1. REAGENT STRIPS Storage Handling
Protect from moisture and excessive heat.
Store at room temp. Do not refrigerate.
Keep container tightly capped.
Do not remove desiccant from container.
Do not use beyond expiration date.
Handling
Review manufacturer’s instructions with each new lot number
Remove strips from bottle for immediate use. Recap.
Check for discoloration.
Keep away from bleach, acids, fumes, etc.

2. REAGENT STRIPS (continued)
Testing
within 30 minutes to 2 hours
fresh, well-mixed, unspun
at room temperature
Do not touch test pad area
Dip briefly, but completely - app. 1 second
Drain off excess urine, avoid runover
Do not lay strip on bench
Compare test areas to color chart on bottle
Read at specified times
Know sources of error, interfering substances, sensitivity, and specificity for each strip.
When automated, follow instrument’s operating manual.

3. Reporting Standard terms must be used Quantitative Qualitative
Concentration mg/dL
Plus system
Qualitative
Small, moderate, large
Negative/positive/WRR (normal)

4. pH
The strip contains the indicators methyl red and bromthymol blue. The give colors over the pH range of 5-9. Colors range from orange through yellow and green to blue.
Reference value on normal diet to 8.0

5. CLINICAL SIGNIFICANCE pH
Acid pH <6.0
Diet (high protein, meat, cranberries)
Acidifying drugs to prevent alk. stone formation
Abnormal crystalluria (bilirubin, cystine, tyrosine, leucine, cholesterol )
Uric acid stone formers
Acidosis and uncontrolled Diabetes mellitus
Hypokalemia
Starvation

6. pH (continued) Alkaline pH >6.5 Diet (vegetarian and citrus fruits)
Alkaline tide produced after a meal
Metabolic and respiratory alkalosis
Renal tubular diseases (Fanconi’s syndrome)
Alkalizing drugs to treat acid calculi formation
Genitourinary tract infections
pH >8.0
Contamination or old urine (not suitable for testing)

7. Protein
Reagent strip testing is based on the principle of protein error of the indicators. Protein in the form of albumin accepts ions from the indicator, which is buffered in a very acid medium. Color changes from shades of green to blue.
Most significant for early detection of renal disease.

8. CLINICAL SIGNIFICANCE PROTEIN
Reference value
Random urine negative (not detectable) <10mg/dL)
24 Hour <150mg or 10 mg/dL
Composition
Albumin, 1/3a
Globulins, 2/3
Tamm-Horsfall mucoprotein (25%)
Pathology >30mg/dL or on daily output
Heavy >3g/day
Moderate >1-3 g/day
Minimal <1g/day

9. CLINICAL SIGNIFICANCE PROTEIN (CONTINUED)
Benign
Functional
exercise, fever, stress, exposure to cold
Orthostatic
positional, renal congestion
Pregnancy
transient, investigate cause
Pathologic
Prerenal: overflow of low mole weight proteins
IgG light chains (Bence Jones proteins)
acute phase
hemoglobin, myoglobin

10. CLINICAL SIGNIFICANCE PROTEIN (CONTINUED)
Pathologic
Renal Glomerular Pattern
Group A strep and SLE glomerulonephritis
Hypertensive and diabetic nephropathy
Nephrotic syndrome, tumors, infections, toxic agents
Renal Tubular Pattern
Acute and chronic pyelonephritis
Interstitial nephritis
Renal tubular acidosis, rejection of kidney transplant
Post Renal
Inflammation/infection bladder, renal pelvis, ureter, prostate, external genitalia

11. SSA Testing for Protein
When mixed with weak sulfosalicylic acid (SSA), all urine proteins will denature and precipitate at room temperature. The degree of which is graded and reported in semi-quantitative terms.
Albumin, globulins, glycoproteins, and Bence-Jones protein are detected.
False positive caused by x-ray contrast media, penicillin, sulfonamides, tolbutamides
False negative caused by highly alkaline urine

12. Microalbuminuria
Protein (albumin) that cannot be detected by routine dipstick for protein
Sensitive methods needed to detect 10-20mg/L or 1-2 mg/dL
Immunochemical
Dye binding
Clinical significance
Early management of kidney disease in diabetes, hypertension, or peripheral vascular disease

13. Glucose
The dipstick determination of glucose is based on a double sequential enzymatic reaction using the specific glucose oxidase/peroxidase reaction in the presence of glucose and a chromogen.
The Clinitest or Benedict’s Reaction is based on the ability of reducing substances to reduce copper sulfate to cuprous oxide in the presence of a chromogen, which changes color from blue to orange. Test is performed to screen for non-glucose reducing sugars in infants and children under 2 years old.

