CLS 1521 AND CLS 1531
                                                      URINALYSIS  (Part 2)

Objectives with narrative and illustrations.

All objectives are cognitive, unless identified as being psychomotor. The student, at the end of each instructional component, whether in the classroom or lab, is responsible for meeting the objectives. All student that achieves a cumulative score of 70% or better on all problem sets, case studies, major exams, quizzes, and library assignments are deemed to have met the objectives of this course.

2-94    DESCRIBE THE TYPES OF PROTEINS FOUND IN URINE.

Urine contains very little protein, up to 150 mg in a 24 hour period. Over the course of 24 hours, urine will contain from 1.0 to 15.0 mg of protein/dL. Proteins that are found in the glomerular filtrate have a molecular weight of 90,000 or less. Larger proteins are non-filterable. One estimate states that a given urine specimen will contain about 34% albumin and 66% globulins. Other resources state that albumin is a true small molecular weight protein and will be the dominate protein in urine. The urinary tract produces three proteins of interest: (1) Tamm-Horsfall protein, (2) urokinase (a fibrinolytic enzyme), and (3) secretory IgA (immunoglobulin of the renal tubular epithelial cells). Other proteins reported are from the prostate, seminal vesicles, and vagina.

2-95    DISCUSS THE CLINICAL SIGNIFICANCE OF PROTEIN FOUND IN URINE.

Protein testing of urine is important because it tends to be denotative of renal disease. The fact that a test is positive does not mean the patient has a renal disorder but that additional testing is necessary. A positive protein test of a random urine specimen is more significant than a first morning specimen. The presence of protein in urine is known as proteinuria. The major reasons for pathologic proteinuria are: (1) damage to the glomerular membranes, (2) tubular disorders characterized by altered tubular reabsorption mechanisms, and (3) increases in the serum levels of low-molecular weight proteins, which gives rise to several types of proteinuria: overflow, renal, glomerular, tubular, post-renal, and orthostatic. The amount of protein loss that can occur in the urine varies from 0.15 grams to 20.0 grams per day. In most cases of proteinuria, protein loss does not exceed 4.0 grams per day.

2-96     DSCUSS ORTHOSTATIC PROTEINURIA.

Also known as functional or postural proteinuria, it is a non-pathogenic condition associated with the upright position and disappearing when the horizontal or supine position is assumed. The daily protein loss usually does not exceed 1.0 gm/dL, but has been reported at 1.5 gm/dL. This is a disorder of young adults. To diagnose this disorder, collect a urine specimen immediately after rising. This will be negative for protein. Collect a second specimen 3 or 4 hours after rising. This will be positive. Patients should be monitored every six months and re-evaluated. This may be due to blood pressure phenomenon in the renal vein when in the upright position.

2-97      DISCUSS OVERFLOW PROTEINURIA.

Overflow proteinuria (also called "pre-renal" or "overload" proteinuria) is the consequence of increased amounts of low-molecular weight plasma proteins passing through the glomerular membranes into the urine. This phenomenon can be the result of a number of conditions: (1) hemolytic transfusion reaction episode, (2) muscle trauma that causes myoglobin to also appear in the urine, and (3) acute-phase reactant proteins due to surgery, myocardial infarctions, or bacterial septicemia.
Note: Acute-phase reactant proteins are: hemoglobin, C-reactive protein, α1-antitrypsin, fibrinogen, and haptoglobin. These are normally occurring proteins. Other proteins are abnormal, low-molecular weight proteins from light-chain diseases (example: multiple myeloma).

2-98      DISCUSS GLOMERULAR PROTEINURIA.

This is the most common and serious of the proteinuria's. Most of the protein found in this disorder is albumin and usually referred to as "albuminuria". There is an increase in glomerular permeability due to injurious effects upon the glomerular capillaries. Causes for such injuries includes (1) immune complexes resultant of multi-systemic diseases as "systemic lupus erythematosus" or "sickle cell anemia"; (2) primary glomerular disease, as "minimal change disease" or "focal glomerulosclerosis"; (3) infectious diseases, as "hepatitis", malaria, or bacterial endocarditis"; (4) drug injury, as seen with penicillin, lithium, or chloramphenicol; (5) pre-eclampsia; and (6) transplant rejection. Glomerular proteinuria can be progressive and if progressive, the amount of protein loss increases, with losses up to 4.0 gm/day possible. If the loss of protein (albumin) >2.0 gm/day, then the patient may experience edema. This disorder can progress and develop into nephrotic syndrome or if the glomeruli are destroyed, then renal failure and proteinuria ceases.

If the loss of protein is <1.0 gm/day and is due to a disturbance in the glomerular apparatus, with no evidence of renal disease, then this condition is designated as "functional proteinuria". Also called "benign proteinuria", this condition is thought to be caused by blood flow changes in the glomerulus or slight changes in permeability. This type of protein loss is seen in pyrexia, exposure to cold, heavy physical activity or exercise, congestive heart failure, emotional stress, hypertension, and atherosclerosis. This type of proteinuria usually resolves itself over time with care and treatment.

2-99     EXPLAIN WHAT IS MEANT BY SELECTIVE PROTEINURIA.

Selective proteinuria is associated with glomerular proteinuria. If the disease is "non-progressive" and the size of protein molecules being lost are correlated to the size and number of lesions in the glomerulus, then glomerular proteinuria is designated as "selective proteinuria". If severe proteinuria is present, with all kinds of sizes of protein molecules, then the proteinuria is designated as "non-selective".

2-100     DISCUSS TUBULAR PROTEINURIA.

Results when the normal tubular protein reabsorptive functions are impaired. Small molecular weight proteins will appear in the urine. Examples of the more common small proteins that are lost are:
        (1) β2-microglobulin [MW = 11,600],
        (2) lysozyme [MW = 14,500],
        (3) α2-microglobulin [MW = 27,000,
        (4) α1-acid glycoprotein [MW = 40,000],
        (5) retinol binding protein [21,000].
The protein loss per day is < 2.5 grams. Common causes of tubular proteinuria include:        
        (1) lupus erythematosus,
        (2) galactosemia,
        (3) heavy metal poisoning (mercury, cadmium, or lead),
        (4) antibiotics (penicillin, sulfonamides, or cephalosporins),
        (5) muscle trauma,
        (6) transfusion reaction,
        (7) renal tuberculosis.
This type of proteinuria can be diagnosed by identifying the presence of lysozyme or β2-microglobulin. If this proteinuria is designated as the acute type, it is usually reversible with treatment. Acute tubular proteinuria may be seen in acute pancreatitis or burns. There is a chronic tubular proteinuria. Its prognosis is more serious and may not clear up with treatment. This form of proteinuria is seen in Fanconi's syndrome, chronic pyelonephritis, or sarcoidosis.

2-101    DISCUSS POST-RENAL PROTEINURIA.

A disorder that is associated with inflammation in any part of the urinary tract other than the kidney. This can result for tissue injury due infections, trauma, or tumors that allow proteins to "leak" into the urinary tract. A diagnostic feature of this proteinuria is the presence of "pus" cells and/or malignant cells in the urine specimen. Red blood cells are not a reliable indicator of this type of proteinuria.

2-102     DISCUSS THE IMPORTANCE OF TESTING FOR MICROALBUMINURIA.

The use of the term "microalbuminuria" implies a proteinuria that is not detected with the usual screening tests used in the urinalysis lab. A patient is designated as having "microalbuminuria" when tests can detect 30 to 300 mg of albumin over a 24 hour period in at least two of three specimens over a six month period. The value of detecting this disorder is its correlation to diabetes mellitus. If renal complications are "silent" and "insidious", being brought on by the presence of glucose in the urine, the detection of microalbuminuria provides the physician a clue to implement corrective treatment and better stabilize the diabetic patient. Failure to intervene will often lead to diabetic neuropathy.

2-103     BRIEFLY DISCUSS TESTING METHODS FOR MICROALBUMINURIA.

Sensitive testing methods include enzyme immunoassay, radioimmunoassay, and fluorescent technique. There are two commercial sensitive screening methods available.
a.     A tablet method (available from Ames) that can detect 4.0 to 8.0 mg
        albumin per dL.  Urine and water are placed on top of the tablet and
        observed for the appearance of a blue-green color. A color chart is provided
        for semi-quantification.
b.     An immunochemical strip reagent test (available from Boehringer-
         Mannhein) can detect 1.0 to 2.0 mg albumin per dL. An antibody-enzyme
         conjugate is used to produce a red color. A color chart is provided for
         semi-quantification.

2-104     DESCRIBE BENCE-JONES PROTEINS AND EXPLAIN WHY IT APPEARS IN THE URINE.

Bence-Jones protein is a low-molecular weight (MW is < 44,000), immunoglobulin para-protein (either a kappa or lambda monoclonal light-chain type) that is abnormally produced in patients with multiple myeloma, primary amyloidosis, lymphoreticular neoplasms, or macroglobulinemia disorder. This protein is readily filtered from the glomerular capillaries and possesses unique solubility properties.

2-105     DESCRIBE AND/OR PERFORM THE CLASSICAL HEAT SCREENING TEST FOR BENCE-JONES PROTEINS.

Begin with clear urine (centrifuging or filtering if necessary) and transfer 5 to 10 mLs to a large test tube. Heat in a water bath to a temperature up to 60 ^oC and observe for turbidity. If Bence-Jones (BJ) proteins are present, flocculation will occur between 40 ^oC and 60 ^oC. Continue heating, bringing the bath to boiling temperature. The flocculated BJ protein will disappear and the urine will be clear. (NOTE: If flocculation is still present, there may be other interfering proteins and the sample should be filtered hot.) Allow the hot, but clear urine specimen to cool. Observe the behavior as the temperature cools to 60 ^oC. BJ proteins will re-flocculate between 40 ^oC and 60 ^oC. Electrophoresis is the best testing method for detecting BJ proteins.

2-106     DISCUSS THE REAGENT STRIP TEST REACTION FOR PROTEIN.

The reagent strip test employs a indicator dye (tetrabromphenol blue or 3',3",5',5"-tetrachlorophenol-3,4,5,6-tetrabromosulfonphthalein) in an acidic buffer to maintain a constant pH of approximately 3.0. Either dye at this pH value is yellow in color. Albumin is the principle protein measured by the strip, the globulins and non-albumin proteins have little or no influence. The strip will not detect BJ proteins. Albumin is a hydrogen ion acceptor and will remove hydrogen ions from the indicator dye causing it to change from yellow to blue-green. The intensity of the color change is proportional to the amount of protein in the urine specimen. This testing procedure is sensitive enough to detect 5 - 10 mg protein per dL. This testing principle is referred to as "the protein error of indicator". This means that at a certain pH, one color appears with protein present, but a different color if protein is absent. Test readings are reported out as: negative, trace (< 30 mg/dL), 1+ (30 mg/dL), 2+ (100 mg/dL), 3+ (300 mg/dL), and 4+ (2000 mg/dL).

