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Approach to laboraratory diagnosis of acute and chronic renal failure
1. Laboratory approach for diagnosis
of Acute and Chronic Renal Failure
Speaker :-Dr. Adrija Pathak
2. Acute renal Failure
Rapid deterioration of renal function (GFR) over a
period of hours to days
Azotemia (accumulation of nitrogenous wastes)
Decreased urine output (usually but not always)
Oliguria: <500 ml urine output in 24 hours
Anuria: <50 ml urine output in 24 hours
Electrolyte and acid base abnormalities
4. Chronic Renal failure
Progressive decline in GFR over 3 months
Uremia - azotemia with symptoms or signs of
renal failure and biochemical abnormalities
Metabolic and endocrine alteration- anemia,
malnutrion, change in plasma level of PTH,
insulin,glucagon, sex hormone, prolactin
Secondary invovement- renal osteodystrophy,
uremic gastroenteritis, fibrinous carditis,
peripheral neuropathy, dermatological
invovement
5. Classification of chronic kidney
disease
stage GFR, ml/min per1.73 m²
0 >90 with risk factor ofCKD
1 >/= 90 with proteinuria,abnormal blood & urine
chemistry, imaging studies
2 60- 89
3 30- 59
4 15- 29
5 <15
Normal GFR- 120ml/min per 1.73m² attained in 3rd decade
with annual mean decline of 1ml/min per 1.73m² reaching a
mean value of 70ml/min per 1.73m² at 70 years
CRF – Irreversible reduction in nephron and corresponds
to Stages 3-5
End-Stage Renal Disease – Stage 5
6. Evaluation of Renal Failure
Is the renal failure acute or chronic?
laboratory values do not discriminate between acute
vs. chronic
oliguria supports a diagnosis of acute renal failure
Clues to chronic disease
Pre-existing illness – DM, HTN, age, vascular
disease.
Uremic symptoms – fatigue, nausea, anorexia,
pruritis, altered taste sensation, hiccups.
Small, echogenic kidneys by ultrasound.
Previous records
7. Routine Kidney Profile Tests
Blood urea nitrogen
Serum Creatinine
Serum total protein, albumin, globulin,A/G ratio
Serum electrolyte
Blood gases, Blood pH and bicarbonate
Plasma and Urine Osmolarity
Creatinine Clearance test
Routine Urine examination
8. Assessment of GFR
Best indicator of kidney function
Based on concept of clearance- rate of urinary
excretion of a substance to the plasma
concentration
Cx = (UxV)/Px
For assessment of GFR the substance used
should be minimally reabsorbed and minimally
secreted
9. Exogenous substance
Clearance of inulin- gold standard
127ml/min/1.73m² in healthy men
118ml/min/1.73m² in healthy women
Not clinically practical- requires iv infusion and timed
urine collection over many hours
Urinary clearance of other radioactive markers- 125 I-iothalamate
and 99Tc-DTPA gives good measure
Endogenous substance- widely used are cretinine&
urea
Other- cystatin C, ß trace protein, ß2 microglobulin
and tryptophan conjugate
10. Creatinine clearance
Most widely used marker because
- Endogenous substance
- Fairly constant rate of production by muscle
- Not bound to plasma protein---freely filtered
- Not reabsorbed by renal tubule
Drawbacks- partially secreted by proximal tubule
- severe muscle wasting or meat ingestion
- Number of chromogens interfere with its
measurement by alkalaline picrate method
Normal value – male: 105± 20 ml/min
female 95± 20 ml/min
11. Formulas to estimate creatinine
clearance as an estimate for GFR
Cockroft-Gault formula (ml/min)
([140-age].[IDW]) / (72 X SCr) multiply by 0.85 if
female
IDW –Male 50kg +2.3 for each inch over 5 feet
- Female 45.5 +2.3 for each inch over 5 feet
- Reduces the variability of cretinine production due
to differerence in muscle mass based on age and
sex
- However it overestimates GFR diseased state,
obese, edematous
12. Modification of Diet in Renal Disease (MDRD)
formula- based on six variables age,sex, serum
urea nitrogen, serum cretinine, race and serum
albumin
Simlified MDRD formula (ml/min/1.73m² )
GFR= 175. Cr ˆ-1.154 . Age ˆ -0.203 x 1.212 for black x 0.742 if
female
These formulas are useful in chronic states when
cretinine production is assumed to be equal to
excretion in urine.
