Urine analysis Urine analysisUrine analysis .pptx

Urine analysis

1. Urine analysis

2. Background Examination of urine is an indispensable part of evaluation of patients with impaired kidney function, particularly proteinuria, hematuria, urinary tract infection, nephrolithiasis and other renal or non renal diseases Examination of urine sediment provides valuable information about renal parenchyma Experience in examining the urine is valuable; studies show that a urinalysis performed by an experienced nephrologist or trained physician is more likely to be better than a urinalysis reported by a clinical chemistry laboratory

3. SPECIMEN COLLECTION AND HANDLING Urine should be collected with minimum contamination . Clean catch midstream collection is performed If not feasible, bladder catheterization is appropriate for adults— risk of contracting a urinary tract infection is negligible for a single catheterization Suprapubic aspiration is used in infants High urine osmolality and low pH favor cellular preservation, hence first voided morning urine is preferred Chemical composition of urine changes withstanding and formed elements degrade over time. Hence, urine is best examined when fresh but a brief period of refrigeration is acceptable Bacteria in urine multiply at room temperature, hence bacterial counts from unrefrigerated urine are unreliable

4. Routine urinalysis Appearance Specific gravity Chemical tests (Dipstick) • pH • Protein • Glucose • Ketones • Blood • Urobilinogen • Bilirubin • Nitrites • Leukocyte esterase • Microscopic examination (formed element ). Crystals - Urate , calcium phosphate, oxalate or carbonate, triple phosphate, cysteine, drugs Cells- Leukocytes, erythrocytes, renal tubular cells, oval fat bodies, transitional epithelium, squamous cell Casts Hyaline, granular, red blood cell (RBC), white blood cell (WBC), tubular cell, degenerating cellular, broad, waxy, lipid-laden Infecting organism- Bacteria, yeast, trichomonas, nematodes Miscellaneous- Spermatozoa, mucous threads, fiber, starch, hair and other contaminants

5. ROUTINE URINALYSIS Appearance Specific Gravity= Weight of urine of measured volume Weight of distilled water of some volume Clinical Importance of Measuring Specific Gravity: In the absence of proteinuria, glycosuria or iodinated contrast administration, a specific gravity more than 1.018 implies preserved concentrating ability of kidneys. Measurement is useful to differentiate prerenal azotemia and acute tubulointerstitial nephritis (ATIN).

6. Protein Composition of Urine

7. Protein Dipstick Method Protein indicator strips are buffered at an acid pH near their color change point; wetting them with a protein containing specimen indicates a color change. Protein reaction may be scored as follows: Trace = 5–20 mg/ dL 1 + = 30 mg/ dL 2 + = 100 mg/ dL 3 + = 300 mg/ dL 4 + = > 2,000 mg/ dL Protein strips are highly sensitive to albumin but less so to globulins, hemoglobin or light chains

8. Chemical Composition of Urine Physiologic urinary pH lies between 4.5 and 8 . pH should be tested properly in freshly voided urine because: Growth of urea splitting bacteria and loss of carbondioxide (CO2) raise the pH Bacterial metabolism of glucose may produce organic acids and lowers the pH These strips are not sufficiently accurate to be used for the diagnosis of renal tubular acidosis (RTA).

9. Chemical Composition of Urine pH - The physiologic urine pH ranges from 4.5 to 8 Bacterial metabolism of glucose may produce organic acids and lower pH Protein - Protein strips are highly sensitive to albumin but less so to globulins, hemoglobin or light chain Urine that is negative by dipstick but positive with sulfosalicylic acid is highly suspicious for light chains The protein indicator used for routine dipstick analysis is not sensitive enough to detect microalbuminuria

10. Chemical Composition of Urine Blood - Reagent strips for blood rely on the peroxidase activity of hemoglobin to catalyze organic peroxide with subsequent oxidation of an indicator dye Free hemoglobin produces a homogeneous color. Intact red cells cause punctate staining. False-positive reactions occur if the urine is contaminated with other oxidants such as povidone-iodine, hypochlorite or bacterial peroxidase Glucose - Modern dipstick reagent strips are specific for glucose They rely on glucose oxidase to catalyze the formation of hydrogen peroxide, which reacts with peroxidase and a chromogen to produce a color change Ketones - Ketone reagent strips depend on the development of a purple color after acetoacetate reacts with nitroprusside