2-107    LIST CAUSES FOR FALSE-POSITIVE AND FALSE-NEGATIVE RESULTS WITH PROTEIN REAGENT STRIP TESTING.

False-positives: (1) an elevated pH (≥9.0) will override the buffer system... urine specimen should be re-adjusted to 7.0 and retested, (2) quaternary ammonium compounds, (3) detergents, and (4) over wetting of the pad (which leaches out the buffer salts).
False-negatives: (1) elevated specific gravity due to increases "salts" may override the buffer system and cause a lowering of the reading, (2) globulins and non-albumin proteins present and albumin is absent, (3) polyuria [dilutes out the protein is non-detectable levels],

2-108     DISCUSS AND/OR PERFORM THE KINGSBURY-CLARK SULFOSALICYLIC ACID SEMI-QUANTITATIVE TURBIDITY METHOD.

Sulfosalicylic acid (SSA) is a weak acid, capable of precipitating protein in urine. This procedure requires the addition of 3.0 mLs of 3.0% SSA to 1.0 mL of clear urine. The sample is mixed and allowed to "sit" for five minutes. There are variations of this procedure and regardless of which procedure used, the final concentration of urine and SSA will be about 15 mg of SSA/mL of total solution. The patient's sample can be graded on a scale of negative (up to 7.5 mg/dL), trace (≈20 mg/dL), 1+ (30 to 100 mg/dL), 2+ (100 to 250 mg/dL), 3+ (200 to 450 mg/dL), and 4+ (> 450 mg/dL). Commercial standards are available that range from 10 mg/dL to 100 mg/dL. This is not a sensitive test, requiring 20 mg protein/dL or higher (regardless of the type of protein present). NOTE: If the pH is ≥8.0, adjust the pH to 5.0 and proceed with the SSA method.

2-109    LIST CAUSES OF FALSE-POSITIVE AND FALSE-NEGATIVE RESULTS FOR THE SSA PROCEDURE.

False-positives: (1) Iodine based x-ray contrast media, (2) plasma volume expanders, (3) intravenous albumin, (4) antibiotics [sulfisoxazole or Gantrisin, penicillin, cephalosporins], (5) tolbutamide and its metabolites.
False-negatives: (1) highly alkaline urine [pH ≥8.0], (2) elevated specific gravity due to high salt concentrations.

2-110    COMPARE AND APPRAISE TEST RESULTS BETWEEN SSA AND THE REAGENT STRIP.

       Test Strip                    SSA                                  Probable cause
        Positive                    Positive                      Proteinuria
        Negative                  Negative                    No proteinuria
            1+                          Negative                   Proteinuria, but likely not pathogenic
        Negative                   Positive                    May be a false positive or Bence-Jones
                                                                             protein. Confirm with appropriate tests.

2-111     LIST WHICH TESTS CAN BE CORRELATED WITH THE TESTS FOR PROTEIN.

[1] Blood,  [2] leukocytes,  [3] nitrites, and  [4] microscopic.

2-112   DISCUSS THE IMPORTANCE OF TESTING FOR BILIRUBIN IN URINE.

Bilirubin is a normal breakdown product of hemoglobin metabolism. This degradation process requires that bilirubin be conjugated in the liver. If clinically significant levels of bilirubin are detected in the urine, then further clinical investigation is recommended to detect hepatitis, cirrhosis, gallbladder disease, and cancer. In healthy individuals, trace amounts of bilirubin may be found in urine, but routine testing methods do not detect these clinically insignificant levels.

2-113     WRITE OR DESCRIBE A BRIEF OVERVIEW OF THE CATABOLISM OF HEMOGLOBIN TO UROBILINOGEN.

The life span of an erythrocyte is about 120 days. When the RBC begins to breakdown, it is removed from circulation by the reticuloendothelial system and hemoglobin liberated. The hemoglobin molecule is broken into heme and globulin. Heme is converted to a protoporphyrin compound and the heme ring is opened to form verdoglobin (choleglobin), Iron is split away and a linear, non-conjugated, tetrapyrrole called biliverdin is formed. A reduction reaction converts biliverdin to bilirubin, an insoluble, yellow pigmented compound. At this stage, bilirubin is a yellow molecule, non-conjugated and alcohol soluble (which cannot be excreted by the kidneys). As bilirubin is released into circulation it spontaneously conjugates with albumin. In the liver, bilirubin is conjugated with glucuronic acid, forming a water soluble, bilirubin diglucuronide or bilirubin monoglucuronide. If this conjugated bilirubin re-enters blood circulation, it is excreted at the kidney. Most conjugated bilirubin is secreted into the bile system, where it is temporarily stored in the gallbladder and released into the duodenum. In the small intestine, bilirubin is reduced to stercobilinogen, mesobilinogen, and urobilinogen by bacterial activity. Collectively these three colorless, water soluble, tetrapyrroles are called urobilinogen. These three “urobilinogens” are oxidized and excreted in the final form as stercobilin, mesobilin, and urobilin. The "bilins" are orange-brown pigments and contribute to the color of the stool. Some of the "urobilinogens" are reabsorbed (about 20%) into the blood, returned to the liver. Approximately 5% is re-excreted in the bile and the remainder excreted in the kidney as "urobilinogen". "Urobilinogen" is oxidized to urobilin, stercobilin, and mesobilin (forming part of the urine pigments).

2-114     EXPLAIN WHY CONJUGATED AND NON-CONJUGATED BILIRUBIN IS CLINICALLY SIGNIFICANT IN URINE TESTING.

Dependent upon the disorder, the presence or absence of conjugated and non-conjugated bilirubin are clues to successful diagnosis. Bilirubin is normally excreted in urine, but in amounts that escape detection by routine screening methods. Conjugated bilirubin will appear in the urine only if the integrity of the bile duct or hepatocytes have been compromised.
A.    If the bile duct is occluded, conjugated bilirubin cannot pass into the small
        intestine and "urobilinogens" are not formed. A build up of conjugated
        bilirubin means that increased amounts will escape into blood circulation
        and be excreted by the kidney in significant amounts. Since conjugated
        bilirubin is not passing into the intestine, "urobilinogens" are absent and are
        not being reabsorbed. A strip test positive for bilirubin and negative for
        urobilinogen is suggestive of a bile duct obstruction.
B.    If the integrity of the liver has been compromised, it does not function as
        efficiently.  Conjugated bilirubin is being transferred by the liver into the bile
        duct, but at a slower rate, allowing this bilirubin to build-up and some will
        pass into the peripheral blood. Conjugated bilirubin is readily excreted by
        the kidney's. The "urobilinogens" that are formed and reabsorbed are not as
        efficiently recovered and returned to the bile. Peripheral blood levels of the
        "urobilinogens" are increased, resulting in more being excreted by the
        kidney's. A strip test positive for bilirubin will also be positive for
        urobilinogen, supportive of some type of liver cell dysfunction (as in hepatitis
         or cirrhosis).
C.    If a patient experiences a transfusion reaction or a hemolytic disease crisis,
        numerous RBC's will hemolyze, releasing increased amounts of hemoglobin
        in the blood. In the degradation of Hemoglobin, large amounts of bilirubin
        are formed, overwhelming the ability of the body to rapidly conjugate it. The
        excess unconjugated bilirubin is absorbed into the tissues giving a jaundice
        appearance to the skin, sclera, body fluids, and tissues. Unconjugated
        bilirubin cannot be excreted by the kidneys. The liver is functioning normally
        and the conjugated bilirubin is entering the intestines in larger quantities.
       This means that more "urobilinogens" are being reabsorbed. This in turn
       means that the kidney's will excrete larger quantities. The strip test will be
       negative for bilirubin, but positive for urobilinogen.
      
2-115     EXPLAIN CHEMICAL REACTION OF THE REAGENT STRIP TEST FOR BILIRUBIN.

The testing strategy is based upon the diazo reaction.  The diazo reaction is dependent upon presence of a diazo group (=N+ or =N-).  In this testing procedure, the pads contain a diazonium salt (2,4-dichloroanailine diazonium salt or 2,6-dichlorobenzene-diazonium-tetrafluoroborate).  The diazonium reagent is stabilized by acidic buffers. When the reagent reacts with bilirubin, an azodye (azobilirubin) is formed that produces colors ranging from tan, to light brown, or pink or purple.  A commercial color chart is used to measure the reaction.  Results are reported as negative, 1+ (small), 2+ (moderate), and 3+ (large).  The reagent strip test can detect as little as 0.4 mg urinary bilirubin/dL.

2-116     DISCUSS AND/OR PERFORM THE ICTOTEST PROCEDURE.

The Ictotest is a tablet test that uses the same strategy as the reagent strip test, but is about four times more sensitive, detecting concentrations bilirubin as low as 0.05 mg/dL. The tablets use p-nitrobenzene-diazonium-p-toluenesulfonate as the diazonium salt. Ten drops of urine are placed on the absorbent mat to "trap" and concentrate the bilirubin in the fibers. The tablet is placed in the center of the moistened spot and a drop of water added. If the drop spills over the edge of the tablet and flows on the mat, a second drop may not be required. A second drop of water is added to the first drop to cause the water/solution to overflow. The drop(s) of water leach the reagents out of the tablet to form a reactive solution. The test is read after 60 seconds. A blue or purple color produced on the mat is a positive test. Any other color is considered to be negative.

2-117    LIST CAUSES FOR FALSE-POSITIVE AND FALSE-NEGATIVE RESULTS IN BILIRUBIN TESTING.

False-positives: (1) urine pigments, (2) Lodine, (3) indican, (4) Medications as chlorpromazine, phenazopyridine, or ethoxazene. (Note: Colors from other chemicals may interfere with test interpretation if bilirubin is also present.)
False-negatives: (1) urine exposed to light, (2) urine exposed to air, (3) elevated levels of ascorbic acid (blocks diazo reaction), (4) elevated nitrate levels (blocks diazo reaction).
NOTE: The Ictotest tends to be free from most interfering substances since the bilirubin is collected on the surface of the mat and the other components of the urine are washed toward the center of the mat.

2-118     DISCUSS THE “FOAM” TEST.

Urine that contains bilirubin takes on a dark yellow or amber color. If the urine is shaken and the foam observed, the foam takes on the color of the urine. This test is not reliable since medications can cause the foam to be colored.

2-119     LIST WHICH TEST CAN BE CORRELATED WITH THE BILIRUBIN TEST.

Urobilinogen

2-120    DESCRIBE THREE CHARACTERISTICS OF THE TWO FORMS OF BILIRUBIN.

                      FREE BILIRUBIN                 CONJUGATED BILIRUBIN
    [1]         non-polar (water insoluble)             polar (water soluble
    [2]             not excreted in urine                        excreted in urine
    [3]              does not stain tissue                              stains tissue

2-121     DISCUSS THE IMPORTANCE OF UROBILINOGEN TESTING.

