Clinical Chemistry III

ASCP Review Course

K+

Anand Dighe, M.D., Ph.D. Director, MGH Core Laboratory Assistant Professor, Harvard Medical School Massachusetts General Hospital Boston, MA

Outline
Pre-analytic variables Liver function testing Serum proteins Pancreas Toxicology/TDM
Instrumentation Spectrophotometry Immunoassays HPLC GC TLC

Appendix: Miscellaneous Methods

Where Do Errors Occur?

Am J Clin Pathol. 2004 Jun;121 S105-12.

Pre-Analytic Variables
Patient factors
Age, gender, pregnancy, menstruation, diet, medications, fasting, exercise, hemolysis, lipemia, time of day

Order and collection variables

Posture, tourniquet application time, order of draw, underfilling of tubes, hemolysis

Transport, storage, and processing

Storage temperature, light, travel/processing time

Postural Effect
When you stand the pressure in your veins is increased and plasma ultrafiltrate flows out of your veins and into the surrounding tissues >> concentrates blood Increase of 5-15% for most cells (RBC, platelets) and large molecules (albumin, cholesterol, immunoglobulins). Drugs that bind large molecules (e.g. Dilantin, cyclosporine, salicylates) will also be increased. Endogenous compounds that are protein bound (bilirubin, calcium, iron, lipids) will also be increased More pronounced in patients with low plasma volumes (cirrhosis, CHF) Small molecules (e.g. glucose, sodium) move freely with the water and are not affected

Collection Technique
Tourniquet application time (>2 min)
Long application times lead to hemoconcentration in same manner as postural changes In addition, prolonged venous stasis leads to anaerobic glycolysis decreased pH
Drives potassium out of cells Increased potassium Increases ionized Ca2+, lactate, magnesium, ammonia

Repeated fist clenching leads to potassium release from muscles

Traumatic draw
Hemolysis increased potassium, LDH, AST

Biologic (Intraindividual Variation) of Various Analytes

Amount of variation Small (<5%) Examples Electrolytes, calcium, albumin, osmolality, total protein, creatinine Alkaline phosphatase, cholesterol, ferritin, fasting glucose, LDH, uric acid, BUN AST, ALT, cortisol, CK, bilirubin, Fe, TSH, triglycerides

Moderate (5-10%)

Marked (>10%)

Collection Tubes
Color Additive None Type Serum Sample Tests Serologies, ANA, antibodies, CRP, tumor markers, serum toxicology Electrolytes, metabolic panels, LFTs, STAT lab PT/PTT, coag. factors CBC/diff, ABO/Rh, Type & Screen, ESR, reticulocytes Glucose, lactate HLA, some DNA based tests

RED

GREEN BLUE PURPLE GRAY YELLOW

Heparin-Na or Heparin-Lithium Citrate (binds Calcium) K2-EDTA NaF/K oxalate (glycolysis inhibitor) Citrate dextrose (preserves cells)

Plasma Plasma Plasma Plasma Plasma

Order of Draw
Order of filling of evacuated blood tubes is essential
1) blood cultures 2) plain tubes 3) citrate tubes 4) other additives (e.g. EDTA)

Not infrequently, if proper order of draw is not followed, contamination of next tube with purple top additive (potassium EDTA) leads to:
Markedly increased potassium Decreased Ca2+, Mg2+ (chelated by EDTA), decreased enzyme activity for enzymes requiring cations (alkaline phosphatase, creatine kinase)

Storage and Transport Temperature

Prolonged storage at room temperature (e.g. delays in transport or processing)
Factor VIII degraded increased PTT (non-heparin containing specimens) Platelets release platelet factor 4 (PF4) which neutralizes heparin decreases PTT (heparin containing specimens) Plain tubes (red top) increased potassium (cellular leak) Increased phosphorus (cellular leak) Increased ammonia (protein breakdown) Glucose falls 3-5% per hour at room temperature (glycolysis)

Storage at 4 degrees
Increased potassium (Na-K pump inhibited) Factor VII activated shortens PT

Miscellaneous Preanalytic Variables

Exercise causes increases in CK, AST, LDH, uric acid Platelets release potassium when clotting occurs
Serum K > plasma K Thrombocytosis can lead to increased serum Ks (~0.1 mmol/L for each 100K of platelets)

Liver Function Testing

Heme

Heme Degradation Pathway

Albumin

Unconjugated Bilirubin UGT

Intestine

Unconjugated bilirubin is a product of hemoglobin catabolism Unconjugated bilirubin is reversibly bound to albumin. In the liver unconj. BR is conjugated to monoand diglucuronides by bilirubin UDP-Glucuronyl Transferase (UGT)

Conjugated Bilirubin
Kidney

Conjugated Bilirubin
Liver

Urobilinogen
Urine

Urobilinogen Conjugated Bilirubin * UGT

Stercobilirubin

*in pathologic states

Unconjugated

Conjugated

Bilirubin Measurement
Bilirubin + Diazotized sulfanilic acid Azobilirubin
Absorbs light at 540 nm

van den Bergh method: Based on diazo reaction with an accelerator Direct: Conjugated Total: Detergent, alcohol or caffeine added (dissociates albumin and unconjugated bilirubin) Indirect:Unconjugated (subtraction) Bilirubin is light sensitive. Levels decrease with light exposure. Gross hemolysis (due to traumatic draw, improper handling) will increase bilirubin levels.

