Chemical Examination
pH urine purpose
Along with the lungs, the kidneys are the major regulators of acid-base content in the body. They do this through the secretion of hydrogen in the form of ammonia ions, hydrogen phosphate, weak organic acids, and the reabsorption of bicarbonate from the filtrate in the tubules of the nephron.
Hematuria
An abnormal amount of intact RBCs in the urine. Most closely related to disorders of renal or genitourinary origin in which bleeding is the result of trauma or damage to the organs of these systems. The lab is frequently requested to perform a urinalysis when patients presenting with severe back and abdominal pain are suspected of having renal calculi.
Bence-Jones proteins
Increased serum protein levels is excretion of Bence-Jones protein by multiple myeloma. This low molecular-weight protein is filtered in quantities exceeding the tubular reabsorption capacity and is excreted in the urine. Coagulates at 40-60 degrees C Dissolves when 100 degrees C. Turbid between 40-60 and clear at 100 can be suspected of Bence-Jones protein. Suspected cases of multiple myeloma must be diagnosed by performing serum elctrophoresis and immunoelectrophoresis.
Bilirubin
Intensely orange-yellow pigment that causes a characteristic coloration of plasma and urine when present in significant amounts. Bilirubin is produced from the catabolism of RBC in the RES system.
pH urine clinical significance
The pH of normal random samples can range from 4.5-8.0. The importance of urinary pH is primarily as an aid in determining the existence of systemic acid-base disorders of metabolic or respiratory origin. The precipitation of inorganic chemicals dissolved in the urine forms urinary crystals and renal calculi (kidney stones). Maintenance of acidic urine can be of value in the treatment of UTI caused by organisms that do not multiply as readily as in acidic medium.
False positive reactions
can be caused by menstrual contamination, strong oxidizing agents, and bacterial enzymes, including Escherichia Coli. Therefore, sediments containing bacteria should be checked closely for the presence of RBCs.
Nitrite reagent strip test
provides a rapid screening test for presence of UTI. Many UTIs are believed to start in bladder as a result of contamination and progress upward through ureters to tubules, renal pelvis, kidney.
Increased urine urobilinogen cause
-Early detection liver disease -Liver disorders -Hepatitis -Cirrhosis -Carcinoma -Hemolytic disorders
False negative reactions
-High levels of ascorbic acid -Crenated RBCs that do not lyse when in contact with reagent pad -Failure to properly mix specimen, which causes RBCs to settle at bottom -Decreased reactivity when formalin is used as preservative.
Myoglobin causes
-Muscular trauma/crush syndromes -Prolonged coma -Convulsions -Alcoholism -Drug abuse -Vigorous exercise -Ischemia
Hematuria causes
-Renal Calculi -Glomerulonephritis -Phleonephritis -Tumors -Trauma -Strenuous exercise -Menstruation
Hemoglobinuria causes
-Transfusion reactions -Hemolytic anemias -Severe burns -Brown recluse spider bites -Malaria -RBC damage
Urobilinogen
Conjugated bilirubin is reconverted to its unconjugated form and reduced by intestinal bacteria to form a colorless substance called urobilinogen. Urobilinogen is reabsorbed from the intestine into the blood, re-circulates to liver, and is excreted back into intestine through bile duct.
pH Meter
Consists of a silver-silver chloride indicator electrode with a pH sensitive glass membrane connected by a salt bridge to a reference electrode. pH meter is calibrated using 2 or 3 standard buffer solutions. pH measurement is temperature dependent and pH decreases with increasing temperature, it is necessary that the pH measurement be adjusted for the temperature of the urine being tested.
Bilirubinuria
Detection of urinary bilirubin provides an early indication of liver disease, but also its presence or absence can be used in determining cause of clinical jaundice. -Hepatitis -Cirrhosis -Biliary obstruction (gallstones, carcinoma)
Acetest Tablets
In cases of severe ketosis, it may be necessary to perform tests to provide more info on the extent of ketosis. Acetest provides sodium nitroprusside, glycine, disodium phosphate, and lactose in tablet form. 1 Tablet on paper towel 1 drop urine on the tablet Wait 30 sec. Compare to color chart and record as neg,small,moderate, or large
Hemoglobin Reagent Strip Test
In presence of hemoglobin, color ranging from yellow (neg) through green to strong blue-green (positive) appears on pad. *In contrast, intact RBCs are lysed when they come in contact with the pad, and liberated hemoglobin produces an isolated reaction that results in a speckled pattern on the pad.