Urobilinogen is a bile pigment, a product of heme degradation. It is formed in the intestines by the reduction reactions of bacteria on bilirubin. Urobilinogen is normally present in the urine in concentrations < 2.5 mg/dL. Urobilinogen testing is basically limited to screening tests that help detect early liver disease and hemolytic disease (including transfusion reactions). There is little value in performing quantitative tests on urobilinogen since there are better and more specific liver function tests.

2-122     DISCUSS THE REAGENT STRIP TEST FOR UROBILINOGEN.

The reagent strip test was originally developed using the Ehrlich's test concept. Ehrlich's reagent is p-dimethylaminobenzaldehyhde (DAB) and is employed in the test pad with an acidic buffer. In the presence of urobilinogen, the pad turns from a light pink to a dark pink color. p-dimethylaminobenzaldehyde is non-selective and will react with a number of substances, termed as "Ehrlich-reactive". Another chemical, 4-methoxybenzene-diazonium-tetrafluoroborate (MDT), is used in other reagent test strips and had been found to be more specific, with fewer false positives. A positive reaction will produce pink to red color. If Ehrlich-reactive substances is present in the urine sample, they will interfere with the DAB reaction, producing a falsely elevated value. Ehrlich-reactive substances generally are non-reactive with the MDT reaction. The reagent strip test should not be used to determine the absence of urobilinogen (seen in bile duct obstruction).

2-123     LIST CAUSES FOR FALSE-POSITIVES AND FALSE-NEGATIVES IN UROBILINOGEN TESTING WITH p-DIMETHYLAMINOBENZALDEHYDE (EHRLICH’S REAGENT).

False Positives: (1) porphobilinogen, (2) indican, (3) bilirubin, (4) indole, (5) sulfonamides, (6) p-aminosalicylic acid, (7) methyldopa, (8) procaine, (9) 5-hydroxy-indole acetic acid, (10) chlorpromazine.
NOTE: The presence of (1) phenazopyridium, (2) azodyes [riboflavin, nitrofurantoin, and ethoxazene), and (3) red beets can mask the urobilinogen pad with a reddish color, causing a false positive.
False-negatives: (1) formaldehyde, (2) improper storage, and (3) high nitrite levels.

2-124   BRIEFLY DESCRIBE HOW THE LABORATORY MAY TEST FOR THE ABSENCE OF UROBILINOGEN.

Since testing urine cannot validate the absence of urobilinogen, fecal testing will verify the absence or presence of urobilinogen. Chemical testing for bilirubin will be helpful

2-125    LIST DISORDERS IN WHICH UROBILINOGEN MAY BE ELEVATED OR ABSENT.

Elevated in (1) hemolytic anemia, (2) cardiac infarction, (3) sickle cell anemia, (4) pernicious anemia, (5) cirrhosis of the liver, and (6) hepatitis.
Absent in (1) cholestasis (bile duct obstruction), (2) starvation, (3) hepatitis with cholestasis, (4) fibrosis.

2-126    EXPLAIN THE CLINICAL SIGNIFICANCE OF PORPHOBILINOGENS.

Porphobilinogens are the precursors of porphyrins which are intermediates in the biosynthesis of hemoglobin. The basic structure of the porphobilinogens is a pyrrole ring and four pyrrole rings will be combined to form a linear molecule which has the capability to undergo cyclization and form uroporphyrinogen which will undergo a series of reactions to form protoporphyrin IX, which can chelate iron. When iron is incorporated into the porphyrin molecule, it is called heme. Refer to the following illustration. 
       

                            Intermediates in the Synthesis of Heme.

The porphobilinogens can spontaneously oxidize and if this occurs, then the oxidized porphobilinogen (biologically non-functional) must be excreted. If there is an enzyme defect in any of the reaction sequences between glycine + succinyl-CoA to protoporphyrinogen, this block in the synthesis pathway will cause a deficit in some of the metabolites and an accumulation of others. There are consequences to the accruing of porphobilinogens, which includes accumulation in certain tissues and a toxic effect upon nerves. Six type of porphyria's have been described: acute intermittent porphyria, congenital erythropoietic porphyria, porphyria cutanea tarda, hereditary coproporphyria, variegate porphyria, and protoporphyria. For example, if the enzyme "uroporphyrinogen cosynthetase" is defective, the condition "Acute intermittent porphyria" will diagnosed. γ-aminolevulinic acid (ALA) and porphobilinogen (PBG) accumulates in the blood stream and because of their low renal threshold, they will quickly appear in the urine. It is the PBG that is detected since screening procedures do not detect ALA. One other point... hemoglobin acts as enzyme inhibitor in porphyrin synthesis. If an enzyme is defective in the metabolic pathway, hemoglobin production is reduced and its rate controlling effect lost which allows for the abnormal accumulation of the porphyrins.

2-127     EXPLAIN THE DIFFERENCE BETWEEN ACQUIRED AND HEREDITARY PORPHYRIAS.

Hereditary porphyria is due to a defective enzyme or the inability of the tissues to produce sufficient amounts of the enzyme. There are two general types of hereditary porphyrias:  erythropoietic, in which the effects are expressed in the hemopoietic tissues. The other type is found in the liver as enzyme defects.

Acquired porphyrias are the result of an external influence upon the liver parenchyma cells. Examples of external influences are (1) antibiotics [penicillin or sulfonamides], (2) alcohol, (3) stilbestrol, (4) lead poisoning or other heavy metals, (5) diabetes mellitus, (6) sedatives, (7) hypnotics, and the disease syphilis.

2-128     DESCRIBE AND/OR PERFORM THE HOESCH TEST.

This is a simple semiquantitative procedure in which 2-3 mLs of modified Ehrlich's reagent is added a large test tube. Two drops of fresh urine is layered onto the reagent. A red (or deep pink) color will form at the interface of the layers and indicates the presence of porphobilinogen. The modified Ehrlich's (or Hoesch) reagent consists of 2.0 gms of p-dimethylaminobenzaldehyde in 6.0 Molar HCL.

2-129     EXPLAIN WHY THE HOESCH TEST IS A GOOD SCREENING TEST FOR PORPHOBILINOGEN.

The Hoesch test is insensitive to urobilinogen, requiring ≥20 mg/dL to produce a positive reaction. It is sensitive to porphobilinogen producing an instant color if present and can detect levels as low as 2.0 mg/dL. Although the color produced is in proportion to the amount of porphobilinogen present, it is not used as a quantitative test.

2-130       DESCRIBE AND/OR PERFORM THE WATSON-SCHWARTZ DIFFERENTIATION TEST.

Equal volumes (2.5 mLs. each) of fresh urine and Ehrlich's reagent are mixed in a large test tube. 5.0 mLs of saturated sodium acetate is added and the tube mixed well. If urobilinogen, porphobilinogen, or other Ehrlich-reactive substances are present, the solution will develop a pink to cherry-red color. If there is no color the test is negative. If the color is present add the test solution to 5 mLs of chloroform in a separatory flask and mix well. The solution will separate into two layers, a top aqueous layer and a bottom chloroform layer. If there is color in the aqueous later, with the chloroform layer clear, then porphobilinogen or other Ehrlich-reactive substances are present. If the color is in the chloroform layer, then urobilinogen is present. A second extraction step is required if the color is in the aqueous layer. Transfer the aqueous layer to a second separatory funnel and add 5.0 mLs of butanol, mixing well. If the color remains in the aqueous layer, then porphobilinogen is present. If the color transfers to the butanol layer, then urobilinogen or other Ehrlich-reactive substances are present. This test is more sensitive, detecting as little as 0.6 mg of porphobilinogen/dL. The results should be reported as (1) positive or negative for urobilinogen, (2) positive for porphobilinogen, or (3) positive for both porphobilinogen and urobilinogen. NOTE: Urobilinogen is soluble in both chloroform and butanol. Porphobilinogen is not soluble in either chloroform or butanol. Generally Ehrlich-reactive substances are soluble in butanol, but not chloroform.

2-131     LIST A MINIMUM OF ELEVEN EHRLICH-REACTIVE SUBSTANCES.

(1) porphobilinogen, (2) indican, (3) bilirubin, (4) indole, (5) sulfonamides, (6) p-aminosalicylic acid, (7) methyldopa or Aldomet, (8) procaine, (9) 5-hydroxy-indole acetic acid, (10) chlorpromazine, (11) urobilinogen

NOTE: The presence of (1) phenazopyridium, (2) azodyes [riboflavin, nitrofurantoin, and ethoxazene), and (3) red beets can mask the tests for porphobilinogen by imparting a reddish color and cause difficulty in interpretation.

2-132     EXPLAIN THE IMPORTANCE OF UROBILIN IN URINE TESTING.

It has little if any importance. It is not found in fresh urine. It is the end product of urobilinogen oxidation by air, light, or bacteria. The addition of Lugol's iodine to a urine specimen will oxidize the urobilinogen to urobilin. The only reason to perform a urobilin test would be to confirm the absence of urobilinogen and there are other methods that are easier and user friendly.

2-133      DISTINGUISH BETWEEN THE TERMS "HEMATURIA" and "HEMOGLOBINURIA".

Both terms imply the presence of blood in the urine. Hematuria means that intact erythrocytes are present. Hemoglobinuria implies that erythrocytes are hemolyzed or absent and only hemoglobin is present.

2-134    EXPLAIN THE IMPORTANCE OF TESTING FOR BLOOD IN URINE.

The presence of blood (RBC and/or hemoglobin) is clinically significant. Evaluation is important to determine if the problem is pathological or non-pathological. Blood can enter the urinary tract at any point in the urinary system.

If the urine is transparent and red, then this would strongly suggestive of hemoglobinuria. Hemoglobinuria may be the result of intravascular hemolysis because of a transfusion reaction or hemolytic anemia. Other causes of this problem are burns, exhausting and intense physical activity, parasite infections, poisoning, proximal nocturnal hemoglobinuria, proximal cold hemoglobinuria, and trauma. If hemoglobin is crossing the glomerulus, renal tubular cells will reabsorb some of the hemoglobin, catabolize it to hemosiderin and ferritin, and excrete it a few days later into the urine. Renal tubular epithelial cells containing hemosiderin can be observed in the urine sediment as-well-as hemosiderin granules. The use of Prussian blue stain will confirm the presence of hemosiderin. True hemoglobinuria is an uncommon occurrence. Most hemoglobin in the urine is the consequence of hemolysis.

Hematuria is characterized by a cloudy, smokey colored urine. Major causes of hematuria are trauma, pyelonephritis, exhausting and intense physical activity, anticoagulants, malaria, appendicitis, leukemia, thrombocytopenia, hemophilia, kidney stones, tumors, catheterization, glomerulonephritis, cystitis, acute febrile episodes, and exposure to toxic chemicals. The presence of RBC's in urine may be an indicator of early renal disease and requires medical follow-up. The presence of a few (very few) RBC's in urine is generally considered to be normal. If RBC’s are present, look for RBC casts.

