on page
375,

Pentose: I indicates pentosria, which is an
Indem error of metaboksm, Pentose- xylose
Is excreted in the urine. Pentosua can aso
‘occur after the ingestion of raw plums or
hemes. ts checked by the ba-orcna test.
Lactose: This sugar may be found inthe urne
in late prognancy. laiaon or in patients on
extomely hgh milk diets. Lactose intolerance
‘th ociosua 1 are metabol disease

BILE PIGMENTS (BILIRUBIN)

‘This esti required for screoning, agnosis and

monitoring “of Iver, Dany and haëmoyie

diseases. Normaly, une birbin is less than

003 mg and i undeteetable by routne test.

may appear below other sans are noticeable.

Babine found nine in cross of te er,

ral hopatits. carcinoma head of pancreas and

‘oer bie duct obstuckens, and haemolysis,

Bun in urine can be detected by.

+ Foam test: Shaking urine specimen and

‘colour of foam (green, yotow or
row) is insensive and is now obsolete.

+ Dye dilution test Methylene blue is added
u rine tus blue, Ils also Intensive
and thus obsolete (detects butin 22
mo.

+ Fouchels test: Barium Chore
preciplates phosphates and concentrates
Bie pigments which are tested or by the
‘oxidation reacton. The pigment is oxdised
lo green bilverdin by Fouchets reagent
prepared by maing sick tichleracete acd
Solution (page 50) equivalent to 25 9. 10 mi
10% aqueous ere Horde and making the
volume to 100 mi wih tl water,
roceduce: Add 19 Darum corde to 10 mi
then a test ube, ma thoroughy and titer.
‘Spread the Mer paper. When party dry, put
a Tow drops of Fouchete reagent. Green
(werd) or ble (cholecyanin) colours
Indicate a postive reacton. The sens}
Varios ftom 0005 lo 10 mgd. False
pose test may be obiained win
Says but th colour produced purple
‘and Pyedium tke substances
(Penazopyrine) give ted colour Parents
Of une also obscure postve reaction:

+ Diazotisation Test In this tos a stabised
‘azo compound reacts with brin 1 form
‘blue colour.

Procedure: To 10 mi urine add and mix 4 mi
(110% barium chloride, Mark upper level of
uid win marker. Contigo and decant
completly Add distild water 10 the mark,
mx contigo, and docant completly. Add

a

(05 mi Diazo reagent, 2 mi absolute alcoho!
‘and 0.3 mi 6% hycralad Asocium hydrogen
prosphate (Na,HPO, 124.0). Mix and
Centriuge. Presence of bilirubin is indicated
by supemalanı fd becoming red due to
fzobirubin. iis sense to 0.08 mg/l and
is specie. For preparaon of Diazo reagent
see on page 327. Mis test's also avaliable
‘on dipsieks in which stable clazoisod sats
Aro used. The tests very sensiive and can
‘detect brub 3s low as 0.2 mg/100 mi. The
test should be performed on fresh urine
‘only. Very largo amounts of phenothiazine
(Chlorpromazine) “motabottes gue_ false
poste result pyidum Ine substances
ro present. they lv red colour

BILE SALTS

Hays test's employed based on the prie

that bie sats lower surface tension because of

that ight powdored sUhur sks to ne bottom.

Procedure: Take 5 mi urine in atest tube and

pre on ts surface a Da of finely powdered

Suphor granules. I À anis, Die sata are

present in tho urine. Falso positve may be

Feperied because of sinking of heavy impurtes

in subhur powder

L000

This can be haematuia, haemoglobinuria, or
myogiotinuña (Table 85). In haematuña intact
red Blood cals (REC) are present in the urine
(lesion of kidnoy or postronal Bleeding. cancer
in urnary tract. unary tract Infections et). In
haemoglobinuria tree haemoglobin is present in
tho uine. K occurs ln invavascular haemoyis.
(ranstusen reactons, autommune hama.
‘anaomia, ete), severe bum and alegic
reactions. In myogobinuta myoglobin (muscle
piment) is present in tho urine. may resul
from trauma (crush ijury. bul beating)

+ Gusiacum reaction
Bol 5 mi urine, cool and add 2 drops of
tincture gualacum. Shake and make a ayer
‘wth ozone ether. Blue rng wi ind
Blood (low sens).

+ Reduced phenolphthalein test
Take 3m of reduced ¡nal and
29 10 dop of HO, and 3 m urine. Sake
‘Wel, Pink colour indicates blood,

6. + Pyramidone ring test
‘Tako 2-3 mi urine and add few drops of
acatc acd. Add slowly equal volume of 5%
rramidone and 5-5 drops of H,O; Amauve.
Colour ing indicates blood.

