Glucose Hemoglobin Iron and Bilirubin

Which of the following is characteristic of type 1 diabetes mellitus?
A. Requires an oral glucose tolerance test for diagnosis
B. Is the most common form of diabetes mellitus
C. Usually occurs after age 40
D. Requires insulin replacement to prevent ketosis

• D. Requires insulin replacement to prevent ketosis
• Type 1, or juvenile, diabetes is also called insulindependent diabetes because patients must be given insulin to prevent ketosis. Type 1 accounts for only about 10%–20% of cases of diabetes mellitus, and is usually diagnosed by a fasting plasma glucose. Two consecutive results ≥126 mg/dL is diagnostic. Approximately 95% of patients produce autoantibodies against the beta cells of the pancreatic islets. Other autoantibodies may be produced against insulin, glutamate decarboxylase, and tyrosine phosphorylase IA2. There is genetic association between type 1 diabetes and human leukocyte antigens (HLA) DR3 and DR4.

Which of the following is characteristic of type 2 diabetes mellitus?
A. Insulin levels are consistently low B. Most cases require a 3-hour oral glucose t
olerance test to diagnose
C. Hyperglycemia is often controlled without insulin replacement
D. The condition is associated with unexplained weight loss

• C. Hyperglycemia is often controlled without insulin replacement
• Type 2, orlate-onset diabetes, is associated with a defect in the receptor site for insulin. Insulin levels may be low, normal, or high. Patients are usually obese and over 40 years of age, although the incidence is increasing in both children and young adults. The American Diabetes Association (ADA) recommends screening all adults for diabetes who are overweight and have one additional risk factor and all adults over age 45, and to retest them every 3 years, if negative. Patients do not require insulin to prevent ketosis and hyperglycemia can be controlled in most patients by diet and drugs that promote insulin release. Type 2 accounts for 80%–90% of all diabetes mellitus.

Which of the following results falls within the diagnostic criteria for diabetes mellitus?
A. Fasting plasma glucose of 120 mg/dL
B. Two-hour postprandial plasma glucose of 160 mg/dL
C. Two-hour plasma glucose of 180 mg/dL following a 75 g oral glucose challenge
D. Random plasma glucose of 250 mg/dL and presence of symptoms

• D. Random plasma glucose of 250 mg/dL and presence of symptoms
• The American Diabetes Association recommends the following criteria for diagnosing diabetes mellitus : fasting glucose ≥ 126 mg/dL, casual (random) glucose ≥ 200 mg/dL in the presence of symptoms (polyuria, increased thirst, weight loss), glucose ≥ 200 mg/dL at 2 hours after an oral dose of 75 g of glucose, and hemoglobin A1c ≥ 6.5%. A diagnosis of diabetes mellitus is indicated if any one or combination of these four criteria is met on more than a single testing event. The fasting plasma glucose test requires at least 8 hours with no food or drink except water. The 2-hour postloading test should be conducted according to the oral glucose tolerance guidelines currently recommended by the World Health Organization.

Select the most appropriate adult reference range for fasting blood glucose.
A. 40–105 mg/dL (2.22–5.82 mmol/L)
B. 60–140 mg/dL (3.33–7.77 mmol/L)
C. 65–99 mg/dL (3.61–5.50 mmol/L)
D. 75–150 mg/dL (4.16–8.32 mmol/L)

C. 65–99 mg/dL (3.61–5.50 mmol/L) Reference ranges vary slightly depending upon method and specimen type. Enzymatic methods specific for glucose have an upper limit of normal no greater than 99 mg/dL. This is the cutoff value for impaired fasting plasma glucose (prediabetes) recommended by the American Diabetes Association. Although 65 mg/dL is considered the 2.5 percentile, a fasting level below 50 mg/dL is often seen without associated clinical hypoglycemia, and neonates have a lower limit of approximately 40 mg/dL owing to maternal insulin.

When preparing a patient for an oral glucose tolerance test (OGTT), which of the following conditions will lead to erroneous results?
A. Administration of 75 g of glucose is given to an adult patient following a 10–12-hour fast
B. Carbohydrate intake is restricted to below 150 g/day for 3 days prior to test
C. No food, coffee, tea, or smoking is allowed 8 hours before and during the test
D. The patient remains ambulatory for 3 days prior to the test

• B. Carbohydrate intake is restricted to below 150 g/day for 3 days prior to test
• Standardized OGTTs require that patients receive at least 150 grams of carbohydrate per day for 3 days prior to the test in order to stabilize the synthesis of inducible glycolytic enzymes. The 2-hour OGTT test is no longer recommended for screening and should be reserved for confirmation of diabetes in cases that are difficult to diagnose, such as persons who lack symptoms and signs of fasting hyperglycemia.

Which of the following 2-hour glucose challenge results would be classified as impaired glucose tolerance (IGT)? Two-hour serum glucose:
A. 130 mg/dL
B. 135 mg/dL
C. 150 mg/dL
D. 204 mg/dL

Which statement regarding gestational diabetes mellitus (GDM) is correct?
A. Is diagnosed using the same oral glucose tolerance criteria as in nonpregnancy
B. Converts to diabetes mellitus after pregnancy in 60%–75% of cases
C. Presents no increased health risk to the fetus
D. Is defined as glucose intolerance originating during pregnancy

• D. Is defined as glucose intolerance originating during pregnancy
• Control of GDM reduces perinatal complications such as respiratory distress syndrome, high birth weight, and neonatal jaundice. Women at risk are usually screened between 24 and 28 weeks’ gestation. The screening test can be performed nonfasting and consists of an oral 50-g glucose challenge followed by serum or plasma glucose measurement at 1 hour. A result ≥ 140 mg/dL is followed by a 2-hour or 3-hour oral glucose tolerance test to confirm gestational diabetes. For the 3-hour test, a 100-g dose of glucose is used and at least two of the following cutoffs must be exceeded : fasting, ≥ 95 mg/dL or higher; 1 hour, ≥ 180 mg/dL or higher; 2 hour ≥ 155 mg/dL or higher; 3 hour, ≥ 140 mg/dL or higher. The same cutpoints are used for the 2-hour test except that a 75-g dose is used. GDM converts to diabetes mellitus within 10 years in 30%–40% of cases. ADA recommends testing persons with GDM for diabetes 6–12 weeks after delivery.