2-135   DISCUSS HOW MYOGLOBIN APPEARS IN URINE AND ITS SIGNIFICANCE.

Myoglobin is a small hemoprotein (MW = 17,000) that is rapidly cleared from blood and excreted in the urine. It is the result of muscle tissue trauma due to surgery, injury crushes, toxic action of ethanol or drug addiction, muscle tissue, electric shock, muscle disease, snake venoms, and idiopathic paroxysmal myoglobinuria. Myoglobin is readily reabsorbed by the proximal tubular cells. Myoglobin has the potential to damage the kidneys. It presence in urine is pathological and should be quickly identified.

2-136     DESCRIBE HOW MYOGLOBIN CAN BE IDENTIFIED AND CONFIRMED.

A simple screening technique requires that 5.0 mLs of centrifuged urine (neutral pH) be placed in a test tube and add 2.8 grams of ammonium sulfate, then mix well. Allow the specimen to sit for five minutes. Filter and test the filtrate with a chemical test for blood. If the test is positive, then myoglobin is presumed to be present. The principle of this test lies in the fact that hemoglobin is precipitated out in an 80% ammonium sulfate solution. The best methods for identifying myoglobin is protein electrophoresis. Other reliable test procedures are radioimmunoassay, absorption spectrophotometry, and immunodiffusion.

2-137      DISCUSS THE REAGENT STRIP TEST FOR BLOOD.

The hemoglobin molecule can break down the peroxide molecule and the test was developed to take advantage of this peroxidase activity. Manufacturers use a peroxide (cumene hydroperoxide or 2,5-dimethyl-2,5-dihydroperoxyhexane) and the pseudoperoxidase activity of hemoglobin reduces the "peroxide", which in turn oxidizes, that is releases oxygen to convert the reduced chromogen (tetramethylbenzidine), with no color to an oxidized chromogen with a blue color. The test is sensitive, detecting as few as 5 RBC's/μL (≈ 0.015 mg hemoglobin/dL) urine.

2-138      WHEN GIVEN DATA, DIFFERENTIATE BETWEEN ERYTHROCYTES, HEMOGLOBIN, AND MYOGLOBIN IN URINE.

The following table of information will illustrate the parameters to differentiate the differences.

Descriptor                                           Hemoglobin    Myoglobin         RBC's
Reagent strip test                                positive                positive           positive
Appearance of urine                            clear/red                clear              smokey/cloudy
Appearance of plasma                         pink/red               normal            normal
RBC's in urine sediment                      0 to few             0 to few              present
Serum creatine kinase (CK)                 elevated (*)     elevated (#)      normal
Serum haptoglobin                                decreased           normal             normal
Serum lactate dehydrogenase (LD)   elevated              elevated           normal
Serum LD-1 and LD-2                         elevated              normal             normal
Serum LD-4 and LD-5                         normal                elevated          normal
        (*) will be ≤ 10 times the upper reference limit.    
        (#) will be ≥ 40 times the upper reference limit.
        Upper reference limit for serum CK in men = 130 IU/L and for women = 115 IU/L.
        Upper reference limit for serum LD for adults range from 40 to 90 IU/L

139    LIST A MINIMUM OF TEN SUBSTANCES THAT INTERFERE WITH THE REAGENT TEST STRIP FOR BLOOD, CAUSING FALSE-POSITIVES AND FALSE-NEGATIVES.

False-positives: (1) myoglobin, (2) oxidizing detergents, (3) leukocyte esterase, (4) menstrual contamination, (5) hemorrhage contamination, (6) bleach, (7) microbial peroxidases.
False-negatives: (1) ascorbic acid (note: iodate is being included in reagent pad to remove ascorbic acid so that it now requires markedly elevated levels to interfere), (2) elevated specific gravity, (3) elevated protein levels, (4) nitrite (≥10 mg/dL), (5) captopril, (6) formaldehyde, (7) vegetable peroxidase.

2-140     IDENTIFY WHAT OTHER PARAMETERS IN URINALYSIS TESTING THAT THE BLOOD TEST CAN BE CORRELATED TO.

[1] Microscopic sediment and [2] protein testing.

2-141     EXPLAIN THE IMPORTANCE OF TESTING FOR NITRITES IN URINE.

Nitrites are not normally detected in the urine of healthy individuals. This test provides a quick and convenient method of screening for urinary tract infections (UTI). One value that is inherent in this test is that a UTI may be asymptomatic or the patient may be complaining of vague symptoms that would not alert an physician to order a urine culture and sensitivity test. A word of caution.... this test depends upon the infecting bacteria to be nitrate reducers (have the enzyme "nitrate reductase) and not all bacteria that can cause a UTI have the nitrate reducing enzyme .

Urine in the bladder is normally a sterile environment and sterility is supported by the frequent flushing of urine from the bladder. Most infections are assumed to begin in the bladder and may be resultant of infrequent urination (oliguria), "malfunction" of the bladder, or some other problem. If a bladder infection (cystitis) occurs, then it is possible for the infection to spread to other part of the urinary system. Infections that are not detected can lead to renal tissue damage.

Testing for nitrites can be a means for assessing the effectiveness of antibiotic therapy; the presence of a UTI (cystitis, urethritis, ureteritis, pyelonephritis, or glomerulonephritis); monitoring patients at high risk for a UTI; and screening urine specimens for UTI.

2-142     EXPLAIN THE CHEMICAL BASIS OF THE STRIP TEST FOR NITRITES.

This test is a modified Griess reaction (1879) and depends upon the presence of nitrites in the urine. The test pad contains an aromatic amine (p-arsanilic acid or sulfanilamide) and buffers to maintain an acidic environment for the reaction to occur. Nitrite and the aromatic amine will chemically interact (called "diazotization") to form an intermediate, a "diazonium salt". The diazonium salt would then react with another chemical in the pad, a chromogen [3-hydroxy-1,2,3,4-tetrahydorbenz-(h)-quinolin] and the end product would a pink "azodye" molecule. The pink color (without regard to intensity) is interpreted as positive. See the reaction sequence in the following illustration.

      Aromatic Amine–NH₂  +  Nitrite (NO₂) ----> Aromatic Amine  +  ⁺N=N  +
      chromogen ------>   Aromatic Amine–N=N - chromogen (a pink Azo-dye)

This test pad is "standardized" to be insensitive to the presence of < 100,000 microorganism/mL in order to eliminate false positive test results. If this test is positive, then there are > 100,000 microorganisms/mL. Any shade of pink represents a clinically significant number of microorganisms. This test will not work if there are no dietary nitrates in the diet of the patient. Fresh urine specimens (in particular a first morning specimen) should always be used when testing for nitrites.

2-143    LIST A MINIMUM OF EIGHT THINGS THAT CAN CAUSE A FALSE-POSITIVE OR FALSE- NEGATIVE NITRITE TEST.

False-positive:   [1] presence of colored medications (pyridium),  [2] contaminating bacteria in a stool that has stood too long or improper collection, or  [3] storage of reagent strip test in an open container.
False-negative:   [1] lack of dietary nitrate,  [2] antibiotic therapy,  [3] ascorbic acid at concentrations of 25 mg/dL or greater,  [4] high urinary urobilinogen,  [5] a urine pH < 6.0,  [6] a urine specimen that did not stay in the bladder a sufficient amount of time, or [7] or bacteria that do not reduce nitrate.

2-144     IDENTIFY WHAT OTHER PARAMETERS IN URINALYSIS TESTING THAT THE NITRITE TEST CAN BE CORRELATED TO.

[1] Microscopic and [2] leukocyte esterase.

2-145     EXPLAIN THE ADVANTAGES OF TESTING FOR THE PRESENCE OF THE ENZYME LEUKOCYTE ESTERASE.

This is a screening strategy for detecting the presence of a urinary tract infection. The enzyme "leukocyte esterase" is produced in the granules of neutrophils and by monocytes. The presence of this enzyme in the urine is an indicator of a significant number of leukocytes. In an inflammatory reaction, white blood cells increase in number and many will "lyse" or are otherwise destroyed in urine. The microscopic examination has been the standard for detecting the WBC activity associated with inflammation. The leukocyte esterase test will detect the presence of WBC's without their having to undergo lysis.

2-146     DISCUSS THE SIGNIFICANCE OF WBC'S IN URINE.

The urine of a healthy adult male will contain from zero to 2 or 3 WBC's/high power field (hpf) in a urine specimen that has been concentrated from 10 to 12 mLs to 0.5 to 1.0 mLs of urine. This will be ≤ 10 WBC's/μL. Adult female urine will contain from zero to five WBC's/hpf. Normal values for children are similar to that of the adult female. If the number of WBC's in a urine specimen 10 to 15/hpf, this is suspicious of a UTI and should be investigated. If the count is ≥ 20/hpf, this is suggestive of a UTI.

2-147     EXPLAIN THE BASIS OF THE REAGENT STRIP TEST FOR LEUKOCYTE ESTERASE AND HOW THE STRIP TEST IS EVALUATED.

The test uses an enzymatic reaction principle that employs the diazo principle as in the nitrite and bilirubin test. The test pad contain an organic ester (indoxylcarbonic acid ester or pyrrole amino acid ester) embedded with a diazonium salt. If the urine specimen contains the leukocyte esterase enzyme, the organic ester will be cleaved an intermediate that will react with the diazonium salt to produce a colored "azodye", which can be measured with standardized color chart. The intensity of the color will correlate to the amount of leukocyte esterase present in the urine specimen. The test result is reported as negative, trace,  1+ (small),  2+ (moderate), and  3+ (large). Examine the following reaction scheme to understand the enzymatic principle.

      Ester  +  leukocyte esterase  ---->   [intermediate]  +  N=N⁺-Diazonium
      salt (in an acidic buffer)   ----->   [intermediate]–N=N-Diazonium salt
      (purple azo dye)

2-148    IDENTIFY WHAT OTHER PARAMETERS IN URINALYSIS TESTING THAT THE LEUKOCYTE ESTERASE TEST CAN BE CORRELATED TO.

[1] Microscopic, [2] protein, and [3] nitrate.

2-149    LIST A MINIMUM OF TEN THINGS THAT WILL CAUSE A FALSE-POSITIVE OR FALSE-NEGATIVE TEST RESULT ON THE LEUKOCYTE ESTERASE TEST OR OTHERWISE OBSCURE THE INTERPRETATION OF THE TEST.