11. Chemical Composition of Urine Urobilinogen - Urobilinogen is a colorless pigment that is produced in the gut from metabolism of bilirubin The urobilinogen test is based on the Ehrlich reaction, in which diethylaminobenzaldehyde reacts with urobilinogen in acid medium to produce a pink color Bilirubin -Bilirubin reagent strips rely on the chromogenic reaction of bilirubin with diazonium salts. Conjugated bilirubin is not normally present in the urine Nitrite - The nitrite screening test for bacteriuria relies on the ability of Gramnegative bacteria to convert urinary nitrate to nitrite, which activates chromogen Leukocytes - Granulocyte esterases can cleave pyrrole amino acid esters, producing free pyrrole that subsequently reacts with a chromogen. The test threshold is five to fifteen white blood cells (WBCs) per high-power field (HPF). False-negative results occur with glycosuria, high-specific gravity, cephalexin or tetracycline therapy

12. Cellular elements in the urine. (A) Nondysmorphic red blood cells (RBCs). They appear as uniform, biconcave disks; (B) Dysmorphic RBCs from a patient with immunoglobulin A nephropathy. Their shape is irregular, with membrane blebs and spicules (C) Urine obtained from a patient with an indwelling bladder catheter. Innumerable white blood cells (WBCs) as well as individual (small arrows), budding (single thick arrow) and hyphal (open arrow) forms are present ( D) Renal tubular epithelial cells. Note the variability of shape. The erythrocytes in the background are much Smaller ( E) Squamous epithelial cells ( F) Transitional epithelial cells in a characteristic clump

13. Proteinuria Normal urine contains small amount of protein usually upto 150mg/24 hrs These include proteins from Plasma ( albumin) Protein from urinary tract ( tamm-horsfall protein, secretory IgA, proteins from tubular epithelial cells, leukocytes and other desquamated cells) Excretion of more than this level >150mg/24hrs causes proteinuria Proteinuria

14. Causes of proteinuria according to pathophysiology Glomerular proteinuria Tubular proteinuria Overflow proteinuria Tissue proteinuria Primary glomerular disease – Minimal change glomerulopathy – Immunoglobulin A nephropathy – Focal segmental glomerulosclerosis (FSGS) – Membranous glomerulonephritis – Membranoproliferative glomerulonephritis (MPGN) – Fibrillary and immunotactoid glomerulopathy – Crescentic glomerulonephritis • Secondary glomerular disease – Multisystem disease: Systemic lupus erythematosus (SLE), vasculitis, amyloid, scleroderma – Metabolic disease: Diabetes mellitus, Fabry’s disease – Neoplasia: Myeloma, leukemia, solid tumors – Infections: Bacterial, fungal, viral, parasitic – Drugs, toxins and allergens: Gold, penicillamine , lithium, nonsteroidal anti-inflammatory drug (NSAID), penicillin – Familial: Congenital nephrotic syndrome, Alport’s syndrome, nephronophthisis – Others: Toxemia of pregnancy, transplant nephropathy, reflux nephropathy • Glomerular proteinuria without renal disease – Exercise induced, orthostatic, febrile proteinuria • Drugs and toxins – Luminal injury: Light-chain nephropathy, lysozyme ( myelogenous leukemia) – Exogenous: Heavy metals (lead, mercury, cadmium), tetracycline, aristolochic acid (Balkan nephropathy) • Tubulointerstitial nephritis – Hypersensitivity (drug, toxin) – Multisystem: SLE, Sjögren’s syndrome, tubulointerstitial nephritis with uveitis • Others – Fanconi’s syndrome Myeloma, light-chain disease, amyloidosis, hemoglobinuria, myoglobinuria • Acute inflammation of urinary tract • Uroepithelial tumors

15. Glomerular proteinuria : Glomerular diseases damage glomerular basement membrane (increased permeability) but tubular function is normal Two types S elective proteinuria---chiefly albumin N onselective proteinuria Causes : acute glomerulonephritis and nephrotic syndrome