+ Benzidine test
‘Take 2 mi urine and add few drops of acetic
ac and knfepoint of benzidne powder
see also. TEST FOR BLOOD IN FAECES
On page 93). Mix to make saturated solution
‘and add few drops of H.O;. A Blue colour
indicate blood (highly sensitive),

+ Commercial dipstick test
“Those Spstcks work on following princi:
Caner sen pero Tie Haein + >
evesyumsre)sOndsedeTeusee Gr Ba)
Tho test is most sensitivo for foo
haemoglobin (0.15 mg/d) or 5-15 intact
Race),
NTAITE

Normal urine contains nirates and many
bacteria conver nates to rites. Detection of
Nites in urine ineates unnary tac infection
or contamination. Early moming specimen gives.
best result. The test is done by commercial
lipstick working on folowing principle:

Ua mate Baer aie te
PAra A Dam coçouré
nera cnn compe Ea)
KETONE BODIES

Ketone bodies aro breakdown products of at
melabolsm. Those aro exhaled from Jungs and
‘Those consist of

‘acetone, These are normally present in
Concentrations of up lo 125 mg in 24 hours urine
but cannot be detected by routine testing. In
Ketosis the quantiy may be as high as 509 in 24
hours. These may appear in urne In starvation,
Uncontoled diabetes | mellus, | proonged
vomiing, “severe dlathoea in chen, low
Carbohydrate de, high fat dit and toxaema of
pregnancy. Ketone bodies are tested by the
following:
+ Rothers tube test

‘Akaline nitroprusside with acetoacetc acid

or acetone gives purple colour but no colour
with B-Hydroxybuyre acd. The tos is also
‘avaiable in commercial dipstcks and
tablets. This test is more sensitive lo
‘caloacaic acid and detects as low as 10
mg of acotoacetic acid/100 m of urine. The
ost must be performed on fresh urine before
aceloacai ack breaks. down to acelono.
Rothera test isnot standardised and vary in
sensiviy depending on the amount of
úreagents and their order of addon. Large
‘amounts of phenyketones of L-dopa
metaboites may cause false positive
resus,

‘+ Gerhardt’ test (Ferric chloride test)
This lost detects acetoacetle acid and is
simple to perform. À few drops of 10%
‘aqueous for chiovide solution are added to
Tm urne. Appearance of red. colour
Indicates prosont of acetoacetc acid. The
test detects 05-10 mmoll. (5-10 mg/d) of
‘cotoacaic acid in urine. Gerhardt test wi
‘Show falso positive osuts with salicylate,
PAS and antipyines (these wil not be
destroyed by boling whereas acetoactic
‘acid evaporates).

UROBILINOGEN

His a pigment produced by bacteria
ecompositon of bibi in intestine, from
there itis reabsorbed and appears in urine.
Trace amounts are normally present. Increased
amounts indeate increased. production of
bilirubin. The test is roqured lo dotoct
haemolysis and in the Giferental diagnosis of
Jaundice. The test must be performed on freshly
voided urine as urobiinegen is converted 10
Goblin on exposure 10 light and al
‘Urobiinogen may be increased in toxic hepatitis.
‚gandular fever. haemoljte anaemia and
Carcinoma head of pancreas. Following methods
are used for ts determination:
+" Spectroscopic examination: Acisy urine
‘wih HCI and examine with spectroscope.
‘Absorption band at juncion of green and
blue indicates presence of urobilinogen (see
‘ection on SPECTROSCOP Yon page 45).
+ Bogomolow's test: To 10 mi urine add 05
m of 20% Copper suphale and 4 mi
“horolorm. Max by inversion. Uroblin tums

chloroform Layer pink or yellow.
+ Emllcva benzaldehyde test: Colouress
ot "converted to coloured

‘Smethyfamaobonzaldonydo i disotved.
Procedure: To 10 mi ut add 1 ml of the

7. agent, mix and let stand for 10 min
Observe colour by looking down into the
{ube held over a white surface, Cherry red
colour incates a postive resul. I no colour
ls produced observe the tubes again ater
heating and if again there is no colour
obinogen is absent, The tos, positive
‘Reeds 10 be repeated on diuted urine un
Only a faint pink colour Is produced. The
su is reported as Increased (postive
reaction in 21/16 dilution), present but not
Increased (positive in dition <1/16) and
absent (no machen even añor heating)
False positive reactons may be soon.
Urobämogen is decreased ot absent in
neuboms when there ls complete
Cestrucion Of the common ble duet,
Starvation, intrahepatc cholestasis and
intestinal storisaton. It is increased. in
Femolyis wih or without junde.