False-positives: (1) formaldehyde, (2) oxidizing agents in the specimen, (3) urine specimens containing vaginal discharges, (4) any medication that can color the pad a violet color in an acid environment
False-negatives: (1) ascorbic acid [high levels], (2) ≥ 500 mg albumin/dL, (3) ≥3.0 gm glucose/dL, (4) elevated specific gravity, (5) cephalosporin antibiotics, (6) oxalic acid, (7) gentamycin, (8) tetracycline
NOTE: If the urine has strong yellow pigmentation, the test pad may take on a green color. This should be interpreted as positive.

2-150     COMPARE THE PRINCIPLE OF THE REAGENT STRIP TEST FOR SPECIFIC GRAVITY TO THAT OF THE REFRACTOMETER.

The reagent strip test is based on a chemical principle that measures the change in the dissociation constant of a polyelectrolyte. This method does not measure the true solute content, only those solutes that are ionic (pertaining to charged molecules). If the strip test technique is used as the means of evaluating the specific gravity over time, then this method will provide clinical data regarding the concentrating and diluting ability of the kidney. Refer to Objectives #55 and #59.

2-151      LIST A MINIMUM OF FIVE THINGS THAT WILL CAUSE AN INCREASE OR DECREASE IN THE REAGENT STRIP SPECIFIC GRAVITY TEST RESULTS OR OTHERWISE OBSCURE THE INTERPRETATION OF THE TEST.

Increase: [1] urea (1.0 gm/dL), [2] a 100 mg/dL protein reading may increase SG by .005, [3] organic acids (lactic acid, acetoacetic acid, etc), [4] ketone bodies.
Decrease: [1] urine pH of 6.5 lowers SG, therefore add .005 to reading

2-152     DESCRIBE THE EFFECTS OF ASCORBIC ACID ON THE STRIP REAGENT TEST PROCEDURE.

Ascorbic acid (vitamin C) is a molecule suggestive of the glucose molecule. It is not synthesized in the human and certain other animals because of a deficiency of L-gulonolactone oxidase, therefore must be included in the diet. Ascorbic acid is a reducing agent, able to donate a hydrogen ion to acceptable recipient molecules. For this reason, it is capable of interfering with one of the reactants in the reagent strip test pads as follows:
[1]     Bilirubin contains diazonium salts which can be reduced causing a
           false-negative reaction.
[2]     Blood contains a pseudoperoxide that can be reduced causing a
           false-negative reaction.
[3]     Glucose contains a pseudoperoxide that can be reduced causing a
           false-negative reaction.
[4]      Nitrate contains a diazonium salt that can be reduced causing a
            false negative reaction.

Ascorbic acid, when it donates its hydrogen ions to appropriate molecules, is reduced to dehydroascorbic acid, which can further degrade to oxalate (oxalic acid). The average diet contains sufficient ascorbic acid for good health and the individual will excrete less that 5 mg/dL ascorbic acid in the urine. There is some research that indicates that as much as 38 mg/dL may be excreted on an average. These levels are clinically insignificant and cause no problems in reagent strip test technology. If an individual supplement their diet with increased vitamin C, then there is a potential risk that one or more of the above urine tests will be altered. Manufacturers of reagent test strips add a substance that will block ascorbic acid reaction and allow an accurate test result. Studies have indicated that individuals may excrete up to 400 mg of ascorbic acid in 100 mLs of urine. Such levels may interfere with strip testing. It may be difficult to detect the interference of ascorbic acid in urine. There are a few clues that will help. For example, if you report a urine specimen with RBC’s in the urine but the strip test pad for blood is negative, then you might have cause to suspect ascorbic acid is present. The simplest method might be to keep reagent strip tests for ascorbic acid as part of the lab inventory and if problems arise with urine testing, then the urine can tested for the presence of ascorbic acid.

2-153     EXPLAIN WHAT HAPPENS WHEN 1%, 2% or 3% ACETIC ACID IS ADDED TO URINE SEDIMENT.

The nucleus of WBC's are accentuated and RBC's undergo lysis.

2-154     DESCRIBE THE QC STEPS FOR PERFORMING A MICROSCOPIC EXAMINATION THAT MEETS QUALITY CONTROL STANDARDS.

[1]    Use fresh urine. If unable to perform within one hour of voiding, refrigerate
          for up to three hours.
[2]    Centrifuge a standard quantity. Use either 10, 12, or 15 mLs.
[3]    Use conical centrifuge tubes
[4]    Centrifuge, using the same centrifuge, rotor, centrifuge speed and force,
          and time.
[5]    Decant urine the same way/same technique, leaving a residual of 0.5 to
         1.0 mLs urine to resuspend the sediment and always be consistent in the
         amount of residual urine remaining.
[6]     If sediment is to be stained, always add the stain to the sediment in the
           tube, not the slide.
[7]     Transfer the sediment to the slide the same way, using the same type of
           transfer pipette to deliver the same size drop. Do NOT invert the tube to
           transfer sediment.
[8]     If using a cover glass, use the same size cover glass since a larger or
          smaller size will affect the way sediment distributes under the cover glass.
[9]      Be consistent in the way that the microscopic evaluation is performed.
[10]    Examine the sediment under low power first to detect the presence or
           absence of casts.
[11]     Use the 45X objective to identify sediment
[12]     Examine the same number of objective fields. Ten fields are acceptable,
             but 20 is preferable.
[13]    Use the same terminology for reporting out sediment.
[14]    Check microscopic findings with physical and chemical results to assure
            accuracy of report.

2-155   STATE EIGHT FACTS THAT RELATE TO SOURCES OF ERROR IN THE EXAMINATION OF URINE SEDIMENT

[1]    Using dirty slides makes accurate differentiation of urine sediment difficult.
[2]    Refrigerating urine will cause the urine specimen to become turbid which
          will make the microscopic examination more difficult. Note: Up to 10
          percent of urines are turbid or cloudy at the time of voiding.
[3]    If dilute acetic acid is used to dissolve amorphous phosphates, then any
         erythrocytes in the urine specimen will be hemolyzed.
[4]    There are a number of look-a-likes that may be found in urine. Oil
          droplets, yeast, certain crystals, and even bubbles have been confused
          with erythrocytes by inexperienced laboratorians.
[5]    Using a sediment stain will facilitate recognition of formed elements in
          the urine.
[6]    Urine that has stood at room temperature for two or more hours will not
          be reliable as some of the constitutents in the urine will have disintegrated
          or disappeared.
[7]    If urine contains myoglobin, it will yield a positive blood test and no
          erythrocytes will be seen.
[8]    If a patient takes mega-doses of ascorbic acid, then there may be a
          suppressive effect upon the blood, nitrate, bilirubin, and glucose reagent
          test pads causing a false-negative test result.

2-156     LIST THE CORRECT PROCEDURE FOR REPORTING URINARY SEDIMENT AND/OR WHEN GIVEN DATA, CORRECTLY COMPLETE A URINALYSIS REPORT.

Report should be filled out according to the following parameters.
[1]     RBC's, WBC's, renal tubular epithelial cells, transitional epithelial cells,
          crystals, and parasites are reported as "none seen" or "#/hpf".
[2]     Casts and squamous epithelial cells are reported as "none seen" or "#/lpf".
[3]     Bacteria, yeast, mucus, fat droplets, spermatozoa, and amorphous crystals
           are reported as "none seen",  ± (trace),  1+ (few),  2+ (moderate),
           3+ (many),  4+  (TNTC/too numerous to count).
[4]     Report casts, crystals, and epithelial cells as to their type.
[5]     Artifacts, as a rule, are not reported. If you see undigested animal or plant
           cells or fibers, this should be investigated to rule out if the urine is actually
           stool water,  poor hygiene habits. or a vesicosigmoid fistula.

2-157     LIST FOURTEEN ARTIFACTS AND STATE HOW THEY APPEAR IN THE URINE.

[1]    plant cells:   via fecal contamination or vesicosigmoid fistula
[2]    starch:   body power from patient or laboratorian gloves
[3]    talc:   body powder from patient
[4]    pollen:   air contamination
[5]    muscle fibers:   via fecal contamination or vesicosigmoid fistula
[6]   cotton fibers:   sifting from patient's clothing during urine collection.
[7]    synthetic fibers:   sifting from patient's clothing during urine collection.
[8]    hair:   from the patient's body.
[9]    paper fibers:   during urine collection or wiping urine container.
[10]   oil droplets:   body lotion, catheter lubricants.
[11]   wood fibers:   wooden applicator sticks.
[12]   plastic shards:   usually sloughing off from centrifuge tube.
[13]   glass chips:   from the cover slip or glass slide due to scratching.
[14]   air bubbles:   appear when transferring the specimen to the slide.
            Examine the following illustrations for examples of artifacts:
   

2-158    LIST THE EXPECTED STAINED COLOR WHEN THE FOLLOWING TEN FORMED ELEMENTS ARE STAINED WITH STERNHEIMER-MALBIN (S-M) STAIN.

[1]     mucus: pale pink or a pale blue
[2]     erythrocytes: neutral pH = pink to purple, alkaline pH = purple, acid
          pH = do not stain. RBC may not stain well with SM stain.
[3]     leukocytes: nucleus = purple, cytoplasm = purple granules.
[4]     bacteria: motile (alive) = not stained, non-motile (dead) = purple
[5]     RTEC: nucleus = dark purple, cytoplasm = light shade of purple.
[6]     transitional cells: nucleus = dark purple, cytoplasm = light shade of
           purple.
[7]     squamous epithelial cells: nucleus = purple, cytoplasm = light shade
          of purple.
[8]     "Trich": Trichomonas vaginalis, when alive, has a light greenish
           appearance with S-M stain.
[9]     hyaline cast: pale pink or pale blue matrix, similar to mucus.
[10]    waxy cast: pale pink or pale blue, similar to the hyaline cast.
NOTE:   most casts with inclusions will demonstrate a similar staining reaction in the matrix. The inclusion may or may not stain. See any reference text or your text book.
NOTE:    Color perception varies among techs.   Cells identified as taking on a purple color may appear blue to another tech.

2-159    WHEN GIVEN DATA, THE NUMBER OF FORMED ELEMENTS IN A VOLUME OF URINE CAN BE CALCULATED.