Unconjugated Jaundice
Normal total bilirubin
Most (>80%) of bilirubin is unconjugated (indirect)

Jaundice
Unconjugated jaundice: Greater than 80% indirect Conjugated jaundice: Greater than 50% direct

Differential Diagnosis of Unconjugated Jaundice TYPE Prehepatic Hepatic CAUSE/ EXAMPLES Increased production: Hemolysis, hematoma, ineffective erythropoiesis. Defective clearance/conjugation: neonatal jaundice, Gilbert syndrome, Crigler-Najjar syndrome.

Prehepatic Unconjugated Jaundice

Differential diagnosis Includes hemolysis, ineffective erythropoiesis, hematoma resorption Characterized by: Increased bilirubin production Production exceeds capacity of liver UGT to conjugate bilirubin Indirect (unconjugated) bilirubin increased Urine bilirubin negative Urine urobilinogen normal or elevated
Albumin

Heme

Unconjugated Bilirubin UGT

Intestine

Conjugated Bilirubin

Conjugated Bilirubin
Liver

Kidney

Urobilinogen
Urine

Urobilinogen Conjugated Bilirubin (NEG)

Stercobilirubin

Hepatic Unconjugated Jaundice

Neonatal Immature conjugating enzymes Gilberts syndrome
Autosomal dominant mutations in the UGT promoter Decreased bilirubin UDPglucuronyl transferase, impaired BR conjugation Affects up to 5% of the population Diagnosis:
Asymptomatic mild unconjugated hyperbilirubinemia (total < 5 mg/dl) No hemolysis, normal LFTs

Heme

Albumin

Unconjugated Bilirubin UGT

Intestine

Conjugated Bilirubin

Conjugated Bilirubin
Liver

Kidney

Urobilinogen
Urine

Urobilinogen Conjugated Bilirubin (NEG)

Stercobilirubin

Hepatic Unconjugated Jaundice: Crigler-Najjar Syndrome

Rare AR diseases: only few 100 cases in world literature. Mutations in coding region of UGT

Type I
Very rare. Usually fatal in infancy Absence of bilirubin glucuronyl transferase Presents in infants with severe jaundice and kernicterus Normal liver function tests and histology Usually benign Typically diagnosed by age 1 Marked deficiency of bilirubin glucuronyl transferase Normal liver function tests and histology

Type II (a.k.a. Arias Syndrome)

Initial Laboratory Approach to Unconjugated Hyperbilirubinemia

Unconjugated hyperbilirubinemia 1. Evaluate for hemolysis 2. If hemolysis absent consider hereditary and other causes

Lab evaluation of hemolysis:

CBC, smear, retics, indirect BR, haptoglobin, LDH, urine hemosiderin, urine hemoglobin, Coombs test

Simplified Differential Diagnosis of Conjugated Jaundice

TYPE CAUSE/ EXAMPLES Hepatocellular damage or hereditary. Includes hepatitis, alcohol induced liver injury, Dubin-Johnson syndrome, Rotor syndrome Drugs (anabolic steroids, erythromycin, chlorpromazine phenothiazines), primary biliary cirrhosis, sclerosing cholangitis, some cases of hepatitis Includes gallstones, obstructing tumors, strictures

Hepatic

Intrahepatic cholestasis

Extrahepatic obstruction

Conjugated Jaundice: Hepatocellular Disease

Examples: Alcoholic liver disease, Albumin cirrhosis (any cause), hepatitis (any cause), ischemic necrosis, drugs and toxins (acetaminophen, isoniazid) Conjugated Laboratory findings (variable) Bilirubin Conjugated jaundice Urine bilirubin elevated Urine urobilinogen low Kidney Hepatocellular enzyme pattern
Elevated AST/ALT Slight/moderate ALP, GGT elevation Alcoholic hepatitis AST:ALT 2:1 Ratio 0.5-0.8 in viral hepatitis

Heme

Unconjugated Bilirubin UGT Conjugated Bilirubin

Liver Intestine

Urobilinogen
Urine

Urobilinogen Conjugated Bilirubin

Stercobilirubin

Conjugated Jaundice: Intrahepatic Cholestatic Disease

Intrahepatic cholestasis Albumin Examples: Infiltrative disorders, drugs (oral contraceptives, androgens), primary biliary cirrhosis, primary sclerosing Conjugated cholangitis Bilirubin Laboratory findings (variable) Conjugated jaundice Kidney Urine bilirubin elevated Urine urobilinogen low Obstructive/cholestatic liver Urine enzyme pattern
elevated ALP, 5NT, GGT slight/moderate AST/ALT elevation