16.   Selective proteinuria - When glomeruli can retain larger molecular weight proteins but allow passage of comparatively lower molecular weight proteins ( albumin and transferrin) Occurs in early stages of disease Non selective proteinuria - With further glomerular damage, this selectivity is lost and larger molecular weight proteins (ƴ globulins) are also excreted along with albumin Both distinguished by urine protein electrophoresis In selective proteinuria, albumin and transferrin bands are seen. In non selective – pattern resembles that of serum

17. Tubular proteinuria Normally, glomerular membrane although impermeable to high molecular weight proteins, allows ready passage to low molecular weight proteins like - β2 microglobulin , retinol binding protein, lysozyme, α1- microglobulin and free immunoglobulin light chains. These low molecular weight proteins are readily reabsorbed by proximal renal tubules. In diseases involving mainly tubules, these proteins are excreted in urine while albumin excretion is minimal

18. Disease : Acute and chronic pyelonephritis, Tb of kidney ,Heavy metal poisoning, Interstitial nephritis, Cystinosis , Fanconi syndrome, Rejection of kidney transplant Purely tubular proteinuria cant be detected by reagent strip test – detected by heat and acetic acid test and sulphosalicylic acid test

19. Overflow proteinuria When concentration of a low molecular weight protein rises in the plasma, it overflows from plasma into the urine. Such proteins are I mmunoglobulin light chains B ence jones proteins (plasma cell dyscrasias ) H emoglobin (intravascular hemolysis ) M yoglobin (skeletal muscle trauma ) L ysozyme ( AML M4 or M5)

20. Hemodynamic proteinuria Alteration of blood flow through glomeruli causes increased filteration of proteins. Protein excretion is transient Seen in High fever , Hypertension , Heavy exercise, CCF Seizures, Exposure to cold, Post renal proteinuria, Inflammation or neoplastic conditions in renal pelvis, ureter, bladder, prostate or urethra

21.  Postural ( orthostatic proteinuria) When subject is standing or ambulatory, but is absent in recumbant position. Seen in adolescents Due to lordotic posture that causes inferior venacaval compression between liver and vertebral column. Amount <1000mg/day Periodic testing is done to rule out renal diseases. 1st morning urine after rising is negative, while another sample collected after patient performs activities is positive

22. Test for proteinuria 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)

23. Reagent strip test Principle- the reagent area of the strip is coated with an bromophenol blue indicator and buffered to an acidic pH which changes color in presence of proteins. Called as protein error of indicators. When dye gets adsorbed to protein, there is change in ionization – change in color of indicator. Intensity of color directly proportional to concentration of protein False negative- alkaline urine, gross hematuria, contamination with vaginal secretions

24. Sulphosalicylic acid test – Principle- addition of sulphosalicylic acid to the urine causes formation of a white precipitate if proteins are present (proteins are denatured by organic acids and precipitate out of the solution). False positive – gross hematuria, highly concentrated urine, radiographic contrast media, excess uric acid, tolbutamide , sulphonamides , salicylates and penicillin. False negatives- very dilute urine.

25. Quantitative estimation of proteins   Indications 1 . Diagnosis of nephrotic syndrome 2 . Detection of microalbuminuria or early diabetic nephropathy 3 . Follow response to therapy in renal diseases Proteinuria - >1500mg/day – glomerular diseases >3500mg/day – nephrotic range proteinuria <1500mg/day- tubular, hemodynamic and post renal diseases

26. Two methods – Quantitative estimation of proteins in a 24 hour urine sample Esbach’s albuminometer method Turbidimetric method Biuret reaction Immunologic method Estimation of protein/creatinine ratio in a random urine sample. Normal protein/creatinine ratio - <0.2 Low grade proteinuria – 0.2-1.0 Moderate- 1.0- 3.5 Nephrotic range proteinuria - >3.5

27. Microalbuminuria Defined as urinary excretion of 30- 300mg/24 hrs of albumin in urine Significance Earliest sign of renal damage in diabetes mellitus (diabetic nephropathy). Independent risk factor for cardiovascular disease in diabetes mellitus.