In acute renal failure when serum cretinine rises
rapidly measurement of cretinine in urine with
timed urine collection is more useful in
13. Urea clearance
As a mesure for GFR is not very good
- Urea production varies widely depending on
protein intake
- Freely filtered but reabsorbed substantially
In normal renal function without volume
depletion, urea clearance is about 50% of
creatine clearance, but in severe volume
depletion it could be 10% of creatinine clearance
In advance renal failure urea clearance
approaches unity with GFR and is better than
creatinine clearance as a measure for GFR
14. Normal value
Maximum urea clearance : 60-95 ml ( average :74 ml)
- When rate of urine excretion is >2ml/min
- Expressed as a percentage of average normal
- Formula is 1.33 x UV/P
Standard urea clearance : 40 -65 ml (average : 54 ml)
- Formula is 1.85 x U√V/P
Two urine sample collected. If difference in clearance
exceed 10% repeat the test
15. Creatinine measurement
Based on Jaffe reaction- reaction of creatinine
with trinitrophenol (picric acid) in alkaline
condition to form a red complex. OD measured at
520nm
Alkaline Picrate Method
Several chromogens – ketones, glucose,
fructose, urea, ascorbic acid , guanidine,pyruvate
(also protein) react with picrate and gives false
high value
Bilirubin and hemoglobin interfere giving false low
value
Without removing chromogens upper limt of
normal measured by jaffe reaction is1.6-1.9 mg/dl
for adults
16.
17. Automated method-based on jaffe reaction principle
using autoanalyser equipped with a thermocuvette
(30˚C)
1st reading recorded at 20 second as most of
interfering chromogen react fast
Creatinine and alkaline picrate react relatively slowly.
Hence 2nd reading noted after 80 seconds
Same procedure is used for a standard
Creatinine (mg/dl)= OD T(80sec) – OD T(20sec) x
conc. of std
OD S(80sec) – OD S(20sec)
Reference Range 1-5 yr 0.3- 0.5 mg/dl
5-10 yr 0.5- 0.8 mg/dl
Adult male 0.6-1.2mg/dl
18. Measurement of urea/ urea nitrogen
Gold standard is isotope dilution mass
spectrometry- used only as reference method
colorimetric method based on reaction of urea
with diacetyl monoxime under strong acidic
condition in the presence of ferric ions and
thiosemicarbazide to form intense red coloured
which is measured at 540nm.
normal range – birth to 1 yr 4-16mg/dl
1-40 yr 7-21 mg/dl
Gradual slight increase over 40 yr
Possible panic range BUN>100mg/dl
19.
20. enzymatic method
- berthelot method- urease splits urea into ammonia
& CO2. ammonia reacts with phenol in presence of
hypochlorite to form indophenol which with alkali
gives a blue coloured compound whose OD can be
measured at 546nm
- UV Kinetic method- after hydrolyses by urease, in
the presence ammonia, æ ketoglutarate &
glutamate dehydrogenase, NADH is oxidised to
NAD+. The rate of decrease of OD is measured at
an interval of 30 sec at 340 nm which is ∝ urea
concentration.
21.
22. Urinanalysis
Physical Chemical Microscopy
Volume (1200-
1500ml/d)
Protein Leukocyte/ Pus cell
Colour Glucose Erythrocyte
Apperance (clear) Ketone bodies Epithelial cells
Sediment Occult blood Casts
Odor Bile pigment Crystals
Reaction/pH (acidic 4.7-
7.5)
Bile salt Yeast/Bacteria/spermato
zoa
Specific gravity (1.003-
1.030)
urobilinogen
23. Specific gravity
Urinometer-vessel is filled 3/4th with urine (min 15ml
is required)urinometer inserted without touching the
side/bottom. Lower meniscus is read.
- Checked daily by measuring sp. Gravity of distilled
water
- Correction for temperature/protein/glucose
Reagent strip
Refractometer
Falling drop method
24.
25. Albuminuria
Helpful in monitoring nephron injury & response
to therapy especially in chronic glomerular
diseases
24 Hour urine collection is gold standard
Albumin/ creatinine ratio in a spot first morning
urine sample is practical and correlates well
Persistence of >17mg albumin/gram of creatinine
in male or >25 mg albumin/gram of creatinine in
female signifies chronic renal damage
26. Microscopic findings
Hyaline Casts:
Better seen with low light.