Heme

Unconjugated Bilirubin

xx xx

xx Conjugated x Bilirubin x xx Liver

UGT

Intestine

Urobilinogen

Urobilinogen Conjugated Bilirubin

Stercobilirubin

Conjugated Jaundice: Extrahepatic Obstruction

Heme

Etiology: Due to obstruction of Unconjugated Albumin biliary tree Bilirubin Causes: UGT Intestine Stones, strictures, tumors Conjugated (biliary tract, ampullary, Conjugated Bilirubin pancreatic) Bilirubin Liver Laboratory findings: Conjugated jaundice Kidney Urine bilirubin elevated Urine urobilinogen low Urobilinogen Obstructive/cholestatic liver enzyme pattern Urine elevated ALP, 5NT, GGT Stercobilirubin Urobilinogen slight/moderate Conjugated Bilirubin AST/ALT elevation

Conjugated Jaundice: Dubin-Johnson and Rotor Syndromes Dubin-Johnson syndrome

Rare in U.S. Highest frequency in Iranian Jews (1:1000) Autosomal recessive: mutation in gene for human canalicular multispecific organic anion transporter (cMOAT) resulting in impaired transport of organic anions Clinical findings: Intermittent jaundice, benign course Histology: Yellow-black pigment in hepatocytes Laboratory tests: Normal liver function tests Urine coproporphyrin I > III (normally III > I by 3-4 x)

Rotor syndrome
Very rare AR disease. Etiology: Defective hepatic uptake and storage of organic anions Other findings: Normal LFTs, normal histology, benign course

Initial Laboratory Approach to Conjugated Hyperbilirubinemia

Conjugated hyperbilirubinemia Measure alkaline phosphatase (ALP), transaminases (ALT/AST) Evaluate for liver disease (labs, imaging) If workup is normal: Consider hereditary and other causes

Transaminases
Alanine Aminotransferase, ALT (SGPT) Aspartate Aminotransferase, AST (SGOT) Widely distributed: Liver, muscle, myocardium, kidney, brain In lab test enzymatic activity is measured, not an immunoassay ALT: Relatively Liver specific. AST: Less specific AST elevated with normal ALT Consider non-hepatic causes (MI, pericarditis, hemolytic anemia, muscle disease, pancreatitis) Both AST and ALT are cytosolic enzymes: Leak from damaged cells Elevated in hepatocellular disease, less elevated in obstructive disorders

Transaminase Measurement
AST Alpha-ketoglutarate + Aspartate ---------> Glutamate + Oxaloacetate Malate Dehydrogenase Oxaloacetate + NADH --------> NAD+ + Malate Measure disappearance of NADH at 340 nm (NADH absorbs light at 340 nm, NAD does not)
NAD+ Abs NADH

250

300 350 400 nm

Spectrophotometry
Cuvet Monochromator Light source
(filters, diffraction gratings, prisms)

Detector & Meter

(photo multiplier tubes, diodes, diode arrays)

Io
Selects specific wavelength of light (e.g 340 nm)

I
Detectors work best when Abs = 0.05-1.0)

e.g. tungsten lamp

Io = incident light I = transmitted light

%T = Transmittance = I/Io * 100 A = Absorbance = 2 log %T

T A 100% 0 50% 0.30 1% 2

Beers Law
Cuvet Monochromatic light Detector & Meter

A = 2-log T Beers Law A = a*b*C

A = absorbance a = molecular absorptivity (fixed property of the molecule)

A = 0.100

Cuvet Monochromatic light Detector & Meter

b = path length of the cuvet (fixed) C = concentration

A = 0.500

Spectrophotometry: Calibration
Cuvet Light source Detector & Meter
Monochromator

Selects specific wavelength of light (e.g 340 nm)

Verify wavelength accuracy with substances with known absorbance peaks (holmium oxide or didymium) Photometer (entire system) accuracy: test with known concentration of a substance of known molar absorptivity (potassium dichromate)

Alkaline Phosphatase
Tissue distribution
High levels in bile ducts, bone, intestine, kidney, placenta each with its own isoenzyme

Clinical utility
Most useful in differential diagnosis of hepatobiliary and bone disease

ALP in hepatobiliary disease

ALP is a cell surface enzyme of bile ducts Obstruction -----> Increased synthesis ----> Release from cells

Measurement: Spectrophotometric assay based on enzymatic hydrolysis of 4nitrophenylphosphate to colored 4-nitrophenol (4-NP) in alkaline pH 4-NPP + H2O 4-NP + Phosphate
ALP

Causes of an Elevated Alkaline Phosphatase

Obstruction or cholestasis
ALP, bilirubin, 5NT, and GGT elevated. AST/ALT may also be elevated

Space occupying lesions and infiltrative disorders

ALP, 5NT, GGT elevated. Bilirubin may be normal in some space occupying lesions. AST/ALT normal or slight elevation

Bone disease
ALP elevated, 5NT, GGT, AST/ALT, bilirubin normal. Differential diagnosis includes Pagets disease (prevalence of 3% in people > 40), hyperparathyroidism, metastatic tumor, healing fracture

Miscellaneous
CHF, pregnancy, intestinal ischemia/infarction, malignancy, sepsis, elderly, children, pulmonary infarction