BENCE JONES PROTEINS (B4P)

These are light chains of globins wih a
molecular weight of 45.000. They are found in
40% casos of muttplo myoloma and thor
Iymphopreferalve disorders wih monoclonal
Grsgobuinseme Since thoy are small
molecules, they are easy cleared trom plasma
by Kidneys. and excreted in the urine. These
Proteins give postive sulosaliylc aci tot for
Proteins but only a weak postive or no reaction
Win pst

Heat precipitation test

Principle: JP precipitate at about 60°C and re-
solve near 100°C. When the urine is cooled
these reappear between 85°C and 60°C.
Procedure: Centrfuge fresh urine and take 10
‘ml of clear urine in a test tube. Check pH and
adjust to 50 wih 25% acetic acid. Paco a
thermometer inne test tube and eat slowly in à
water bath. If BAP are present clouding wil
begin at 40°C and precipitation wil be complete
at 60°C. Now take out the thermometer and bol
{ho urne in the test tubo. The precipitate wil
isappear. Replace thermometer in test tube
and coo. Preciptate reappears and ten fades
10 disappear at temperature below 40°C. This
test should be confirmed by electrophoresis of
oncentated une

(QUANTITATIVE TEST FOR PROTEINS

+ Estach test
“The test ls based on protein pracplaion by
Pie acid’. Esbach’s reagent consists of

Fe ms rn de wr. er ech
‘hea fie a sd dl poe à
a or noe

1% plete acid. The instrument
used is called” Esbachs

Feacton shoud be changed o acid
wit 1-2 drops of 2% aca ac.
Fil Esbach’s tubo to mark U. Add Esbache
reagent to mark R. MX by geile inversion
about 12 mes, Replace inthe case, stopper
‘and leave for 24 hous. Read ne height of
‘white protein precipiate in grams perlite.
Correct or any däubon. This, however, not
an accurate method of protein estimation

+ Pyrogaliol red dye test

Prndole The pyrogalal red molybdate
‘complexed with protein at pH 2.5 gives vilo!
‘coloured compound measured at 600 nm.
‘whic is proportanal to the concentration of
proteins. The method is sensve tothe mg
Fänge suitable for oth urine and CSF protein
measurement. The method can also be used
10 measure microalbuminuria (See
microalbuminaria on page 325). This method
has also been automates
Pyragalel_tud de Dissolwe 10 mg of
sodium mayodate, 5.9 9 of succinic aid,
134 mg of sodium oxalate and 430 mg of
sodium benzoate in about 800 mi of diles
water To ths add 25 mg of pyroga red
‘dye and mix wall is completly solved.
Make up to IL. Store in an amber boto.
Stable at 28°C or 3 months.
Procedure: To 3 m regent add 50 i sample.
Handard and contol Mx all tubos weil
Leave at 255°C for 15 minutes. Set the
‘pectrophotemete to zero using blank at 600
iim (ed fine) and measure the absorbance
of standards, test and conto

PHENYLKETONURIA

In this disease there is increased concentration
ol phenylalanine. in blood. and CSF due 10
defcieney of hepatic phenylalanine hydroryase.
‘Phenykatones ao excreted in urine and can be
‘elected with Farc corde test

Procedure: To 5 mi rsh urine, add 35 drops of
10% aqueous ferie chiorido, Greyish green to
blue green colour appears within 90 seconds
‘and isappears añer sometime

8. PORPHOBILINOGEN

Watson-Schwartz test
lis based on the principle hat Ehrlich reagent
{ums porphebilnogen Io à red coloured
compound. which ers in sohbity fom red

Pdimeihylamı
fal concentated HCI added to 100 mi dl
water),

Procedure: Mx 25 mi fresh wine with 2.5 mi
Erich reagent, shake for 30 seconds
(immediate ted colour ls due to porpho-
Bünogen) Add 5 mi saturated sodium acetate
and mix wel. Adjust pH to 5.5 with more sodium
acetato if requred. 1 colour appears afer
Addition of sodum acetato, is mos! Ikly duo
lo urobiinogen. I colour appears, add 5 mi
Chloroform 1 reacton mature, shake well and
low to stand. Porphebiinogen will remain in
‘aqueous upper layer while uroblinogen is
extracted inthe lower chloroform layer. To
Confirm, separate upper aqueous layer and mix
it with equal volume of butanol Alow to
Separate the Colour s due to porphobinogen
it wil separate with lower layer (see
ao Tho PORPHYRIAS on page 344).