14. Clinical Significance of Glucose
Reference value
No detectable amount present in urine by dipstick method (<50 mg/dl)
Positive values found when renal threshold for glucose is exceeded ( mg/dl)
Diabetes mellitus (DM)
Impaired tubular reabsorption
Pregnancy with latent DM

15. COMPARISON of REAGENT STRIP vs. CLINITEST
Strip Clinitest Cause
Positive Negative Sensitivity of methods
Oxidizing contaminants/bleach
Deteriorated Clinitest tablets
Negative* Positive Non-glucose reducing substance
Deteriorated reagent strips
Reagent strip interferences *Ascorbic acid (vitamin C)

16. Ketones
Conditions that result in increased and or incomplete fat metabolism can produce metabolic intermediary fat products in the urine and blood. The three ketone bodies present in urine are acetoacetic acid (20%), acetone (2%), and betahydroxybutyric acid (78%).
Acetoacetic acid and acetone react with nitroprusside in an alkaline medium to form a violet dye complex. Basis of dipstick.
Betahydroxybutyric acid is not detected with dipstick.

17. Clinical Significance Acetone
No detectable ketones present in normal urines.
Positive values
Diabetic ketosis (ketonuria)
Loss of carbohydrates due to fever, vomiting, weight loss, starvation, diarrhea, stress
Lactic acidosis caused by liver/renal failure, salicylate overdose
Interfering factors
False positive: pigmented urines
False negative: delay in testing

18. Nitrite
This test depends on the conversion of nitrate to nitrite by the action on Gram negative bacteria that contain reductase enzymes in the urine.
At the acid pH of the reagent area, nitrite in the urine reacts with an aromatic amine to form a diazonium salt, followed by a coupling reaction with benzoquinoline to produce a pink color.
Three factors must be present
Reductase producing bacteria must be present
Urine must be retained in bladder long enough to convert nitrates to nitrites (4 hours)
Nitrates must be present

19. CLINICAL SIGNIFICANCE NITRITE
Reference value
No nitrites present
Urine must be FRESH
Improper storage will result in false positive
Positive nitrites
Screen symptomatic and asymptomatic UTI
Common infecting organisms
Enterobacter, Citrobacter, Escherichia, Proteus, Klebsiella, Pseudomonas
Definitive diagnosis made by urine culture
Non-reductase producing microorganisms will be negative for nitrites

20. Leukocyte Esterase
Granulocytic leukocytes contain esterase activity that catalyze the hydrolysis of an amino acid ester to form an aromatic compound which reacts with a diazonium salt to produce a color change from beige to purple on the dipstick pad.
All positive reactions require a microscopic exam of the sediment.

21. CLINICAL SIGNIFICANCE LEUKOCYTE ESTERASE
Reference value
0-5 white cells/hpf
females 0-8 WBC/hpf or app. 10 WBC/uL (vaginal discharge can cause false positive)
Screens for urinary tract inflammation
kidney (pyelonephritis)
bladder (cystitis)
urethra (urethritis)
Leukocyturia can occur with or without bacteria

22. LEUKOCYTE-NITRITE Combination on FRESH urine is
Cost effective tool to screen for UTI
Provides 97% predictive value for negative culture when both tests are negative
Improved care in asymptomatic patient

23. Blood
Dipstick will detect blood by sensing heme that is present in red cell, hemoglobin, and myoglobin. Based on the pseudoperoxidase activity of heme in the presence of an organic peroxide and a benzidine chromogen.
Hematuria: in tact red cells present in urine (scattered green dots)
Hemoglobinuria: presence of hemoglobin from lysed red cell in urine (diffuse green color)
Myoglobinuria: presence of heme protein from muscles in urine (diffuse green color)

24. CLINICAL SIGNIFICANCE BLOOD
Reference value
0-5 erythrocytes/mL or 0-2 RBC/hpf
Hematuria - intact red cells
renal disease, calculi, tumors, infections
bleeding in kidneys or lower urinary tract
Hemoglobinuria - free hemoglobin
intravascular hemolysis as seen in incompatible blood transfusions, AIHA, G6PD, etc.
Myoglobinuria - heme muscle protein
acute destruction of muscle fibers (rhabdomyolysis)
crush/trauma injuries
Excessive exercise can cause all above

25. COMPARISON of URINE FINDINGS
Findings Hematuria Hemoglobinuria Myoglobinuria
Color pink, red, smoke clear pink, red, brown clear red, brown
UA Blood strip positive dots positive diffuse positive diffuse
UA Protein strip Renal 4+ pos/neg pos/neg
Nonrenal +/-
UA RBCs many occasional occasional
UA Casts Renal: RBC Hemoglobin Myoglobin Nonrenal: none

26. Bilirubin
The heme released from red cells is converted to the yellow bile pigment biliribin by a series of complex reactions in liver. A small amount is excreted under normal circumstances and is not detected in the urine with the dipstick.
When present, the Diazo Reaction is based on the coupling of bilirubin with a diazonium salt in an acid medium to form a colored azo-dye complex.