Other Cholestatic Liver Enzymes

Gamma glutamic transpeptidase (GGT) 5 Nucleotidase (5NT) Leucine aminopeptidase (LAP) Cell surface enzymes with relative specificity to biliary tract (not in bone or placenta) Levels increase with bile duct obstruction GGT and 5NT can be used interchangeably in most situations GGT may be more sensitive for alcoholic liver disease
Chronic EtOH exposure increases GGT levels GGT present in SER so toxins that cause microsomal proliferation (alcohol, barbs, Dilantin) increase GGT

GGT may be elevated in infectious mononucleosis, pancreatic cancer, acute pancreatitis

Approach to Patient with Elevated Alkaline Phosphatase

ALP elevated Pursue non-hepatic causes (bone, etc.) If no symptoms recheck in 3-6 months Rarely needed tests: Heat stable ALP Liver US without biliary dilation Consider AMA, liver biopsy (Bone isoenzyme is heat labile) ALP isoenzymes (electrophoresis)

GGT elevated
YES

NO

ALP, GGT > 2x

YES

NO

Liver Ultrasound Liver US shows biliary dilation

Consider ERCP

Summary of Liver Function Patterns

Pattern Hemolysis Hepatocellular Obstruction/cholestasis Mass lesion Passive Congestion Bilirubin Inc (Unconj) Inc (Conj) Inc (Conj) Normal sl Inc ALP Normal N/Inc Inc Inc sl Inc ALT Normal Inc N/Inc N/Inc sl Inc

Caveats 1. Patterns assume pure disorders 2. Sepsis, congestive heart failure, multisystem organ failure, and DIC may be associated with variable abnormalities in LFTs

Ammonia
Derived from metabolism/ deamination of proteins Detoxified by liver to urea Major sites of production: colon, kidney, liver, muscle Normal Colonic ammonia Portal circulation Liver Disease Hepatic detoxification

Shunting, impaired metabolism: elevated blood ammonia

Ammonia
Ammonia may be elevated in liver disease, certain urea cycle deficiencies, Reyes syndrome, and HHH syndrome (hyperammonemia, homocitrullinuria, hyperornithinemia) Poor correlation between ammonia level and hepatic coma Factors that may increase ammonia production and precipitate hepatic encephalopathy: Dietary protein Blood protein: GI hemorrhage, transfusions Uremia Increased catabolism of muscle protein (fever, malnutrition)

Ammonia Measurement
Principle: Most assays based on conversion of NADPH to NADP (NADPH absorbs at 340 nm) Alpha-ketoglutarate + Ammonium + NADPH ----------------------> Glutamate + NADP + H2O
Glutamate dehydrogenase Sample must be kept on ice
Blood ammonia increases rapidly at room temperature

Testing should be performed within 60 min of venipuncture Hemolysis increases ammonia levels (reject specimen)

Child-Pugh Classification of Hepatic Status

Parameter
Bilirubin (mg/dL)* PT (sec prolonged) Albumin (gm/dL) Ascites Encephalopathy (grade)

1 Point
<2 1-4 >3.5 Absent None

2 Points
2-3 4-6 2.8-3.4 Mild 1-2

3 Points
>3 >6 <2.8 Moderate 3-4

Total points =score (5-15)

*For primary biliary cirrhosis: 1 point=1-4, 2 points=4-10, and 3 points >10

Child class: A: 5 - 6, B: 7 - 9, C: 10-15 Child class C predicts a survival of less than 12 months

Laboratory Studies That May Suggest an Etiology for Cirrhosis Cause

Alcoholic Viral Wilsons disease AAT deficiency Hemochromatosis PBC Autoimmune

Lab Tests
AST:ALT > 2, elevated GGT Viral serologies, DNA/RNA testing Ceruloplasmin, serum and urine copper Serum AAT and phenotyping Transferrin saturation (screening test) Serum ferritin, DNA testing Antimitochondrial antibody, markedly elevated ALP, cholestatic enzyme pattern, elevated IgM, hyperlipidemia ANA, anti-SM antibody (smooth muscle), hypergammaglobulinemia

Serum Proteins
Total protein Albumin Prealbumin Ceruloplasmin

Total Protein (Albumin plus Globulins)

Total protein
Total protein = ~ 60% albumin and ~ 40% globulins

Globulins Globulin = Total protein albumin Total protein not a good index of hepatic function May increase in some types of liver disease
IgA (alcoholic liver disease) IgM (primary biliary cirrhosis) IgG (autoimmune disease, infection, many others)

Albumin a better index of hepatic synthetic function than total protein Albumin to globulin ratio (A:G): May be useful to detect shifts in albumin or globulins

Total Protein Measurement

A. Kjeldahl nitrogen method: Reference method B. Biuret Widely used colorimetric method Peptide bonds react with copper ions in alkaline solution to form blue color Read absorbance at 540 nm Easily automated Lacks sensitivity for low concentration samples
C. Lowry method

Not widely used. Two stage modification of Biuret method

D. Dye binding Widely used Based on change in absorbance spectrum of dye upon binding to protein Dyes include: Coomassie blue, amido black Easily automated E. Others: OD 280, refractometry