Ketone Clinical Significance
Insulin dosage monitoring: Ketonuria shows a deficiency in insulin, indicating the need to regulate dosage. Strenuous exercise: Frequent strenuous exercise can cause overuse of available carbs and produce ketonuria. Vomiting: Inadequate absorption of carbs Starvation: Inadequate intake of carbs and eating disorders.
Ketone bodies
Ketones represent 3 products of fat metabolism: acetone, acetoacetic acid, and beta-hydroxybutyric acid. When use of available carbs as major source of energy becomes compromised, body stores of fat must be metabolized to supply energy. Ketones are then detected in urine, known as ketonuria.
Protein in urine Purpose
Most indicative of renal disease is the protein determination. The presence of protein in the urine, also known as proteinuria, is often associated with early renal disease. Due to its low molecular weight, albumin is the major serum protein found in abnormal urine. Other proteins found include: small amounts of serum and tubular microglobulins Tamm-Horsfall protein produced by tubules of nephron prostatic, seminal, vaginal secretions
Controls
Must be checked with both positive and negative controls minimum of every 24 hours. Resolve control results that are out of range by further testing. Testing also performed when new bottle is opened, questionable results are obtained, or there is a concern about the integrity of the strips. All results and reagent lot numbers must be recorded. Non-reagent strip testing using tablets and liquid chemicals are available when questionable results or highly pigmented specimens are encountered.
Handling and Storage of Reagent Strips
Must be protected from deterioration caused by moisture, volatile chemical, heat, and light. Packed in opaque containers with a desiccant to protect from light and moisture. Stored at room temperature below 30 degrees C
Greiss reaction
Nitrite is detected by the Greiss reaction, in which nitrite at an acidic pH reacts with an aromatic amine to form a diazonium compound that then reacts with tetrahydrobenzquinolin compounds to produce a pink (positive) colored azodye. Results pos or neg.
Ictotest Tablets
Questionable results can be repeated using the Ictotest. This test is less subject to interference and is sensitive to 0.005 to 0.10 mg/dL of bilirubin, whereas the reagent strips have a lower sensitivity level of 0.40. Consist of testing mats and tablets containing p-nitrobenzene-diazonium-p-toluenesulfonate, SSA, sodium carbonate, and boric acid. 10 drops urine onto square of mat Using forceps, 1 tablet into center of square 1 drop water onto tablet and wait 5 sec 2nd drop of water onto tablet so water runs off tablet onto mat Observe color of mat around tablet at end of 60 sec. Presence of blue-to-purple indicates bilirubin. slight pink or red should be ignored. Report pos or neg
Automated reagent strips
Reagent strips currently provide a simple, rapid means for performing medically significant chemical analysis of urine including: -pH -Protein -Glucose -Ketones -Blood -Bilirubin -Urobilinogen -Nitrite -Leukocytes -Specific Gravity
Hemoglobinuria
Results from the lysis of RBC and indicates the presence of hemoglobin in the urine. Can result from the intravascular hemolysis and subsequent filtering of hemoglobin through the glomerulus.
Bilirubinuria Reagent Strip Test
Routine testing for urinary bilirubin uses the diazo reaction. Qualitative results reported as neg, small,moderate, large. *Reagent strip color reactions for bilirubin are more difficult to interpret than other reagent strip reactions and are easily influenced by other pigments present in the urine.
Glucose Reagent Strip Method
Strips use the testing glucose oxidase testing method by impregnating the testing area with a mixture of glucose oxidase, peroxidase, chromogen, and buffer to produce a double sequential enzyme reaction. Specific for glucose; false positive reactions are not obtained from other urinary substances, including other sugars.
pH Test Papers
The indicator papers do not add impurities to the urine and produce sharp color changes for comparison to a supplied color chart of pH values.
Protein Confirmation Test
The sulfosalicyclic acid (SSA) test is a cold precipitation test that reacts equally with all forms of protein. 5 drops urine, 5 drops SSA to a 13x100 test tube. Mix by inversion and observe. Grade degree of turbidity.
