Examination Of Body Fluids (urinalysis)

Exa minati on of Body F luid s Woon Sung Thong, FMIMLS Past President, Malaysian Institute of Medical Laboratory Sciences Laboratory Manager, Biolife Lab Sdn Bhd

Body Fluids • • • • • • •

Urine Seminal Fluid Amniotic Fluid Cerebrospinal Fluid Synovial Fluid Pleural, Pericardial and Peritoneal Fluid Fecal analysis

Urine Collection First morning *voids before going to bed *formed elements are more stable *unsuitable for cytology studies

Random • ? Second voiding

Collect ion Techniques Routine Midstream Midstream “Clean Catch” *for eliminating contamination *for culture

Urine Container • • • • •

Wide mouth (4 - 5 cm) Sufficient volume (50 ml preferred) Glass or plastic with no additives Leak-proof Sterile, if specimen is stored for a period of time before testing

Why bot her w ith ur inaly sis ? • At pre-employment medical examinations • Routinely at physician office and medical clinics • Annual medical examination An indicator of health or disease, especially with metabolic and renal disorders.

What are the potenti al changes in unpreserved u rine?

Potentia l C hanges in Unpre serv ed Ur in e Physical changes Colour- bilirubin - biliverdin hemoglobin - methemoglobin urobilinogen - urobilin Clarity - Decreased due to bacterial proliferation,solute precipitation Odour - Increased due to bacterial proliferation and decomposition

Potentia l C hanges in Unpre serv ed Urine Chemical Changes pH - increased or decreased Glucose - decreased Ketones - decreased Bilirubin - decreased Urobilinogen - decreased Nitrite - increased or decreased

Potentia l C hanges in Unpres erv ed Urine Microscopic Changes RBC, WBC, Casts *decreased due to disintegration especially in alkaline urine RBC decreased after 6 hours WBC decreased 50% within 3 hours Hyaline and granular casts decreased after 2 hours

Bacteria *increased due to bacterial proliferation

Potentia l C hanges in Unpre serv ed Urine Microscopic Changes • Precipitation of uric acid, calcium phosphate and calcium oxalate • Yeast cells develop pseudo-mycelia • Spermatozoa become immobile • Trichomonas become immobile, maybe counted as WBC • Contamination by air borne particles

Urine Examinatio n

Preservatives Most preservatives prevent bacterial growth and loss of glucose (eg. Stabilur, formalin) No preservatives can prevent destruction of bilirubin, urobilinogen or occult blood. Use of preservatives may increase SG, minor effects on pH and may inhibit leukocyte esterase reaction.

Urine Examinatio n No single urine preservative is available NCCLS recommends that it be analyzed within 2 hours. Refrigeration can induce precipitation of amorphous urates and phosphate crystals that can interfere substantially with microscopic examination

What is FE ME?

Urine FEME • Physical Examination • Chemical Examination • Microscopic Examination

Urine - Phys ical Examination • Colour - urochrome, urobilin, uroerythrin • Clarity Clear Slightly cloudy Cloudy Turbid

• Odour

Physical Tests • Color Normal color range from straw, pale yellow, to amber. Abnormal color: red - RBCs beer-brown - bilirubin orange, blue, green - drug, dye or food

Colour

Colorless, Light Yellow Yellow Amber Dark amber Orange

Red

CO LO UR ITEMS

Constituent

Dilute urine

Comments

Fluid ingestion: polyuria

Normal Urine Concentrated urine Dehydration, fever Urobilin No yellow foam Bilirubin Yellow foam Biliverdin Imparts green blue Bilirubin Yellow foam if sufficient bilirubin Urobilin No yellow foam Medications Hemoglobin, red blood cell Myoglobin Muscle injury Porphyrins Beets Genetic

COLOU R IT EMS Colour

Constituent

Pink

Hemoglobin Porphyrins Hemoglobin Myoglobin Methemoglobin Muscle Homogentisic acid Acid pH Melanin Melanin Upon standing: rare Homogentisic acid Upon standing: alkaline urine Indican Infections of small intestine Chlorophyll Breath deodorizers Pseudomonas infectiion Dyes and medications