Albumin
Multifunctional ~60% of total plasma protein. Contributes 80% of colloid oncotic pressure Transport functions ( bilirubin, some lipids, drugs, hormones, others) Genetic variants (e.g. bisalbuminemia) Hepatic function indicator Albumin a better index of hepatic synthetic function than total protein Albumin half life 19 days so may not decline for weeks after acute hepatocellular injury

Albumin
Hyperalbuminemia
Dehydration

Hypoalbuminemia
Volume expansion Decreased synthesis: Liver disease, malnutrition Increased catabolism Losses: Skin, gastrointestinal tract, kidney

Albumin: Measurement
Subtraction: Total protein - globulins (rarely used) Serum protein electrophoresis: Densitometric scanning and quantitation using the total protein value Immunoassays: Expensive and not widely used except for low concentration applications (e.g. urine microalbumin) Dye binding Widely used, inexpensive, and easy to automate Albumin binds to dye, causing shift in dyes absorbance Bromocresol green (BCG) and bromocresol purple (BCP) BCP more accurate for low albumin levels (<2.0)

Prealbumin
a.k.a serum transthyretin, thyroid-binding prealbumin Forms 1:1 complex with vitamin A carrier protein retinol binding protein Good for monitoring response to treatment in protein-calorie malnutrition Mutations in prealbumin can cause familial neuropathic or cardiopathic amyloid (AF amyloid, 1-5% of amyloid cases) 1-2 d half-life Measurement: Immunoassay

Acute Phase Response (APR)

Definition: Acute phase proteins: Proteins that change concentration in response to inflammatory states Infection Trauma, post-surgery, myocardial infarction Malignancy Any condition associated with tissue necrosis Clinical utility: To detect or monitor inflammation (ex: rheumatic fever, rheumatoid arthritis) Measurement: ESR Specific acute phase reactants especially C-reactive protein

Acute Phase Reactants

Positive APRs
Alpha-1-antitrypsin Haptoglobin Ceruloplasmin Fibrinogen Ferritin C3 C-reactive protein Serum amyloid A protein

Negative APRs
Albumin Transferrin (rises late) Retinol binding protein Prealbumin

Wilsons Disease and Ceruloplasmin

Characterized by Autosomal recessive inheritance Toxic accumulation of copper in the liver and brain A cause of chronic liver disease in childhood (rare before age 6, almost always presents before age 30) Not due to an inborn error in Ceruloplasmin (CER)
CER is a polypeptide with 6 copper atoms CER may have role in copper transport: Controversial Hypoceruloplasminemia no copper deficiency

Wilson gene on chr 13. CER gene on chr 3

Defect:

Defective P-type ATPase (ATP7B) on Chr 13 Defective incorporation of copper into CER Reduction of CER concentration

Wilsons Disease
Major manifestations (onset age 8-50 years, most < 30) Liver: Variable: Mild liver disease to cirrhosis CNS: Copper accumulation in basal ganglia (bradykinesia, tremor, ataxia, rigidity) Cornea: Kayser-Fleischer rings (absent in 45% overall, absent in 9% of patients with neurologic findings) Other : Renal tubular damage, nephrolithiasis, osteoporosis, arthropathy, cardiomyopathy, hyperparathyroidism Treatment Penicillamine: Chelates copper, enhances urinary excretion Early diagnosis essential: Most cases go undetected

Wilsons Disease: Lab Diagnosis

Screening tests Most patients: Exhibit low serum CER (<10 mg/dl). Measurement: Immunoassay. However, this test is not specific (poor liver function, nephrotic syndrome can also lead to reduced levels) Low plasma copper: Due to low CER Elevated urine copper: Due to increase in filterable non-ceruloplasmin copper in plasma Penicillamine challenge: Augments urinary copper excretion Confirmatory tests Liver biopsy: With quantitative copper measurement: Usually essential for diagnosis, not definitive Genetic testing for ATP7B (limited - many diff mutations) Radioactive copper studies: Rarely required

Pancreas: Serum Amylase

Useful in the workup of abdominal pain Elevated within 2-12 hours of onset of pancreatitis in 95% of patients Persists 3-5 days Serum amylase is cleared by the kidneys
Urinary amylase can be measured as well (dipstick-type test)

Significant increases are 3-5 X upper limit of normal Often measured with serum lipase

Amylase
Amylase isoenzymes Pancreatic and salivary type (P type, S type) Can separate by electrophoresis or isoelectric focusing (ref lab test) Immunoassays now available Adds very little to diagnosis of pancreatitis Amylase elevations in pancreatic disease Acute pancreatitis Pancreatic pseudocyst Pancreatic trauma Pancreatic carcinoma (25%)

Serum amylase elevation in non-pancreatic disease

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. Renal insufficiency (amylase cleared by kidneys) Salivary gland lesions (salivary isoform) Tumor (lung, esophagus, ovary, breast) (salivary isoform) Diabetic ketoacidosis Burns Pregnancy Renal transplantation Drugs (morphine, codeine) Macroamylasemia Other abdominal disorders Biliary tract disease (cholecystitis, choledocholithiasis) Perforated peptic ulcer Intestinal obstruction or infarction Postoperative hyperamylasemia Peritonitis Ruptured ectopic pregnancy (salivary isoform***)