Copper Reduction Method
This method relies on the ability of glucose and other substances to reduce copper sulfate to cuprous oxide on the presence of alkai and heat. Color change progressing from negative blue through green, yellow, orange/red occurs when reaction takes place. Urine is added to a solution of copper sulfate, sodium carbonate, and sodium citrate buffer. Heat applied and resulting precipitate observed for color change.
Clinitest
Uses a tablet that contains copper sulfate, sodium carbonate, sodium citrate, and sodium hydroxide. As water and urine is added to the tablet, heat is produced by the hydrolysis of sodium hydroxide and its reaction with sodium citrate, and CO2 is released from the sodium carbonate to prevent room air from interfering. Tubes placed in rack not held in hand. At end of reaction, tube should be shaken, and the color ranging from blue to orange/red can be compared with color chart to determine amount of reducing substance. 5 drops urine, 10 drops water to a 15x85mm test tube. Add 1 Tablet Wait 15 sec, until boiling stops. Mix gently Compare to chart and record as mg/dL or percent.
Protein strip test
Uses the principle of protein error of indicators to produce a visible colorimetric reaction. Reported in terms of neg, trace, +1,2,3,4, or 30,100,300,2000 mg/dL corresponding to color change. Major source of error with reagent strips occurs when highly buffered alkaline urine that overrides the buffer system. Pad remains in urine too long, which removes buffer system.
Ketone Reagent Strip Test
Uses the sodium nitroprusside reaction to measure ketones. In this reaction, acetoacetic acid in an alkaline medium reacts with sodium nitroprussude to produce a PURPLE color. Reported as neg, trace, 1,2,3+ or semi-quantitatively as neg, trace, 15, 40, or 80-160 mg/dL
Other reducing substances in urine
Various circumstances can result in the urinary excretion of other sugars such as galactose, fructose, lactose, maltose, or pentose.
Pyelonephritis
an inflammatory process of the kidney is a frequent complication of untreated cystitis and can lead to renal tissue damage, impairment of renal function, hypertension, and even septicemia.
Conjugated bilirubin
appears in the urine when the normal degradation cycle is disrupted by obstruction of the bile duct or when the integrity of the liver is damaged, allowing leakage of it into the circulation. Hepatitis and cirrhosis are common examples of conditions that produce liver damage, resulting in bilirubinuria.
Leukocyte esterase test
detects presence of esterase in the granulocytic WBCs (neutrophils, eosinophils, and basophils) and monocytes. *Neutrophils are the leukocytes most frequently associated with bacterial infections. Lymphocytes, erythrocytes, bacteria, and renal tissue cells do not contain esterases. *Requires the longest time of all the reagent strip reactions of 2 minutes. Reactions reported as trace, small,moderate,large.
Glucose test
most frequent chemical analysis performed on urine, because of its detection and monitoring of diabetes mellitus. The presence of glucose in the urine is termed glucouria (or glycosuria). When the glucose plasma concentration becomes elevated (hyperglycemia) and reaches the renal threshold (160-180 mg/dL), reabsorption of glucose will stop in the nephron and increased glucose appears in the urine.
Leukocyte Esterase
offers a standardized means of detection of leukocytes. Normal values for leukocytes are based on the microscopic sediment examination and vary from 0-2 to 0-5 per hpf. Women tend to have higher numbers than men as a result of vaginal contamination. Increased urinary leukocytes are indicators of UTI.
Myoglobin
a heme-containing protein found in the muscle tissue involved in the transport of oxygen in muscles. Easily passes the glomerular filtration barrier and is reabsorbed by the proximal tubules. Abnormal level of myoglobin in urine is called myoglobinuria.
Galactosemia
a pathologic condition characterized by a congenital enzyme deficiency to metabolize galactose to glucose. This sugar is the most significant and severe with irreversible consequences. If left untreated, will cause cataract formation, hepatic dysfunction, and severe mental retardation.
Nitrite test can also be used
to evaluate the success of antibiotic therapy and to periodically screen persons with recurrent infections and pregnant woman all of whom are considered to be high risk of UTI.
Leukocyte regeant strip test
uses the action of LE to catalyze the hydrolysis of an acid ester embedded on the reagent pad to produce an aromatic compound and acid.
