3. Formation of Urine
Three processes of
urine formation:
glomerular filtration
tubular reabsorption
tubular secretion
The nephron:
allows for •
reabsorption of
water and
electrolytes
plays a vital role in •
maintaining normal
fluid balance
4. Physical Composition and
Chemical Properties
Urine
95% water
5% waste products
Other dissolved
chemicals
Urea, uric
acid, ammonia, calcium,
creatine, sodium, chlorid
e, potassium, sulfates, ph
osphates, bicarbonates, h
ydrogen
ions, urochrome, urobilino
gen
5. Obtaining Specimens
General guidelines:
Use the type of specimen container indicated by
the lab
Label the specimen container before giving it to
patient
Explain the procedure to patient
Wash your hands before and after procedure
Complete all necessary paperwork
6. Specimens Types
It Varies in method used and in time frame in which to
collect specimen
Types of specimens:
Random
First morning
Clean catch midstream
Timed
24 hour
7. Specimens Types (cont.)
Random – most common, taken anytime of day
First morning – has a greater concentration of
substances, taken in morning
Clean catch midstream – genitalia is cleaned, urine
is tested for microorganisms or presence of infection
Timed – specific time of day, always discard first
specimen before timing
24 hour – used for quantitative and qualitative
analysis of substances
8. Urinalysis
Evaluation of urine to obtain information about body
health and disease
Four types of testing:
Physical
Chemical
Microscopic
Culture and sensitivity( beyond the scope of lecture)
9. Preservation and Storage
Changes that affect
the chemical or
microscopic
properties of urine
occur if urine is kept
at room temperature
for more than 1 hour
Refrigeration – most
common method for
storing and preserving
urine
It prevents bacterial
growth for 24 hours.
After 24 hours use
chemical preservation
10. Normal Values of Urine
Normal values of
various elements have
been established
A routine vol. of 12 mL
urine is analysed
Average adult urine
output is 1250 mL/24
hours(>1mL/Kg/hour)
11. I- Physical Examination of Urine
Visual examination of
physical characteristics
1.
2.
3.
4.
Volume
Color and turbidity
Odor
Specific gravity/
Osmolality
12. Urinary volume
Normal = 600-1550 mL/d
Polyuria- > 2000mL
Oliguria-< 400 mL
Anuria-complete cessation of urine(< 200
mL)
• Nocturia-excretion of urine by a adult of >
500 ml with a specific gravity of < 1.018 at
night (c.c. of chronic GN)
•
•
•
•
18. Urine Appearance
Turbidity means:
Cellular elements,
Bacteria(which clear by centrifugation), and
Crystals(which clear by addition of acids or
bases)…
It’s the microscopic examination which will determine
which type…
20. Urine Sp. Gr. / Osmolality
• Specific Gravity depends on the concentration of various solutes
in the urine.
• N.Sp.Gr. = 1.016 – 1.022
• Hyperosthenuria: Dehydr., D.M.,..
• Hyposthenuria: Polyuria(except diabetes)
• Isosthenuria: Fixed at 1.010 in CRF
//////////////////////////////////////////////
There is a linear relationship between Sp. Gr. &
Osmolality; ; ; Except in Glycosuria, or Excretion of
contrast ----- In this case, the Sp. Gr. Will be >
Osmolality
21. Measurement of Specific Gravity
It’s measured by:
-urinometer
-refractometer
-dipsticks
22. Urinometer
Method for use:
Take 2/3 of urinometer container with urine
Allow the urinometer to float into the urine
Read the graduation at the lowest level of urinary
meniscus
*Correction of temperature & albumin is a
must.*
Urinometer is calibrated at 15 or 200c
So for every 3oc increase/decrease add/subtract
0.001
For 1gm/dl of albumin add 0.001
26. Dipsticks Use
The main advantage of
dipsticks is that they
are
1. convenient,
2. easy to interpret,
3. and cost-effective
•
The main disadvantage
is that
1.Not very accurate (the
test is time-sensitive).
2. It is a qualitative and
not a quantitative test
(no precise information
about the severity of
the abnormality) .
27. II- Chemical Examination of Urine
Usually done with reagent strips or tablets
Used to determine body processes such as CHO
metabolism, liver or kidney function or acid-base balance.