Urine Amylase, Macroamylasemia

Urine amylase is elevated early and reaches high levels in acute pancreatitis Main use now is in workup of macroamylasemia. 1% of normal population have macroamylasemia. Macroamylasemia is caused by amylase bound to an antiamylase antibody Occurs in higher frequency in patients with autoimmune disease Usually < 5X ULN, asymptomatic Macroamylasemia characterized by high serum amylase with normal urine amylase (normal lipase) Can also calculate amylase:creatinine clearance ratio Can directly measure macroamylase in some ref labs
Via size exclusion chromatography

Amylase Clearance Ratio

Random simultaneously collected serum and urine samples analyzed for both creatinine and amylase UAm x PCr 100 x -------------PAm x UCr

Amylase Clearance Ratio =

Normal ratio 1-5% Acute pancreatitis can be normal or elevated In macroamylasemia ratio is < 1% in patients with normal renal function

Lipase
Sensitive (95%) and specific marker for pancreatitis Stays elevated longer than amylase (7-14 d vs. 2-5 d) Useful along with amylase in patients where acute pancreatitis suspected. Especially helpful for late presentations No lipase activity in urine (enzyme inactivation) In patients with chronic pancreatitis (usually caused by alcohol abuse), lipase may be elevated in the presence of a normal serum amylase level Amylase/lipase ratio no longer considered useful to distinguish alcoholic from non-alcoholic pancreatitis Elevated lipase with normal amylase reported in acute cholecystitis, hypertriglyceridemia, hemodialysis Renally cleared. Elevated in renal failure

Clinical Laboratory Toxicology

Pharmacokinetics Recognizing/categorizing toxicity Toxicology testing basics Serum toxicology testing Drugs of abuse testing

Bioavailability
Describes the fraction of administered drug absorbed into the systemic circulation
F = Absoral / AbsIV

Expressed at 0-1.0 or as a percentage In general, PO drugs have 70% or greater bioavailability

Two Compartment Pharmacokinetic Model

Drug

Central Distribution Peripheral

Elimination

Factors Affecting Plasma Drug Concentration

Drug
Absorption

Kidneys

Liver
Drug Metabolism

Plasma protein bound drug

Excretion Reabsorption

Free drug

Urine
Site of action Receptor bound

Tissue
Tissue Bound

Metabolites

Free versus Bound Drug

Free drug typically most relevant (free drug binds it receptors) Drug binding proteins
Drugs with high protein binding: phenytoin (albumin), carbamazepine (albumin), valproate (albumin), lidocaine (orosomucoid, an acute phase reactant) Decrease in drug binding protein decreases total drug and increases free drug Increase in drug binding protein increases total drug and decreases free drug
Bound

Free

When albumin < 3.0 measure free drug for drugs that bind albumin

Free versus Bound Drug

Free drug typically most relevant (free drug binds it receptors) Drug binding proteins
Drugs with high protein binding: phenytoin (albumin), carbamazepine (albumin), valproate (albumin), lidocaine (orosomucoid, an acute phase reactant) Decrease in drug binding protein decreases total drug and increases free drug Increase in drug binding protein increases total drug and decreases free drug
Bound

Free

When albumin < 3.0 measure free drug for drugs that bind albumin

Free versus Bound Drug

Free drug typically most relevant (free drug binds it receptors) Drug binding proteins
Drugs with high protein binding: phenytoin (albumin), carbamazepine (albumin), valproate (albumin), lidocaine (orosomucoid, an acute phase reactant) Decrease in drug binding protein decreases total drug and increases free drug Increase in drug binding protein increases total drug and decreases free drug
Bound

Free

When albumin < 3.0 measure free drug for drugs that bind albumin

Volume of Distribution
Volume of distribution (Vd). The hypothetical volume necessary to dilute the drug if the total amount of drug was distributed at the same concentration as the measured plasma concentration Usually expressed in L/kg Depends primarily on the lipid vs. water solubility of the drug (i.e. water soluble drugs have lower Vd since they tend to remain in blood) Also influenced by extent drug binds to plasma proteins Uses:
Estimation of the quantity of drug ingested Estimation of blood concentration to be expected based on dose Determination of whether dialysis will be effective Determination of whether blood levels will be useful to monitor

Volume of Distribution for Various Drugs

Plasma Level Correlates With Effects Drug Acetaminophen Digitoxin Ethanol Ethylene glycol Ibuprofen Methotrexate Salicylates Vd (L/kg) 0.9 0.6 0.6 0.8 0.2 0.15 0.58 Plasma Levels Unrelated To Effects Drug Amphetamine Alprazolam Amitriptyline Chlorpromazine Cocaine Heroin Phencyclidine Vd (L/kg) 4.4 1.6 8 9 2.3 25 6.2

Body Water Compartments

Body is 60% water Total body water in 70 kg person = 70 kg x 0.60 = 42 L 2/3 is ICF, 1/3 is ECF. 3/4 of ECF is interstitial, 1/4 is plasma Lithium (Vd = 0.5 L/kg) distributes in 0.5 L/kg x 70 kg = 35 L = Vd Amitriptyline (Vd = 8 L/kg) distributes in 8 L/kg x 70 = 560 L = Vd

Peak Blood Concentration

Peak blood concentration (Cp). Peak levels occur when the rate of absorption equals the rate of clearance/elimination
Cp = dose/Vd Example: 70 kg patient took 20 tablets of 100 mg phenytoin (Vd = 0.6 L/kg). What is the expected peak concentration (assume 100% bioavailablity)? Cp = Dose/Vd = 2000 mg / (0.6 L/kg x 70 kg) = 47.6 mg/L = 47.6 mcg/ml

Half-life. Time required to reduce the blood concentration in half.