PORPHYRIN

To 5 mi of tesh urine add 0.75 mi glacial acetic
acid and 15 mi amyl alcohol. Mix well and
Centriugo to separate the layers, Examine under
Utravolet Ight Salmon pink fuorescence in
upper layer of aml alcohol indicates porphyrin
in excess of norma

caLcium

Calcium in urine is screened by Sukowitch test.
Calcium is preciptated as calcium oxalate by
‘oxalic aci reagent prepared by dissolving 2.5 9
‘uae acid, 25 g ammonium oxalate and 5 mi
‘glacial acote 006 in Tk dtitod water. À 24
hours urine sample s required. Mix 5 mi urine
with 5 mi reagent and observo for bit. No
precpiato is due 10 normal calcium, whereas
Bcn ges a hn pect (abe

CHLORIDE

CChiorde is tested by Fontana’ test in which
chloride is preciptated wih siver nate, excess
Of which then produces recdish precipitate of

‘ser chromate wih potassium chromate, À

20% soliton of potassium chromate and 29%
Solution of iver nitrate are required

Procedure na test tube place 10 drops of une
and one drop of potassium chromate. Add sver
‘irate drop by drop ul permanent distinct red
‘brown colour appears. Same dropper should be
sed for urine and reagenis. Number of drops
required to produce the colour change ts equal
0 number ol gram of sodum chloride per ive of
ine, Normal urine requires 6-12 drops.

MICROSCOPIC EXAMINATION

Microscopic examination is an essential
component of urnalyses. Following examination
procedures are cared out

Light microscopy
is camed out lo see ova or parasitos
(Tichomonas, "Schistosoma, Echinococcus,
Faria ao), RBCS, Toukocytes, cass,
‘pitta! coll and crystals. Bacteria, yeast
GHindroids. spermatozoa, mucous, iat and
relate can alo be soon

Preparation of deposit: Cenifuge 10-15 mi
ol maxed urine at 1000 rpm for 3 mn. Invert
the tube lo pour off the supematant. Noe the
‘iment wth de small amount of ino et in
‘the tube. Pour a drop on a clean glas sde and
put a cover sip. Examine under subdued ight
frst ‘scanning whole area wih the low power
{Bice ad fn a gn pone cer,
‘Amorphous deposit may caver
Aucas, bare ay hou mcd y
‘2dcing a small drop of 10% acetic acd, which
‘ssolves the deposit Too much acd should be
‘veided, as it wil dssoWve the casts. The
eures 10 be noted under low power aro cast,
‘spermatozoa, mucous rends, yous fat
Gropeis and ova of parastes. Casts are
‘reported as number por low power fold and rest
‘ofthe elements a few, moderate or many.

Loucocytes
"Normal urine from males does not contain moro

than 1 leucocyte per high power feld (HPF).
le from females à contains 1.5 calisHPF,
‘These are usvally polymorphs and may show
‘amoebod movements in fresh spécimen.
Increased "number (pyuria) “indicates
inflammation and occurs ‘almost in al ronal
diseases (Figure 9.1 and Figure 2.2). Leukocyte
casts are present. infection is of renal ori.
Some causes of pyuria (pus in the urine) are
acute oF chronic pylonephiis, acute or chronic
ysis, renal tuberculosis and bladder trauma,
Ceukocytes. are rapidly sed in hypotonic
lalo ino. Approximately 50% may be ost in
2 to 3 hours al room temperature. Therefore,

9. {ins should be examined as eat as posite
‘fer cocon

Enthrocytes

‘These appear as Moy relacio, round,
yolowish sructures (Figure 9.3). Normal urine
{tom males does not contain any REC except
the specimen is colected by cateterisaton.
Urine from female may show a few RECs fom
vaginal "contamination 0 many dung
‘menstruation. Wih these wo exceptions
presence of RECS in the urine (haematura) is a
‘Significant fring, Increased numberof red cols
‘may originate in any part ofthe urinary system.
In case {renal ong, urine wil have RECS, red
cell easts, protonuria and dysmerphic RBCs.
Some causes of ronal Aaematura are
lomeruionephitis, lupus. nephiits, calculus,
tumour, trauma, acute infection, ot. I org is
lower unary rat (acute and chronic infection.
caleuus, tumour of unnary bladder and sñcturo
Of Ureta) unne wil have red cells but no cast
‘and no pote.