Non-specific.
Composed of Tamm-
Horsfall mucoprotein.
27. Granular Casts:
Represent degenerating
cellular casts or
aggregated protein.
Nonspecific.
Waxy Casts:
Smooth appearance.
Blunt ends.
Felt to be last stage of
degenerating cast –
representative of chronic
disease.
28. Muddy Brown
Casts:
Highly
suggestive of
ATN.
Pigmented
granular casts as
seen in
hyperbilirubinemi
a can be
confused for
these.
29. Fatty Casts:
Seen in patients with
significant proteinuria.
Refractile in appearance.
May be associated with
free lipid in the urine.
Can see also “oval fat
bodies” – RTE’s that
have ingested lipid.
30. Hematuria
Nonglomerular hematuria:
Urologic causes.
Bladder/Foley trauma.
Nephrolithiasis.
Urologic malignancy.
May be “crenated” based upon
age of urine, osmolality – NOT
dysmorphic.
31. Dysmorphic Red Cells:
Suggestive of
glomerular bleeding as
seen with
glomerulonephritis.
Blebs, buds, membrane
loss.
Rarely reported in other
conditions – DM, ATN.
Red Blood Cell
Casts:
Essentially diagnostic
of vasculitis or
glomerulonephritis.
32. Crystals
Uric acid crystals:
Seen in any setting of
elevated uric acid and an
acidic urine.
Seen with tumor lysis
syndrome.
Calcium oxalate crystals:
Monohydrate – dumbell
shaped, may be needle-like.
Dihydrate – envelope
shaped.
Form independent of urine
pH.
Seen acutely in ethylene
glycol ingestion.
33. Prerenal ARF sediment is characteristically acellular
and contain hyaline cast
Post renal failure may present with inactive
sediment, although pyuria and hematuria are
common
Casts
Pigmented “muddy brown cast” cast containing
tubular epithelial cell characteristic of ATN
RBC cast- glomerular injury/ acute tubulointerstitial
nephritis
WBC cast & nonpigmented granular cast- interstitial
nephritis
Broad granular cast- chronic renal disease
34. Eosinophiluria (>5% of urine leukocyte) is a
common finding in antibiotic induced allergic
interstitial nephritis and can be detected by Hasel’s
stain
Atheroembolic RF have eosinophill-rich
inflammatory rxn but normal urinanalysis or few
eosinophils seen
Lymphocyte may predominate in allergic interstitial
nephritis by NSAIDs, ampicillin, rifampicin,
interferone alpha.
Proteinurea >1g/d suggests injury to gl.
Ultrafiltration/ excretion of myeloma light chain
Hemoglobinuria or myooglobinurea should be
suspected if urine is strongly +ve for heme by
dipstick but show few RBC
35. Fractional excretion
Quantity of substance excreted in urine expressed as
fraction of filtered load of same substance
FE = (Ux/Px). (Pcreat/ Ucreat)
When the subtance excreted in urine has clearance
less than that of creatinine FE< 1
FE of sodium is used to distinguish between
ATN(>1%) and prerenal azotemia (<1%)
36. BUN/serum creatine ratio
Normaly observed to be 14-24
In retention of urine due to prerenal causes upto
40
Early stage of renal disease ratio may be normal
Post renal condition cause simultaneous and
proportional increase in both BUN & creatinine so
the ratio will be below 14 depending on
percentage of obstruction
37. Laboratory finding in ARF
INDEX PRERENAL
AZOTEMIA
OLIGURIC ARF
BUN/P Cr Ratio >20:1 10 -15:1
Urine sodium (U Na), meq/L <20 >40
Urine Osmolality, mosmol/L
H2O
>500 <350
Fractional Excretion of Na <1% >2%
Urine Cr /Plasma Cr >40 <20
39. Determination of serum Na & K
Method- Flame photometry
Principle- test solution passed under controlled
conditions as a very fine spray in the air supply to
non luminous flame. The solution evaporates and
salt dissociates to give neural ions which emit
light of characteristic wavelength.