Which of the following findings is characteristic of all forms of clinical hypoglycemia?
A. A fasting blood glucose value below 55 mg/dL
B. High fasting insulin levels
C. Neuroglycopenic symptoms at the time of low blood sugar
D. Decreased serum C peptide

• C. Neuroglycopenic symptoms at the time of low blood sugar
• Clinical hypoglycemia can be caused by insulinoma, drugs, alcoholism, and reactive hypoglycemia. Reactive hypoglycemia is characterized by delayed or excessive insulin output after eating and is very rare. Fasting insulin is normal but postprandial levels are increased. High fasting insulin levels (usually > 6 μg/L) are seen in insulinoma, and patients with insulinoma almost always display fasting hypoglycemia, especially when the fast is extended to 48–72 hours. C peptide is a subunit of proinsulin that is hydrolyzed when insulin is released. In hypoglycemia, low levels indicate an exogenous insulin source, whereas high levels indicate overproduction of insulin.

Which statement regarding glycated (glycosylated) Hgb (G-Hgb) is true?
A. Has a sugar attached to the C-terminal end of the βchain
B. Is a highly reversible aminoglycan
C. Reflects the extent of glucose regulation in the 8- to 12-week interval prior to sampling D. Will be abnormal within 4 days following an episode of hyperglycemia

• C. Reflects the extent of glucose regulation in the 8- to 12-week interval prior to sampling
• G-Hgb results from the nonenzymatic attachment of a sugar such as glucose to the N-terminal valine of the βchain. The reaction is nonreversible and is related to the time-averaged blood glucose concentration over the life span of the RBCs. There are three G-Hgb fractions designated A1a, A1b, and Alc. Hemoglobin A1c makes up about 80% of glycated hemoglobin, and is used to determine the adequacy of insulin therapy. The time-averaged blood glucose is approximated by the formula (G-Hgb × 33.3) – 86 mg/dL, and insulin adjustments can be made to bring this level to within reference limits. Also, glycated protein assay (called fructosamine) provides similar data for the period between 2 and 4 weeks before sampling.

What is the American Diabetes Association recommended cutoff value for adequate control of blood glucose in diabetics as measured by glycated hemoglobin?
A. 5% B. 6.5% C. 9.5% D. 11%

• B. 6.5%
• The ADA recommends that 6.5% be used as the cutoff for determining the adequacy of treatment for diabetes. A glycated hemoglobin test should be performed at the time of diagnosis and every 6 months thereafter if the result is < 6.5%. If the result is 6.5% or more, the treatment plan should be adjusted to achieve a lower level, and the test performed every 3 months until control is improved.

Which statement regarding measurement of Hgb A1c is true?
A. Levels do not need to be done fasting
B. Both the labile and stable Hgb A1c fractions are measured
C. Samples should be measured within 2 hours of collection
D. The assay must be done by chromatography

• A. Levels do not need to be done fasting
• Since Hgb A1C represents the average blood glucose 2–3 months prior to blood collection, the dietary status of the patient on the day of the test has no effect upon the results. Refrigerated whole-blood samples are stable for up to 1 week. Hgb A1C is assayed by cation exchange high-performance liquid chromatography or immunoassay (immunoturbidimetric inhibition) because both methods are specific for stable Hgb A1C, and do not demonstrate errors caused by abnormal hemoglobins, temperature of reagents, or fractions other than A1c.

Which stationary phase is used for the measurement of hemoglobin A1c by high performance liquid chromatography?
A. Octadecylsilane (C18)
B. Cation exchanger
C. Anion exchanger
D. Polystyrene divinylbenzene

• B. Cation exchanger
• HPLC methods for measuring Hgb A1c are performed by diluting whole blood with an acid buffer that hemolyzes the sample. Normal hemoglobin A has a weak positive charge at an acidic pH and binds weakly to the resin. Glycated hemoglobin has an even weaker positive charge and is eluted before hemoglobin A. Abnormal hemoglobin molecules S, D, E, and C have a higher positive charge than hemoglobin A and are retained longer on the column. Elution is accomplished by increasing the ionic strength of the mobile phase. Cations in the buffer displace the hemoglobin pigments from the column.

Evaluate the following chromatogram of a whole-blood hemolysate, and identify the cause and best course of action.
A. The result is not reportable because hemoglobin C is present and interferes
B. The result is reportable; neither hemoglobin F or C interfere
C. The result is not reportable because labile hemoglobin A1c is present
D. Result is not reportable because hemoglobin F is present and interferes

• B. The result is reportable; neither hemoglobin F or C interfere
• The chromatogram is from a person with hemoglobin AC; however, hemoglobin C is completely separated from Hgb A1c and does not interfere. Hgb F is also present, but does not interfere unless its concentration is > 30%. Labile hemoglobin is formed initially when the aldehyde of glucose reacts with the N-terminal valine of the βglobin chain. This Shiff base is reversible but is converted to Hgb A1c by rearrangementto a ketoamine. It is called labile A1c and produces a peak (LA1c) after HgF and before Hgb A1c. Therefore, it does not interfere.

Peak Calibrated Retention Peak % Area % Area Time Area Alb 0.60 0.25 12500 F 0.50 0.50 11300 LA1c 0.75 0.70 15545 A1c 6.2 0.90 45112 P3 2.6 1.60 57489 Ao 48.0 1.8 994813 C 43.0 2.00 926745
IGNORE

The ADA now recommends that the hemoglobin A1c test be used for both diagnosis and monitoring blood glucose levels. The cutpoint for diabetes is an A1c of 6.5. Persons with an A1c of 5.7%–6.4% are classified as being at high risk for diabetes within 5 years. An A1c between 4.0%–5.5% is defined as within normal limits.

According to American Diabetes Association criteria, which result is consistent with a diagnosis of impaired fasting glucose?
A. 99 mg/dL
B. 117 mg/dL
C. 126 mg/dL
D. 135 mg/dL

What is the recommended cutoff for the early detection of chronic kidney disease in diabetics using the test for microalbuminuria?
A. >30 mg/g creatinine
B. >200 mg/g creatinine
C. >80 mg/L
D. >80 mg/g creatinine

• A. >30 mg/g creatinine
• Microalbuminuria is the excretion of small quantities of albumin in the urine. In diabetics, excretion of albumin that is within allowable limits for healthy persons may signal the onset of chronic kidney disease. The term microalbuminuria is defined as albumin excretion ≥ 30 mg/g creatinine but ≤ 300 mg/g creatinine. The use of the albumin to creatinine ratio is preferred to measures of albumin excretory rate (μg/min) because the latter is subject to error associated with timed specimen collection. ADA recommends the test be done annually for all type 2 diabetics and type 1 diabetics who have had the disease for > 5 years.