The following information and example demonstrates how this can be accomplished.
[1]    Determine the area of the objective being used for the calculation. For the
         low power (10×) objective, you may use an area of 2.545 mm². For the high
         power (45×)  objective, use an area = 0.196 mm².
[2]    Determine the number of low-power or high-power fields possible on the
          cover glass being used for the microscopic examination.
          a.    The Kova slide area is circular and ≈32 mm²
          b.    An 22 mm (×) 22 mm square cover glass ≈484 mm²
          c.    Other examples of cover glass sizes are 18 mm (X) 18 mm and 22 mm
                 (×)  40 mm.
         d.    To calculate the number of fields, divide the area of the coverslip by
                 the area of the objective. Example: for a high power objective and a
                 22 mm (×) 22 mm cover glass, divide 484 mm² by 0.196 mm².  The
                 answer is 2,469 high-power fields.
[3]    The volume of sediment that is viewed under the objective is required. For
          the Kova slide, the volume under the 45× objective is 0.006 μL. The
          disposable glass slide and coverslip create a problem in reproductivity.
          Even under the best of techniques, consistent and exact standardization is
           not possible. Ideally for a 22 mm (×) 22 mm, exactly 20 μL of resuspended
           sediment concentrate should be placed on a glass slide and the coverslip
          placed over it. This means that 0.008 μL of sediment is being viewed
          under the 45× objective. If a 18 mm (×) 18 mm cover glass is used then
          0.012 μL is being viewed by the 10× objective.
[4]    Determine the concentration factor. In most labs, 12 mLs of urine is
          concentrated down to 1.0 mLs by first centrifuging 12 mLs of urine and
          removing 11 mLs of supernatant, then resuspending the sediment in the
          remaining 1.0 mLs. For this exercise, the concentration factor the volume
          of urine concentrated, will be 12 mLs and mLs is dropped and only the
          number 12  is used.
[5]    Use the following formula to calculate the number of fields viewed per mL.
         This step may be called the "field conversion factor".

           # possible fields                          # of fields viewed per
---------------------------        =    --------------------------
total vol. of sediment viewed               1 mL urine tested
     (in mLs)  × Conc. factor

[6]    To set up problem, use 22 mm (X) 22 mm cover glass data.

      2 ,469 [45×] fields of view                2,469
------------------------------     =    --------   =     10,288 hpf/mL of urine
             0.020 mLs (×) 12                         0.24

[7]   To find the number of formed elements/mL urine, determine the average
         number of formed elements observed in 20 fields of view. For example: if
        you determine that there is an average of 4 WBC's/hpf, then calculate as
       follows:

Formula:
   # formed elements/hpf (×) "field conversion factor"  =   # formed elements
                                                                                                        per mL urine.

      4 WBC's/hpf (×) 10,288/hpf    =     41,152 WBC's/mL urine.

2-160        DEFINE A SUPRAVITAL STAIN AND LIST TWO SUCH STAINS, DISADVANTAGES, HOW THEY WORK, AND/OR DEMONSTRATE HOW TO PROPERLY STAIN URINE SEDIMENT.

A supravital stain will stain living tissue by perfusing through the cell and distinguishing cell components. It provides more detailed images of leukocytes, epithelial cells, and casts. The Sternheimer-Malbin stain and the 0.5% toluidine stain are good stains for urine sediment. SM stain has a tendency to precipitate in strongly alkaline urine. If you see crystals in urine to which SM stain has been added, such crystals will be brown or purple stellate crystals.

2-161     DESCRIBE HOW THE LABORATORY CAN DEMONSTRATE FAT/LIPIDS IN URINE.

The polarizing microscope provides an excellent way to demonstrate lipids.  It provides greater contrast of urinary sediment and the presence of cholesterol lipids are characterized by the presence of the Maltese cross.  Neutral fats do not produce the cross phenomenon.  Lipid stains (Sudan III, Sudan IV, and Oil Red 4 are used by labs to stain the neutral fats/triglyceride lipids.  Polarized light or lipid stains may be used to demonstrate the presence of free fat globules, oval fat bodies, and fatty casts.

2-162    EXPLAIN WHY A LABORATORY WOULD USE GRAM STAIN TO STAIN URINE SEDIMENT.

Gram stain is used in microbiology to differentiate bacteria into gram positive or gram negative groups. The gram stain in urinalysis would be for this purpose.

2-163    EXPLAIN THE PURPOSE OF HANSEL’S STAIN AND WHEN A LAB WOULD USE SUCH A STAIN.

Hansel's stain (as is Hinkleman's stain and Manner's stain) is used for the staining of eosinophils.   Eosinophils will appear in the urine in response to hypersensitivity reactions.   If the hypersensitivity is associated with the kidney, there may be an adverse reaction to a drug. The presence of increased numbers of eosinophils is a good indicator that hypersensitive condition is present.   Failure to treat hypersensitivity may result in renal damage.

2-164     EXPLAIN THE PURPOSE OF PRUSSIAN BLUE STAIN.

This stain colors free hemosiderin in urine or that bound in epithelial cells. Prussian blue actually binds with the iron molecule in the form of hemosiderin to produce a distinctive blue color.

2-165    DESCRIBE AND/OR ILLUSTRATE ERYTHROCYTES IN HYPOTONIC, ISOTONIC, AND HYPERTONIC URINE SPECIMENS.

The normal RBC is a round, biconcave disk that appears refractile in urine.  The cell will have a colorless or yellow tinge in unstained urine.  It diameter will be about 7 μ. If viewed from the side it will take on an hour glass appearance and when viewing from the top, there will be a central area of pallor.  RBC's are osmotically sensitive. In hypotonic urine, water will be absorbed into the  RBC (causing it to swell) until it lysis. In isotonic urine, the osmotic forces are equal and the RBC will retain it normal configuration. In hypertonic urine, the osmotic forces will cause fluid to diffuse from the RBC, leaving the cell to shrink and wrinkle.  This cell is called a "crenated cell". See the following illustration.
   
      
2-166     IDENTIFY FOUR ERYTHROCYTE "LOOK-A-LIKES" AND HOW TO DISTINGUISH THEM.

[1]    Yeast cells: Both oval and round form are noted. There is variation in size
          and are characterized by "budding". Yeast cells are resistant to acetic
          acid lysis.
[2]    Oil droplets: May be uniform in appearance, but will vary significantly in
          size.   Neutral lipids will stain with lipid stains.
[3]    Air Bubbles: Will be variable in size and demonstrate dark ring phenomenon.
[4]    Calcium Oxalate: The monohydrate form of calcium oxalate may contain
          oval/round forms. Other (dihydrate) calcium oxalate forms are present
          and easily eliminates this as a "look-a-like).

2-167      DESCRIBE DYSMORPHIC RBC’S AND EXPLAIN THEIR APPEARANCE IN URINE.

Dysmorphic erythrocytes are irregular RBC's with distorted appearances.  Their appearance has been reported as bizarre and consequently their presence may go unreported.  These cells will contain variable amounts of hemoglobin which may be randomly distributed contributing to the appearance of the erythrocyte. The dysmorphic RBC is associated with glomerulonephritis.  The RBC will traverse the length of the nephron, subjected to the osmotic and physical forces, which produces the dysmorphism. In the microscopic, look for irregular cell membranes, ring-like forms, blebs, target cells, buds, and other strange configurations.  See the following illustration.
           

2-168    DESCRIBE WHAT OTHER TEST PARAMETERS THE MICROSCOPIC CAN BE CORRELATED TO WHEN RBC’S ARE REPORTED IN THE SEDIMENT.

[1]     Appearance: should to hazy to cloudy. If <400 RBC/mL, the urine
          specimen will be clear.
[2]     Color: look for pink to red to smokey coloration.
[3]     Sediment button: presence of a red button.
[4]     Reagent strip test:
           a.     blood pad will be positive.
           b.     protein pad may be positive. If bleeding from glomerulus, albumin
                    is being lost from glomerular capillaries.

2-169   DESCRIBE THE APPEARANCE OF RBC’S FROM NON-GLOMERULAR BLEEDING.

Hemoglobin is homogeneously (evenly) distributed. The cells are normal in appearance and crenated contours may be observed. Refer to the illustration in Learning Objective 2-165.

2-170    DISCUSS THE CLINICAL SIGNIFICANCE OF ERYTHROCYTES IN URINE.

Their presence if pathological if greater than 5 RBC/hpf.  Normal values are 2 RBC/hpf which is equivalent to ≤ 12 RBC/μL.   If patient is catheterized or experiencing menses, then RBC's in urine are not significant. Pathological implications may be any of the following: (1) bacterial infection, (2) presence of stones, (3) tumors, (4) trauma, or (5) toxic reactions to drugs/medications.  The increased presence of RBC's in urine is called hematuria.

2-171    DESCRIBE THE TYPE OF LEUKOCYTES AND OTHER PHAGOCYTES THAT MAY BE FOUND IN URINE.

[1]    Neutrophils: The most common WBC encountered. This cell contains a
          multi-lobed nucleus, is similar in size to a renal tubular epithelial cell
          (RTEC), and ranges in size from 10 to 14 μM. These cells produce the
           enzyme esterase that cleaves the ester In the reagent strip test pad.
          They are increased in all inflammatory responses.   Report these cells as
           WBC's or pus cells.
[2]    Lymphocytes: Are seen in urine specimens and should be reported as
          small, mononuclear cells-probably lymphocytes. They do not produce
          esterase and will not affect the reagent strip pad if present in large
          numbers. Large numbers are reported in viral infections and acute
          glomerulonephritis.

[3]    Monocytes: These are similar in size to RTEC's and are easily confused with
          them. These cells can attain diameters up to 40 μM and are easily identified
          if differentially stained. Report their presence as large, mononuclear cells-
          probably monocytes.
[4]    Eosinophils: These cells are difficult to distinguish from neutrophils. Unless
         differentiated with Wright's stain, report these cells as you would
         neutrophils.   Eosinophils are increased in acute interstitial nephritis,
         allergies, and acute allograph rejection.
[5]    Macrophages: Also called histocytes, these cells range usually ranges from
         30 to 40 μM, but may attain diameters of 100 μM. The nucleus may be
         round, irregular, or kidney shaped. There is an abundance of cytoplasm and
         vacuoles are present.   When seen in urine, they are in most cases, spherical.
         Reporting these cells as macrophages.

2-172    DISCUSS THE NEUTROPHIL AS THE TYPICAL WBC IN URINE.

This cell should be referred to as a white blood cell, leukocyte, or pus cell. In a fresh urine specimen, neutrophils will display neutrophil features and may exhibit ameboid movement. If nuclear features are important to discern, add a drop of dilute acetic acid (≤3%).

As neutrophils "age", disintegration quickly set in and the cell becomes increasingly granular in appearance. The nuclei will fuse and the cell appear as a mononuclear cell. When this occurs, it is easily confused with a renal tubular epithelial cell (RTEC). Blebs may appear on the inner cell membrane, the detach from the cell and float free. As the cells continue disintegrate, filaments form and extend outward from the membrane surface. When this occurs the cell membrane breaks down and the cell ruptures.   Note the following illustration.
   
In dilute urine, at room temperature, the WBC will swell to form spheres, then lysis occurs. Within 2-3 hours, up to 50% of the WBC's will have lysed. During the time that the WBC is in its swollen state, cytoplasmic granules exhibit Brownian movement and are refractive. When this occurs, the cell is called a "glitter cell". Such cells have no clinical/pathological significance. When stained with Sternheimer-Malbin stain, the "glitter cell" will appear pale blue. Glitter cells are usually seen in urines with a specific gravity less than 1.019. See above illustraton.