Hyponatremia- salt loosing nephritis, addison ds
Hypernatremis- DI, dehydration, post renal
obstruction
Hypokalemia- cushing ds, tubular damage
Hyperkalemia- renal glomerular disease, anuria,
addison ds
40. Determination of serum Cl
Method- Schales and Schales
Principle – protein free filtrate of the specimen is
titrated with merrcuric nitrate in presence of
diphenylcarbazone as an indicator. The free Hg++
combine with the Cl- to form soluble but
nonionized HgCl2
- After all Cl- have reacted with Hg++ any excess
Hg++ combine with indicator to form blue
complex. Colour change is the end point of
titration
Low Cl- salt loosing nephritis,, burns
High Cl- dehydration, renal tubular ds
41. Determination of bicarbonate
Method- Titrimetric
Principle- Serum is added to standard 0.001N
HCl and loss of strength of standard acid due to
bicarbonate is determined by titrating the strengh
of acid against 0.001N sodium hydroxide
Metabolic acidosis- decrease in bicarbonate
fraction with no /relatively smaller change in
carbonic acid fraction. Compensation is by
elimination of more CO2 by hyperventilaton
42. Determination of blood pH, pCO2,
pO2
Normal value
- Arterial pCO2 : 35-45 mm Hg
- Arterial pO2 : 95- 100 mm Hg
- pH : 7.4
Compensated Metabolic acidosis
pH, pCO2, pO2, Bicarbonate -- decrease
43. Total serum protein
Normal range : 6-8 g/dl
Method : Biuret method
Principle : protein reacts with cupric ions in
alkaline medium to form a violet complex.
Intensity of colour measured at 530nm is ∝
protein present
Decrease- nephrotic synd, malnutrition
Increase- dehydration, multiple myeloma
44. Determination of serum albumin
Normal range : 3.3 -4.8 g/dl
Method : Bromocresol green method
Principle : albumin present in serum binds
specifically with BCG at pH 4.1 to form green
complex. OD measured at 640nm
Determination of globulin : Total protein –albumin
- Normal range : 1.8-3.6g/dl
- A/G Ratio 1.2:1 – 2:1
45. Fractionation of serum proteins by
Electrophoresis
Principle : serum proteins are colloidal in nature.
At pH 8.6 when subjected to electrical current all
serum proteins behave like anions and move
towards anode. The rate of migration depends
upon their molecular weight and size.
- Thus albumin is fastest moving component
followed by alpha1, alpha2, beta & gamma
globulin
46. Osmolarity of urine and serum
Simultaneous determination of serum & urine
osmolarity is a more accurate way of measuring
the concentrating ability of tubule
Normal ratio of urine to serum osmolarity is 3 or
more
Osmometer- based on colligative property which
depends upon number of particle in solution and
not on nature of these particle
- 3 types : freezing point, vapour pressure and
colloidal osmometer.
48. Biomarker for acute kidney injury
These urinary biomarkers are produced by the
kidneys as a result of kidney injury or may be
filtered by glomerulus but not absorbed by tubule
because of injury to tubule
Advantage over conventional markers (Cr/urea) is
their level increase long before any change in
serum creatinine/ BUN.
- Also reveal primary location of injury
1. Kidney injury molecule-1
2. Neutrophil gelatinase-associated lipocalin
3. Interleukin-18
4. Fatty acid binding protein
49. Other investigations in CRF
Anemia: Hb concentration, iron, folate, B12
Calcium, phosphorus and PTH
Serum and urine electrophoresis
Appropriate tests for SLE and vasculitis
In presence of glomerulonephritis underlying
infectious etiologies- hepatitis B&C / HIV should
be assessed
Serial measurement of renal function
50. Role of biopsy
In ARF- biopsy is reserved for patients in whom
prerenal and postrenal ARF have been excluded
and cause of intrinsic ARF is unclear
Useful when clinical/lab investigation suggest
diagnoses other than ischemic or nephrotoxic
injury that respond to disease specific therapy—
glomerulonephritis, vasculitis & allergic interstitial
nephritis
In CRF with b/l small kidney biopsy is not advised
because
- technically difficult, bleeding & other adverse
cosequences
- Scarring- underlying ds may not be apparent
- Window of opportunity for therapy has passed
- Other C/I- hypertension, morbid obesity, uti, bleeding
51. References
Harrison’s principles of internal medicine 17th
edition
Henry’s clinical diagnosis and management
by laboratory methods 22nd edition
Godkar’s textbook of medical laboratory
technology 2nd edition