Clin Path Lab 6 Urinalysis Part 2

ROUTINE URINALYSIS USTMED ’07 Sec C - AsM Components of a Urinalysis Physical Examination of Urine 1. color 2. transparency 3. specific gravity 4. pH Chemical Examination of Urine (using a multi-parameter reagent strip) 1. Protein 2. Glucose 3. Ketone 4. Bilirubin 5. Hemoglobin 6. Urobilinogen 7. Nitrite 8. Leukocyte esterase Microscopic Examination of Sediments

Appearance and Color of Urine Apprnce Colorless Cloudy

Milky

Collection and Handling of Urine Specimen Urine must be collected in a clean, dry container. Disposable containers are recommended in order to reduce bacterial contamination. Label the specimen container w/ the patient’s name, date and time of collection. Place the label on the container and not on the lid. Urine collected should be sent to the lab as soon as possible and should be tested w/in 1 hr. Refrigeration is necessary if the urine cannot be delivered immediately to the laboratory or cannot be tested w/in 1 hr. The addition of a chemical preservative can also be done. Types of urine specimens 1. random specimen – most commonly received specimen 2. First morning urine the ideal screening specimen used for routine screening, pregnancy tests and for determining orthostatic proteinuria 3. Fasting Specimen (Second Morning Specimen) second voided urine specimen used for glucose monitoring 4. 24-hour specimen timed specimen is used to determine concentration of a particular substance Instruction: o Day 1 – 7am - patient voids and discards specimen - patient collects all urine for the next 24 hours o Day 2 – 7am - patient voids and adds this urine to the previously collected urine 5. Catheterized Specimen used for bacterial culture 6. Midstream Clean-Catch Specimen an ideal specimen for routine screening and for bacterial culture patient is instructed to cleanse thoroughly the genitalia and is asked to collect the midstream portion of urine. When collecting, patient should be instructed to separate the labia in females or retract the foreskin in uncircumcised males. 7. Suprapubic Aspiration used for bacterial culture and cytology sterile needle is introduced into the bladder to collect sample that is free of contaminants 8. Pediatric Specimen soft, clear plastic bag w/ adhesive is attached to the genital portion PHYSICAL EXAMIANTION OF URINE Odor usually not a part of routine urinalysis ammoniacal odor of urine is due to breakdown of urea Odor Pathologic Nonpathologic Ammonia UTI Old urine Sweet Diabetes mellitus Starvation, dieting, strenuous exercise, vomiting, diarrhea Mousy Phenylketonuria Maple syrup Maple syrup disease Distinctive Garlic, onions, asparagus

Cause Very dilute urine Phosphates, carbonates Urates, uric acid Leukocytes Red cells (“smoky”) bacteria, yeasts spermatozoa prostatic fluid mucin, mucous threads calculi, “gravel” clumps, pus, tissue fecal contamination radiographic dye Many neutrophils (pyuria) Fat Lipiduria,opalascent

Yellowgreen Yellowbrown Red

Dissolve in 60oC and in alkali Insoluble in dilute acetic acid Lyse in dilute acetic acid Insoluble in dilute acetic acid Insoluble in dilute acetic acid May be flocculent Phosphates, oxalates Rectovesical fistula In acid urine Insoluble in dilute acetic acid

Bilirubin-biliverdin

Nephrosis, crush injury – soluble in ether Lymphatic obstruction – soluble in ether Vaginal creams Green Fluorescence Dehydration, fever No yellow foam Yellow foam, if sufficient bilirubin Yellow foam

Bilirubin-biliverdin

“beer” brown, yellow foam

Hemoglobin

Positive reagent strip for blood Positive reagent strip for blood Positive reagent strip for blood May be colorless Foods, candy Yellow alkaline, genetic Clots, mucus