Brown

Black Green blue

Comments

Colour change due to oxidation RBC

methemoglobin

oxidizes brown

urobilinogen colourless

oxidizes urobilin orange-brown

porphobilinogen colourless

bilirubin amber

black

oxidizes

porphobilin red/purple

oxidizes

biliverdin greenish

Ur in e colo ur changes with commonly u sed d rugs Drug

Colour

Alcohol, ethyl

pale, diuresis

Anthraquinone laxatives

reddish-alkaline, yellow brown-acid

Chlorzoxazone

Red

(muscle relaxant) Deferoxamine mesylate

Red

(Desferal) Furazolidone

Brown

(an antibacterial, anti protozoal nitrofuran) Indigo carmine dye

Blue

(renal function, cytoscopy) Iron sorbital (Jectofer)

Brown on standing

Levodopa (parkinsonism)

Red then brown, alkaline

Urine color changes with commonly used drugs • • • • • • •

Alcohol Desferal Paraflex (muscle relaxant) L-dopa (for parkinsonism) Flagyl Nitrofurantoin Riboflavin

• • • • • • •

Pale Red Red Red then brown Reddish brown Brown-yellow Bright yellow

Physical Tests • Turbidity Normal is essentially clear. Cloudy urine: amorphous salts - non pathologic bacteria, blood cells - pathologic

Causes of Tu rbidity Pathologic RBC WBC Bacteria Yeast, Trichomonas Renal Epithelial Cells Fat (lipids, Chyle) Abnormal crystals Calculi Pus

Non pathologic Normal crystals (eg. Urates, phosphates) Radiographic media Mucus, Mucin Squamous epithelial cells Sperm/prostatic fluid Salves, lotions, cream Powders, talc

Chemical Examination

Ur in alysis Analysis technique Urinalysis Physical examinations Volume - average of 1.0 to 1.5L of urine excreted per day Amount excreted is an indicator for diuretic disorder Polyuria: urine/day

More than 2000ml

Oliguria: Less than 500ml urine/day Anuria : Less than 200ml urine/day Dysuria: No urinary excretion

Chemical Examination Dipstick method (manual and machine) Bayer- Ames Multistix Roche - BM Combur 10 SD UroColor 11 Teco Diagnostics - URS - 10 Yeongdong - Uriscan - Gen 11

Chemical Examination Dipstick method Manual : Subjective Machine : Sandardized Reflectance photometer measures scattered or reflected light multiple channels (LED) compensator pad

Instrumentation

2.5 Concentration Table - (Program Chip Card I) The Miditron(R) Junior prints the test results in the following concentration ranges:

2.5 Concentration Table - (Program Chip Card I) The Miditron(R) Junior prints the test results in the following concentration ranges:

Chemical examination Dips ticks

Dipstick - Care and Storage • • • • •

Store in original container Do not expose to light, heat and moisture If there is any colour change, discard Do not use pass expiration date. Store at manufacturer recommended temperatures

Dipstick - Testing Procedure (Manual) • • • •

Well-mixed uncentrifuged urine sample Dip strip into urine briefly Remove excess urine Read colour development according to manufacturer’s instruction • Read in a well lit area

Dipstick – Testing Shortfall

• Aware of false-positive and falsenegative!

