Clinical Biochemistry

1. RENAL FUNCTION TEST
DR. MOHAMMAD SHIBLEE ZAMAN
Lecturer (Biochemistry)
Dhaka Medical College

2. Functions of the kidney
 Regulation of water, electrolyte
and acid–base balance
 Excretion metabolic waste eg.
urea, creatinine, UA, urobilinogen,
PO4
-, SO4
-, oxalic acid etc.
 Formation of renin, erythropoietin
and calcitriol
2

3. Indications of testing
 Diagnosis of renal disease
 Assessment of severity and
prognosis of renal disease
 Differential diagnosis of edema and
proteinuria
 Differential diagnosis electrolyte
abnormalities
 Differential diagnosis of acid base
disorders
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4. Tests
 Routine & bacteriological
examination of urine
(Urine RME and urine C/S)
 Blood biochemistry
 Glomerular function tests
 Tubular function tests
4

5. Routine & bacteriological
examination of urine
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6. Urine RME and urine C/S
 Physical examination of urine
 Chemical examination of urine
 Microscopic examination of urine
 Bacteriological examination of
urine
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7. Collection of urine
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8. Collection of urine
 Random...anytime
eg. spot urinary ACR
 Timed...at particular time
eg. during OGTT
 24 hrs...whole urine of last 24 hour (8am – 8am
next morning)
eg. 24hrs UTP, 24hrs urinary electrolyte, CCR
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9. 24hrs urine collection
 The bladder should be emptied at the beginning
of collection and urine discarded (If starting time
is 8 am; voided urine of 8 am has to be
discarded).
 Fecal contamination should be avoided. Urine
should be collected before the act of defecation.
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10. 24hrs urine collection contd.
 Voided urine should be collected in a separate
container and then added to the main container
containing acid preservatives to avoid splashing.
 Starting time has to be noted in the main
container and stored at ~4°C.
 All subsequent voiding of 24hrs is collected
including that of 8 am next morning. No portion
should be discarded.
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11. Changes in urine on long time storage
 Bacterial contamination
 Chemical decomposition of analyte
Bacterial fermentation of glucose
Conversion of urea to NH4
+
 Precipitation of analytes –
PO4
- precipitates
 Oxidation of unstable components –
Urobilinogen is oxidized to urobillin
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12. Preservatives used
 Toluene
 Formalin
 HCl
 Boric acid
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13. Routine microscopic examination of urine (Urine RME)
13

14. Physical examination
Characteristics Normal Abnormalities with cause
Volume* 500 – 2500ml/day Polyuria...in DM & DI
Oligouria...in RF, UT
obstruction, dehydration etc.
Colour Straw – slight hazy Dark yellow...in jaundice
Red...in hematuria
Appearance Clear Turbid...in UTI
Osmolarity 600 – 900mosm/L ↑...DM, dehydration
↓...DI
Specific gravity 1.010 – 1.040
*requires 24hrs urine
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15. Chemical examination
Characteristics Normal Abnormal constituents
pH 4.5 – 8.0 (≈6.0)
Protein Nil or trace (<150mg/day) Excess protein
Inorganic Na+, K+, Cl-, Ca2+, Mg2+,
PO4
-
Any electrolyte in excess
Organic NH4
+, urea, UA,
creatinine, urobilinogen
Sugar/ glucose, ketone
bodies, blood, bilirubin/
bile pigment, bile salts,
excess urobilinogen
15

16. Detection of abnormal constituent
Constituent Test Cause of presence
Sugar/ Glucose Benedict’s test DM, renal glycosuria
Protein Heat coagulation test Proteinuria due to any cause
Ketone bodies Rothera’s test DKA
Blood Benzidine test Hemolytic disease
Bilirubin/ Bile
pigment
Fouchet’s test Obstructive jaundice
Bile salts Hay’s sulfur test Obstructive jaundice
Urobilinogen Ehrlich’s test Hemolytic jaundice
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17. Microscopic examination
Components Normal Picture Abnormalities
Epithelial cells 1 – 5/HPF ↑...in ATN
RBC 0 – 2/HPF ↑...in renal stone, tumor
Pus cells 0 – 5/HPF ↑...in UTI
Cast Absent RBC, WBC, granular, hyaline
etc.
Crystal Absent Ca-oxalate, uric acid, triple
phosphate
Microbes Absent Bacteria, yeast, parasite
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18. Casts and crystals
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19. 19