27. Ictotest for Bilirubin
Highly pigmented urines can cause false positive reactions. Confirmation is required by testing with the Ictotest tablet test for bilirubin.
This diazo tablet method is very sensitive to low levels of bilirubin.
Pigments will be removed by the absorbent pad supplied with the test.

28. CLINICAL SIGNIFICANCE BILIRUBIN
Reference value
not detected with reagent strips <0.02 mg/dL
Positive findings
obstruction to bile flow from liver
gallstones and neoplasms of pancreas
inflammation and swelling of liver cells
acute viral hepatitis, drug indued cholestatsis
acute alcoholic hepatits/cirrhosis
congenital hyperbilirubinemias
Dublin-Johnson and Rotor

29. Urobilinogen
Collectively referred to as the end products of bilirubin metabolism. Colorless reduction product of bilirubin which is oxidized by normal intestinal bacteria to brown pigment that is excreted in the feces.
Based on the Ehrlich Reaction in an acid medium to form a red color.

30. CLINICAL SIGNIFICANCE UROBILINOGEN
Reference value
up to 1 mg/dL or 1 Ehrlich Unit
greater in PM (alkaline tide after meals)
up to 2 mg/dL transition from normal to abnormal
decrease or absence cannot be determined with strip
Increased values
liver damage: viral hepatitis, cirrhosis, drugs, toxins
infections of biliary tree (cholangitis)
hemolytic anemias and intravascular hemolysis
increased enteric production
Absent
obstruction of bile duct
absence of intestinal flora

31. UA BILIRUBIN & UROBILINOGEN in UNCOMPLICATED JAUNDICE
Condition Bilirubin Urobilinogen
Normal Negative up to 2 EU/dL
Hepatic Disease Positive Increased (+/-)
Obstructive Disease Positive (+/-) Absent
Hemolytic Disease Negative Increased

32. QUALITY ASSURANCE Facilities and Resources Proficiency Testing
OSHA compliance
Proficiency Testing
Personnel
Qualifications, education and training, competency
Review
Procedure Manual
NCCLS GP2-A2
Controls, Standards, Reagents
Equipment and Instruments
Reporting of Results

33. COMPETENCY ALERTS REAGENT STRIP TESTING
Directly Observe
Followed SOP and manufacturer’s instruction
labeled date received, opened, expired
Removed strip immediately before test run
Replaced cap
Performed test on well mixed, unspun urine
Performed daily maintenance/function checks
Interpretation of color changes for strips or tablets
Performed confirmatory testing as indicated
Performed Clinitest on nursery or pediatric urines
Followed SOP step-by-step

34. COMPETENCY ALERTS (continued)
Monitor and Review
Compliance with QC as defined in SOP
Results logged on scheduled frequency of use
Parallel testing
PM and service logs signed
Critical values reported on interim worksheets: WHO, WHAT, WHEN.
Standard units of measure defined in SOP are used to report qualitative and quantitative results.

35. COMPETENCY ALERTS (continued)
Assessment of Test Performance
Proficiency testing
Internal blind samples of known chemical concentration
Problem Solving
Resolve discrepant results
Investigate and resolve delta checked results
Specimen referred for definitive testing (UA culture) based on reagent strip results. Policy defined in SOP.
Resolution of “out-of-control” results for known reference controls.

36. MICROSCOPIC EXAM of URINARY SEDIMENT
CLIA’88 Complexity
Moderate
Provider Performed Microscopy (PPM)
physician, midlevel practitioner, or dentist
brightfield or phase microscopy
Specimen of Choice
Fresh first morning, midstream, clean catch
Examine within 2 hours
Specific gravity >1.010
pH acid

37. IDENTIFIABLE SEDIMENT ENTITIES
Hematopoietic cells
Red blood cells (RBC)
dysmorphic
white blood cells (WBC)
glitter
eosinophils
lymphocytes , histiocytes and macrophages
Epithelial cells
Transitional (urothelial)
Squamous
Renal tubular
oval fat bodies

38. Casts Crystals Microorganisms Miscellaneous Matrix : Hyaline and Waxy
Cellular: RBC, WBC, Epithelial, Mixed, Bacteria
Inclusion: Granular, Fatty, Hemosiderin, Crystal
Pigment: Hemoglobin, Myoglobin, Bilirubin
Size: Broad or Wide
Crystals
Microorganisms
bacteria, yeasts, parasites
Miscellaneous
spermatozoa, mucus, artifacts and contaminants