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Bilirubin Information and Courses from MediaLab, Inc.

These are the MediaLab courses that cover Bilirubin and links to relevant pages within the course.

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Alpha Thalassemia
Serum Chemistry Results

The following chemistry test results were obtained on the same patient:TestResultsReference RangesSerum iron250 g/dL26 -170 g/dLIron binding capacity130 g/dL250 - 400 g/dLBilirubin (unconjugated)2.6 mg/dL0.2 - 1.0 mg/dLLactate dehydrogenase320 U/L100 - 190 U/LHaptoglobin52 mg/dL40 - 330 mg/dL

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Summary

The normal RBC count (4.84 x 1012/L) in this case, together with the decreased hemoglobin (8.4 g/dL) and MCV (59 fl) is an indicator of ineffective erythropoeisis that often points to thalassemia.The RBC morphology shows slight hypochromic microcytosis with codocytes, schistocytes, and basophilic stippling. Schistocytes form by several mechanisms, one being the removal of RBC inclusions.This patient's elevated bilirubin correlates with her presentation of sclera icterus; her splenomegaly is consistent with increased RBC destruction.The Hb electrophoresis demonstrated a normal pattern, initially, but the unstable Hemoglobin H was revealed upon repeat electrophoresis with reduced incubation time. Hemoglobin H is the result of beta globin chain tetramer formation due to the insufficient supply of alpha globin chains in alpha thalassemia intermedia.People with Hemoglobin H disease (alpha thalassemia intermedia) usually have a normal life expectancy without treatment. However, hemolysis may lead to moderate anemia that may be treated with splenectomy.

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Serum Bilirubin

Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of bilirubin in the serum of adults is 0.2-1mg/dl. Bilirubin levels increase with liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dl.Persons with alpha thalassemia intermedia usually have an increased bilirubin level, because of ongoing hemolysis. This bilirubin is typically the unconjugated fraction of bilirubin.

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Authentic and Spurious Causes of Thrombocytopenia
Laboratory Findings

These laboratory findings are associated with TTP and HUS:Thrombocytopenia -- Platelet count is often less than 20 x 109/L in TTP, but may not be as low in HUS. Schistocytes (red blood cell fragments, as indicated by the arrows in the image to the right) may be observed on the peripheral blood smear. Schistocytes are the result of erythrocytic membrane damage caused by sheering of red blood cells as they pass through a fibrin mesh of clot formation occurring in the blood vessels. LDH, serum bilirubin, and reticulocyte counts are elevated. Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are usually normal. Proteinuria and hematuria may be present.

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Beta Thalassemia
Serum Bilirubin

Bilirubin is formed as a result of hemoglobin degradation. Normally, senescent red blood cells are removed from circulation and the bilirubin that is formed is processed by the liver. The normal level of serum bilirubin for adults is 0.2-1mg/dL.Bilirubin levels increase with some liver disorders and also in anemia that is a result of a hemolytic process. Patients may display jaundice when serum bilirubin levels exceed 2mg/dL.Persons with beta thalassemia major usually have an increased bilirubin level. This bilirubin is typically the unconjugated fraction of bilirubin.

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Cerebrospinal Fluid (retired 7/17/2012)
An Example of Xanthochromia

Two to four hours after a subarachnoid hemorrhage, the supernatant of a CSF sample will be pale pink to pale orange. The source of this color is oxyhemoglobin from lysed red cells present in the CSF before the puncture. Xanthochromia from the lysed red cells reaches its peak 24 - 36 hours after the hemorrhage and gradually disappears after four to eight days. In the same type of hemorrhage, after 12 hours yellow xanthochromia begins to appear due to the presence of bilirubin. The bilirubin is the breakdown product of oxyhemoglobin from the original lysed red cells. The yellow color in the supernatant reaches its peak in about two to four days and disappears after two to four weeks.

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Other Causes of Xanthochromia

Examples of sources of pigment other than oxyhemoglobin and bilirubin that can cause xanthochromia include: methemoglobinincreased CSF protein (> 150 mg/dL)contamination by skin antiseptic (iodine or merthiolate)

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Causes of Xanthochromia in Premature Infants

Xanthochromia may also be present in the cerebrospinal fluid of premature infants. Reasons for this include: elevated bilirubin in the bloodimmaturity of the blood-brain barrierelevated protein in CSF

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Chemical Screening of Urine by Reagent Strip
Chemical Urinalysis Reagent Strips