Old" leukocytes tend to be smaller in size, nuclear features are more indistinct, increased granulation, and in differing states of disintegration. "Fresh" leukocytes appear larger, distinct nuclear features, abundant cytoplasmic granules, and absence of disintegration.  
        NOTE: A normal and healthy urine does not "shed" leukocytes.

2-173     COMPARE AND CORRELATE THE URINE MICROSCOPIC FINDINGS OF LEUKOCYTES TO PHYSICAL AND CHEMICAL PARAMETERS.

[1]     Appearance will be hazy to cloudy if ≥400 WBC/mm3.
[2]     Odor may be strong, pungent, or foul.
[3]     A grey button will appear in the bottom of the centrifuge tube in pyuria.
[4]     Clumping will be a characteristic feature in pyuria.
[5]     Leukocyte esterase test = positive ( WBC ≥100,000/mm³)
[6]     Nitrite test = positive (if infective bacteria is a nitrate reducer).
[7]     Blood test = positive (if lesions are present due to infection).
[8]     If there are many WBC’s present, look for WBC casts.

2-174    DESCRIBE THE SQUAMOUS EPITHELIAL CELL OBSERVED IN URINE.

This is the most frequently found epithelial cell in urine and has the least clinical significance. It is derived from the vagina, prepuce of uncircumcised men, and urethra. The squamous cell is characterized by an abundance of cytoplasm and a small eccentric nucleus (which is about the size of a RBC). The edges of the cell can roll and fold, causing the cell to present in a variety of configurations. The cytoplasm may appear finely granular and also have a few large granules scattered throughout it. It diameter ranges from 40 to 60 μM. As the cell deteriorates, granulation becomes more prevalent. It is easy to identify and should be enumerated with 10X objective. If large numbers of squamous cells are noted in a urine specimen from a female, this is an indicator of vaginal contamination. Normal amounts of sloughing will be characterized by ≤6 cells/lpf. When examining the squamous cell, the cell for any aberrant features in the cytoplasm, cell, or nucleus. Comparing the cytoplasm and nuclear ratios will help to identify the squamous cell, being ≥ 12 to 1. Note the following illustration.
                       

2-175    DESCRIBE THE TRANSITIONAL EPITHELIAL CELL OBSERVED IN URINE.

Also called urothelial or bladder cells, these cells originate from the bladder, urethra of males, ureters, renal pelvis, and the major and minor calyces. The size is variable dependent upon which later the cell sloughed from. Cells from the outer, superficial layer will be larger and flatter, with a diameter ranging from 30 to 40 μM, whereas cells from the inner, intermediate layers are smaller and plumper have a diameter of 20 to 30 μM. Transitional cells from the bladder may be larger and closely resemble squamous cells. Review the following illustrations.
            

To recognize the transitional cell;
[1]     look for larger cytoplasmic granules that tend to accumulate around the
          nucleus.   This is called nuclear distribution.
[2]     increased number of inclusions are the rule.
[3]     look for distinct peripheral borders on the cytoplasm and nucleus.
[4]     shape variations are as follows:
  
[5]     The nucleus will be eccentric, round or oval, and about the size of a small
           WBC.
[6]     It is not abnormal to find cells with two nuclei.
[7]     they have the tendency to absorb water and this will alter their appearance.
[8]     there will be about 6 to 7 times more cytoplasmic area than nuclear area.

2-176    DISCUSS THE CLINICAL SIGNIFICANCE OF TRANSITIONAL CELLS.

Usually there are no clinical significance. In urinary tract infections (UTI) increased numbers are sometimes encountered. If an increased number of transitional cells are noted, take time to review the morphology of the cells. If abnormal morphology is noted, the specimen should be referred to a pathologist for cytologic review. If the cell originates from the trigone of the bladder or the renal pelvis, there will often be a tail-like extension. These cells are called caudate cells and are not diagnostic of anything.   Refer to the previous learning objective (2-175)

2-177    DESCRIBE A "DECOY" CELL AND ITS SIGNIFICANCE.

The "decoy" cell is a transitional cell that is thought to be infected with polyoma virus. This cell is so called because it displays unique staining phenomenon in Sternheimer-Malbin stain. The cytoplasm stains red and the nucleus is irregular, very large, vacuolated, and has dense peripheral chromatin. It is called a pseudo-malignant cell.  Examine the following illustration.
                               
                   
2-178    DESCRIBE "MULTI-NUCLEATED GIANT" CELLS AND THEIR SIGNIFICANCE.

This is a cell generally thought to be derived from the superficial layers of the renal pelvis and ureters. It is characterized with a vacuolated cytoplasm, multi-nucleated (with up to 1 00 nuclei reported), nucleoli present, and diameters up to 300 μM. Cell shapes vary from round to polygonal. The cells are observed in heavy metal poisoning, inflammation, and trauma. They have been reported in normal urine (voided and catheterized).  Examine the following illustrations.
       
   
2-179    DESCRIBE "HISTOCYTES" AND THEIR SIGNIFICANCE.

A large phagocytic cell that is usually seen in inflammatory conditions (glomerulopathy) . The cell is larger than WBC's with a foamy appearing cytoplasm containing distinct vacuoles.  The cytoplasmic borders are often poorly defined. The round to bean-shaped nucleus is eccentrically placed in the cell. It is not unusual to observe multinucleated histocytes.  Refer to the following illustrations.
       
2-180    DESCRIBE "CLUE CELLS" AND THEIR SIGNIFICANCE.

Clue cells are squamous epithelial cells of vaginal origin. They are unique in that they are coated with the coccobacilli (Gardnerella vaginalis). Their presence is an indicator of a vaginal infection. Look for squamous cells that have a granular appearance with shaggy borders. Bacteria may be lightly or densely "crusted" across the squamous cells and will even extend across the cytoplasmic margins. Be cautious because some squamous cells will contain irregular keratohyaline granules which makes them a "clue cell look-a-like". See the following illustration:
           

2-181   DESCRIBE AN “UMBRELLA" CELL AND ITS SIGNIFICANCE.

Umbrella cells are best observed with Sternheimer-Malbin stain. Look for concave cells with foamy cytoplasm. These are multi-nucleated, sloughed-off disintegrating cells from the renal pelvis, ureters, and bladder. It is the largest of the transitional cells and it is clinically insignificant. Examine the following illustration:
            

2-182     DESCRIBE RENAL TUBULAR EPITHELIAL CELLS (RTEC).

The RTEC is the most significant of the epithelial cells. They can originate from any part of the nephron and collecting duct. Most RTEC's appear in urine sediment as a consequence of natural process of cell replacement. These cells can be differentiated by size and shape. Since it is difficult to differentiate these cells into proximal, distal, and collecting duct types, most laboratories, these cells are nor differentiated into their three categories, but simply referred to as RTEC's. To differentiate, slides should be made from the sediment and stained with Wright's stain or other good cellular stain. Actually there is no real clinical advantage in urinary screening procedures to categorize into different types. Since some laboratories do differentiate between RTEC's, they will be reviewed in this course. RTEC's are significant only if their numbers are significantly increased. Based on the studies of G. B. Schumann, if ≥15 RTEC's are observed per high power field, this is a strong indication for renal pathology. The specimen should then be re-evaluated according to lab policy.

General morphology describes this cell as being round to oblong in shape with a size that is about 1½ to two or three times larger than leukocytes. Diameters will be as up to 25 μM. A dense, round, often eccentric nucleus is typical. The cytoplasm is granular. These cells are not prone to absorb water therefore do not swell and change shapes as do transitional cells. They tend to retain their original shape. See Figures 15 and 16.
Refer to the following illustrations:
          

2-183     DESCRIBE AND/OR DIFFERENTIATE BETWEEN PROXIMAL CONVOLUTED TUBULE CELLS, DISTAL CONVOLUTED TUBULES CELLS, AND COLLECTING DUCT CELLS.

Proximal convoluted tubule cells. These are elongated or oval and polyhedral in shape with a granular cytoplasm. The nucleus tends to be eccentric, dense, and small. Diameters range from 20 to 60 μM.  Multinucleated forms are not unusual nor abnormal. One side of the cell is usually (but not always) flattened. They are reported to be elongated in form and when unstained, may appear to resemble a small granular cast.

Distal convoluted tubule cells.  They resemble the proximal convoluted tubule cell, but tend to be smaller. Diameters range from 14 to 25 μM.

Collecting tubule cells. These cells are either columnar, cuboidal or polygonal; but never round. To identify this cell, look for straight sides and corners. This cell is characterized by a single, large nucleus that may occupy as much as 2/3  of the cytoplasm. The smaller of the collecting tubule cells range from 12 to 20 μM and become larger as and more columnar as the tubule approaches its minor calyx.   Note the following illustration:
               

2-184    DISCUSS THE CLINICAL SIGNIFICANCE OF THE RENAL EPITHELIAL CELLS WHEN OBSERVED IN INCREASE NUMBERS.

Increased in toxic renal tubular damage due to heavy metal poisons (lead, mercury, etc.), drugs (cytotoxins), graft rejection, pyelonephritis (sepsis), nephrosclerosis, trauma, shock, ischemic necrosis, or any renal disorder characterized by heavy proteinuria.

2-185   DESCRIBE THE COURSE OF ACTION WHEN COLUMNAR TUBULAR CELLS ARE PRESENT AND A RENAL PATHOLOGY MAY BE SUSPECT.

Evaluate the microscopic sediment for the presence of RBC's, granular casts, RTEC casts, and waxy casts. Retest for protein using the sulfosalicylic acid procedure if the strip test is negative.

2-186   DESCRIBE AND EXPLAIN THE SIGNIFICANCE OF OVAL FAT BODIES.

Oval fat bodies (OFB) are renal tubular cells that contain lipids (refer to the following illustration).  These cells are formed when the intracellular lipids degenerate and coalesce into lipid globules.  If lipids appear in the glomerular filtrate due to glomerular dysfunction and plasma leakage, they will be readily absorbed by the renal tubular cells. Lipids, as the coalesce in the RTEC's, will form variable size droplets and demonstrate high refractile properties. They are more easily recognized with the brightfield microscope than the phase microscope. Under low power, they will resemble brownish spheres.  The phase microscope provides a positive identification if the fat globules (with cholesterol and cholesterol esters present) demonstrates the maltose cross effect. If the fat globules contains only neutral fats, then verification must be by a fat stain (Sudan III or Oil Red 4).  The presence of OFB are pathologically significant and are to be reported out in numbers per high power field. Their presence may suggest any of the following: (1) trauma with release of bone marrow fat, (2) lipid storage disease (Fabry's disease, Gaucher's disease, Niemann-Pick disease, etc.), (3) toxemia of pregnancy, (4) diabetes mellitus, (5) pyelonephritis, (6) polycystic kidney disease, (7) nephrotic syndrome, and (8) congestive heart failure. When you observe OFB in urine sediment, evaluate the sediment for free floating fat globules. Also look for casts and re-check the protein test (which should be positive).
   