Used to determine presence of drug, toxic environmental
substances or infections
30. 1- Urinary pH/ reaction
Reaction reflects ability of kidney to maintain normal hydrogen
ion concentration in plasma & ECF
Normal= 4.6-8
Tested by :- 1.litmus paper
2. pH paper
3. dipsticks
Other Tests:
Titrable acidity
Blood gases
32. •
Buffers from the protein area of the strip
(pH 3.0) spill over to the pH area of the
strip and make the pH of the sample
appear more acidic than it really is
Dipstick for
pH
Limitations:
• Interference:
Bacterial overgrowth
•
Run-Over Effect:
Protein pad effect on
PH pad
33. 2- Urinary Glucose detection
Detection of reducing sugars by:
Benedict’s Test
Urinary dipsticks
Benedict: Semi-quantitative)
Principle-Benedict’s reagent contains cuso4.In the presence of •
reducing sugars cupric ions are converted to cuprous oxide which is
hastened by heating, to give the color.
Method- take 5ml of benedict’s reagent in a test tube, add 8drops of •
urine. Boil the mixture.
Blue-green = negative
Yellow-green = +(<0.5%)
Greenish yellow = ++(0.5-1%)
Yellow = +++(1-2%)
Brick red = ++++(>2%)
N.B: Renal threshold must be passed in order for glucose to spill into urine
34. Dipsticks for Glucose
However, Benedict detects all
reducing substances like
glucose, fructose, & other
reducing sustances such as:
Sugar
Disease
Galactose
Galactosemia
Lactose
Lactase def. or
intolerance
Fructose
Fructose intolerance
Pentose
Essential pentosuria
Maltose
Non-pathogenic
N.B: Sucrose is not a reducing substance
To confirm it is
glucose, dipsticks can be
used (glucose oxidase)
35. Causes of glycosuria
Glycosuria with
hyperglycaemia• Diabetes,
• Acromegaly,
• Cushing’s
disease, Hyperthyroidis
m,
• Drugs like
corticosteroids
Glycosuria without
hyperglycaemia• Renal tubular
dysfunction(ex.
Fanconi syndrome; also
with: a.a.uria & po4
uria)
• Renal Glycosuria
• TTT with SGLT(sodium
glucose transport
inhibitors used to treat
DM)
36. 3- Urinary ketone detection
There are 3 types of ketone bodies:
Acetone
Acetoacetate
Beta-hydroxy-butyrate
Detection of ketones by:
Rothera’s Test
Dipsticks
• Rothera’s t. principle:Acetone & acetoacetic acid react with
sodium nitroprusside in the presence of alkali to produce
purple colour.
• Method- take 5ml of urine in a test tube & saturate it with
ammonium sulphate. Then add one crystal of sodium
nitroprusside. Then gently add 0.5ml of liquor ammonia along
the sides of the test tube.
• Change in colour indicates a positive result
37. Dipsticks For Ketones
Significance:
Diabetes
Starvation
Severe vomiting/diarrhea
High fever
Limitations:
Measure only acetoacetate
and not other ketones
>>>Cannot detect alcoholic
KA(with ↑BHB >AA)
Reagents can undergo
degradation with exposure
to moist of air
38. 4- Urinary protein detection
--Normally, up to 150 mg total
proteins may be found in urine per
24 hours
--More than 300 mg per 24 hours is
termed “ Frank Proteinuria “
N.B
Test-thermal method:water-bath)
Proteins has an unusual and peculiar
property of precipitation at 400 -600c
& then dissolving when urine is
brought to boiling at 1000c & then
reappearing de novo on cooling of
sample.
Protein
Max.
(mg/day)
% of
Total
Albumin
60
40
TammHorsefall
60
40
Igs
24
12
Secretory 6
IgA
3
Others
5
10
39. Tests for proteins
Test – heat & acetic acid test
Principle-proteins are denatured & coagulated on heating to
give white cloud precipitate.
Method-take 2/3 of test tube with urine, heat only the upper part
keeping lower part as control.
Presence of phosphates, carbonates, proteins gives a white
cloud formation. Add acetic acid 1-2 drops, if the cloud persists it
indicates it is protein(acetic acid dissolves the
carbonates/phosphates)
Other Tests:
-Sulphosalicylic acid SSA turbidity test
-Dipsticks
-Esbach-albuminometer- for quantitative estimation of proteins
-Urine protein electrophoresis(UPEP)
40. Albumin Excretion:
Alternative Methods for expressing the normal range
Sample
Normal Value
24-h urine collection
< 30 mg / 24 hrs
Timed sample from ambulant pt.