A chemical urinaylsis reagent strip, also called a dipstick, for screening urine is a narrow band of paper which has been saturated with chemical indicators for specific substances or properties. Depending on the product being used, chemical urinalysis reagent strips may include test indicators for glucose, bilirubin, ketones, specific gravity, blood, pH, protein, urobilinogen, nitrite, and leukocyte esterase. The results obtained from urine screening using chemical urinalysis strips can indicate the patient's carbohydrate metabolism status, kidney and liver function, urinary tract infection, and acid-base balance. Most chemical urinalysis reagent strips can be read visually and do not require instrumentation for automatic reading, though many laboratories utilize instruments for this purpose. When performing chemical urinalysis reagent strip analysis, the directions must be performed exactly. Accurate timing is paramount in order to achieve appropriate and optimal results. In addition, the reagent strips must be stored properly in their containers with the lid tightly closed to maintain reagent reactivity. It is always essential to utilize well-mixed urine which has been collected within 2 hours of analysis.Always read the package insert for your particular brand of chemical urinalysis reagent strip, as each manufacturer may have slightly different instructions and interpretations.

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Precautions in Urinalysis Chemical Reagent Strip Analysis

The following precautions should be observed when working with urinalysis reagent strips: Store strips according to the manufacturer's recommendation.DO NOT expose strips to moisture, volatile fumes, or direct sunlight. Remove only enough strips for immediate use and immediately recap the bottle.Avoid contamination of test strips. Do not touch the test areas with fingers or do not lay the test strips directly on the workbench.DO NOT use discolored strips. Compare the color of the unused strip to the negative area on the color chart provided by the company. The color should be similar.Check the expiration date. Re-label the container with a revised expiration date if the manufacturer states a shortened usage period once the container has been opened.Procedural PrecautionsAlthough the procedure is simple to perform, accurate results depend on careful adherence to manufacturer's directions and adequate quality control.Normal and abnormal controls should be tested whenever a new lot of strips is opened, and at the frequency defined by the laboratory's procedure.If quality control results do not correspond to the published control values, the problem must be resolved before patient samples are tested.High levels of ascorbic acid (Vitamin C) in the urine may inhibit some reagent strip reactions, such as glucose, blood, bilirubin, nitrate and leukocyte esterase. The urine dipstick's package insert will provide information about potential interfering substances, including ascorbic acid.Intensely colored urine may make it difficult to correctly interpret color reactions on the dipstick, as illustrated below. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

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Overview of Bilirubin

Bilirubin, a product of hemoglobin catabolism, is characterized by its distinctive yellow pigment. The presence of bilirubin in urine is always abnormal. In most healthy individuals the amount of conjugated bilirubin excreted is not detected by the strips. In cases when bilirubin is elevated and is conjugated, it will be detected by the test strip.It is important to note that unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water. In the liver, bilirubin combines with glucuronic acid through the action of a glucuronyl transferase to form water soluble bilirubin diglucuronide. Under normal circumstances, conjugated bilirubin passes from the bile duct and then to the intestinal tract. Intestinal bacteria reduce conjugated bilirubin to urobilinogen. Approximately half of the urobilinogen is excreted in the feces; most of the other half is recirculated through the liver. A small amount of urobilinogen bypasses the liver and is excreted in the urine.

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Testing for Urine Bilirubin

The test for bilirubin on the urine chemical reagent strip is based on the formation of an azobilirubin compound resulting from a reaction of bilirubin in an acid medium with diazotized 2, 4 dichloroaniline. The color of this compound ranges through various shades of tan. Some sources describe the colors produced as shades of tan-to-pink-to-violet.Since other pigments in the urine may influence the test results, this test strip is more difficult to interpret than the others. Colors which are unlike either the positive or negative color blocks on the color chart may be due to the presence of bilirubin -derived bile pigments. Any urine which demonstrates an atypical color on the bilirubin test strip should be tested further. Even a slight change in color should be considered significant since bilirubin is never present in normal urine.

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False Positive and Negative Urine Bilirubin Results

False Positive BilirubinFalse positive results may occur when patients are on large doses of chloropromazine, and may occur in the presence of metabolites of phenazopyridine. When these compounds are present, the urine becomes red. Metabolites of Lodine® (etodolac) may cause false positive or atypical results. False Negative BilirubinFalse negative bilirubin dipstick results are often due to testing a specimen that is not fresh. Bilirubin breaks down when exposed to light. Indoxyl sulfate (Indican) can produce a yellow orange-to-red color response which may interfere with the interpretation of a positive or negative reaction. Positive nitrites due to a urinary tract infection may also cause a false negative result.

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Confirmatory Testing for Urine Bilirubin

Confirmatory testing using an alternative method, such as Ictotest® reagent tablets, can be performed when positive results are seen on the dipstick strip, when a red color forms on the strip, or when atypical color changes occur that are the result of bilirubin-derived bile pigments in the urine masking the bilirubin reaction. The Ictotest® employs the same diazotization reaction as the reagent strip, but should not give a false positive result with colored urines.