Blood

• Reaction: – Pseudoperoxidase action of Hgb myoglobin catalyzes the oxidation of chromogens to produce a color change

• False negatives: – formalin, excess nitrites (>2.2 mmol/l), elevated SG, ph <5.1, captopril, ascorbic acid

• False positives: – oxidizing detergents, microbial peroxidase (UTI), dehydration, exercise, hemoglobinuria, myoglobinuria, menstrual contaminants, proteinuria (>5 g/l)

Blood • Hematuria - presence of an abnormal number of blood cells in urine as microhematuria or gross hematuria (0.5ml or 2500 RBC/µl) • occurs with disease or trauma anywhere in the kidneys or urinary tract • can be seen in healthy persons undertaking excessive exercise (marathon runners) in whom bleeding emanates from the bladder mucosa. Repeat urinalysis after 48 – 72 hours should be negative

Blood • Separate scale is given: - green dots (intact RBC) - homogenous green color scale (for lysed RBC)

Blood • Even one episode of hematuria must be investigated •Cancer •Trauma •Stones •Infections •Obstructions •Viral infections

•Inflammation of kidneys •Benign prostrate enlargement •Warfarin therapy

Blood • Calculi - Ca oxalate = 60% Uric acid = 25% Phosphate = 20% • Tumors - painless hematuria • Glomerulonephritis - hematuria with proteinuria • UTI

Bilirubin

• Reaction: – Bilirubin in the urine couples with a diazonium salt in an acid medium

• False negatives: – samples exposed to light, excess levels of ascorbic acid.and nitrite, selenium, chlorpromazine

• False positives: – highly colored metabolites of drugs eg pyridium

Bilirubin • Breakdown product of hemoglobin formed in the in the RES, liver, and bone marrow • carried in the blood by protein • Normal adult urine contains 1 mg/dL and this is not detected by usual tests.

Glucose

• Reaction: – double sequential enzyme reaction of glucose oxidase and peroxidase-reacts with a chromogen to produce the final color.

• False negatives: – elevated specific gravity, uric acid, ascorbic acid

• False positives: – presence of oxidizing agents, ketones, levodopa

Glucose • May appear in the urine and is influenced by: – blood glucose levels – glomerular blood flow – tubular reabsorption rate

• often regarded as a hallmark of disease and requires a patient to receive a workup for diabetes mellitus.

Ketones

•

Reaction: (Legal or Rothera’s test) – Reaction with nitroprusside or sodium nitroferricyanide and glycine to produce a color change. β-hydroxybutyerate 78%, acetoacetate 20%,acetone 2%

•

False negatives: - delay in examination False positives: – highly pigmented urines; some drug metabolites, acidic urine, elevated SG

Ketones • Products of incomplete fat metabolism • presence is indicative of acidosis • low carbohydrate diet for weight reduction will produce ketonuria • exposure to cold and severe exercise

Leukocytes • Reaction: – Leukocyte esterase, present in granulocytes, catalyzes the reaction of the chromogens to produce a color change.

• False negatives: – cephalexin and gentamicin concentrations; elevated SG, glucose, ketone and protein concentrations, ascobic acid

• False positives: – vaginal contaminants, drugs or foods that color the urine red

Nitrites

• Reaction: – Nitrates in the urine are converted to nitrites by the action of gram-negative bacteria. These nitrites then react to form a diazonium salt which in turn reacts with a chromogen to produce the final color.

• False negatives: – Elevated SG, urobilinogen, pH <6.0, excess ascorbic acid

• False positives: – presence of red dyes or other chromogens, contamination

pH

• Reaction: – double indicator system detects the amount of hydrogen ions in the urine to produce a color change.

• Interferences: – If excess urine is left on the reagent strip, a phenomenon known as “runover” may occur. The urine from one reagent area carries reagent onto the pH test area and changes the result erroneously.

pH • Reflection of the ability of the kidney to maintain normal hydrogen ion concentration in plasma and extracellular fluid • Normal adult: 4.6 - 8.0 pH - Hypertonic urine < 6.0 - crenated RBC -

Hypotonic urine > 7.5 - Lysis of cells

• Acid urine: diet high in meat protein • Alkaline urine: diet high in citrate or vegetables

pH • RTA type I (renal tubular acidosis) - serum is acidic, urine is alkaline • RTA type II - urine initailly alkaline but becomes more acidic due to decrease in bicarbonate load • Useful in diagnosis and management of UTI and calculi - alkaline urine in UTI suggests presence of ureasplitting organisms - magnesium-ammonium phosphate crystals can form staghorn calculi - uric acid calculi associated with acidic urine

