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Lab diagnosis in renal diseases
This document discusses laboratory diagnosis of renal diseases. It covers renal function tests like glomerular filtration rate (GFR) and clearance tests which are used to detect early renal impairment. GFR is estimated using creatinine clearance tests or formulas using serum creatinine. Urine analysis and renal biopsy are also used to diagnose and characterize renal diseases by examining features under light and electron microscopy. Renal biopsy can identify conditions affecting the glomeruli, tubules, interstitium or blood vessels. Recent advances include use of genomics and proteomics in renal disease diagnosis and classification.
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Lab diagnosis in renal diseases
- 1. LABORATORY DIAGNOSIS IN RENALDISEASES - Dr. Meghana P
- 2. INTRODUCTION Kidney is ahighly specialized organ. Functions: • ECF volume and composition • Metabolic waste products • Blood pressure • Erythropoietin • Vit D3
- 3. FACTORS AFFECTING RENAL FUNCTION •Diffuse renal disease • Pre renal conditions – dehydration, CCF, shock • Post renal conditions – obstruction
- 4. INVESTIGATIONS • Renal functiontests • Urine analysis • Renal biopsy
- 5.
- 6. INDICATIONS • Early identificationof renal impairment in patients at risk. • Diagnosis of renal disease. • Assess response to treatment. • Adjust dosage of certain drugs. • Plan renal replacement therapy in advanced renal diseases.
- 7. CLASSIFICATION GLOMERULUS PROXIMAL TUBULES DISTAL TUBULES Clearance testsGlycosuria Specific gravity S. Creatinine Aminoaciduria Osmolality BUN Tubular proteinuria Water deprivation test BUN/S.Creatinine Urinary excretion of sodium Water loading test Proteinuria Fractional sodium excretion Ammonium chloride loading test
- 8. GLOMERULAR FILTRATION RATE • Rateat which a substance is cleared from the plasma in unit time by the glomeruli. • ml/min
- 9. GFR - RATIONALE •Varies according to age, sex, BSA. • Normal GFR is 120 – 130 ml/min/1.73m2. • Declines with age. • Fall in GFR leads to accumulation of waste products. • GFR < 60ml/min/1.73m2 - >50% loss of renal function.
- 10. CHRONIC KIDNEY DISEASES STAGEDISEASE GFR VALUE (ml/min/1.73m2 ) Stage I Kidney disease with Normal GFR >90 Stage II Kidney disease with Mildly reduced GFR 60-89 Stage III Kidney disease with Moderately reduced GFR 30-59 Stage IV Kidney disease with Severely reduced GFR 12-29 Stage V Kidney failure <15
- 11. METHODS TO MEASUREGFR • Direct assessment - Clearance tests • Indirect assessment from S. Creatinine
- 12. CLEARANCE TESTS • Volumeof plasma that is completely cleared of that substance per minute. • C = UV/ P • C - Clearance (ml/min) U - Concentration of substance in urine (mg/dl) V - Volume of urine per min (ml/min) P - Concentration of substance in plasma (mg/dl)
- 13. AGENT FOR CLEARANCE STUDIES •Not bind to plasma proteins. • Freely filtered across glomeruli. • Not reabsorbed • Not secreted. • Not metabolized by kidney. • Excreted only through the kidney.
- 14. AGENTS USED EXOGENOUS ENDOGENOUS InulinCreatinine Radiolabelled EDTA Urea Radiolabelled 125I-iothalamate Cystatin C
- 15. INULIN CLEARANCE TEST •Gold standard for measurement of GFR. Method: • Bolus dose – 25 ml of 10 % solution. • 500 ml of 1.5 % solution – 4 ml/ min. • Timed urine and blood samples are collected.
- 16. INULIN CLEARANCE TEST Disadvantage: •Time consuming • Expensive • Need to maintain steady plasma levels Normal values: • Males – 125 ml/min/1.73 m2 • Females – 110 ml/min/1.73 m2
- 17. CREATININE CLEARANCE TEST Rationale: • Mostcommonly used. • Produced from creatine in muscle. • Secreted in small amount – overestimation of GFR.
- 18. CREATININE CLEARANCE TEST Method: • 24hour urine sample. • A blood sample is obtained at midpoint of urine collection. • Final calculation is by the formula UV/P. • Cimetidine can be used to prevent overestimation.
- 20. CREATININE CLEARANCE TEST Disadvantages: • Smallamounts of creatinine secreted by renal tubules can increase even further in advanced renal failure. • Collection of urine is often incomplete. • Creatinine levels are affected by - - meat and muscle mass. - certain drugs (cimetidine, trimethoprim)
- 21. CYSTATIN-C CLEARANCE TEST Rationale: •Protease inhibitor produced by all nucleated cells. Advantages over creatinine: • More sensitive and specific. • Not affected by sex, diet and muscle mass.