In addition to measuring blood glucose, Hgb A1c, and microalbumin, which test should be done on diabetic persons once per year?
A. Urine glucose
B. Urine ketones
C. Plasma fructosamines
D. Estimated glomerular filtration rate

• D. Estimated glomerular filtration rate
• While urinary glucose can identify persons who may have diabetes, it is not sensitive enough to manage glucose control on a daily basis, and has been replaced by whole-blood glucose monitoring or continuous glucose monitoring. While the urinary ketone test is a useful screening test for diabetic and other forms of ketosis, the plasma βhydroxybutyrate test should be used to identify and monitor ketosis in diabetic persons. Fructosamine is a useful adjunct to Hgb A1c to identify poor control of blood glucose in the past 2–4 weeks, but has not been recommended for routine use in all diabetic patients.

Which testing situation is appropriate for the use of point-of-care whole-blood glucose methods?
A. Diagnosis of diabetes mellitus
B. Monitoring of blood glucose control in type 1 and type 2 diabetics
C. Screening for type 2 diabetes mellitus
D. Monitoring diabetics for hyperglycemic episodes only

• B. Monitoring of blood glucose control in type 1 and type 2 diabetics
• The ADA does not recommend the use of whole-blood glucose monitors for establishing a diagnosis of diabetes or screening persons for diabetes. The analytical measurement range of these devices varies greatly, and whole blood glucose is approximately 10% lower than serum or plasma glucose. In addition, analytical variance is greater and accuracy less than for laboratory instruments. Whole blood glucose meters should be used by diabetics and caregivers to monitor glucose control and can detect both hyper- and hypoglycemic states that result from too little or too much insulin replacement. Therefore, postprandial monitoring with such a device is recommended for all persons who receive insulin therapy.

Which of the following is the reference method for measuring serum glucose?
A. Somogyi–Nelson
B. Hexokinase
C. Glucose oxidase
D. Glucose dehydrogenase

• B. Hexokinase
• The hexokinase method is considered more accurate than glucose oxidase methods because the coupling reaction using glucose-6-phosphate dehydrogenase (G-6-PD) is highly specific. The hexokinase method may be done on serum or plasma collected using heparin, EDTA, fluoride, oxalate, or citrate. The method can also be used for urine, cerebrospinal fluid, and serous fluids.

Polarographic methods for glucose analysis are based upon which principle of measurement?
A. Nonenzymatic oxidation of glucose
B. The rate of O2 depletion
C. Chemiluminescence caused by formation of adenosine triphosphate (ATP)
D. The change in electrical potential as glucose is oxidized

• B. The rate of O2 depletion
• Polarographic glucose electrodes measure the consumption of O2 as glucose is oxidized. Glucose oxidase in the reagent catalyzes the oxidation of glucose by O2 under first-order conditions, forming hydrogen peroxide (H2O2). As the dissolved O2 decreases, less is reduced at the cathode, resulting in a decrease in current proportional to glucose concentration. It is important that the H2O2 not breakdown to re-form O2. This is prevented by adding molybdate and iodide that react with H2O2, forming iodine and water, and by adding catalase and ethanol that react with H2O2, forming acetaldehyde and water.

In addition to polarography, what other electrochemical method can be used to measure glucose in plasma?
A. Conductivity
B. Potentiometry
C. Anodic stripping voltammetry
D. Amperometry

• D. Amperometry
• In some critical care analyzers, amperometric measurement of glucose is used. The glucose oxidase is impregnated into the membrane covering the electrode. It reacts with glucose in the sample, forming H2O2. This diffuses across the membrane to the anode of the electrode, where it is oxidized to O2.The electrons produced are used to reduce oxygen at the cathode, completing the current path. At the anode (usually platinum), 2H2O2 →4e–+ 2O2+ 4H+. At the cathode(usually silver), O2 + 4H++ 4e–→2H2O. The net equation is 2H2O2 →O2 + 2H2O.

Select the enzyme that is most specific for β-D-glucose.
A. Hexokinase
B. G-6-PD
C. Phosphohexisomerase
D. Glucose oxidase

• D. Glucose oxidase
• Glucose oxidase is the most specific enzyme reacting with only β-D-glucose. However, the peroxidase coupling reaction used in the glucose oxidase method is subject to positive and negative interference. Therefore, hexokinase is used in the reference method.

Select the coupling enzyme used in the hexokinase method for glucose.
A. Glucose-6-phosphate dehydrogenase
B. Peroxidase
C. Glucose dehydrogenase
D. Glucose-6-phosphatase

• A. Glucose-6-phosphate dehydrogenase
• The hexokinase reference method uses a protein-free filtrate prepared with barium hydroxide (BaOH) and zinc sulfate (ZnSO4). Hexokinase catalyzes the phosphorylation of glucose in the filtrate using ATP as the phosphate donor. Glucose-6-phosphate (glucose-6-PO4) is oxidized to 6-phosphogluconate and NAD+ is reduced to NADH using G-6-PD. The increase in absorbance at 340 nm is proportional to glucose concentration. Although hexokinase will phosphorylate some other hexoses including mannose, fructose, and glucosamine, the coupling reaction is entirely specific for glucose-6-PO4 eliminating interference from other sugars.

Which glucose method is subject to falsely low results caused by ascorbate?
A. Hexokinase
B. Glucose dehydrogenase
C. Trinder glucose oxidase
D. Polarography

• C. Trinder glucose oxidase
• Although glucose oxidase is specific for β-D-glucose, the coupling (indicator) reaction is prone to negative interference from ascorbate, uric acid, acetoacetic acid, and other reducing agents. These compete with the chromogen (e.g., o-dianisidine) for peroxide, resulting in less dye being oxidized to chromophore. The choice of chromogen determines the specificity and linearity. 4-aminophenazone and phenol is more resistant to interference from azo compounds and proteins than is o-dianisidine

Which of the following is a potential source of error in the hexokinase method?
A. Galactosemia
B. Sample collected in fluoride
C. Ascorbic acid
D. Hemolysis

• D. Hemolysis
• The hexokinase method can be performed on serum or plasma using heparin, EDTA, citrate, or oxalate. RBCs contain glucose-6-PO4 and intracellular enzymes that generate NADH, causing positive interference. Therefore, hemolyzed samples require a serum blank correction (subtraction of the reaction rate with hexokinase omitted from the reagent).

Which statement about glucose in cerebrospinal fluid (CSF) is correct?
A. Levels below 40 mg/dL occur in septic meningitis, cancer, and multiple sclerosis
B. CSF glucose is normally the same as the plasma glucose level
C. Hyperglycorrhachia is caused by dehydration
D. In some clinical conditions, the CSF glucose can be greater than the plasma glucose

• A. Levels below 40 mg/dL occur in septic meningitis, cancer, and multiple sclerosis
• High glucose in CSF is a reflection of hyperglycemia and not central nervous system disease. The CSF glucose is usually 50%–65% of the plasma glucose. Low levels are significant and are most often associated with bacterial or fungal meningitis, malignancy in the central nervous system, and some cases of subarachnoid hemorrhage, rheumatoid arthritis, and multiple sclerosis.

