patholgy

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

3. Types of ARF
 Prerenal
- Hypovolemia : hemorrhage, diarrhea, vomiting, diuretics,
dehydration, burn, pancreatitis
- Altered renal hemodynamics : low cardiac output state,
systemic vasodialation(sepsis), renal
vasocontriction(hypercalcemia), hepatorenal syndrome
 Intrinsic
- ATN : ischemia, infection, toxin (exogenous- radiocontrast,
chemotherapy, ethylene glycol / endogenous-rhabdomyolysis,
hemolysis)
- Renovascular obstruction
- Diseases of glomeruli/ vasculature
- Interstitial nephritis
- Intratubular obstruction
 Post renal –uretric/bladder/urethral obstruction

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

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

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.

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

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

38. Serum electrolyte
 Normal level
- Sodium 133- 148 mEq/l
- Potassium 3.8-5.6 mEq/l
- Chloride 95- 106 mEq/l
- Bicarbonate 21- 28 mEq/l
- Calcium 9-11mg/dl
- Phosphorus 2.5-4.5mg/dl

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.

47. Other investigations in ARF
 Renal artery stenosis- elevated LDH, mild
proteinuria,occasional hematuria, renal
angiogram
 Atheroembolic diseases- Eiosinophilia,
hypocomplementemia, skin/renal biopsy
 Glomerulonephritis- ANA, ANCA, anti-BM Ab,
hepatitis serology, cryoglobulin, blood cultures,
ASO, complements
 Hemolytic uremic syndrome- Schistocyte on PBS,
elevated LDH, anemia, thrombocytopenia
 Rhabdomyolysis- hyperkalemia,
hyperphosphatemia, hypocalcemia, increase uric
acid level and creatine kinase

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