Elimination
Most drugs are excreted via the kidneys Rate of excretion depends on RBF, GFR, extent of protein binding, tubular secretion/reabsorption, urine pH, drug pKa Drugs that are charged or polar are excreted directly by the kidneys Drugs that are highly lipid soluble are first metabolized by the liver and made polar so they can be excreted

Biotransformation
Phase I reactions
Mainly occur in liver by cytochrome p450 (CYP) enzymes (microsomal oxygenases) Hydroxylation, dealkylation, deamination, epoxide formation

Phase II reactions
Glucuronidation (morphine) Glutathione conjugation (acetaminophen) Acetylation (isoniazid)

Hepatic Mixed Function Oxidase System

Cytochrome p450 (CYP) most important. 63 different CYP genes with numerous isoforms of each gene For drug metabolism most important CYP is CYP3A4 (most prevalent, metabolizes the most drugs, ~50% of drugs) Polymorphisms in CYP isoforms responsible for some drug responses
Persons lacking CYP2D6 not able to activate codeine (codeine is a pro-drug) so codeine has no effect Persons with certain CYP2C9 polymorphisms have increased sensitivity to Warfarin

Inhibition or Induction of CYP

Inhibition/induction of cytochrome p450 enzymes is common and affects the metabolism of many drugs Inhibit CYP (increase levels of many other drugs)
Cimetidine, Warfarin, Disulfuram, Izoniazid, Allopurinol, Chloramphenicol, Oral Contraceptives, Erythromycin

Induce CYP (decrease levels of many other drugs)

Phenobarbital, other barbiturates, Carbamazepine, Phenytoin, Rifampin, Ethanol (chronic)

First Order Elimination

Decline in the drug level to a constant percentage of the remaining amount per unit time. Half life for first order elimination is a constant. After 5-6 half lives there will be no significant drug effect (10050 25 12.5 6.25 3.125 1.56) Most drugs in the therapeutic range follow first order kinetics Drugs eliminated by the kidneys often follow first order kinetics
Log Concentration

Time

Concentration

Time

Zero Order Elimination

Decline in the drug is a constant amount per unit time Half life for zero order elimination is dependant on the initial concentration Acetaminophen, dilantin, salicylates, theophylline, ethanol are examples For these drugs elimination depends on the concentration of hepatic enzymes, which are limiting Monitoring of drug levels is often done since levels not easily predictable for given dose In overdose, liver enzymes can become limiting, causing a drug that is normally eliminated with first order kinetics to show zero order elimination
Log Concentration

Time

Concentration

Time

Toxicology Testing Methods

Color/spot testing Thin layer chromatography Gas/liquid chromatography
Non-automated, but suitable for broad spectrum toxicology testing Good for separation of drug mixtures

Immunoassays
Automated, commercially available assays

Mass spectroscopy
Gold standard method

High Pressure Liquid Chromatography (HPLC)

Solvent reservoir Mobile phase

Standard phase HPLC has polar stationary phase and non-polar mobile phase Reversed phase HPLC has non-polar stationary phase and polar mobile phase (non-polar compounds retained longer on the column, like dissolves like) Retention time gives information about the compound ID and is a constant for a given set of conditions

Sample Introduction

Stationary phase

Detector

Reversed Phase HPLC

Reversed Phase HPLC

Measure absorbance (at 214 nm in this example for benzodiazepines) From retention (elution) time and the use of standards the preliminary identity of the compound can be determined Drugs that are the most non-polar will elute last In reversed phase HPLC metabolites elute prior to parent drug

Chromatography Resolution
Resolution is defined as the ability to separate different compounds Factors improving resolution
Increasing column length, increasing number of particles (longer column or smaller particles), decreasing difference in polarity between stationary and mobile phase

Unresolved

No effect on resolution
Temperature, flow rate, pressure. These can improve the speed of the run but do not improve resolution Resolved

GC Basic Instrumentation

Nitrogen or Helium are typical mobile phases Stationary phase consists of non-volatile support particles packed into a column Compounds separate based on their volatility and/or polarity Applicable to volatiles (such as alcohols) or non-volatile compounds that can be derivitized into volatile ones, often with tetramethyl silane.