In peroxidase-coupled glucose methods, which reagent complexes with the chromogen?
A. Hydroxide
B. Tartrate
C. Nitroprusside
D. Phenol

• D. Phenol
• The coupling step in the Trinder glucose oxidase method uses peroxidase to catalyze the oxidation of a dye by H2O2. Dyes such as 4-aminophenozone or 4-aminoantipyrine are coupled to phenol to form a quinoneimine dye that is red and is measured at about 500 nm.

Point-of-care-tests (POCTs) for whole-blood glucose monitoring are based mainly on the use of:
A. Immunochromatography
B. Glucose oxidase as the enzyme
C. Amperometric detection
D. Peroxidase coupling reactions

• C. Amperometric detection
• All POCT devices for monitoring blood glucose use either glucose dehydrogenase (GDH) or glucose oxidase and are amperometric. For glucose oxidase methods, the electrons derive from the oxidation of hydrogen peroxide. For GDH, the electrons are transferred from one of several coenzymes that are reduced when glucose is oxidized, FAD+, NAD+, or PQQ (pyrroloquinoline quinone). Interferences depend upon which enzyme/coenzyme pair are used. For example, maltose and xylose interference can be pronounced with GDH/PQQ-based strips, but not with other GDH or glucose oxidase strips. Uric acid depresses glucose oxidase reactions but has no effect on GDH reactions.

What effect does hematocrit have on POCT tests for whole-blood glucose monitoring?
A. Low hematocrit decreases glucose readings on all devices
B. High hematocrit raises glucose readings on all devices
C. The effect is variable and dependent on the enzyme/coenzyme system
D. Low hematocrit raises readings and high hematocrit lowers readings unless corrected

• D. Low hematocrit raises readings and high hematocrit lowers readings unless corrected
• Hematocrit affects POCT glucose measurements. High hematocrit lowers the glucose because RBC glucose concentration is lower than plasma concentration. Other factors include binding of oxygen to hemoglobin and the slower diffusion of glucose onto the solid phase—both of which occur when the hematocrit is high. Bias due to an abnormal hematocrit can be avoided by simultaneously measuring the conductivity of the sample. The hematocrit is calculated and used to mathematically correct the glucose measurement.

Which of the following is classified as a mucopolysaccharide storage disease?
A. Pompe’s disease
B. von Gierke disease
C. Hers’ disease
D. Hurler’s syndrome

• D. Hurler’s syndrome
• Hurler’s syndrome is an autosomal recessive disease resulting from a deficiency of iduronidase. Glycosaminoglycans (mucopolysaccharides) accumulate in the lysosomes. Multiple organ failure and mental retardation occur, resulting in early mortality. Excess dermatan and heparin sulfate are excreted in urine. Other mucopolysaccharidoses (MPS storage diseases) are Hunter’s, Scheie’s, Sanfilippo’s, and Morquio’s syndromes.

Identify the enzyme deficiency responsible for type 1 glycogen storage disease (von Gierke’s disease).
A. Glucose-6-phosphatase
B. Glycogen phosphorylase
C. Glycogen synthetase
D. β-Glucosidase

• A. Glucose-6-phosphatase
• Type 1 glycogen storage disease (von Gierke’s disease) is an autosomal recessive deficiency of glucose-6-phosphatase. Glycogen accumulates in tissues, causing hypoglycemia, ketosis, and fatty liver. There are seven types of glycogen storage disease, designated type 1 through type 7, involving deficiency of an enzyme that acts on glycogen. Types 1, 4, and 6 cause deficient glycogen breakdown in the liver. Types 2, 5, and 7 involve skeletal muscle and are less severe. Type 3 usually involves both liver and muscle, although an uncommon subtype (3B) involves only the liver

Which of the following abnormal laboratory results is found in von Gierke’s disease?
A. Hyperglycemia
B. Increased glucose response to epinephrine administration
C. Metabolic alkalosis
D. Hyperlipidemia

• D. Hyperlipidemia
• Von Gierke’s disease (type 1 glycogen storage disease) results from a deficiency of glucose-6-phosphatase. This blocks the hydrolysis of glucose-6-PO4 to glucose and Pi, preventing degradation of glycogen to glucose. The disease is associated with increased triglyceride levels because fats are mobilized for energy and lactate acidosis caused by increased glycolysis. A presumptive diagnosis is made when intravenous galactose administration fails to increase serum glucose, and can be confirmed by demonstrating glucose-6-phosphatase deficiency or decreased glucose production in response to epinephrine.

The D-xylose absorption test is used for the differential diagnosis of which two diseases?
A. Pancreatic insufficiency from malabsorption B. Primary from secondary disorders of glycogen synthesis
C. Type 1 and type 2 diabetes mellitus
D. Generalized from specific carbohydrate intolerance

• A. Pancreatic insufficiency from malabsorption
• Xylose is a pentose that is absorbed without the help of pancreatic enzymes and is not metabolized. In normal adults, more than 25% of the dose is excreted into the urine after 5 hours. Low blood or urine levels are seen in malabsorption syndrome, sprue, Crohn’s disease, and other intestinal disorders, but not pancreatitis.

Which of the following statements about carbohydrate intolerance is true?
A. Galactosemia results from deficiency of galactose-1-phosphate (galactose-1-PO4) uridine diphosphate transferase
B. Galactosemia results in a positive glucose oxidase test for glucose in urine
C. Urinary galactose is seen in both galactosemia and lactase deficiency
D. A galactose tolerance test is used to confirm a diagnosis of galactosemia

• A. Galactosemia results from deficiency of galactose-1-phosphate (galactose-1-PO4) uridine diphosphate transferase
• Galactose is metabolized to galactose-1-PO4 by the action of galactokinase. Galactose-1-PO4 uridine diphosphate (UDP) transferase converts galactose-1-PO4 to glucose. Deficiency of either enzyme causes elevated blood and urine galactose. Lactase deficiency results in the presence of urinary lactose because it is not broken down to glucose and galactose. Tests for reducing sugars employing copper sulfate are used to screen for galactose, lactose, and fructose in urine. Nonglucose-reducing sugars are not detected by the glucose oxidase reaction. A positive test is followed by TLC to identify the sugar, and demonstration of the enzyme deficiency in RBCs. The galactose tolerance test is used (rarely) to evaluate the extent of liver failure since the liver is the site of galactose metabolism.