< 20 micro-g / min
Timed sample for recumbent
pt.(or over-night sample)
< 10 micro-g / min
Albumin / creatinine ratio on a
random urine sample
< 2.5 mg / mmol in male)
< 3.5 mg / mmol (in female)
41. Dipsticks for proteins
Limitations:
Interference: Highly alkaline urine
-Almost all dipsticks detect proteins if present
in an amount more than 300 mg / 24 Hs
-They cannot detect micro-albuminuria (30150 mg) alb./24-h urinary sample >>>>>
Esbach –albuminometer can be used …
Bences- Jones proteins are light chain
globulins present in multiple myeloma,
macroglobulinemias and lymphomas
They are detected by: UPEP
N.B.: Usually +1 ptn. correlates with 30 mg alb.
And +3 ptn. Correlates with > 500 mg alb.
42. Importance of micro-albuminuria
It is an early indicator of subclinical nephropathy
either due to on intrinsic kidney disease or due to a
cardiovascular disease..
It may be an important prognostic marker..?
It is considered as a routine check-up in all cases of
diabetes mellitus or in hypertension (every 6
months)..
Serial rise in micro-albuminuria during the first 48
hours after admission to an intensive care unit can
predict elevated risk for acute respiratory failure
, multiple organ failure , and overall /CV mortality
(as a bad prognostic criterion.. )
45. 5-Urine blood detection
Test- BENZIDINE TEST
Principle-The peroxidase activity of hemoglobin
decomposes hydrogen peroxide releasing nascent
oxygen which in turn oxidizes benzidine to give blue
color.
Method- mix 2ml of benzidine solution with 2ml of
hydrogen peroxide in a test tube. Take 2ml of urine &
add 2ml of above mixture. A blue color indicates +
reaction.
47. Causes of hematuria
Pre renal- bleeding
diathesis, hemoglobinopathies, malignant hypertension.
Renal- Trauma, calculi, ac. & chr.
glomerulonephritis, pyelonephritis, renal TB, renal tumours, Goodpasture syndrome and Henoch-shonlein purpura
Post renal – severe UTI, calculi, trauma, tumors of
urinary tract
48. The Urine Dipstick:
Negative
Trace (non-hemolyzed)
Moderate (non-hemolyzed)
Trace (hemolyzed)
+ (weak)
++ (moderate)
+++ (strong)
Blood
Chemical Principle
Lysing agent to lyse red blood cells
Diisopropylbenzene dihydroperoxide +
Tetramethylbenzidine
Heme
------------> Colored Complex
Read at 60 seconds
RR: Negative
Analytic Sensitivity: 10 RBCs
49. 6- Urine bilirubin or Urobilinogen
Bilirubin
Test- fouchet’s test.
Causes
•
•
Liver diseases, injury, hepatitis
Obstruction to biliary tract
Significance
It correlates with D. serum
bilirubin
Limitations
Urobilinogen
Test- Ehrlich test
Causes- hemolytic anemia's and
hepatocellular jaundice
Significance
- High: increased hepatic processing of
bilirubin
- Low: bile obstruction
- Interference: prolonged
exposure of sample to light
- Only measures direct
bilirubin--will not pick up
indirect bilirubin
Limitations
- Ictotest (more sensitive
tablet version of same assay)
Other Tests
Other Tests
- Interference: prolonged exposure of
specimen to oxygen (urobilinogen --> urobilin)
- Cannot detect low levels of
urobilinogen
- Serum total and direct bilirubin
51. 7- Urinary detection of nitrites
Significance:- Gram negative bacteriuria
Limitations
- Interference: bacterial overgrowth
- Only able to detect bacteria that reduce nitrate to
nitrite
Other Tests
- Correlate with leukocyte esterase and urine
microscopic examination (bacteria)
- Urine culture
52. The Urine Dipstick for
nitrite:
Chemical Principle
Negative
Positive
Acidic
Nitrite + p-arsenilic acid -------> Diazo compound
Diazo compound + Tetrahydrobenzoquinolinol
----------> Colored Complex
Read at 60 seconds
RR: Negative
60. III- Microscopic Examination of Urine
• Centrifuge the urine sample for a few minutes (10-20 fold
conc.)
• Discard the supernatant.
• The solid part left in the bottom of the test tube (the urine
sediment) is mixed with the remaining drop of urine in the test
tube and one drop is analyzed under a microscope
• FOV(field of view): What is seen through the ocular lens
A normal urine contains few epithelial
cells, occasional RBC’s, few crystals.