2-187    DISCUSS THE SIGNIFICANCE OF FINDING INCLUSION BODIES IN RENAL TUBULAR EPITHELIAL CELLS.

Renal tubular epithelial cells that contain inclusion bodies are difficult to see and are easily missed unless the sediment is stained. Most inclusions are the result of a viral infection, usually the herpes or rubella (German measles). Other inclusions may be the result of heavy metal poisoning. An infection with cytomegalovirus (a herpes virus) causes inclusions in the nucleus.

2-188     DESCRIBE CASTS AND HOW THEY ARE FORMED.

Casts are elements of solidified protein that may or may not contain inclusions that are found in both normal and abnormal urine. The distal convoluted tubules and collecting tubules secrete a muco-protein (Tamm-Horsfall protein) that appears first in the form of fibrils. These fibrils stick to the lumen walls and as more protein fibrils are secreted, an interweaving occurs and the cast takes forms. Cast formation is augmented if plasma proteins are present, solutes are increased, the pH is acidic, and filtrate flow through the lumen is slow. Casts are formed in the tubules and conform to the shape and structure of the tubule. The cast can undergo changes in the tubule and undergo transitional changes. Casts have parallel sides but tend to be thicker in the middle and more slender toward the ends. Casts can be long, short, thin, thick, convoluted, curved, or straight. The cast can be fragile (easily broken) or resilient (resistant to breaking).

2-189    EXPLAIN HOW pH, SOLUTE CONCENTRATION, AND URINARY STASIS IN THE NEPHRON CONTRIBUTE TO CAST FORMATION.

(1)    An acidic environment contributes to solute and protein precipitation,
         which favors cast formation. This occurs most often in the distal and
         collecting tubules.
(2)    Solute concentration.... favors crystal precipitation and protein precipitation.
(3)    Urinary stasis... usually occurs for some type of pathological disease or
          obstruction or congenital abnormality. Stasis facilitates accumulation and
          concentration of substances that contribute to cast formation. The
          presence of plasma proteins (albumin and/or globulins), hemoglobin, or
          myoglobin enhances cast formation.

2-190     DISCUSS THE CLASSIFICATION OF CASTS.

Casts are classified according to their matrix and the "stuff" (inclusions) observed in the matrix. Inclusions includes: bacteria, leukocytes, erythrocytes, renal tubular epithelial cells, lipids, granules, hemosiderin, and crystals. Casts may be classified as follows:
(a)    Homogeneous/non-inclusion:   hyaline, waxy.
(b)    Inclusion/cellular:   leukocytes, erythrocytes, renal tubular epithelial cells,
          bacteria.
(c)    Inclusion/non-cellular:   lipids, granules, hemosiderin, crystals.
(d)    Pigmented:   bilirubin, hemoglobin, myoglobin, drug pigments.
Note:  Another category of casts are the broad casts. These casts are
              extra-large and can be classified as identified in (a) through (d).

2-191    DESCRIBE "FALSE" CASTS AND HOW TO RECOGNIZE THEM.

This is a phenomenon that occurs during an "alkaline tide" which facilitates the precipitation of amorphous phosphates.  The precipitate tends to form in slender, cylindrical appearing arrangements. To differentiate from true casts, look for the absence of a protein matrix border.  A true cast will present a hyaline-like matrix. Examine the surround area. If this is a pseudo-cast, there will also be indiscriminate masses of precipitate scattered about.  False-casts can also include aggregates of groups of bacteria, cells, or crystals. Remember to be a true cast, the cells or “whatever” must be embedded in a mucoprotein matrix.

2-192    DISTINGUISH BETWEEN "NARROW" AND "BROAD" CASTS AND BRIEFLY EXPLAIN THE SIGNIFICANCE OF THEIR APPEARANCE IN URINE SEDIMENT.

Most casts tend to have a diameter that similar, however the length may vary. Narrow casts usually form during an inflammatory process in which the tubules are swollen, causing the lumen to be more narrow. Most casts reported out as narrow casts are hyaline and the prognosis is less serious. Broad casts are formed in dilated renal tubules or the collecting tubules. These are the more pathological of the casts and indicate a severe renal problem. Broad casts tend to form in chronic renal disorders or obstruction which produces stasis. Most broad casts that are reported are the waxy type. Broad casts are also called "renal failure" casts.

2-193     DISCUSS THE HYALINE CAST, HOW TO RECOGNIZE IT, AND ITS CLINICAL SIGNIFICANCE.

This is the most commonly observed cast and has the least clinical significance. It consists of a congealed mass of Tamm-Horsfall protein and may contain no or a few inclusions. The refractive index is low and may be easily overlooked if in low numbers. These casts tend have rounded ends and present with a variety of sizes and shapes. Staining with Sternheimer-Malbin stains enhances their visualization. They take on a pink coloration and borders are more distinct. Numbers are increased during severe exercise, dehydration, heat exposure, and stress. They are also observed to accompany the pathological casts during a variety of renal disease, congestive heart failure, and febrile illnesses. Normal values reported by most laboratories are 0-2/lpf. When identifying the hyaline cast, do not confuse it with mucus threads. It is not uncommon see these casts containing very fine granules, in which case, they are still reported out as hyaline casts. Refer to the following illustrations.
           

2-194    DESCRIBE CYLINDEROIDS.

Cylinderoids are hyaline casts, except that one end (as the rule) will be tapering or serpentine in appearance. Cylinderoids are to be reported out as hyaline casts.  Refer to the illustration of hyaline casts in previous learning objective.

2-195    DISCUSS THE STABILITY AND SOLUBILITY OF CASTS.

The Tamm-Horsfall protein matrix is stable in a acidic urine with an elevated specific gravity (≥1.010). The cast matrix is soluble in water and the solubility increases as the pH increases. If the kidney's fail to concentrate the urine and hold an acidic pH, casts may not form. Casts tend to be fragile, breaking easily. Casts tend to disintegrate in urine that sits out at room temperature over a period of time.

2-196    DISCUSS THE RBC CAST AND IT CLINICAL SIGNIFICANCE.

The presence of these casts are always pathological. These casts form when the RBC becomes trapped in the Tamm-Horsfall protein matrix in a random manner, not in rows or columns. If there is glomerular damage with bleeding, the plasma proteins and fibrinogen will contribute to the formation of the cast's matrix. These casts form whenever any disorder damages the glomerulus, nephron, or parenchyma tissue of the kidney. These casts are very fragile and urine should be fresh and carefully handled to increase the success of finding these casts. Since this cast is not stable, the RBC's will quickly deteriorate and the cast becomes unrecognizable as a RBC cast. It is then referred to as a blood cast. As erythrocytes lyse in the cast, the cast takes on a more homogeneous appearance with little color variation. RBC casts are refractile and have a color ranging from yellow to red-brown. Look for intact and clearly identifiable erythrocytes. Note the margin of the cast, the edge of the hyaline matrix should be seen about the cast. If you see 2 or 3 RBC's in a cast, it is acceptable to call it a RBC cast. If RBC casts are present in the sediment, examine the "neighborhood", free erythrocytes should also be present which helps in the identification of the RBC cast. RBC casts are observed in (1) glomerulonephritis, (2) Wegner's granulomatosis, (3) polyarthritis, (4) sickle cell anemia, (5) sub-acute bacterial endocarditis, (6) systemic lupus erythematosus, (7) renal infarction, and (8) after strenuous physical exertion. RBC casts should be reported as number you see per lpf. A rare RBC cast may be observed in normal urine. When RBC casts are observed in urine sediment, it is probable that the blood values for uric acid, blood urea nitrogen (BUN), and creatinine will be increased. If RBC casts are present, the strip reagent test pads should be positive for blood and possibly protein.  Refer to the following illustrations of erythrocyte casts.
           

2-197     DISCUSS THE WBC CAST AND ITS CLINICAL SIGNIFICANCE.

The presence of leukocyte casts indicate the presence of a renal inflammation or infection. Specific disorders are (1) pyelonephritis, (2) post streptococcal acute glomerulonephritis, (3) nephrotic syndrome, (4) systemic lupus erythematosus, and (5) polyarteritis nodosa. These casts should be reported as number per lpf. Synonyms are leukocyte cast or pus cast. A positive leukocytes strip test and protein help verify the presence of these casts. If a bacterial infection is present, the nitrate test should be positive. Staining the sediment helps to visualize the nuclear detail in the leukocytes in the cast. The consensus among laboratorians is that casts with leukocytes scattered throughout the cast matrix seldom occurs. It is thought that the WBC's adhere to the outside of a hyaline cast to form a WBC cast by means of it proteinaceous fibrils. WBC's attach to the cast matrix in a random manner and do nor form rows or columns. If WBC casts are present, it might be appropriate to recommend a culture and sensitivity. Caution: do not confuse WBC casts with RTEC casts.   Refer to the following examples of WBC casts.
                 

2-198     DISCUSS THE RTEC CAST AND ITS CLINICAL SIGNIFICANCE.

Renal tubular epithelial casts (RTEC) are formed when RTEC's are damaged and sloughed into the lumen of the nephron. These cells can be incorporated into the matrix of the cast or they can adhere to the surface of the hyaline matrix. Since RTEC's slough, they are likely to do so in sheets, and in the cast will appear as rows and lines. Renal tubular casts are found in viral diseases (cytomegalovirus or hepatitis), nephrotoxins (heavy metals, salicylate toxicity), glomerulonephropathies, lipoid necrosis, and renal parenchymal disease.  The RTEC disintegrates rapidly which may result in the laboratorian miscalling the cast as a granular cast. These casts have been confused with WBC casts.  Report RTEC casts as number per lpf.    Note the following examples of RETC casts.
           

2-199    DISCUSS THE HEMOGLOBIN CAST, HOW TO DISTINGUISH IT, AND ITS CLINICAL SIGNIFICANCE.

Hemoglobin casts have the same clinical significance as RBC casts and appear in urine under the same causes. See Objective #189. It is generally conceded that hemoglobin casts are formed by the breakdown of RBC casts. The three additional conditions that contribute to hemoglobin cast formation are transfusion reactions, Clostridium bacteremia, and hemolytic anemia. Hemoglobin casts should not be confused with pigmented casts (See Objective #205). An examination of the "neighborhood" helps to distinguish pigmented casts. The pigment that stains the cast also stains the cells and other elements around the cast. Staining the sediment with Puchtler method (Buffalo black stain) causes hemoglobin casts to turn dark blue and Ralph's method (a nuclear fast red stain) will turn hemoglobin casts brown. If the reagent test pad for blood is positive and RBC’s are observed in the sediment, look for hemoglobin and RBC casts.

Revised for spring 2006

This web site is maintained by Whitney Williams, wwilliam@astate.edu

This page last updated 07/28/08