- 22. INDIRECT ASSESSMENT OF GFR Convenientbut insensitive. Creatinine clearance (140 – age in years) x Body weight in kg ( 72 x serum creatinine in mg/dl) =
- 23. BLOOD UREA NITROGEN(BUN) • Biochemical parameter. • Adults: 7-18 mg/dl
- 24. PRODUCTION OF UREA Proteins Aminoacids Synthesis of tissue proteins Energy Ammonia Urea Cycle Excretion in urine Urea
- 25. DISADVANTAGES - BUN •Less sensitive. • Completely filtered by glomeruli and 30-40 % is reabsorbed. • Affected by - - Protein diet - Upper G.I haemorrhage - Liver function • Considerable destruction of renal parenchyma - blood urea elevation.
- 26. METHODS 1. Diacetyl monoxaminemethod + DAM Yellow diazine derivative High temp, strong acid, oxidizing agent Intensity of color is measured Urea
- 27. METHODS 2. Urease Berthelotreaction Iodophenol Intensity of color is measured Urea 370 C Urease Ammonia Alkaline hypochlorite Phenol
- 28. CAUSES OF INCREASEDBUN PRE RENAL RENAL POST RENAL • Shock • CHF • Dehydration • High protein diet • Trauma • Burns • GI haemorrhage • Impairment of renal function • Obstruction of urinary tract
- 29. SERUM CREATININE Rationale: • Creatinineis produced in muscles at a constant rate. • Production is proportional to muscle mass and body weight. Normal range: 0.7 to 1.3 mg/dl
- 30. METHODS 1. Jaffe’s method CreatinineColored product Alkaline reagent + Picric acid Spectrophotometer
- 31. METHODS 2. Enzymatic method CreatinineH2O2 + phenol + dye Colored product enzymes Spectrophotometer
- 32. SERUM CREATININE INCREASED DECREASED •Azotemia • Dietary meat • Acromegaly • Gigantism • Pregnancy • Old age
- 33. BUN/SERUM CREATININE RATIO • Discriminatedifferent types of azotemia. • Normal value - 12:1 to 20:1
- 34. BUN/SERUM CREATININE RATIO RATIO >20:1RATIO <10:1 ↑BUN WITH NORMAL CREATININE ↑CREATININE WITH NORMAL BUN High protein diet Increased protein catabolism GI haemorrhage Dehydration Starvation Low protein diet Severe liver disease ↑BUN & CREATININE, BUT ↑IN BUN IS MORE ↑BUN & CREATININE, BUT ↑ IN CREATININE IS MORE Post renal obstruction Acute tubular necrosis
- 35. PROTEINURIA Rationale: • A verysmall amount of albumin is excreted in urine. • Microalbuminuria (30 to 300 mg/24 hrs) • Clinical or overt albuminuria ( >300mg/24 hr)
- 36. TESTS TO ASSESSPROXIMAL TUBULAR FUNCTION
- 37. GLYCOSURIA, AMINOACIDURIA, TUBULAR PROTEINURIA Rationale: •Proximal tubules reabsorb 99% of glomerular filtrate. • If PCT are non functioning (or these substances are in excess) they will appear in urine.
- 38. URINARY CONCENTRATION OF SODIUM Rationale: •Pre renal azotemia – normal sodium absorption. • Acute tubular necrosis – decreased sodium absorption. Values: • Pre renal azotemia: < 20 mEq/L • Acute tubular necrosis: > 20 mEq/L
- 39. FUNCTIONAL EXCRETION OF SODIUM(FENa) Rationale: • Better indicator. • Distinction between pre renal failure and renal failure.
- 40. Values: • Pre renalfailure: <1% • ATN: >1% FENa Urine Na+ x Plasma Creatininex 100 Urine CreatininePlasma Na+ = x FUNCTIONAL EXCRETION OF SODIUM (FENa)
- 41. TESTS TO ASSESSDISTAL TUBULAR FUNCTION
- 42. SPECIFIC GRAVITY • Normalvalue: 1.003 to 1.030 • Depends on – hydration and fluid intake.
- 43. SPECIFIC GRAVITY INCREASED DECREASEDFIXED (1.010) • Proteinuria • Glycosuria • Glomerulonephritis • Urinary tract obstruction • Decreased renal perfusion • Diabetes insipidus • Increased water intake • Diseases of tubules • CRF
- 44. URINE OSMOLALITY Rationale: • Mostsensitive and most commonly employed method. • Depends on number of dissolved particles. • Normal value: 500 - 800 mOsm/kg of water.
- 45. URINE OSMOLALITY Application: • ↓urine : plasma osmolality - Acute renal failure • ↑ urine : plasma osmolality - Pre renal azotemia
- 46. WATER DEPRIVATION TEST Rationale: •Measures concentrating ability of kidney with fluid restriction. • Differentiate between central DI and nephrogenic DI.
- 47. WATER DEPRIVATION TEST Method: Risein specific gravity and urine osmolality Urinary concentrating ability maintained No rise in specific gravity and osmolality Administer desmopressin Rise No rise Nephrogenic DICentral DI Restriction of water intake
- 48. WATER LOADING –ADH SUPPRESSION TEST Rationale: • Measures ability of kidney to dilute urine after water loading.