Chyluria, milky Yellow Yelloworange

Remarks Polyuria, diabetes insipidus Soluble in dilute acetic acid

Emulsified paraffin Acriflavine Concentrated urine Urobilin in excess Bilirubin

Erythrocytes Myoglobin Porphyrin Fuscin, aniline dye Beets Menstrual contamination Red-purple Porphyrins Red-brown Erythrocytes Hemoglobin on standing Methemoglobin Myoglobin Bilifuscin (dipyrrole)

May be colorless

Acid pH Muscle injury Result of unstable hemoglobin Blood, acid pH On standing, alkaline On standing, rare Small intestine infection

Brownblack

Methemoglobin Homogentisic acid Melanin Blue-green Indicans Pseudomonas infections Chlorophyll

Mouth deodorants

Color Normal urine color – Yellow (due to urochrome) Color Orange

Pathologic Bilirubin

Dark Yellow

Bilirubin Urobilin Oxidized bilirubin Biliverdin Pseudomonas RBCs Hemoglobin Myoglobin Porphyrins Biliary pigments Melanin Hmogentisic acid Diabetes mellitus Diabetes insipidus

Green

Red or Pink

Brown or Black

Pale Yellow

NonPatho Rhubarb Carrots Carrots Concentrated Vit B complx

Drugs Phenazopyridine

Beets

Benzene Acetophenetidin Phenindione

Rhubarb

Phenol derivatives Methyldopa Levodopa

Large fluid intake

Diuretics

Fluorescein Dithiazanine Nitorfurans Phenol

Transparency (clarity) Normal appearance freshly voided urine is usually clear white cloudiness may be caused by precipitation of amorphous phosphates and carbonates Causes of turbidity presence of phosphates, carbonates, crystals red blood cells, leukocytes, epithelial cells, etc. Specific Gravity defined as the density of a substance compared with the density of a similar volume of distilled water at a similar temperature used to assess kidney’s ability for reabsorption Normal values = 1.015-1.025 ROUTINE URINALYSIS PROCEDURE Urine in specimen container

Formula: Au x Cs = Cu in mg/dl As

0.16 x 50 = 40 mg/dl 0.20

Urine Total Protein (mg/24 hrs or g/24 hrs) Urine Total Protein = Au x Cs x Volume in dl (24 hr urine) As To convert mg/dl to g/L: Divide computed value by 100 or multiply by 0.01 ExampleAu = 0.12 As = 0.08 Cs – 50 mg/dl Urine volume = 10 dl or 1 L

Transfer urine in a clean tube Urine Total Protein = Step 1 Physical Examination *note color & transparency *specific gravity & pH will be done using the Multistix (Step)2 Step 2 Chemical Examination * use Multistix Reagent Strip and test for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, Urobilinogen, nitrite & leukocytes

0.12 x 50 mg/dl x 10 dl 0.08

=

1.5 x 50 mg/dl x 10 df

=

750 mg/24 hrs

to convert to g/24 hrs: Divide by 1000 =

0.75 g/24 hrs

Centrifuge for 5 mins Normal Values Decant supernatant & place a drop of the sediments onto a slide Step 3 Microscopic Examination *examine under LPO & HPO and write your findings in the result form QUANTITATIVE DETERMINATION OF TOTAL PROTEIN IN URINE A. Pyrogallol Red-Molybdate Method Clinical Significance: Urinary protein determinations are important in the evaluation of kidney function. The detection of protein in urine is considered evidence of renal disease and usually of glomerular disease. A minor evaluation of urinary albumin excretion from about 15-30 mg/day is now recognized to be a useful indicator of renal disease. Principle: The Total Protein Test for Urine is based on the procedure developed by Watanabe et al which is a dye-binding colorimetric method utilizing pyrogallol red-molybdate complex. The pyrogallol red is combined w/ molybdenum acid, forming a red cplx w/ maximum abs at 467nm. When this cplx is combined w/ protein in acidic conditions, a blue-purple color develops w/ an inc in abs to 598nm. Reagents: 1. PRM color reagent • pyrogallol red-molybdate soln buffered at pH 2.2 2. Microprotein standard • (50 mg/dl) a soln of bovine serum albumin with preservative Specimen Collection & Handling: Urine – random or 24 hr urine collection. Keep specimen cold during collection. Analyze fresh. Stable frozen at -20oC for up to one year. Procedure: Unknown Standard Blank PRM reagent 3.0 ml 3.0 ml 3.0 ml Serum 0.1 ml Protein Standard 0.1 ml Distilled water 0.1 ml Allow to stand at room temperature for 20 mins Set wavelength of the photometer at 600 nm and zero with the BLANK. Read and record readings of remaining vials. Computation: ExampleAu = Abs of Unknown As = Abs of Standard Cu = Concentration of Unk Cs = Conc of Standard