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Clinical Significance of Urine Bilirubin

Liver damage or an obstructed bile duct allows conjugated bilirubin to enter the circulation and ultimately to appear in the urine. Patients with clinical jaundice due to hepatitis or cirrhosis will have bilirubinuria. If the jaundice is due to red cell destruction, there is an increase in unconjugated bilirubin which the kidneys cannot excrete.

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Which of the following may cause a false positive bilirubin result on a urine chemical reagent strip?View Page
When a patient has a bile duct obstruction, the bilirubin test portion of the reagent strip is:View Page
Introduction to Urobilinogen

Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal levels of urobilinogen and the patient should be evaluated further. It is important to note that the chemical reagent strip cannot determine the absence of urobilinogen, so a negative result is impossible.

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Clinical Significance of Urobilinogen in Urine

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction, as shown in the table below:ConditionUrine Bilirubin ResultUrine Urobilinogen ResultHemolytic diseaseNegativeIncreasedHepatitic diseasePositive or negativeIncreasedBiliary obstructionPositiveNormal* *Urine chemical reagent strip methods cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

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In hemolytic disease, the urine bilirubin test result is negative and the urine urobilinogen test result is:View Page
To screen for urinary tract infections, leukocyte esterase results should be evaluated along with the results from which of these other reagent strip tests?View Page

Chemical Screening of Urine by Reagent Strip (retired March 2012)
Which of the following tests included on a urine reagent strip would never be reported out as negative?View Page
Excessive carbohydrate loss that may occur due to vomiting, or rapid weight loss may result in the presence of which of following substances not normally contained in the urine?View Page
Match the following reagent strip tests to the disease or disorder that would most likely cause a positive test result.View Page
Sulfosalicylic acid can be used to confirm the result of which of the following tests included on a urine reagent strip?View Page
Procedural Considerations

Although the procedure is simple to perform, accurate results depend on careful adherence to manufacturer's directions and adequate quality control. Normal and abnormal controls should be tested whenever a new lot of strips is opened, and at the frequency defined by the laboratory's procedure. If quality control results do not correspond to the published control values, the problem must be resolved before patient samples are tested. High levels of ascorbic acid (Vitamin C) in the urine may inhibit some reagent strip reactions, such as glucose, blood, bilirubin, nitrate and leukocyte esterase. The urine dipstick's package insert will provide information about potential interfering substances, including ascorbic acid. Intensely colored urine may make it difficult to correctly interpret color reactions on the dipstick, as demonstrated in the image on the right. The affected tests should not be reported from the dipstick. It would be necessary to use an alternative method of testing if available.

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Bilirubin Characterization

Bilirubin, a product of hemoglobin breakdown, is characterized by its yellow pigment. The presence of bilirubin in urine is always abnormal. It is important to note that unconjugated bilirubin cannot be excreted by the kidneys because it is bound to albumin and is not soluble in water. In the liver, bilirubin combines with glucuronic acid through the action of a glucuronyl transferase to form water soluble bilirubin diglucuronide. Under normal circumstances, conjugated bilirubin passes from the bile duct and then to the intestinal tract. Intestinal bacteria reduce conjugated bilirubin to urobilinogen. Approximately half of the urobilinogen is excreted in the feces; most of the other half is recirculated through the liver. A small amount of urobilinogen bypasses the liver and is excreted in the urine.

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Test for Bilirubin

The test for bilirubin on the urine reagent strip is based on the formation of an azobilirubin compound resulting from a reaction of bilirubin in an acid medium with diazotized 2, 4 dichloroaniline. The color of this compound ranges through various shades of tan. Some sources describe the colors produced as shades of tan-to-pink-to-violet. Since other pigments in the urine may influence the test results, this test strip is more difficult to interpret than the others. Colors which are unlike either the positive or negative color blocks on the color chart may be due to the presence of bilirubin -derived bile pigments. Any urine which demonstrates an atypical color on the bilirubin test strip should be tested further. Even a slight change in color should be considered significant since bilirubin is never present in normal urine.

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False Negative Results

False negative bilirubin dipstick results are often due to testing a specimen that is not fresh. Bilirubin breaks down when exposed to light. Indoxyl sulfate (Indican) can produce a yellow orange-to-red color response which may interfere with the interpretation of a positive or negative reaction. Positive nitrites due to a urinary tract infection may also cause a false negative result.

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Confirmatory Testing

Confirmatory testing using an alternative method, such as Ictotest reagent tablets, can be performed when positive results are seen on the dipstick strip, when a red color forms on the strip, or when atypical color changes occur that are the result of bilirubin-derived bile pigments in the urine masking the bilirubin reaction.