20. Blood biochemistry
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21.  Estimation of serum creatinine, blood urea &
BUN
 Estimation of serum uric acid
 Estimation of serum albumin
 Estimation of serum electrolytes, calcium &
phosphate
Blood biochemistry
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22. Parameters Normal range
Serum creatinine2 : 0.7 – 1.4mg/dL
Blood urea1 : 15 – 40mg/dL
BUN : 10 – 20mg/dL
Serum uric acid : 3 – 7mg/dL
Serum albumin : 3 – 4gm/dL
Blood biochemistry
22
1varies with high protein intake, trauma, severe infection
2relatively constant with constant muscle mass. That’s why more reliable

23. Blood biochemistry
Parameters Normal range (in mmol/L)
Na+ : 135 – 145
K+ : 3.4 – 5
Cl- : 95 – 107
HCO3
- : 22 – 28
Ca2+ : 2.2 – 2.6
Inorganic PO4
- : 1 – 2
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24. “
Glomerular function tests
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25. Glomerular function tests
 Renal clearance tests
a) Inulin clearance test (normal: 125ml/min)
b) Creatinine clearance test (normal: 70 – 140ml/min)
c) Urea clearance test (normal: 60 – 70ml/min)
 Measurement of GFR through
Radioisotope clearance study – 99mTc-DTPA, 51Cr-
EDTA
Formula based estimated GFR (eGFR)*
 Detection of albumin
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26. Clearance tests
Based upon
C (ml/min) = U x V
P
C = Renal clearance of substance in ml/min
U = Concentration of substance in urine (mg%)
V = Volume of urine in ml excreted per min
P = Concentration of substance in plasma (mg%)
“Endogenous” creatinine clearance test is preferred over
intravenously injected inulin clearance test
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27. Formula based estimation of GFR (eGFR)
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Cockroft–Gault equation
eGFR (mL/min/1.73/m2) = (140 – age) x weight (kg) x (constant)
Serum creatinine [μmol/L]
Constant = 1.23 for male & 1.04 for female
Modification of diet in renal disease (MDRD) equation
eGFR (mL/min/1.73 m2) = 1.86 x (SCr)−1.154 x (age)−0.203 x constant (0.742 if female) x
(1.21 if black African)
To convert creatinine values in μmol/L to mg/dL multiply by 0.0113
CKD-EPI equation
eGFR (mL/min/1.73 m2) = 141 x min(SCr/κ, 1)α x max(SCr/κ, 1)–1.209 x 0.993Age x 1.018 [if
female] x 1.159 [if black]
SCr is serum creatinine in mg/dL, κ is 0.7 for female & 0.9 for male, α is –0.329
for female & –0.411 for male, min indicates minimum of SCr/κ or 1 & max
indicates maximum of SCr/κ or 1