Au = 0.16 As = 0.20 Cu = ? Cs = 50 mg/dl

Random urine = 0.01 – 0.14 g/L Urine Total protein = up to 0.15 g/24 h4s B. Microalbumin An immunological, semi-quantitative mcroalbuminuria up to 100 mg/L

determination

of

Principle: Immunological detection of human albumin. The absorbed urine enters a zone on the strip containing a soluble antibody-goldconjugate which specifically binds to urine albumin. Excess conjugate is retained in a separation zone containing immobilized human albumin so that only the albumin-loaded gold conjugate reaches the detection zone. The color produced (white to red) is directly related to the albumin content of the urine. Crossreactions with other human proteins such as hemoglobin, transferring, Bence-Jones protein, α1-antitrypsin, acidic-α1glycoprotein, α-amylase, Tamm-Horsfall protein and retinolbinding protein, as well as with IgG, IgA, human leukocytes and erythrocytes have been found to be 1% Elevated S.G. readings presence of moderate 91.0-7.5 g/L) qty of protein. S.G. readings correlate with values obtained by the refractive index method. A lower S.G. reading, relative to the other methods, may be caused by highly buffered alkaline urines and urines containing glucose or urea at conc of > than 1%. Elevated S.G. readings, relative to other methods, may be obtained in the presence of moderate (1.0-7.5 g/L) qty of protein. Random urines may vary in S.G. between 1.003-1.040. This S.G. test permits determination of urine S.G. between 1.000-1.030. For increased accuracy, 0.005 may be added to readings from urines with pH equal or greater than 6.5. Reagent Composition: Bromthymol blue, poly (methyl vinyl ether maleic anhydride), sodium hydroxide VII. pH (may be read immediately – timing not critical)

protein and mucoprotein, so a negative result does not rule out the presence of these proteins. False positive results may occur w/ alkaline, highly buffered urines or in the presence of contaminating quaternary ammonium compounds. IX. Nitrite (60 seconds)

Principle: Greiss reaction Conversion of nitrate (derived from diet) to nitrite by the action of principally Gram negative bacteria in the urine Reagent Composition: p-arsanilic acid, tetrahydrobenzo(h)-quinolin-3-ol This test depends upon the conversion of nitrate (derived from diet) to nitrite by the action of principally Gram negative bacteria in the urine. The test is specific for nitrite and will not react w/ any other substance normally excreted in urine. Pink spots or pink edges should not be interpreted as positive result. Any degree of uniform pink color development should be interpreted as a positive nitrite test suggesting the presence of 105 or more organisms per mL, but color development is not proportional to the number of bacteria present. A negative result does not in itself prove that there is not significant bacteriuria. Negative results may occur when UTI are caused by organisms w/c do not contain reductase to convert nitrate to nitrite; when urine has not been retained in the bladder long enough (4 hrs or more) for reduction of nitrate to occur; or when dietary nitrate is absent, even if organisms containing reductase are present and bladder incubation is ample. Sensitivity of the nitrite test is reduced for urines w/ high specific gravity. Ascorbic acid conc of 1.42 mmol/L or greater may cause false negative results w/ specimens containing small amts of nitrite ion (13 umol/L or less). X. Leukocytes (2 minutes)