Which of the following statements regarding iron metabolism is correct?
A. Iron absorption is decreased by alcohol ingestion
B. Normally, 40%–50% of ingested iron is absorbed
C. The daily requirement is higher for pregnant and menstruating women
D. Absorption increases with the amount of iron in the body stores

• C. The daily requirement is higher for pregnant and menstruating women
• For adult men and nonmenstruating women, approximately 1–2 mg/day of iron is needed to replace the small amount lost mainly by exfoliation of cells. Because 5%–10% of dietary iron is absorbed normally, the daily dietary requirement in this group is 10–20 mg/day. Menstruating women have an additional requirement of 1 mg/day and pregnant women 2 mg/day. Absorption efficiency will increase in iron deficiency and decrease in iron overload. Iron absorption is enhanced by low gastric pH and is increased by alcohol ingestion.

Which of the following processes occurs when iron is in the oxidized (Fe3+) state?
A. Incorporation into protoporphyrin IX to form functional heme
B. Reaction with chromogens in colorimetric assays
C. Absorption by intestinal epithelium
D. Binding to transferrin and incorporation into ferritin

• D. Binding to transferrin and incorporation into ferritin
• Intestinal absorption occurs only if the iron is in the reduced (Fe+2) state. After absorption, Fe+2 is oxidized to Fe+3 by gut mucosal cells. Transferrin and ferritin bind iron efficiently only when in the oxidized state. Iron within Hgb binds to O2 by coordinate bonding, which occurs only if the iron is in the reduced state. Likewise, in colorimetric methods, Fe+2 forms coordinate bonds with carbon and nitrogen atoms of the chromogen.

Which of the following is associated with low serum iron and high total iron-binding capacity (TIBC)?
A. Nephrosis
B. Iron deficiency anemia
C. Hepatitis
D. Noniron deficiency anemias

• B. Iron deficiency anemia
• Iron-deficiency anemia is the principal cause of low serum iron and high TIBC because it promotes increased transferrin. Pregnancy without iron supplementation depletes maternal iron stores and also results in low serum iron and high TIBC. Iron-supplemented pregnancy and use of contraceptives increase both iron and TIBC. Nephrosis causes low iron and TIBC due to loss of both iron and transferrin by the kidneys. Hepatitis causes increased release of storage iron, resulting in high levels of iron and transferrin. Noniron deficiency anemias may cause high iron and usually show low TIBC and normal or high ferritin.

Which condition is associated with the lowest percent saturation of transferrin?
A. Hemochromatosis
B. Anemia of chronic infection
C. Iron deficiency anemia
D. Noniron deficiency anemia

• C. Iron deficiency anemia
• Percent saturation = Serum Fe × 100/TIBC. Normally, transferrin is one-third saturated with iron. In iron deficiency states, the serum iron falls but transferrin rises. This causes the numerator and denominator to move in opposite directions, resulting in very low percent saturation (about 10%). The opposite occurs in hemochromatosis and sideroblastic anemia, resulting in an increased percent saturation.

Which condition is most often associated with a high serum iron level?
A. Nephrosis
B. Chronic infection or inflammation
C. Polycythemia vera
D. Noniron deficiency anemias

• D. Noniron deficiency anemias
• Anemia associated with chronic infection causes a low serum iron, but unlike iron deficiency, causes a low (or normal) TIBC and does not cause low ferritin. Noniron deficiency anemias such as pernicious anemia and sideroblastic anemia produce high serum iron and low TIBC. Nephrosis causes iron loss by the kidneys. Polycythemia is associated with increased iron within the RBCs and depletion of iron stores.

Which of the following is likely to occur first in iron deficiency anemia?
A. Decreased serum ferritin
B. Increased TIBC
C. Decreased serum iron
D. Increased transferrin

• A. Decreased serum ferritin
• Body stores must be depleted of iron before serum iron falls. Thus, serum ferritin falls in the early stages of iron deficiency, making it a more sensitive test than serum iron in uncomplicated cases. Ferritin levels are low only in iron deficiency. However, concurrent illness such as malignancy, infection, and inflammation may promote ferritin release from the tissues, causing the serum ferritin to be normal in iron deficiency.

Which formula provides the best estimate of serum TIBC?
A. Serum transferrin in mg/dL × 0.70 = TIBC (μg/dL)
B. Serum transferrin in mg/dL × 1.43 = TIBC (μg/dL)
C. Serum iron (μg/dL)/1.2 + 0.06 = TIBC (μg/dL)
D. Serum Fe (μg/dL) × 1.25 = TIBC (μg/dL)

• B. Serum transferrin in mg/dL × 1.43 = TIBC (μg/dL)
• Transferrin, a β-globulin, has a molecular size of about 77,000. Transferrin is the principal iron transport protein, and TIBC is determined by the serum transferrin concentration. One mole of transferrin binds two moles of Fe+3, so the transferrin concentration can be used to predict the TIBC. Since the direct measurement of TIBC requires manual pretreatment to remove the excess iron added and is prone to overestimation if all of the unbound iron is not removed, some labs prefer to measure transferrin immunochemically and calculate TIBC. This formula may underestimate TIBC because albumin and other proteins will bind iron when the percent iron saturation of transferrin is abnormally high.

Which statement regarding the diagnosis of iron deficiency is correct?
A. Serum iron levels are always higher at night than during the day
B. Serum iron levels begin to fall before the body stores become depleted
C. A normal level of serum ferritin rules out iron deficiency
D. A low serum ferritin is diagnostic of iron deficiency

• D. A low serum ferritin is diagnostic of iron deficiency
• Serum iron levels are falsely elevated by hemolysis and subject to diurnal variation. Levels are highest in the morning and lowest at night, but this pattern is reversed in persons who work at night. A low ferritin is specific for iron deficiency. However, only about 1% of ferritin is in the vascular system. Any disease that increases ferritin release may mask iron deficiency.

Which statement about iron methods is true? A. Interference from Hgb can be corrected by a serum blank
B. Colorimetric methods measure binding of Fe2+ to a ligand such as ferrozine
C. Atomic absorption is the method of choice for measurement of serum iron
D. Serum iron can be measured by potentiometry

• B. Colorimetric methods measure binding of Fe2+ to a ligand such as ferrozine
• Atomic absorption is not the method of choice for serum iron because matrix error and variation of iron recovered by extraction cause bias and poor precision. Most methods use HCl to deconjugate Fe3+ from transferrin followed by reduction to Fe2+. This reacts with a neutral ligand such as ferrozine, tripyridyltriazine (TPTZ), or bathophenanthroline to give a blue complex. Anodic stripping voltammetry can also be used to measure serum iron. Hemolysis must be avoided because RBCs contain a much higher concentration of iron than does plasma.