Protein

• Reaction: – based on “protein error of indicators” - because protein carries a charge at physiologic pH, their presence will elicit a pH change

• False negatives: - acidic or diluted urine, primary protein is not albumin

False positives: – Alkaline or concentrated urine, quaternary ammonia compounds

Protein • High levels in urine indicates renal disease: – Glomerular disease – Tubular disease

• Functional proteinuria – after strenuous exercise

Protein Other methods of detection: • Heat • Acid (SSA- sulfosalicylic acid precipitation test) sensitivity: 5-10 mg/dL of protein

Specific Gravity

• Reaction: ionic SG – based on the change of an indicator color in the presence of high concentrations of various ions.

• False negatives: – highly alkaline urine

• False positives: – Proteinuria, Dextran solutions,IV radiopaque dyes,

Specific Gravity

• • • • •

Random SG - 1.015 - 1.025 SG< 1.010 indicates relative hydration SG > 1.020 indicates relative dehydration SG - 1.000 should be checked SG - 1.040 physiologically impossible Other methods: Urinometer. Refractometer

Specific Gravity

- Correlates with urine osmolality

- insight to patient’s hydration status - reflects concentration ability of the kidneys

Urobilinogen

• Reagent: – urobilinogen reacts with a chromogen to form an azo dye which appears as various shades of pink or purple.

• False negatives: – excess nitrites; presence of formalin

• False positives: – presence phenazopyridine; very warm urine, elevated nitrite levels

Urobilinogen • Elevated urobilinogen found in hemolysis and hepatocellular diseases • Decrese robilinogen levels can be due to antibiotic use and bile duct obctruction

Ascorbic Acid Interference with dipstick Tests affected Needed Blood Glucose Bilirubin Nitrite

Ascorbic Acid Conc 9 mg/dl 50 mg/dl 25 mg/dl 25 mg/dl

Bridgen (1991): 22.8% positive (4379) Mean: 37.2 mg/dl (7.1 - 333.5 mg/dl)

“Test Strip Sieve Technique” • • • • •

Leucocyte RBC Protein Nitrite pH > 7.0

Micr oscopic Examination

Sediment examination • Most common laboratory procedure utilized for the detection of renal and/or urinary tract disease • numerous morphologic entities – blood cells, epithelial cells, organisms

• correlate with the biochemical results – dipsticks – clinical condition of the patient

Microscopic Components in Urine Sediment • Cells – blood cells; RBCs and WBCs – epithelial cells; renal, transitional, squamous • Casts – Hyaline – Waxy – Inclusion casts; Granular, Fatty – Cellular; RBC, WBC, and Epithelial casts • Bacteria, Fungi, and Parasites • Crystals

Methods for Examining Urine sediment • Bright field Microscopy of unstained urine and w/ Supravital staining • Phase Contrast Microscopy • Polarized Microscopy • Interference Contrast Microscopy • Cytodiagnostic Urinalysis • Quantitative and Differential Counts

Red blood cells

• appear as pale discs • can be confused with yeast cells • yeast cells do not stain and are not lysed by the addition of acetic acid

Dysmorphic RBCs

• Increased numbers in conjunction with RBC cast bleeding assumed to be renal in origin • Absence of casts and protein - bleeding assumed to be non-renal

DRBC

Leukocytes

• Increased numbers are seen: – renal diseases – urinary tract infection

• when accompanied by casts: – renal in origin

Squamous Epithelial cells

• Line the distal 1/3 of the urethra • Large numbers in women maybe a source of contamination

Transitional Epithelial cells

• Line the urinary tract from the renal pelvis to the proximal 2/3 of the urethra • few are present in normal urine

Renal Tubular Epithelial cells • Small numbers maybe seen in normal urine – sloughing of aging cells

• Increased numbers are seen: – acute tubular necrosis – certain drug or heavy metal toxicity

Casts • Formed when an increased numbers of proteins enter the tubules. • Formation increases with: – lower pH – increased ionic concentration