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Clinical Significance

Liver damage or an obstructed bile duct allows conjugated bilirubin to enter the circulation and ultimately to appear in the urine. Patients with clinical jaundice due to hepatitis or cirrhosis will have bilirubinuria. If the jaundice is due to red cell destruction, there is an increase in unconjugated bilirubin which the kidneys cannot excrete.

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Diazotized 2, 4, dichloroaniline reacts with bilirubin in an ___________ medium.View Page
Which of the following may cause a false positive bilirubin result on a urine reagent strip?View Page
Which of the following may cause false negative bilirubin results on a urine reagent strip? (Choose ALL of the correct answers)View Page
When a patient has a bile duct obstruction, the bilirubin test portion of the reagent strip is:View Page
Urobilinogen

Urobilinogen is a byproduct of hemoglobin breakdown. It is produced in the intestinal tract as a result of the action of bacteria on bilirubin. Almost half of the urobilinogen produced recirculates through the liver and then returns to the intestines through the bile duct. Urobilinogen is then excreted in the feces where it is converted to urobilin. As the urobilinogen circulates in the blood to the liver, a portion of it is diverted to the kidneys and appears as urinary urobilinogen. Up to 1 mg/dL or Ehrlich unit of urobilinogen is present in normal urine. A result of 2.0 mg/dL represents the transition from normal to abnormal and the patient should be evaluated further. It is important to note that the reagent strip cannot determine the absence of urobilinogen.

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Clinical Significance

Urinary urobilinogen may be increased in the presence of a hemolytic process such as hemolytic anemia. It may also be increased with infectious hepatitis, or with cirrhosis. Comparing the urinary bilirubin result with the urobilinogen result may assist in distinguishing between red cell hemolysis, hepatic disease, and biliary obstruction. Urobilinogen is increased in hemolytic disease and urine bilirubin is negative. Urobilinogen is increased in hepatic disease, and urine bilirubin may be positive or negative. Urobilinogen is low with biliary obstruction, and urine bilirubin is positive. Reagent strips methods however, cannot distinguish normal urobilinogen from absent urobilinogen, as might be seen in complete biliary obstruction.

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To screen for urinary tract infections leukocyte esterase should be coupled with: (Choose ALL of the correct answers)View Page

Chemistry / Urinalysis Question Bank - Review Mode (no CE)
Elevation in conjugated bilirubin is most likely to be found in which of the following conditions:View Page
A spectrophotometric scan of amniotic fluid may be valuable in the determination of which of the following conditions:View Page
Most common methods for measuring bilirubin are based on the reaction of bilirubin with:View Page
Which of the following conditions would be suggested by a marked rise in alkaline phosphatase, jaundice, and a moderate rise in ALT:View Page
Match urine color with substance that might have been responsible:View Page

Confirmatory and Secondary Urinalysis Screening Tests
Urine Bilirubin

Bilirubin is a degradation product of hemoglobin. When red blood cells (RBCs) have reached the end of their normal life span (approximately 120 days), they are destroyed in the spleen and liver. Hemoglobin that is freed in the process is further broken down into iron, protein, and protoporphyrin. Protoporphyrin is converted to bilirubin and released into the circulation. Bilirubin binds to albumin and is transported in the blood to the liver. This unconjugated bilirubin is insoluble in water and cannot be filtered through the glomerulus of the kidney. Bilirubin is then conjugated with glucuronic acid in the liver. This conjugated bilirubin is water soluble and is excreted by the liver through the bile ducts and into the duodenum; bilirubin does not normally appear in the urine. However, if the normal degradation cycle is disrupted, as happens with cirrhosis, hepatitis, and other conditions that damage the liver, conjugated bilirubin will appear in the urine. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma bilirubin level is increased.

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A urine specimen is received in the laboratory late in the afternoon. The specimen was collected early in the morning and was accidentally left in bright sunlight and at room temperature on a counter in the outpatient clinic. The test order is for urine bilirubin screening. Which of the following could occur as a result of the storage conditions?View Page
Urine Bilirubin

Normally, small amounts of conjugated bilirubin, regurgitate back from the bile duct and enter the blood stream, so small amounts of conjugated bilirubin can be found in the plasma. Since conjugated bilirubin is not bound to protein, it is easily filtered through the glomerulus and excreted in the urine whenever the plasma level is increased. Normally, no detectable amount of bilirubin (sometimes referred to as "bile") is found in the urine.

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Bilirubinuria

Bilirubin may be present in the urine when bile duct obstruction, liver disease, or liver damage is present. Bilirubinuria can be detected before other clinical symptoms, such as jaundice, are present or recognizable. The detection of small quantities of bilirubin is very important in early diagnosis of obstructive and hepatic jaundice. The urine bilirubin test is also useful in the differential diagnosis of obstructive jaundice (positive for bilirubinuria) vs. hemolytic jaundice (negative for bilirubinuria).

