URINALYSIS & BODY FLUIDS (MLT 305) CHEMICAL EXAMINATION LECTURE ONE

Dr. Essam H. Aljiffri

THE CHEMICAL EXAMINATION OF URINE

REAGENT STRIPS

- Reagent strips currently provide a simple, rapid means

for performing 10 medically significant chemical analyses, including pH, protein, glucose, ketones, blood, bilirubin, urobilinogen, nitrite, specific gravity, and leukocytes.

THE CHEMICAL EXAMINATION OF URINE

- The two major types of dipsticks are manufactured under the trade names Multistix and Chemstrip. - Reagent strips consist of chemical impregnated absorbent pads attached to a plastic strip. - A color-producing chemical reaction takes place when the absorbent pad comes in contact with urine.
3

THE CHEMICAL EXAMINATION OF URINE

- Color reactions are interpreted by comparing the color produced on the pad with a chart supplied by the manufacturer. - Several colors or intensities of a color for each substance being tested appear on the chart. - By careful comparison of the colors on the chart and the strip, a semiquantitative value of trace, 1+, 2+, 3+, or 4 + can he reported.
4

THE CHEMICAL EXAMINATION OF URINE

REAGENT STRIP TECHNIQUE

1. 2. 3. 4. 5. 6. 7. 8. 9.
5

Mix specimen well. Dip completely, but briefly, into specimen. Remove excess urine when withdrawing strip from specimen. Holding the strip horizontally Compare reaction colors with manufacturer's chart under a good light source at the specified time. The amount of time needed for reactions to take place varies between tests and manufacturers and ranges from an immediate reaction for pH to 120 seconds for leukocytes. Perform confirmatory tests when indicated. Be alert for the presence of interfering substances. Understand the principles and significance of the test. Relate chemical findings to each other and to the physical and microscopic urinalysis results.

THE CHEMICAL EXAMINATION OF URINE

Care of Reagent Strips 1. Store with desiccant in an opaque, tightly closed container. 2. Store in a cool place, but do not refrigerate. 3. Do not expose to volatile fumes. 4. Do not use past the expiration date. 5. Use within 6 months after opening. 6. Do not use if chemical pads become discolored.
6

THE CHEMICAL EXAMINATION OF URINE

Quality Control 1. Test open bottles of reagent strips which known positive and negative controls during each laboratory shift. 2. Resolve control results that are out of range by further testing. 3. Test reagents used in confirmatory tests with positive and negative controls. 4. Perform positive and negative controls on new reagents and newly opened bottles of reagent strips. 5. Record all control results and reagent lot numbers.
7

THE CHEMICAL EXAMINATION OF URINE

AUTOMATION IN URINALYSIS - Clini-Tek measures light reflected from a reagent strip that has been manually dipped in urine and inserted into the machine. - Light reflection from the test pads decreases in proportion to the intensity of color produced by the concentration of the test substance.

- The instrument compares the amount of light reflection with that of known concentrations and displays or prints concentration units.
8

THE CHEMICAL EXAMINATION OF URINE

YELLOW IRIS - The newest addition to automated urinalysis is the Yellow IRIS (International Remote Imaging), capable of performing specific gravity tests, routine chemical analysis, and a slideless microscopic analysis from an uncentrifuged specimen.

pH
- The lungs, and the kidneys are the major regulators of the acid-base content in the body. - A healthy individual will usually produce first morning specimen with a slightly acidic pH of 5.0 to 6.0, the pH of normal random samples can range from 4.5 to 8.0.

10

pH
Reagent Strip Reactions
- Both the Multistix and Chemstrip brands of reagent strips measure urine pH between pH 5 and 9. - A double-indicator system of methyl red and bromthymol blue is used by both manufactures.

11

pH
CLINICAL SIGNIFICANCE OF URINE pH

1. Respiratory or metabolic aciclosis 2. Respiratory or metabolic alkalosis 3. Defects in renal tubular secretion and reabsorption of acids and bases 4. Precipitation of crystals and calculi formation 5. Treatment of urinary tract infections 6. Determination of unsatisfactory specimens
12

Protein
- As a routine chemical tests performed on urine the most indicative of renal disease is the protein determination. - The presence of proteinuria is often associated with early renal disease. - Normal urine contains very little protein; usually, less than 10 mg/dl or 150 mg per 24 hours is excreted. - This protein consists primarily of low-molecular-weight serum proteins that have been selectively filtered by the glomerulus, albumin is the major serum protein found in normal urine.
13

Protein
ORTHOSTATIC (POSTURAL) PROTEINURIA

- Benign proteinuria is usually transient and can be produced by conditions such as: exposure to cold, strenuous exercise, high fever, dehydration, and in the acute phase of sever illnesses.