casts
Casts are cyindical structures with paralel
sides and blunt rounded ends that quckyy
dissolve in akalne urine. These are formed in
tubules and may even be present when tests for
‘abumin aro negalve. They are vanslucent,
Colouess ges. Their size and shape depends
‘on tubules where hey were formed. They
Indicate widespread kidney disease. These ofen
‘cca intermitenty and may not be seen in al
‘Specimens. They are basicaly composed of
‘mucus protein, called Tamm-Horsfll protein,
forming a mat i whieh are incorporates other
elements depending upon the type of cast
Casis are increased in ac. urnary stass,
increased plasma protens, and high solute
concantraion. À morphological varian is called
fylindroi. ts structure is similar to cast buts
‘shape is diferent I tapers al one end and may
thin down into a read at that end. Another
‘merphelogical variant 1 à very broad cast wich
is formed in colecing tubules. I is also called
‘renal ale cast Diforet types of casts sen in
rine are
+ Hyaline casts in which no other elements
are mixed in the base structure (Figure 9.4).
= Rod cel casts when RECS are tapped in
the matrix Figure 95).
‘+ Pus casts when pus cols are present in the
‘ast (Figure 96 and Figure 97)
+ Epihelal casts contain epihelal ces
(igure 98)
= Fine granular cast, when fine
‘granules ro present in the cast (Figure 9.9
‘and Figure 3.10)

.s

+ Coarse granular casts when the granules
Fatty casts contain fat droplets in matrix or
aro formed when tho contents undergo fay
Segeneraton (Figure 9-11)
+ Bie casts contain bäruin in matrix
© Haomoglebin casts aro brown and are
formed einer due to presence of
haemogiobin in the cast or due 10
generation of are cal cast (Figure 9.12).
+ Wary casts are formed in amylod disease,
‘They aro structureless and colouress Ike
hyaline casts but are more transparent
Epithelial Cols
‘Squamous opihelal cls are normaly present
in'smal numbers. In females, very large number
indicates vaginal contamination. Tubular
epihoa eels appear in renal disease. These
Fesemble leucocytes but have a, prominent
uciecus in a centaly located nuceus (Figure
8.19). These cels may contain bilnbin or
heemosider (Figure 914).
‘Amorphous Deposits
‘Amorphous (ine granuar) urates are seen
‘id uine while amorphous phosphates. aro
Common in alain une (Figure 919),
Crystals
These are not seen in fresh warm urine but form
aer sometime. Except or cystine. uric acid,
leucine and tyrosine crystals. they have le
Significance. The type of cysials seen dopend
upon the pH of urine, Alkane urine contains
tiple “phosphate (ammonum-magnesium
hosphate) (Figure 9.16). calcum carbonate
and ammonium burae crystals. Ac urine may,
contain caloum oxalate (Figure 9.17). une acd
(Figure 918), cystine (Figure 9.19. Figure 920
and Figure 921), tyrosine (igure 922) and
leucino (Figure 9.23) eysas. Other crystals
include cholesterol (Figure 924) and drugs tke
subha, which erystalise in urine (Figure 925
‘and Figure 9.26),
Miscellaneous
Besides tose, many other things aro seen in
the urnary sediment. These include ova of
‘Schistosoma haematobium (page 93), malgnant
cols, bacteria, yeast cal (Figure 827),
tichomonas aria (page 114), mucous threads
(Figure 928) te
Dark ground illumination is required when
organisme Ike Leptospira are expected.
Staining of urine sediment’ Folowing stains
at used fr urine microscopy:
+ Methylene blue
2 Stemheimer-Mabin stain
Poymorphs are orange purple, Hyaine cast

10. pale blue or coluress, RBCS lavender collar
East ik to purple; chomonas ight lue nude
of bladder ‘epithelial cats blue and. vaginal
‘pital cal nucel purple). Stans for Fat Cols
inciado Sudan Il, Sudan IV and Où Red O. Acid
Fast stan Include Zen-Nocison and Kinyoun
sans.

AUTOMATED INSTRUMENTATION.
There are many automated equipments (Ike

‘AUTOMATED URINE STRIP READER on page

ss)
MICROSCOPIO TEST FOR FAT

‘This toa is based on staining of fa with Sudan
me

Procedure: Mix a few drops of 36% acetic acid
‘na sie with few drops from the top surface of
Conriuges une. Add several” drops of
‘Saturated soliton of Sudan In 95% ethanol
nd heat 10 boing for few seconds. Examine
Under the microscope. Fat appears as deep
orange globules that become spiked on coding.
To see neutral fats use 95% ethanol in place of
‘acetic acid, Neural fat appear as yellow to pale
Grange globules (Figur 629)