28. Detection of microalbuminuria Cannot be detected by routine tests for proteinuria. Methods are Measurement of albumin-creatinine ratio in a random urine sample . Measurement of albumin in an early morning or random urine sample Measurement of albumin in 24 hr sample. Test strips Exact quantitation by radioimmunoassay or enzyme linked immunosorbent assay

29. Bence Jones proteinuria Presence of bence jones protein (with unusual thermosolubility ) in the urine, usually indicative of a neoplastic process such as multiple myeloma, amyloidosis or Waldenstrom's macroglobulinaemia . The classic method of identifying bence -jones protein is precipitation from urine in the range of 4o-60 c. It goes into solution at temprature above or below this range. Electrophoresis- The presence of B.J protein & clonal production of Ig is indicated by single sharp peak in the globulin region . Heat test- If albumin & B.J protein are present , boil the urine to 100 degree & filter ppt - albumin will be filtered out , take the rest urine heat it to 45-60 deg - B.J protein will ppt then dissolve on boiling False positive– other protein e.g globulin are ppted by heat test. False negative – when B.J protein is too conc - ppt does not resolve on boiling

30. Precitipation test for BJ protein

31. Evaluation of proteinuria

32. Hematuria T he presence of abnormal number of intact red cells in urine is called hematuria. Causes Diseases of urinary tract – 1. Glomerular diseases 2. Non glomerular diseases Hematological conditions- Coagulation disorders , sickle cell anemia

33. Tests for detection Microscopic examination of urinary sediment Chemical tests- ◦ These tests detect both intracellular and extracellular hemoglobin ( intact and lysed red cells) as well as myoglobin Principle – Heme proteins in hemoglobin act as peroxidase , which reduces hydrogen peroxide to water . This process needs a hydrogen donor ( benzidine , orthotoluidine or guaiac). Oxidation of hydrogen donor leads to development of color . Intensity of color development is proportional to amount of hemoglobin present

34. Benzidine test Make saturated solution of benzidine in glacial acetic acid. Mix 1ml of this solution with 1 ml of hydrogen peroxide in a test tube Add 2ml of urine. If green or blue color develops within 5 min, the test is positive. Reagent strip test- uses chromogens- o-toluidine. False positive- contamination of urine by oxidizing agents or microbial peroxidase in UTI False negative- presence of a reducing agent like ascorbic acid in high concentration, formalin as preservative

35. Evaluation of hemoglobinuria

36. Pyuria The term “pyuria” literally means “pus in the urine” but, in common usage, the focus is not on the presence of pus but on the number of white blood cells (WBCs) or amount of leukocyte esterase (LE) that exceeds a threshold and suggests a urinary tract infection (UTI ) Sterile pyuria- Pyuria without bacteuria

37. Pyuria

38. MANAGEMENT History and examination Urinary tract symptoms such as dysuria and haematuria should alarming to the clinician. Furthermore, a detailed clinical examination is mandatory General findings like hypertension and pallor are important to consider. Skin rashes, oedema , muffled heart sounds, organomegaly , swollen joints, and lymphadenopathy are signs of more serious underlying pathology. Abdominal and pelvic examination, including a digital rectal examination and vaginal examination in females (except during pregnancy), is recommended.

39. Investigations and treatment  U rinalysis is the prime investigation for SP Importantly, SP is not always sterile, so repeating cultures often yields a positive result on subsequent testing . S amples should always be collected as a midstream clean catch   Routine haematological tests including a full blood count, renal, and liver function tests are of paramount importance . Swabs for  Chlamydia  and  Gonorrhoea  are recommended for sexually active patients . If urinary tuberculosis is suspected, three consecutive first-void morning samples are required for acid-fast bacilli and polymerase chain reaction (PCR) testing.

40. Eosinophilia is an important marker of drug-induced interstitial nephritis, but may also be seen in schistosomiasis . In suspected schistosomiasis, a terminal urine sample should be collected between noon and 3 pm, to coincide with maximal egg excretion . Imaging : A renal tract ultrasound scan or computerised tomography is recommended when renal stones, masses, or nephritis are differential diagnoses. Endourological procedures such as rigid or flexible cystourethroscopy and tissue sampling are undertaken if tumours are suspected, but they also have the advantage of diagnosing and treating benign pathologies such as bladder stones.