61. Types of microscopy:
1. Phase contrast
2. Polarized
3. Bight field with special staining
N.B:Cells and casts begin to disintegrate in 1 - 3 hrs. at room
temp( refrigeration for up to 48 hours is a must to limit cell loss).
Presence of the following is considered “abnormal”:
o Fungal hyphae or yeast, parasite, viral inclusions
o Mononuclear cells(transplant rejection), eosinophils (all.
Interstitial nephritis, vasculitis, prostatitis & atheroembolic
dis.)
o Sperms(post-vasectomy),starch, mucus, fibres
o Pathological crystals (cystine, leucine, tyrosine)
o Large number of uric acid or calcium oxalate crystals
62. Abnormal Microscopical Findings
Per high power field(x40)
> 3 erythrocytes
> 5 leukocytes(glitter cells)
Eosinophils: Giemsa or
Hansel stain
> 2 renal tubular cells
> 1 bacteria
Mononuclear cells
Yeast
Trichomonas
Crystals
Per low power field(x10)
> 3 hyaline casts
> 1 granular cast
> 10 squamous cells
(contaminated specimen)
Any other cast (RBCs,
WBCs)
83. Urinary Casts
Urinary casts are cylindrical
aggregations of particles that form in
the distal nephron, dislodge, and
pass into the urine.
In urinalysis they indicate kidney
disease.
They form via precipitation of TammHorsfall mucoprotein which is
secreted by renal tubule cells.
Cast formation is enhanced by:
• PH of urine
• Solute conc.
• Presence of plasma
proteins(albumin, globulin, hemoglo
bin, myoglobin,..
100. Case 1
A 35-year old man undergoing routine
pre employment drug screening.
Glucose
Negative
Bilirubin
Negative
Ketones
Negative
S.G.
1.001
Blood
Negative
pH
5.5
Protein
Negative
Urobilinogen
0.2 mg/dL
Nitrite
Negative
L.E.
Negative
Physical characteristics: Clear.
. Microscopic: Not performed
Drugs Identified: None
Questions :
- What is your differential diagnosis?
- What would you do next to confirm
your suspicion?
- Would you order a microscopic
analysis on this sample?
101. Answer 1
Diluted urine with a low Sp. Gr.>>>>>
Request a morning urine sample>>>>>
If persisting low Sp.Gr.>>>>>
Possible diagnosis of diabetes insipidus
102. Case 2
A 42-year old woman presents with “dark
urine”
Glucose
Negative
Bilirubin
+++
Ketones
Negative
S.G.
1.020
Blood
Negative
pH
5.5
Protein
Negative
Urobilinogen
0.2 mg/dL
Nitrite
Negative
L.E.
Negative
Physical characteristics: Red-brown.
Microscopic: Not performed.
Questions
- What is your differential diagnosis?
- Could this be a case of hemolytic
anemia?
- How would you rule it out?
- What tests would you order next? Why?
- Would you order a microscopic analysis?
103. Answer 2
Possible gallbladder or hepatic disease.
No hemolytic anemia.
Perform Serum assessment for total and direct
bilirubin
Microscopic exam. is unlikely to provide additional
information for diagnosis
104. Case 3
A 27-year old woman presents with severe
abdominal pain.
Glucose
++
Bilirubin
Negative
Ketones
Trace
S.G.
Physical characteristics: clear-yellow.
Microscopic: Not performed.
1.015
Blood
Negative
pH
6.0
Protein
Negative
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
Negative
Questions
- What is the most likely diagnosis?
- What do you make of the ketone result?
- What do you expect to happen to the
ketone measurement when treatment
begins?
105. Answer 3
Diabetes
May be associated with ketoacidosis
Ketones should become negative after treatment
106. Case 4
8-year old boy presents with discolored
urine
Glucose
Negative
Bilirubin
Negative
Ketones
Negative
S.G.
1.015
Blood
+++
pH
6.5
Protein
+
Urobilinogen
1.0 mg/dL
Nitrite
Negative
L.E.
Negative
Physical characteristics: Red, turbid.
Microscopic: erythrocytes = >100 per HPF
(almost all dysmorphic)
Red cell casts present.
Questions:
- What is the most likely diagnosis in this
case?
- Does the presence of red cell casts help
you in any way?
- If the erythrocytes were not dysmorphic
would that change your diagnosis?