• Tamm-Horsfall (TH) protein forms the matrix of all casts – glycoprotein secreted by cells in the ascending loop of Henle

Casts - Some Definitions • If cast contain 3 or more cells e.g. RBC, WBC, then it is RBC cast, WBC cast • If it contains 1/3 or more granules granular cast • If a cast is about 60 µm or more - broad cast RBC 7-8 µm, WBC 8-22 µm

Hyaline cast

• Translucent with brightfield microscopy • increased numbers: – Pyelonephritis, chronic renal disease – transiently with exercise – May be a normal finding

Waxy cast

• Associated with tubular inflammation and degeneration • observed frequently with chronic renal failure

Granular casts

• Appear with glomerular and tubular diseases • accompany: – pyelonephritis – viral infections – chronic lead poisoning

Fatty casts

• Commonly seen when there is heavy proteinuria • a feature of nephrotic syndrome • hypothyroidism

Red Blood Cell casts

• Diagnostic of glomerular disease • glomerular damage allows RBCs to escape into the tubules

White Blood Cell casts

• WBCs enter the tubular lumen through and between tubular epithelial cells • associated with pyelonephritis and tubulointerstitial disease

Epithelial Cell casts

• To differentiate from leukocyte cast, supravital staining and phase -contrast microscopy are helpful. • Associated with: tubular necrosis, viral disease (CMV), heavy metal ingestion

Bacteria, Fungi, and Parasites

Bacteria

Yeast

Trichomonas

Yeast

Clue Cells

Crystals • Limited clinical significance • phosphates, urates, and oxalates are common and occur in normal urine • Alkalization and refrigeration promotes crystals formation • few crystals are important:

Inherited metabolic disorders – cystine – tyrosine – bilirubin - hepatic and biliary tract diseases – cholesterol - nephrotic syndrome

Crystals • few crystals are important: – Cystine – Tyrosine Inherited metabolic disorders – Leucine – bilirubin - hepatic and biliary tract diseases – cholesterol - nephrotic syndrome

Crystals found in Normal Urine

Amorphous urates/phosphates

Uric acid

Calcium oxalates

Triple phosphates

Crystals found in Abnormal Urine

Cystine

Bilirubin

Tyrosine

Cholesterol

Procedure for Urine Microscopy

sample collection

centrifugation

decantation

Report slide preparation writing report

microscopy

Quantitative ME Issue 1: To centrifuge or not to centrifuge? “After a 5 min centrifugation of the urine at 3500rpm, only 48% of RBC and 40% of WBC found to be present could still be detected under the microscope” Gadeholt, Brit Med J,1964, 1.1547

Quantitative ME Issue 2: To report in /µl or /LPF, /HPF ? Issue 3: To stain or not to stain?

JCCLS / NCCLS / ECLM Methods J CCLS

NCCLS

ECLM

RCF

400 G

500 G

400 G

Time

5 min

5 min

5 min

Urine volume

10 mL

S.V.W.I.

10 or 12 mL

Sediment vol.

0.2 mL

S.V.W.I.

0.5 or 0.6 mL

aspiration

After metering

aspiration

Centrifugation

Discard supernatant

S.V.W.I. - Standardized Volume within the Institute

JCCLS / NCCLS /ECLM Methods Centrifugation: RCF (g) = 1.118 x 10-5 x radius in cm x RPM2 or RPM = 1000 x (

RCF )1/2 11.18 x R

eg. R = 20 cm, speed = 1500 rpm R = 16 cm, speed = 1700 rpm R = 10 cm, speed = 2100 rpm Swing type, balanced, sample should stop naturally

JCCLS / NCCLS / ECLM Methods Slide Preparation

J CCLS

NCCLS

ECLM

Staining

If needed

Sediment volume

15 uL

Specific volume

Known Volume

Coverslip

18 x 18 mm

Standard size

Defined size

If needed P.C.- no (SM stain) B.F.- S stain

P.C. - Phase contrast microscope B.F. - Brightfield microscope * NCCLS recommends standardized commercial system