Principle: Granulocytic leukocytes contain esterases that catalyze the hydrolysis of the derivatized pyrrole amino acid ester to liberate 3-hydroxy-5-phenyl pyrrole. This pyrrole then reacts w/ a diazonium salt to produce a purple product. Reagent Composition: Derivatized pyrrole amino acid ester, diazonium salt MICROSCOPIC ANALYSIS OF URINE

Principle: Double indicator system Gives a range of colors from orange through yellow and green to blue and permits differentiation to w/in 0.5 pH units in the range pH 5 to pH 8.5. Reagent Composition: methyl red, bromthymol blue, nonreactive ingredients This test is based on the double indicator system principle. Readings are not affected by variations in urinary buffer concentration, but contamination by reagent washed from an overwetted adjacent reagent area may affect results.

1.

VIII. Protein (may be read immediately – timing not critical) 2.

Fresh or adequately preserved urine specimen (approximately 10-15 m) is placed preferably on a conical tube and centrifuged for 5 minutes. Decant supernatant and the sediment is resuspended with the remaining urine in the tube (usually 0.5 mL or 1.0 mL)

Principle: Protein-error-of-indicators The presence of protein results in development of a green color Reagent Composition: Tetrabromphenol blue, buffer, non-reactive ingredients. Note: contamination of the urine sample w/ chlorhexidine antiseptic will give a false-positive reading for protein. The test is based on the protein-error-of-indicators principle. The presence of protein results in development of a green color (echo!). The test yields negative results w/ normal urines, so any positive result greater than “Trace” indicates significant proteinuria. Clinical judgment may be used to interpret “Trace” results, w/c may occur w/ urines of high specific gravity w/ nonsignificant protein-uria. The “trace” result corresponds to 0.05-0.2 g/L albumin, but the test is less sensitive to globulin, Bence-Jones

3. 4. 5.

Using a pipet, place a drop of resuspended sediment on a clean slide and cover with a cover slip Examine the sediment first using the LPO Shift to HPO to identify specific types of cells, casts, bacteria and crystals

Manner of Reporting: Elements RBCs and WBCs Casts Epithelial cells Crystals Other elements (Bacteria, yeasts)

Report As Average of RBC or WBC seen in 10 high power fields (HPF) Average casts seen per cover slip ( __/cs) Report as: few, occasional, moderate, many or +,++,+++,++++ Report as: few, occasional, moderate, many or +,++,+++,++++

2. 3.

4.

Read under LPO Report As Casts Hyalin Granular Waxy Pus cell RBC Cells Squamous cells Renal cells Mucus threads Crystals Amorphous urates Uric acid Calcium oxalate Amorphous PO4 Triple PO4 Read under HPO RBC WBC Yeast cells Bacteria

# of casts seen/ cover slip

Few, occasional, moderate or many OR +, ++, +++, ++++ Few, occasional, moderate or many OR +, ++, +++, ++++

Average of RBC/WBC seen in 10 fields/HPF Few, occasional, moderate or many OR +, ++, +++, ++++

ROUTINE FECALYSIS There is no substitute for careful visual inspection of the patient’s stool by the physician. Patient’s descriptions of their feces are subjective and inaccurate. Routine Fecal Analysis: Laboratory examination of feces is most commonly employed for: 1. Detection of gastrointestinal bleeding 2. Confirmation of steatorrhea 3. Detection and identification of parasites Specimen Collection: A well-ringed bed pan is a convenient collecting vessel. A cleaned, rinsed and boiled glass jar provides a satisfactory alternative. Female patients must be warned not to contaminate the specimen w/ urine since urine has harmful effects on protozoa. Patients must also be instructed not to overfill and contaminate the container. Stool collected w/in 72 hrs after barium enema or swallow is unsatisfactory. Similarly, stool from patient taking mineral oil is undesirable.

porcelain plate. Apply a small portion of a collected stool specimen to the HEMATEST filter paper w/ an applicator stick. The smear should be a thin, narrow streak. Do not use an emulsion or suspension. place the tablet on the specimen so that part of the tablet is directly touching the clean filter paper Place one drop of distilled water on the tablet. Allow 510 secs for the water to penetrate the tablet. Then add a second drop so that the water runs down the side of the tablet onto the specimen filter paper. If necessary, gently tap side of plate once or twice to dislodge water from top of tablet. For up to two minutes, observe filter paper for appearance of any trace of blue color surrounding the tablet. Ignore color that develops on or directly under the tablet itself and any color appearing on the filter paper after the 2-minute time.