Which of the following statements regarding the TIBC assay is correct?
A. All TIBC methods require addition of excess iron to saturate transferrin
B. All methods require the removal of unbound iron
C. The chromogen used must be different from the one used for measuring serum iron
D. Measurement of TIBC is specific for transferrinbound iron

• A. All TIBC methods require addition of excess iron to saturate transferrin
• All TIBC methods require addition of excess iron to saturate transferrin. Excess iron is removed by ion exchange or alumina gel columns or precipitation with MgCO3 and the bound iron is measured by the same procedure as is used for serum iron. Alternatively, excess iron in the reduced state can be added at an alkaline pH. Under these conditions, transferrin will bind Fe2+ and the unbound Fe2+ can be measured directly.

Which of the following statements regarding the metabolism of bilirubin is true?
A. It is formed by hydrolysis of the αmethene bridge of urobilinogen
B. It is reduced to biliverdin prior to excretion C. It is a by-product of porphyrin production D. It is produced from the destruction of RBCs

• D. It is produced from the destruction of RBCs
• Synthesis of porphyrins results in production of heme and metabolism of porphyrins other than protoporphyrin IX yields uroporphyrins and coproporphyrins, not bilirubin. Reticuloendothelial cells in the spleen digest Hgb and release the iron from heme. The tetrapyrrole ring is opened at the αmethene bridge by heme oxygenase, forming biliverdin. Bilirubin is formed by reduction of biliverdin at the γmethene bridge. It is complexed to albumin and transported to the liver.

Bilirubin is transported from reticuloendothelial cells to the liver by:
A. Bilirubin-binding globulin
B. Haptoglobin
C. Transferrin
D. Albumin

• D. Albumin
• Albumin transports bilirubin, haptoglobin transports free Hgb, and transferrin transports ferric iron. When albumin binding is exceeded, unbound bilirubin, called free bilirubin, increases. This may cross the blood–brain barrier, resulting in kernicterus.

In the liver, bilirubin is conjugated by addition of:
A. Vinyl groups
B. Methyl groups
C. Hydroxyl groups
D. Glucuronyl groups

• D. Glucuronyl groups
• The esterification of glucuronic acid to the propionyl side chains of the inner pyrrole rings (I and II) makes bilirubin water soluble. Conjugation is required before bilirubin can be excreted via the bile.

Which enzyme is responsible for the conjugation of bilirubin?
A. β-Glucuronidase
B. UDP-glucuronyl transferase
C. Bilirubin oxidase
D. Biliverdin reductase

• B. UDP-glucuronyl transferase
• UDP-glucuronyl transferase esterifies glucuronic acid to unconjugated bilirubin, making it water soluble. Most conjugated bilirubin is diglucuronide; however, the liver makes a small amount of monoglucuronide and other glycosides. β-Glucuronidase hydrolyzes glucuronide from bilirubin, hormones, or drugs. It is used prior to organic extraction to deconjugate urinary metabolites (e.g., total cortisol). Biliverdin reductase forms bilirubin from biliverdin (and heme oxygenase forms biliverdin from heme). Bilirubin oxidase is used in an enzymatic bilirubin assay in which bilirubin is oxidized back to biliverdin and the rate of biliverdin formation is measured at 410 nm.

The term δ-bilirubin refers to:
A. Water-soluble bilirubin
B. Free unconjugated bilirubin
C. Bilirubin tightly bound to albumin
D. Direct-reacting bilirubin

• C. Bilirubin tightly bound to albumin
• HPLC separates bilirubin into four fractions : α= unconjugated, β= monoglucuronide, γ= diglucuronide, and δ= irreversibly albumin bound. δBilirubin is a separate fraction from the unconjugated bilirubin, which is bound loosely to albumin. δBilirubin and conjugated bilirubin react with diazo reagent in the direct bilirubin assay.

Which of the following processes is part of the normal metabolism of bilirubin?
A. Both conjugated and unconjugated bilirubin are excreted into the bile
B. Methene bridges of bilirubin are reduced by intestinal bacteria forming urobilinogens
C. Most of the bilirubin delivered into the intestine is reabsorbed
D. Bilirubin and urobilinogen reabsorbed from the intestine are mainly excreted by the kidneys

• B. Methene bridges of bilirubin are reduced by intestinal bacteria forming urobilinogens
• Most of the conjugated bilirubin delivered into the intestine is deconjugated by β-glucuronidase and then reduced by intestinal flora to form three different reduction products collectively called urobilinogens. The majority of bilirubin and urobilinogen in the intestine are not reabsorbed. Most of that which is reabsorbed is re-excreted by the liver. The portal vein delivers blood from the bowel to the sinusoids. Hepatocytes take up about 90% of the returned bile pigments and secrete them again into the bile. This process is called the enterohepatic circulation.

Which of the following is a characteristic of conjugated bilirubin?
A. It is water soluble
B. It reacts more slowly than unconjugated bilirubin
C. It is more stable than unconjugated bilirubin
D. It has the same absorbance properties as unconjugated bilirubin

• A. It is water soluble
• Conjugated bilirubin refers to bilirubin mono- and diglucuronides. Conjugated bilirubin reacts almost immediately with the aqueous diazo reagent without need for a nonpolar solvent. Historically, conjugated bilirubin has been used synonymously with direct-reacting bilirubin, although the latter includes the δ-bilirubin fraction when measured by the Jendrassik–Grof method. Conjugated bilirubin is excreted in both bile and urine. It is easily photo-oxidized and has very limited stability. For this reason, bilirubin standards are usually prepared from unconjugated bilirubin stabilized by the addition of alkali and albumin.

Which of the following statements regarding urobilinogen is true?
A. It consists of a single water-soluble bile pigment
B. In hemolytic anemia, it is decreased in urine and feces
C. It is measured by its reaction with p-aminosalicylate
D. It is formed in the intestines by bacterial reduction of bilirubin

• D. It is formed in the intestines by bacterial reduction of bilirubin
• Urobilinogenis a collective term given to the reduction products of bilirubin formed by the action of enteric bacteria. Urobilinogen excretion is increased in extravascular hemolytic anemias and decreased in obstructive jaundice (cholestatic disease). Urobilinogen is measured using Ehrlich’s reagent, an acid solution of p-dimethylaminobenzaldehyde.