JCCLS / NCCLS / ECLM Methods J CCLS

NCCLS

ECLM

brightfield

brightfield

eyepiece

x 10

view # of eyepiece objective

20

determined value determined number determined value determined # of fields determined # of fields /mL

phase contrast x 10–12.5

Microscopy microscope

Examined LPF

LP: x 10 HP: x 40 whole field

examined HPF

at least 10

unit

/LPF/HPF

18-22 LP: x 10-16 HP: x 40 whole field at least 10 /uL, /mL,/LPF /HPF

Neubauer counting chamber 3 mm

1 mm

W

W

W

W

Depth of chamber = 0.1mm

Fuchs-Rosenthal counting chamber

Small square Medium square

Large square 1 medium square = 0.2 uL 16 middle square 1 large square = 3.2 uL

Depth of chamber = 0.2 mm

1 mm

1 mm

Fuch Rose nth al vs Neubauer fo r Qu antita tive Urin e Mic roscopy

• Advantage of Fuch Rosenthal over Neubauer – analysis volume of Neubauer is half of Fuch Rosenthal • concentration of urine formed elements is very low compared with those of hematology. • For urinalysis, the more volume, the better results

– some urine formed elements are large (ie casts), these might clog the chamber Depth of chamber • the deeper the depth, the better Cells /uL = total cells counted / [mm2 counted x 0.1 mm or 0.2mm] How many mm squares were counted

neubauer chamber

fuch rosenthal chamber

/HPF, /LPF to /uL conversion • This depends on the following conditions. – Real View ( Diameter ) – Magnification# ( x10 or x40 ) – Original Urine Volume before Centrifugation – Sediment Volume after Centrifugation – Loaded Sediment Volume on the Slide – Area of Cover Slip

Urine ME - Commercial Systems Features

Count- 10

Kova

Urisystem

Initial vol

12 ml

12 ml

12 ml

Final vol

0.8 ml

1.0 ml

0.4 ml

Sediment Conc

15 : 1

12 : 1

30 : 1

6 µl

6 µl

16 µl

Vol of urine used

Urine ME - Commercial Systems Features Area of viewing

Count- 10 36 sq mm

Kova 32 sq mm

Urisystem 90 sq mm

No of LPF

11

10

28

No of HPF

183

163

459

Coverslip Type

Acrylic

Acrylic

Glass

Procedure for ME Low power microscopy • Ensure uniform distribution of urine sediment • If uneven distribution, make a new preparation • Reduce light intensity • Scan whole area. Note: sediments tend to gather along sides of cover-slip

Procedure for ME High power microscopy • Examine 20 - 30 fields (optimal) But not less than 10 fields

Procedure for ME Microscopy with staining Sediment to stain ratio: 4:1 Types of stains 1.Sternheimer-Malbin Stain (SM Stain) 2.Sterheimer Stain (S Stain) 3.0.5% Toluidine Blue 4. Sudan III and Oil Red O

Procedure for ME Reporting Format Blood cells Less than 1cell/HPF 1 - 4 cells/HPF 5 - 9 cells/HPF 10 - 19 cells/HPF 20 - 29 cells/HPF 30 - 49 cells/HPF 50 - 99 cells/HPF Numerous -100 cells and more

Procedure for ME Reporting Format Casts - :0 + : 1 cast/100LPF ++ : 1 cast/LPF +++ : 10 casts/LPF ++++: 100 casts/LPF

or 1 cast/WF or 100 casts/WF or 1,000 casts/WF or 10,000 casts/WF or 6 casts/HPF

Procedure for ME Reporting Format Bacteria and Yeast - :0 +/- : scatter in several fields + : seen in each foeld ++ : many or scatter in cluster +++ : numerous

Procedure for ME Reporting Format Crystal and amorphous materials - :0 + : 1 ~ 4/HPF ++ : 5 ~ 9/HPF +++: 10 ~ /HPF