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Confirmation of Urine Bilirubin Result

The reagent strip test for bilirubin may not be sensitive enough to detect small amounts of bilirubin in urine, which may be present in the earliest phases of liver disease or viral hepatitis. The Ictotest® reagent tablet is more sensitive and is recommended when bilirubin in the urine is particularly of interest. False-positive results can occur in screening procedures for bilirubin due to color interference from large amounts of blood in the urine, very concentrated urine, or drugs that discolor the urine, such as phenazopyridine (Pyridium). Because of this, it is important to verify positive or questionable bilirubin results with a confirmatory method, such as the diazo tablet test, available commercially as the Ictotest®. Ictotest® will detect as little as 0.05-0.10 mg bilirubin/dL urine, making it the procedure of choice for confirming bilirubin in urine specimens.

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Reporting Ictotest® Results

Since detectable amounts of bilirubin are not normally present in urine, results of the Ictotest® are reported as "positive" or "negative", there is no quantitation. The sensitivity of Ictotest® is better than dipstick methods or the Harrison test. Ictotest® will detect as little as 0.05-0.10 mg bilirubin/dl urine, making it the procedure of choice for confirming bilirubin in urine specimens.

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Diazo Tablet Test

The diazo tablet test is available commercially as the Ictotest®. The test is based on the coupling of a diazonium salt with bilirubin, which is also the basis of the urine bilirubin reagent strip test. However, the Ictotest method is more sensitive than the reagent strip method. When bilirubin is present, a blue or purple color is produced.Since detectable amounts of bilirubin are not normally present in urine, results of the Ictotest® are reported as "positive" or "negative;" there is no quantitation. Any amount of blue or purple color on the absorbent pad should be interpreted as "positive." Any other color, such as red, pink, or orange, should be reported as "negative." The results shown on the right would both be reported as positive for bilirubin.

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What is the basis of both the reagent strip test and Ictotest® for detection of bilirubin?View Page
Limitations of the Procedure

The product profile for Ictotest® points out that bilirubin is very light sensitive, so urine specimens should be protected from excessive light exposure and examined as soon as possible after specimen collection. On standing, bilirubin, which has a goldish color, is oxidized to biliverdin, which is a green color. Many of the procedures used to detect bilirubin will not react with biliverdin, so false-negative results may occur if urine is not fresh when tested.

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Dermal Puncture and Capillary Blood Collection
Protect Me From the Light

Some specimens routinely collected for testing by using a capillary puncture are adversely affected by exposure to light. One example is a specimen collected for bilirubin testing that is obtained from a newborn. When obtaining the specimen for this testing, it is important for the phlebotomist to recognize the effect of light on the specimen. Room light or sunlight can metabolize the bilirubin in the specimen to a different compound. This will cause a falsely lower bilirubin level. A neonatal bilirubin specimen should be obtained in a dark-colored (amber) container. Alternately, a clear or white container can be immediately wrapped in aluminum foil following the blood collection, preventing the blood from exposure to light.

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General Laboratory Question Bank - Review Mode (no CE)
Match urine color with substance that might have been responsible:View Page
Match the urine sediment or crystal to the correct description.View Page

Hematology / Hemostasis Question Bank - Review Mode (no CE)
Which of the following tests on amniotic fluid would be included when assessing fetal maturity:View Page
Which of the following tests would be employed in order to detect neural tube defects:View Page

Hemoglobinopathies: Hemoglobin S Disorders
Hemolytic Crisis

Sickle cell anemia, in addition to being a hemoglobinopathy, is a hemolytic anemia. Hemolysis is both intravascular (about one-third) and extravascular (about two-thirds). Common markers of hemolysis include elevated LDH, bilirubin, and reticulocyte levels.The hemoglobin that is released into the plasma during intravascular hemolysis combines with nitric oxide (NO). The resulting decrease in NO availability contributes to the vaso-occlusive crisis by stimulating vasoconstriction, endothelial adhesiveness, and thrombosis.Hemolytic crisis also involves splenic sequestration, which occurs in an effort to remove damaged red blood cells. This can result in hypovolemia, which may lead to shock, especially in children. Children can also exhibit splenomegaly due to intrasplenic pooling of blood.Adults in hemolytic crisis may experience autosplenectomy. This occurs when the spleen has multiple infarctions, followed by fibrosis, which renders the spleen nonfunctional.