Which statement regarding bilirubin metabolism is true?
A. Bilirubin undergoes rapid photo-oxidation when exposed to daylight
B. Bilirubin excretion is inhibited by barbiturates
C. Bilirubin excretion is increased by chlorpromazine
D. Bilirubin is excreted only as the diglucuronide

• A. Bilirubin undergoes rapid photo-oxidation when exposed to daylight
• Samples for bilirubin analysis must be protected from direct sunlight. Drugs may have a significant in vivo effect on bilirubin levels. Barbiturates lower serum bilirubin by increasing excretion. Other drugs that cause cholestasis, such as chlorpromazine, increase the serum bilirubin. Although most conjugated bilirubin is in the form of diglucuronide, some monoglucuronide and other glycosides are excreted. In glucuronyl transferase deficiency, some bilirubin is excreted as sulfatides.

Which condition is caused by deficient secretion of bilirubin into the bile canaliculi?
A. Gilbert’s disease
B. Neonatal hyperbilirubinemia
C. Dubin–Johnson syndrome
D. Crigler–Najjar syndrome

• C. Dubin–Johnson syndrome
• Dubin–Johnson syndrome is an autosomal recessive condition arising from mutation of an ABC transporter gene. It produces mild jaundice from accumulation of conjugated bilirubin that is not secreted into the bile canaliculi. Total and direct bilirubin are elevated, but other liver function is normal. Rotor syndrome is an autosomal recessive condition that also results in retention of conjugated bilirubin. The mechanism in Rotor syndrome is unknown, and like Dubin–Johnson syndrome it is commonly asymptomatic. It can be differentiated from Dubin–Johnson syndrome by the pattern of urinary coproporphyrin excretion and because it produces no black pigmentation in the liver.

In hepatitis, the rise in serum conjugated bilirubin can be caused by:
A. Secondary renal insufficiency
B. Enzymatic conversion of urobilinogen to bilirubin
C. Failure of the enterohepatic circulation
D. Extrahepatic conjugation

• C. Failure of the enterohepatic circulation
• Conjugated bilirubin is increased in hepatitis and other causes of hepatic necrosis due to failure to re-excrete conjugated bilirubin reabsorbed from the intestine. Increased direct bilirubin can also be attributed to accompanying intrahepatic obstruction, which blocks the flow of bile.

Which of the following is a characteristic of obstructive jaundice? A. The ratio of direct to total bilirubin is greater than 1:2
B. Conjugated bilirubin is elevated, but unconjugated bilirubin is normal
C. Urinary urobilinogen is increased
D. Urinary bilirubin is normal

• A. The ratio of direct to total bilirubin is greater than 1:2
• Obstruction prevents conjugated bilirubin from reaching the intestine, resulting in decreased production, excretion, and absorption of urobilinogen. Conjugated bilirubin regurgitates into sinusoidal blood and enters the general circulation via the hepatic vein. The level of serum direct (conjugated) bilirubin becomes greater than unconjugated bilirubin. The unconjugated form is also increased because of accompanying necrosis, deconjugation, and inhibition of UDP-glucuronyl transferase

Which of the following would cause an increase in only the unconjugated bilirubin?
A. Hemolytic anemia
B. Obstructive jaundice
C. Hepatitis
D. Hepatic cirrhosis

• A. Hemolytic anemia
• Conjugated bilirubin increases as a result of obstructive processes within the liver or biliary system or from failure of the enterohepatic circulation. Hemolytic anemia (prehepatic jaundice) presents a greater bilirubin load to a normal liver, resulting in increased bilirubin excretion. When the rate of bilirubin formation exceeds the rate of excretion, the unconjugated bilirubin rises.

Which form of hyperbilirubinemia is caused by an inherited absence of UDP-glucuronyl transferase?
A. Gilbert’s syndrome
B. Crigler–Najjar syndrome
C. Rotor syndrome
D. Dubin–Johnson syndrome

• B. Crigler–Najjar syndrome
• Crigler–Najjar syndrome is a rare condition that occurs in two forms. Type 1 is inherited as an autosomal recessive trait and causes a total deficiency of UDP-glucuronyl transferase. Life expectancy is less than 1 year. Type 2 is an autosomal dominant trait and is characterized by lesser jaundice and usually the absence of kernicterus. Bilirubin levels can be controlled with phenobarbital, which promotes bilirubin excretion. Gilbert’s syndrome is an autosomal recessive condition characterized by decreased bilirubin uptake and decreased formation of bilirubin diglucuronide. It is the most common form of inherited jaundice. UDP glucuronyl transferase activity is reduced owing to an increase in the number of AT repeats in the promoter region of the gene. Dubin–Johnson and Rotor syndromes are autosomal recessive disorders associated with defective delivery of bilirubin into the biliary system.

Which statement best characterizes serum bilirubin levels in the first week following delivery?
A. Serum bilirubin 24 hours after delivery should not exceed the upper reference limit for adults
B. Jaundice is usually first seen 48–72 hours postpartum in neonatal hyperbilirubinemia
C. Serum bilirubin above 5.0 mg/dL occurring 2–5 days after delivery indicates hemolytic or hepatic disease
D. Conjugated bilirubin accounts for about 50% of the total bilirubin in neonates

• B. Jaundice is usually first seen 48–72 hours postpartum in neonatal hyperbilirubinemia
• Bilirubin levels may reach as high as 2–3 mg/dL in the first 24 hours after birth owing to the trauma of delivery, such as resorption of a subdural hematoma. Neonatal hyperbilirubinemia occurs 2–3 days after birth due to increased hemolysis at birth and transient deficiency of the microsomal enzyme, UDP-glucuronyl transferase. Normally, levels rise to about 5–10 mg/dL but may be greater than 15 mg/dL, requiring therapy with UV light to photo-oxidize the bilirubin. Neonatal jaundice can last up to 1 week in a mature neonate and up to 2 weeks in prematures babies. Neonatal bilirubin is almost exclusively unconjugated.

Which form of jaundice occurs within days of delivery and usually lasts 1–3 weeks, but is not due to normal neonatal hyperbilirubinemia or hemolytic disease of the newborn?
A. Gilbert syndrome
B. Lucey –Driscoll syndrome
C. Rotor syndrome
D. Dubin–Johnson syndrome

• B. Lucey –Driscoll syndrome
• Lucey–Driscoll syndrome is a rare form of jaundice caused by unconjugated bilirubin that presents within 2–4 days of birth and can last several weeks. It is caused by an inhibitor of UDP-glucuronyl transferase in maternal plasma that crosses the placenta. Jaundice is usually severe enough to require treatment.