28. Staging of kidney upon eGFR
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29. Staging of kidney upon eGFR
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30. Tubular function tests
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31. Tubular function tests
 Urine concentration and dilution test
 Urine concentration test
a) Water deprivation test
b) Urine concentration test with ADH
 Urine dilution test (excess water intake test)
 Detection of LMW protein (α2 or β2 microglobulin)
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32. Water deprivation test
 Restriction of water/fluid for certain period (8 –
14hrs; with meal of <200ml fluid content)
supposed to excrete concentrated urine with
high osmolarity and specific gravity (≥1.025).
 Failure to increase osmolarity and specific
gravity (remains ≤1.020) indicate tubular
damage or ADH deficiency.
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33. Urine concentration test with ADH
 S/C injection of ADH following evening meal
followed by overnight fasting and measurement
of next morning 2 urine sample specific gravity
 In normal kidney, at least one of two sample
specific gravity ≥1.020
 In tubular impairment, failure to concentrate
urine >1.020
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34. 34
সুয োগ থোকযে CCR করয ো, নো হযে
eGFR, eGFR-এ S.
Creatinine ফ্রি পেযে োয ো, তো নো
হযে শুধু S. Creatinine, আর তোও
ফ্রি নো হে তোহযে Urine R/E-ই
করয ো।
আমি খামির
মিডমি টেস্ট
িরব া। অবিি
টেস্ট!!
িার মিডমি িযার?
আিার িা
আপিার?
ফ্রিফ্রনেোস!! এিদি টেি
ফ্রকডফ্রনর ডোক্তোর!!
এিদি
আঁবেল
এিো!!
একটি মাত্র ককডকি টেস্ট
ককিবে? টকািো ককিবে?
োাঁ ইচ্চা টেমি!!
আিাবর ধবর
িাই!

35. Proteinuria
Excretion of protein >30 mg/day
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36. Origin of urinary protein
Filtered
Albumin
Pre-albumin
β2-microglobulin
Ceruloplasmin
Haptoglobin
Transferrin
Tubular secretion or
desquamation
Tamm-Horsfall protein
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37. Types
Macroproteinuria
•24hrs UTP >300mg/day
•Dipstick positive
•Detected by heat
coagulation test
Microproteinuria
•Mainly albumin
•24hrs UTP 30-
300mg/day
•Dipstick negative
•Not detected by heat
coagulation test
eg. Early DN, HTN
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38. Types of macroproteinuria
Functional
eg. PE
Physiological
eg. fever,
trauma, stress,
pregnancy, high
protein intake
Glomerular
eg. AGN, NS
Tubular
eg. ATN
Overflow
eg. multiple
myeloma
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39. Dipstick testing
 Strips impregnated with chemical
reagents
 Urinalysis strip for quick
determination of urobilinogen,
bilirubin, ketone, creatinine, blood,
protein, microalbumin, nitrite,
leukocytes, glucose, specific
gravity, pH and ascorbic acid in
urine.
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40. Dipstick testing
 Strip is manually immersed in the
urine specimen
 Reagents in each block react with
a specific component of urine
 Block changes colour if the
component is present
 Colour change proportional to
concentration of component being
tested for
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41. Dipstick testing
41

42. Heat coagulation test
 2/3rd of test tube filled with urine
 Heating of upper 1/3rd of tube
inclining at an angle by low flame
 Turbidity develops in heated
portion of urine
 1-2 drops of 5% acetic acid is
added and reheated
 Persistence of turbidity indicates
presence of protein
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43. Need to keep in mind
 Turbidity may appear due to protein or phosphate
 After addition of 5% acetic acid turbidity due to
phosphate disappears but that due to protein
persists
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44. Benedict’s test
 5ml of Benedict’s qualitative
solution taken in a test tube and
heated
 8 drops of urine is added and
reheated to boiling point followed
by cooling
 Change of colour indicates
presence of reducing substance
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45. Appearance Colour change Grade
Blue (No color change) Absent
Green Trace
Yellow Low
Orange Moderate
Brick red High
Benedict’s test: interpretation
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46. Need to keep in mind
 The test determines presence of reducing substance,
not glucose or sugar
 Reducing sugar: All mono and disaccharides except
sucrose
 Reducing substance: All reducing sugar,
homogentisic acid, nalidixic acid, uric acid, ascorbic
acid, aspirin etc.
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47. Rothera’s test
 5ml of urine taken in a test tube
followed by addition of ammonium
sulphate till saturation
 Addition of 2 drops of 5% sodium
nitroprusside solution followed by
few drops of ammonia water
 After mixing the content, tube is kept
for 5min
 In presence of ketone body, a purple
colour ring appears
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48. Need to keep in mind
 Acetone is volatile, not present in urine
 β-hydroxybutyrate does not give this test
positive
 Only acetoacetate give Rothera’s test positive
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49. “ Thank you.
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