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Which of these blood levels will increase during hemolysis?View Page

Hemolytic Disease of the Fetus and Newborn
Symptoms and Laboratory Findings in Severe HDFN Due to Anti-D

Anti-D causes the most severe HDFN. Symptoms and laboratory findings in HDFN due to anti-D typically include:1. Anemia: Cord Hb can be less than 10 g/dL (100 g/L) and as low as 3–5 g/dL (30–50 g/L).2. Jaundice (icterus gravis): Jaundice occurs after delivery, as fetal bilirubin is cleared by the mother during pregnancy. Extravascular fetal red cell destruction by maternal antibody produces high bilirubin levels. The newborn, who is unable to produce adequate amounts of the liver enzyme glucuronyl transferase, is unable to conjugate the bilirubin into its water-soluble, excretable form.3. Kernicterus: If indirect bilirubin levels reach approximately 20 mg/dL (340 mmol/L) the fat soluble unconjugated bilirubin deposits in the fat-rich brain cells causing brain cell damage. Cerebral palsy, deafness, mental retardation, and other serious disorders can result.4. Hydrops fetalis: Gross edema occurs in severely affected infants, and often results in stillbirth or death soon after birth. Liver failure and hypoproteinemia also play a role in this syndrome.5. Enlarged organs, e.g., liver, spleen and heart6. Laboratory findings include a positive direct antiglobulin test (DAT), low hemoglobin (as above), increased reticulocyte count, and increased nucleated red cells.

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Postnatal Treatment: Exchange Transfusion

Whenever possible, a hallmark of HDFN treatment is to induce labor as early as possible once lung maturity has been attained so that the newborn will be able to survive. Once the infant is born, the main treatment for severe HDFN due to anti-D (and other antibodies causing severe disease) is exchange transfusion. In exchange transfusions, up to 85–90% of the infant's blood can be exchanged with donor blood by a process of removing 5–20 mL of blood at a time, and injecting an equivalent amount until the exchange is complete. An exchange transfusion accomplishes the following: Removes bilirubin and thus helps prevent kernicterus; Removes sensitized red cells that have not been broken down yet; Removes circulating maternal antibody; Provides antigen-negative red cells that will not be destroyed by the maternal antibody, thus will survive and provide oxygen to the tissues.

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Which symptom of HDFN is associated with low levels of glucuronyl transferase?View Page
Which symptom of HDFN does phototherapy help prevent?View Page
Other Postnatal Treatment

Besides exchange transfusion, postnatal treatment of HDFN may include the following:RBC TransfusionMany infants who have received IUTs also require simple RBC transfusions in the first few weeks of life to treat ongoing hemolysis caused by persistent maternal antibody in the newborn's circulation.Phototherapy Phototherapy is used to treat jaundice in preterm infants without HDFN and in infants with mild HDFN. Intensive phototherapy has also been used to treat moderate and severe HDFN and decrease the need for exchange transfusion. The newborn is placed under a "blue light" which chemically alters the bilirubin in the surface capillaries to a harmless substance. Human Serum AlbuminHuman serum albumin can also be transfused, either separately or as part of an exchange transfusion in place of FFP. Albumin binds unconjugated bilirubin, thus preventing its deposition in the fat-rich brain cells. Albumin must be used judiciously, because it can aggravate congestive heart failure.

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Fetal Monitoring: Amniocentesis

Amniocentesis is a procedure used to obtain amniotic fluid for diagnostic tests.As applied to diagnosing severity of HDFN, amniocentesis can be done beginning at approximately 28 weeks and repeated to provide serial estimates of the amount of bile pigment (bilirubin) in amniotic fluid. The process measures the difference in optical density at a wavelength of 450 nm (DOD450) between the observed density and an extrapolated baseline (if no excess bilirubin was being produced).Serial DOD 450 values are plotted on charts (e.g., Queenan chart or the extended Liley* chart), which categorize HDFN into 3 zones of severity.See examples of Liley and Queenan charts (from e-Medicine/WebMD)

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ABO HDFN - Treatment

Prenatal treatment Prenatal management and treatment of ABO HDFN is not routinely done because: Titers of anti-A and anti-B do not correlate well with severity of disease; The risks of fetal monitoring (e.g., amniocentesis, cordocentesis) and fetal transfusion are greater than the risk of ABO HDFN since it is usually mild and subclinical. However, if a woman has a history of infants with moderate to severe ABO HDFN requiring treatment, she may be monitored so that the infant can be treated for possible HDFN as soon as possible. Postnatal TreatmentTreatment of ABO HDFN usually consists of phototherapy in which the newborn is placed under a "blue light" that chemically alters bilirubin in the surface capillaries to a harmless substance.For more severe cases, exchange transfusion may be performed. Donor RBC for exchange transfusion in cases of ABO HDFN must meet these criteria: Group O; Rh compatible with infant; Less than or equal to 7 days old (or fresher); Reconstituted with AB FFP to obtain a prescribed hematocrit; CMV negative (or equivalent, e.g., leukoreduced by filtration); Negative for hemoglobin S to prevent blood from sickling under conditions of reduced oxygen concentration in the newborn; Irradiated to prevent graft-versus-host disease. Exchange transfusion is also discussed later in the course in the section related to HDFN due to anti-D and other antibodies. Red Blood Cells are crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