- 49. Method: Over night fast Drink20 ml/kg of water in 15-20 min • <80% excreted • >1.003 • >100 mOsm/kg • ADH fails to decrease • >90% excreted • Specific gravity <1.003 • Urine osmolality <100 mOsm/kg • Decreased plasma osmolality and ADH levels Normal diluting ability of kidney Renal function impairment Collect urine for next 4 hours
- 50. AMMONIUM CHLORIDE LOADING TEST Rationale: •Diagnose type 1 renal tubular acidosis.
- 51. • Urine pH<5.4 •Plasma HCO3 - normal/ high • Urine pH > 5.4 • ↓ Plasma HCO3 - Inconclusive results Give NH4Cl (0.1 gm/kg) Normal renal acidifying ability RTA Collect urine samples for next 6-8 hrs If pH <5.4 - Acidifying ability maintained Urine pH and plasma HCO3 - levels Overnight fast Method:
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- 53. URINE ANALYSIS Physical Examination Volume Color Turbidity Odor pH Specificgravity Biochemical Examination Proteins Sugars Ketone bodies Bile salts Bile Pigments Blood Microscopy Cells Crystals Casts Microorganism Parasites
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- 55. INDICATIONS • Nephrotic syndrome •Acute renal failure • Systemic diseases • Sub-nephrotic proteinuria • Hematuria • Post transplant
- 56. CONTRAINDICATIONS • Solitary kidney •Multiple cysts • Acute infection • Renal neoplasm • Bleeding diathesis • Severe hypertension
- 57. COMPLICATIONS • Haemorrhage • AVfistula • Infection • Accidental biopsy of another organ or perforation of viscus • Death
- 58.
- 59. ADEQUACY • Two biopsycylinders • Length - 1 cm • Diameter - 1.2 mm • 10–15 glomeruli
- 60. • Light microscopy- neutral buffered formaldehyde. • Electron microscopy - 2-3% glutaraldehyde. • Immunofluorescence - do not need any fixative.
- 61. 1. H&E -General 2. PAS - Basement membrane and matrix 3. Trichrome - Fibrosis 4. Silver - Basement membrane and matrix 5. Congo red - Amyloid STAINING
- 62. EVALUATION • Glomeruli • Tubules •Interstitium • Vessels
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- 65. GLOMERULUS: NO /MINIMAL HYPERCELLULARITY DISEASE LM IF EM MCD Normal glomeruli, lipid vacuolation in tubules Negative Loss of foot processes, no deposits FSGS Focal and segmental sclerosis and hyalinosis IgM, C3 Loss of foot processes, electron dense deposits MGN Diffuse thickening of capillary wall Granular IgG, C3 Subepithelial deposits (spikes)
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- 71. GLOMERULUS: WITH HYPERCELLULARITY DISEASE LMIF EM MPGN Double contour of GBM I – IgG, C3 II – C3, properdin III – C3, IgG, Ig M I - Sub endothelial deposits II – Dense intra membranous deposits III – Sub endothelial and sub epithelial deposits. Acute GN Diffuse proliferation, leukocytic infiltration Irregular IgG, C3 Subepithelial deposits (humps) Crescentic GN Proliferation, crescents I- Linear Ig G, C3 II- Granular Ig G, C3 III - Negative I - Linear deposits along GBM II - Sub epithelial deposits III - No deposits
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- 73. MPGN I - SubendothelialII - Intramembranous
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- 78. TUBULO-INTERSTITIAL DISEASE LIGHT MICROSCOPY ATNDilated, necrosed tubules Acute pyelonephritis Extensive acute inflammation, destruction of the tubules Chronic pyelonephritis Thyroidization, chronic inflammation, periglomerular fibrosis
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- 81. VASCULAR CONDITIONS DISEASE LIGHTMICROSCOPY Benign nephrosclerosis Hyaline arteriosclerosis, intimal thickening Malignant nephrosclerosis Necrotising arteriolitis, hyperplastic intimal sclerosis Thrombotic microangiopathy Fibrinoid necrosis, thrombi in renal microvasculature
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- 85. RECENT ADVANCES • Genomicsand proteomics. • Laser-capture micro dissection - selection of glomeruli or a specific tubular segment for study. • ISH Advantages: • Identification of specific mRNA expression pattern and their correlation with diagnosis, prognosis and response.
- 86. SUMMARY • Renal diseasescauses no symptoms until 50-75% of kidney function is destroyed. • Lab tests often provide the initial recognition of renal impairment and can be used to direct further therapy. • The simplest diagnostic test is the examination of the urine. • Renal biopsy is performed to establish the specific diagnosis of renal disease.
- 87. REFERENCES • U.Satyanarayana, U.Chakrapani,Biochemistry, Arunabha Sen Books Pvt. Ltd., 2010. • Gaw A, Murphy MJ, Cowan RA, O’Reilly DSJ, Stewart MJ, Shepherd J. Clinical Biochemistry: An illustrated colour text (3rd Ed). Edinburgh: Churchill Livingstone, 2004. • Stevens LA, Levey AS. Measurement of kidney function. Med Clin N Am 2005;89:457-73. • Pathological basis of disease : Robbins and Cotran, 9th edition.
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