Results: A positive rxn is indicated by the appearance of any trace of blue color on the filter paper around the tablet w/in the 2minute reading time. Ignore any color that develops on or directly under the tablet itself and any color appearing on the filter paper after the two-minute time. Neutral Fat Estimation (Sudan III Staining) Procedure: 1. mix stool specimen thoroughly w/ an applicator stick 2. place a small amt (2-3 mm) on a clean glass slide. Emulsify w/ a drop of ethyl alcohol. 3. add 2 drops of sudan stain 4. mix and apply cover slip 5. let stand for 5 mins and examine microscopically Interpretation: Presence of neutral fat droplets is indicated by yelloworange to red globules which tend to collect at the edges of the cover slip. In normal stools, there are less than 50 fat globules/highly power field by microscopic examination. Fecal Trypsin Determination Principle: x-ray film strips are immersed in diluted fecal emulsions. Proteolytic activity (of trypsin and chymotrypsin) causes digestion of the gelatin emulsion on x-ray film Procedure: 1. mix duodenal content or a pea-sized fecal material in a test tube w/ 2% sodium carbonate (about 0.5 ml) 2. place a small amount of gelatin square (xray film) into the mixture and incubate at 37oC

Principle: The test is based on the peroxidase-like activity of hemoglobin in catalyzing the oxidation by peroxide of a chromogen, tetramethylbenzidine, to form a blue color.

Interpretation: Complete digestion of gelatin is recognized by complete clearing of x-ray film (the blue tint of the film base should be apparent). Partial digestion is indicated by mixed clear and cloudy film. When there is no digestion, the gelatin remains intact.

Reagent: Tetramethylbenzidine, strontium peroxide

-fin-

Detection of Fecal Occult Blood Using Hematest Reagent Tablets

Specimen Collection and Preparation: Collect fecal specimen uncontaminated w/ urine and test as soon as possible. When testing collected specimens, it is recommended that several segments or portions of the stool be tested; blood from colo-rectal bleeding may be predominantly on the outer surface of the formed stool, and blood from upper portions of the GI tract is not always uniformly dispersed throughout the specimen. A meat-free diet prior to specimen collection is desirable to minimize false positive reactions that can result from ingestion of raw or rare meat. Intake of Vit. C should be restricted because ingestion of large quantities may result in false negative results. Procedure: 1. Place HEMATEST filter paper on a clean glass or

arrrrgggggghhhhhh!!!!!!

Sample Urine Results with corresponding diagnosis

Urinalysis Result Form Urinalysis Result Form Physical Characteristics Physical Characteristics

Microscopic Findings

Microscopic Findings

Color: reddish Transparency: turbid pH: 6.0 Specific Gravity: 1.026

Casts: Hylaine Granular Waxy Pus Cell RBC Chemical Test Cells: Glucose: negative RBC: 50-60 / HPF Bilirubin: negative Pus Cell: 1-30 / HPF Ketone: negative Yeast Cell Blood: ++ Squamous Cells: few Protein: ++ Renal Cells Urobilinogen: negative Bacteria Nitrite: negative Mucus Threads Leucocyte esterase: negative Crystals: Amorphous urates: few Uric Acid: ++++ Calcium Oxalate Amorophous PO4 Triple PO4