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Kernicterus due to high levels of unconjugated bilirubin can cause brain damage in newborns suffering from severe HDFN.View Page

Hereditary Hemochromatosis (retired 2/13/2014)
References

1. Beutler E. Iron storage disease: Facts, fiction and progress. Blood Cells Mol Dis. 2007;39:140-7.2. Higgins T, Beutler E, Doumas BT. Hemoglobin, iron, and bilirubin. In: Burtis CA, editor. Teitz Fundamentals of Clinical Chemistry. 6th ed. Saunders Elsevier, 2008.3. Ganz T. Hepcidin, a key regulator of iron metabolism and mediator of anemia and inflammation. Blood 2003;102(3):78-8.4. Andrews NC, Schmidt PJ. Iron homeostasis. Annu Rev Physiolo. 2007;69:69-85.5. Murtagh LJ, Whiley M, Wilson S, et al. Unsaturated iron binding capacity and transferrin saturation are equally reliable in detection of HFE hemochromatosis. Am J Gastroenterol. 2002;97(8):2093-9.6. Haddy TB, Castro OL, Rana SR. Hereditary hemochromatosis in children, adolescents, and young adults. Am J Pediatr Hematol Oncol 1988;10:23-4.7. Edwards CQ, Ajoika RS, Kushner JP. Hemochromatosis: A genetic definition. In Barton JC, Edwards CQ, eds. Hemochromatosis: Genetics, Pathophysiology, Diagnosis and Treatment. Cambridge, UK:Cambridge Univ Pr 2000:8-11.8. Whitlock EP, Garlitz BA, Harris EL , et al. Screening for Hereditary Hemochromatosis: A Systematic Review for the U.S. Preventive Services Task Force. Ann Intern Med. 2006; 145: 209-23.9. Wallace DF, Subramaniam VN. Non-HFE haemaochromatosis. World J Gastroenterol. 2007;13(35):4690-8.10. Tavill AS. Diagnosis and management of hemochromatosis. Hepatology. 2001;33:1321-811. Qaseem A, Aronson M, Fitterman N, Snow V, Weiss KB, Owens DK, et al. Screening for hereditary hemochromatosis: a clinical practice guideline from the American College of Physicians. Ann Intern Med. 2005;143:517-21.12. Phatak PD, Bonkovsky HL, and Kowdley KV. Hereditary Hemochromatosis: time for targeted screening. Ann Intern Med. 2008; 149(4): 270 – 2.13. Brissot P, deBels F. Current approaches to the management of hemochromatosis. Hematology Am Soc Hematol Educ Program. 2006:36-41. 14. Guidance for industry: Variances for blood collection from individuals with hereditary hemochromatosis. http://www.fda.gov/cber/gdlns/hemchrom.htm Accessed 12/17/08.

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Phlebotomy
Case

Marcie Moore was a phlebotomist at a community hospital in Atlanta. It was her week to collect the pediatric unit and she was on her way to the room of a newborn for which she had just received orders to draw a STAT BMP (chem-7) and bilirubin. After informing the mother of the baby about the test she needed to perform, Marcie set up to perform a heel stick on the baby. Marcie chose a site on the outer edge of the heel on the bottom of the baby's foot ( the correct area for a heel stick) and made a small incision with a Tenderfoot lancet after cleaning the site well with alcohol.She immediately began collecting the blood in the correct tube for the BMP and bilirubin. Blood flow was not strong so Marcie squeezed the baby's foot a little to help the blood come out faster – the newborn was screaming and Marcie could tell it was making the mother uncomfortable. She wanted to hurry and get done so the mother could hold the baby.After the chemistry tech ran the blood tests on the tube, she informed Marcie that the newborn had a panic potassium level which did not coincide with the previous blood work on the newborn. Also the chemistry instrument could not perform the bilirubin due to hemolysis. Marcie was asked to recollect the specimen.