- A more persistent benign proteinuria occurs frequently in young adults and is termed orthostatic, or postural, proteinuria.
14

Protein
BENCE JONES PROTEIN

- In multiple myeloma, marked elevated levels of monoclonal immunoglobulin light chains (Bence Jones protein). Unlike other proteins, which coagulate and remain coagulated when exposed to heat, Bence Jones protein coagulates at temperature between 400C and 600C and dissolve when the temperature reaches 1000C.

- The specimen that appears turbid between 400C and 600C and clear at 1000C can be suspected of containing Bence Jones protein.
15

Protein
CLINICAL SIGNIFICANCE OF URINE PROTEIN

1.Glomerular membrane damage a. Immune complex disorders b. Amyloidosis c. Toxic agents 2. Impaired tubular reabsorption 3. Multiple myeloma 4. Orthostatic or postural proteinuria 5. Preeclampsia 6. Diabetic nephropathy
16

Glucose
- Glucose test is the most frequent chemical analysis performed on urine because of its value in the detection and monitoring of diabetes mellitus. - Early diagnosis of diabetes mellitus through blood and urine glucose tests provides a greatly improved prognosis.

17

Glucose
REAGENT STRIP (GLUCOSE OXIDASE) REACTION

- Two very different tests are utilized by laboratories to measure urinary glucose, the glucose oxidase procedure provides a specific test for glucose.

1. Glucose + O2 (air)

glucose oxidase Peroxidas

gluconic acid + H2O2 oxidized chromogen + H 20

2. H2O2 + chromogen

- Urine glucose may be reported in terms of trace, 1+, 2+, 3+ and 4+; however, the color charts also provide quantitative measurements ranging from 100 mg per dl to 2 g per dl, or 0.1 percent to 2 percent.
18

Glucose
COPPER REDUCTION TEST (Clinitest)
- The test relies on the ability of glucose and other substances to reduce copper sulfate to cuprous oxide in the presence of alkali and heat. - A color change progressing from a negative blue through green, yellow, and orange to red occurs when the reaction takes place. - The classic Benedict's solution was developed in 1908 and contained copper sulfate, sodium carbonate, and sodium citrate buffer.

19

Glucose
CLINICAL SIGNIFICANCE

- Under normal circumstances, almost all of the glucose filtered by the glomerulus is reabsorbed in the proximal convoluted tubule; therefore, urine contains only minute amounts of glucose. - The blood level at which tubular reabsorption stops is termed the "renal threshold," which for glucose is between 60 and 180 mg per dl.
20

Glucose
SUMMARY OF CLINICAL SIGNIFICANCE OF URINE GLUCOSE

1. Diabetes mellitus 2. Impaired tubular reabsorption a. Fanconi's syndrome b. Advanced renal disease 3. Central nervous system damage 4. Pregnancy with possible latent diabetes mellitus

21

KETONES
- The term ketones represents; three intermediate products of fat metabolism, namely, acetone, acetoacetic acid, and beta-hydroxybutyric acid.

- Normally, measurable amounts of ketones do not appear in the urine, because all of the metabolized fat is completely broken down into carbon dioxide and water.

22

KETONES
REAGENT STRIP REACTIONS

- The three ketone compounds are not present in equal amounts in urine, both acetone and betahydroxybutyric acid are produced from acetoacetic acid, and the proportions of 78 % beta-hydroxybutyric acid, 20 % acetoacetic acid and 2% acetone are relatively constant in all specimen.

23

KETONES
REAGENT STRIP REACTIONS - Reagent strip tests utilize the sodium nitroprusside (nitroferricyanide) reaction to measure ketones, in this reaction, acetoacetic acid in an alkaline medium will react with sodium nitroprusside to produce purple color.

- The test does not measure beta-hydroxybutyric acid and is only slightly sensitive to acetone.
24

KETONES
TUBE TESTS - The nitroprusside reaction is referred to as Rothera's test.

- Tests to measure the individual ketones were also performed and included Gerhardt's test for acetoacetic acid and Hart's test for beta-hydroxybutyric acid. - The removal of acetone and acetoacetic acid by boiling and the reaction of the remaining beta-hydroxybutyric acid with hydrogen peroxide constitute Hart's test.
25

KETONES
CLINICAL SIGNIFICANCE OF URINE KETONES

1. Diabetic acidosis 2. Insulin dosage monitoring 3. Starvation

26