Color: reddish Transparency: turbid pH: 7.0 Specific Gravity: 1.030

Casts: Hylaine: 3 / cs Granular: 12 / cs Waxy Pus Cell RBC Chemical Test Cells: Glucose: negative RBC: 0-2 / HPF Bilirubin: negative Pus Cell: 1-3 / HPF Ketone: negative Yeast Cell Blood: +++ Squamous Cells: few Protein: ++ Renal Cells Urobilinogen: negative Bacteria Nitrite: negative Mucus Threads Leucocyte esterase: negative Crystals: Amorphous urates: few Uric Acid: Calcium Oxalate Amorophous PO4 Triple PO4 Diagnosis: Acute Immune-mediated Blood Transfusion Reaction

Diagnosis: Ureterolithiasis (Uric Acid), Right Urinalysis Result Form

Urinalysis Result Form

Physical Characteristics

Microscopic Findings

Physical Characteristics

Color: dark yellow Transparency: turbid pH: 6.0 Specific Gravity: 1.030

Casts: Hylaine: 20 – 25 /cs Granular: 15-20 / cs Waxy Pus Cell RBC > 50 / cs Cells: RBC: 50-60 / HPF Pus Cell: 2-4 / HPF Yeast Cell Squamous Cells: few Renal Cells: few Bacteria Mucus Threads Crystals: Amorphous urates: few Uric Acid: Calcium Oxalate Amorophous PO4 Triple PO4

Color: yellow brown Transparency: turbid pH: 6.0 Specific Gravity: 1.030

Chemical Test Glucose: negative Bilirubin: negative Ketone: negative Blood: + Protein: +++ Urobilinogen: negative Nitrite: negative Leucocyte esterase: trace

Microscopic Findings

Casts: Hylaine Granular Waxy Pus Cell RBC Chemical Test Cells: Glucose: negative RBC: 0-1 / HPF Bilirubin: negative Pus Cell: 0-2 / HPF Ketone: negative Yeast Cell Blood: negative Squamous Cells: few Protein: trace Renal Cells Urobilinogen: negative Bacteria Nitrite: negative Mucus Threads: few Leucocyte esterase: negative Crystals: Amorphous urates: few Uric Acid: Calcium Oxalate Amorophous PO4 Triple PO4

Diagnosis: Acute Glomerulonephritis

Diagnosis: Cholelithiaasis (obstructive jaundice)

Urinalysis Result Form

Urinalysis Result Form

Physical Characteristics

Microscopic Findings

Physical Characteristics

Microscopic Findings

Color: yellow Transparency: turbid pH: 5.0 Specific Gravity: 1.035

Casts: Hylaine: 2-4 / cs Granular: 4-6 / cs Waxy Pus Cell: > 50 / cs RBC Cells: RBC: 2-4 / HPF Pus Cell: 50-60 / HPF Yeast Cell: few Squamous Cells: few Renal Cells: +++ Bacteria: +++ Mucus Threads: + Crystals: Amorphous urates: few Uric Acid Calcium Oxalate Amorophous PO4 Triple PO4

Color: yellow Transparency: turbid pH: 6.0 Specific Gravity: 1.028

Casts: Hylaine: 0-1 / cs Granular: 0-1 / cs Waxy Pus Cell RBC Cells: RBC: 0-3 / HPF Pus Cell: 50-60 / HPF Yeast Cell Squamous Cells: few Renal Cells Bacteria: ++ Mucus Threads: few Crystals: Amorphous urates: + Uric Acid: Calcium Oxalate Amorophous PO4 Triple PO4

Chemical Test Glucose: ++++ Bilirubin: negative Ketone: negative Blood: negative Protein: ++ Urobilinogen: negative Nitrite: ++ Leucocyte esterase: ++

Diagnosis: Acute Pyelonephritis and Diabetes Mellitus

Chemical Test Glucose: negative Bilirubin: negative Ketone: negative Blood: negative Protein: trace Urobilinogen: negative Nitrite: ++ Leucocyte esterase: ++

Diagnosis: Urinary Tract Infection (E. Coli)