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Hepatic function panel

Albumin (Alb) Bilirubin (Bili) Alkaline phosphatase (Alk Phos) Total protein (TP) Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)

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Comprehensive metabolic panel (CMP)

Consists of a basic metabolic panel, plus:Albumin (Alb) and Bilirubin (Bili) Alkaline Phosphatase (Alk Phos) Total protein (TP) Alanine aminotransferase (ALT) Aspartate aminotransferase (AST)

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Red Cell Disorders: Peripheral Blood Clues to Nonneoplastic Conditions
A 5-year-old girl was brought to the emergency department with bloody diarrhea and severe abdominal pain. A complete blood count produced these results:CBC ParameterPatient ResultReference IntervalWBC9.6 x 109/L4.3 - 10.8 x 109/LHemoglobin9.1 g/dL11.5 - 13.5 g/dLHCT28%37 - 48%MCV80 fL86 - 98 fLRDW13.111 - 15Platelets90.1 x 109/L150 - 450 x 109/LThe peripheral blood smear is represented in the image to the right. Which of the following condition(s) could be present in this patient when considering the information above and the cells indicated by the arrows on the peripheral smear?View Page

Rh negative female with anti-D at delivery: A case study
The positive DAT on the newborn means that the infant probably has clinically significant hemolysis.View Page
Newborn's Clinical Status

The newborn showed no clinical evidence of HDFN or early newborn hyperbilirubinemia, with related laboratory tests as follows (laboratory's reference ranges for newborns in brackets): Test USA SI Hemoglobin 16 g/dL (13.5 - 21.0 g/dL) 160 g/L(130.5 - 226.0 g/L)* Hematocrit 52% (43-62%) 0.52 (0.43-0.62) Total bilirubin (cord blood) 2.1 mg/dL (<2.5 mg/dL) 35.9 µmol/L (<43 µmol/L) *Most countries that adopted SI do not use the official SI unit for Hb (mmol/L), but rather use g/L.

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The Disappearing Antibody: A Case Study
Delayed HTR - Signs and symptoms

Delayed HTR often go undetected as the symptoms are usually mild and subclinical (death has occurred, but rarely). Symptoms may not occur until days after transfusion when the patient has left the hospital. Donor red cell destruction is usually by extravascular hemolysis (EVH). Signs and symptoms can include: Fever with or without chills Unexplained drop in hemoglobin and hematocrit Transient jaundice due to elevated serum bilirubin

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The Urine Microscopic: Microscopic Analysis of Urine Sediment
Abnormal crystals that can be found in urine include: (Choose all that apply.)View Page
Which of the following abnormal crystals may indicate liver disease?View Page
Crystals of Clinical Significance

Crystals of clinical significance include leucine, tyrosine, cystine, cholesterol, and bilirubin.

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Bilirubin Crystals

Bilirubin crystals are seen in the urine when the serum bilirubin level is increased. The macroscopic appearance of urine with bilirubin crystals is orange to almost black in color. The crystals themselves appear as gold-orange needle-like forms that are sometimes clumped together.

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Transfusion Reactions
Additional Testing

If preliminary testing suggests hemolysis or if the results are misleading, additional testing may be required. If human error has been ruled out during the clerical check, repeat ABO/Rh testing should be performed on the unit of blood or its segment and the pretransfusion sample to detect any sample mix ups and clerical errors. Antibody detection studies should be performed on the pre- and post-transfusion samples to look for any unidentified antibodies. If an antibody is identified, the donor cells should be tested for the corresponding antigen. The crossmatch should be repeated with pre-and post-tranfusion specimens using the indirect antiglobulin test (IAT). An incompatible crossmatch with the pretransfusion sample indicates an original error, either clerical or technical. Incompatibility with only the post-transfusion sample indicates a possible anamnestic response, as in a delayed hemolytic transfusion reaction (DHTR), or sample misidentification. The patient's first voided urine specimen should be examined for the presence of free hemoglobin. The patient's bilirubin levels may also be evaluated. A change from normal pale yellow serum to a post-transfusion bright or deep yellow serum should prompt an investigation for hemolysis. The maximum concentration of bilirubin following hemolysis is not usually detectable until 3 to 6 hours after transfusion. The hemoglobin and hematocrit can be tested to detect a drop in hemoglobin or failure of the hemoglobin to rise after transfusion. Important information about physical or chemical hemolysis may be gained from examining the returned unit bag. If hemolysis is present in the bag or tubing, a process that affected the blood should be suspected, such as inappropriate warming or a faulty infusion pump. If bacterial contamination is suspected, the unit should be cultured. A positive culture indicates a reaction due to bacterial contamination.

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Clinical Laboratory Tests

A post transfusion specimen should be sent to the laboratory for work-up. A clerical check should be performed to investigate possible errors in specimen labeling, blood product issuance, or patient identification. The plasma must be examined for hemolysis. A direct antiglobulin test must be performed. The patient's ABO, Rh and antibody screen should be repeated and confirmed. The blood product ABO/Rh can be confirmed. Other laboratory tests include: complete blood count (CBC), urinalysis, serum bilirubin, creatinine, coagulation profile, and disseminated intravascular coagulation (DIC) evaluation. The full laboratory work-up and details of other laboratory tests will be discussed later in the course.

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