Which statement regarding total and direct bilirubin levels is true?
A. Direct bilirubin exceeds 3.5 mg/dL in most cases of hemolytic anemia
B. Direct bilirubin is normal in cholestatic liver disease
C. Total bilirubin level is a less sensitive and specific marker of liver disease than the direct level
D. The ratio of direct to total bilirubin exceeds 0.40 in hemolytic anemia

• C. Total bilirubin level is a less sensitive and specific marker of liver disease than the direct level
• Direct bilirubin measurement is a sensitive and specific marker for hepatic and posthepatic jaundice because it is not elevated by hemolytic anemia. In hemolytic anemia, the total bilirubin does not exceed 3.5 mg/dL, and the ratio of direct to total is less than 0.20. Unconjugated bilirubin is the major fraction in necrotic liver disease because microsomal enzymes are lost. Unconjugated bilirubin is elevated along with direct bilirubin in cholestasis because some necrosis takes place and some conjugated bilirubin is hydrolyzed back to unconjugated bilirubin.

A lab measures total bilirubin by the Jendrassik–Grof bilirubin method with sample blanking. What would be the effect of moderate hemolysis on the test result?
A. Falsely increased due to optical interference
B. Falsely increased due to release of bilirubin from RBCs
C. Falsely low due to inhibition of the diazo reaction by hemoglobin
D. No effect due to correction of positive interference by sample blanking

• C. Falsely low due to inhibition of the diazo reaction by hemoglobin
• The sample blank measures the absorbance of the sample and reagent in the absence of azobilirubin formation and corrects the measurement for optical interference caused by hemoglobin absorbing the wavelength of measurement. However, hemoglobin is an inhibitor of the diazo reaction and will cause falsely low results in a blank corrected sample. For this reason, direct bichromatic spectrophotometric methods are preferred when measuring bilirubin in neonatal samples, which are often hemolyzed.

Which reagent is used in the Jendrassik–Grof method to solubilize unconjugated bilirubin?
A. 50% methanol
B. N-butanol
C. Caffeine
D. Acetic acid

• C. Caffeine
• A polarity modifier is required to make unconjugated bilirubin soluble in diazo reagent. The Malloy–Evelyn method uses 50% methanol to reduce the polarity of the diazo reagent. Caffeine is used in the Jendrassik–Grof method. This method is recommended because it is not falsely elevated by hemolysis and gives quantitative recovery of both conjugated and unconjugated bilirubin.

Which statement about colorimetric bilirubin methods is true?
A. Direct bilirubin must react with diazo reagent under alkaline conditions
B. Most methods are based upon reaction with diazotized sulfanilic acid
C. Ascorbic acid can be used to eliminate interference caused by Hgb
D. The color of the azobilirubin product is independent of pH

• B. Most methods are based upon reaction with diazotized sulfanilic acid
• Unconjugated bilirubin is poorly soluble in acid, and therefore, direct bilirubin is assayed using diazotized sulfanilic acid diluted in weak HCl. The direct diazo reaction should be measured after no longer than 3 minutes to prevent reaction of unconjugated bilirubin, or the diazo group can be reduced using ascorbate or hydroxylamine preventing any further reaction.
• Unconjugated bilirubin is poorly soluble in acid, and therefore, direct bilirubin is assayed using diazotized sulfanilic acid diluted in weak HCl. The direct diazo reaction should be measured after no longer than 3 minutes to prevent reaction of unconjugated bilirubin, or the diazo group can be reduced using ascorbate or hydroxylamine preventing any further reaction.

Which statement regarding the measurement of bilirubin by the Jendrassik–Grof method is correct?
A. The same diluent is used for both total and direct assays to minimize differences in reactivity
B. Positive interference by Hgb is prevented by the addition of HCl after the diazo reaction C. The color of the azobilirubin product is intensified by the addition of ascorbic acid
D. Fehling’s reagent is added after the diazo reaction to reduce optical interference by hemoglobin

• D. Fehling’s reagent is added after the diazo reaction to reduce optical interference by hemoglobin
• The Jendrassik–Grof method uses HCl as the diluent for the measurement of direct bilirubin because unconjugated bilirubin is poorly soluble at low pH. Total bilirubin is measured using an acetate buffer with caffeine added to increase the solubility of the unconjugated bilirubin. After addition of diazotized sulfanilic acid and incubatiion, the diazo group is reduced by ascorbic acid, and Fehling’s reagent is added to alkalinize the diluent. At an alkaline pH the product changes from pink to blue, shifting the absorbance maximum to 600 nm where Hgb does not contribute significantly to absorbance.

In the enzymatic assay of bilirubin, how is measurement of both total and direct bilirubin accomplished?
A. Using different pH for total and direct assays
B. Using UDP glucuronyl transferase and bilirubin reductase
C. Using different polarity modifiers
D. Measuring the rate of absorbance decrease at different time intervals

• A. Using different pH for total and direct assays
• Enzymatic methods use bilirubin oxidase to convert bilirubin back to biliverdin, and measure the decrease in absorbance that results. At pH 8, both conjugated, unconjugated, and delta bilirubin react with the enzyme, but at pH 4 only the conjugated form reacts.

What is the principle of the transcutaneous bilirubin assay?
A. Conductivity
B. Amperometric inhibition
C. Multiwavelength reflectance photometry
D. Infrared spectroscopy

• C. Multiwavelength reflectance photometry
• Measurement of bilirubin concentration through the skin requires the use of multiple wavelengths to correct for absorbance by melanin and other light-absorbing constituents of skin and blood. More than 100 wavelengths and multiple reflectance measurements at various sites may be used to derive the venous bilirubin concentration in mg/dL. Such devices have been shown to have a high specificity. They can be used to identify neonates with hyperbilirubinemia, and to monitor treatment.

A neonatal bilirubin assay performed at the nursery by bichromatic direct spectrophotometry is 4.0 mg/dL. Four hours later, a second sample assayed for total bilirubin by the Jendrassik–Grof method gives a result of 3.0 mg/dL. Both samples are reported to be hemolyzed. What is the most likely explanation of these results?
A. Physiological variation owing to premature hepatic microsomal enzymes
B. δ-Bilirubin contributing to the result of the first assay
C. Falsely high results from the first assay caused by direct bilirubin
D. Hgb interference in the second assay

• D. Hgb interference in the second assay
• The Jendrassik–Grof method is based upon a diazo reaction that may be suppressed by Hgb. Because serum blanking and measurement at 600 nm correct for positive interference from Hgb, the results may be falsely low when significant hemolysis is present. Direct spectrophometric bilirubin methods employing bichromatic optics correct for the presence of Hgb. These are often called “neonatal bilirubin” tests. A commonly used approach is to measure absorbance at 454 nm and 540 nm. The absorbance contributed by Hgb at 540 nm is equal to the absorbance contributed by Hgb at 454 nm. Therefore, the absorbance difference will correct for free Hgb. Neonatal samples contain little or no direct δ-bilirubin. They also lack carotene pigments that could interfere with the direct spectrophotometric measurement of bilirubin.