Gas Chromatography

Standards

Gas Chromatography
Patient with 0.21% EtOH

Thin Layer Chromatography

Same principle as HPLC Separation based on polarity and absorption to solid phase We calculate the retention dS factor (Rf) to compare samples
dB Solvent Front

A
dA

Rf of A = dA/dS
Origin

Rf of B = dB/dS

Ethylene Glycol Poisoning

Ingredient of antifreeze Not detected with typical serum toxicology panels Active metabolites oxalic acid and glycolic acid cause damage (CNS, pulmonary, cardiovascular and renal) Measured by gas chromatography Support the diagnosis: Increased Anion gap (Na + K) - (Cl + [HCO3-]) Osmolar gap (Measured minus calculated) Calc Osm = 2(Na+) + BUN/2.8 + glucose/18 + (ethanol/4.6 + ethylene glycol/6.2) Calcium oxalate crystals in urine Decreased serum calcium

Oxalate crystals

Organophosphate or Carbamate Poisoning

Organophosphates and carbamates are common in pesticides. Both inhibit cholinesterase and overdose results in the stimulation of muscarinc (urination, miosis, salivation, etc) and nicotinic (tachycardia, hypertension, muscle cramping) acetylcholine receptors. Cholinesterases
Acetylcholinesterase (a.k.a. true cholinesterase): Present in RBCs, nerve endings, gray matter Pseudocholinesterase (a.k.a. plasma cholinesterase). Present in serum. An acute phase protein made in the liver and decreased in liver disease and malnutrition.

Organophosphate poisoning decreases pseudocholinesterase and RBC cholinesterase.

RBC cholinesterase is the more accurate of the 2 measurements. However, falsely increased levels of RBC cholinesterase may be found in cases of hemolytic states (RBC cholinesterase levels normally decrease as RBCs age) and falsely decreased levels may be found in pernicious anemia. Specimens should be collected on ice and frozen until analyzed.

Definitive antidote for organophosphate poisoning is 2-PAM (pralidoxime) regenerates acetylcholinesterase.

Acetaminophen
Average toxic dose 15 g, max toxicity 3-4 days after ingestion Half life > 12 h correlates with hepatic injury Toxicity occurs when glutathione is depleted Glutathione is depleted by EtOH abuse and starvation >> can increase toxicity Treatment N-acetylcysteine (Mucomyst) regenerates glutathione

Rumack-Matthew nomogram

Phenytoin (Dilantin)
Metabolism: zero order kinetics due to limiting enzymes in liver that inactivate Often monitored. Typical assays measure total drug (immunoassays) Highly protein (albumin) bound (90%) Free fraction is active component. Can measure free phenytoin by performing immunoassay on plasma ultrafiltrate. Low albumin, renal failure (possibly due to endogenous binding inhibitors), hyperbilirubinemia, drugs that bind albumin (high dose salicylates, valproate, sulfamethoxazole, ceftriaxone) all cause increased FREE phenytoin, decreased TOTAL Common indication for measuring free Dilantin level is a low albumin level
Bound

90%

Free 10%

Lithium
Narrow range of safety
Side effects: tremor, polydipsia/polyuria (nephrogenic DI), hypercalcemia (changes PTH response to Ca), ataxia, non-toxic goiter (hyper or hypothyroidism)

Does not bind proteins, eliminated in the urine (dosage must be adjusted for creatinine clearance) Vd = 0.6 L/kg Treatment for OD is hemodialysis Can be measured by ion-selective electrodes, flame photometry, or newer spectrophotometric assays.

Drugs of Abuse Testing

Two types of tests: SCREENING and CONFIRMATORY
SCREENING
High sensitivity, adequate specificity, rapid, inexpensive

CONFIRMATORY
Greater specificity (correct false positives), generally more technically complex, time consuming and expensive

Urine Drugs of Abuse Testing

For routine drug surveillance URINE is the specimen of choice

ADVANTAGES
Ease of collection Stable, ample sample Long detection period Availability of rapid, inexpensive methods Affected by fluid intake Easily substituted, adulterated Qualitative results Cannot always discern parent drug

DISADVANTAGES

Toxicology: Urine Immunoassays

Rely on the specificity of one or two monoclonal antibodies often only classspecific (e.g. benzodiazepines not valium). Most do not identify specific agents within a class. Urine immunoassays typically qualitative Subject to interferences and false positives Common urine assays Amphetamines Benzodiazepines Barbiturates Cannabinoids Cocaine metabolite Methaqualone 6-Monoacetyl morphine Opiates/metabolites Oxycodone Phencyclidine Propoxyphene Lysergic acid

Immunoassays
Many formats. Can be categorized as heterogeneous or homogeneous. Traditional assays (heterogeneous) require a washing and separation step
In heterogeneous assays the physical properties of the label are not changed in the binding process Measure the total remaining signal

Heterogeneous immunoassay

Homogeneous Immunoassays
Homogeneous assays do not require a separation step In homogenous assays the labeled reactant behaves differently when bound Fluorescence polarization, EMIT assay, CEDIA immunoassays are examples of homogeneous assays Measure modulation of the signal.

Homogeneous immunoassay

The Hook Effect

Response

Reportable range

Response in error range: dilute and retest

Hook effect: Response looks to be in reportable range Underestimate true concentration

Hormone Concentration

Interference by Human Anti-Animal Antibodies: False Positives/Over Estimation

Wash

Wash

Human anti-Mouse Antibody (HAMA)

Read

Wash

Wash

Urine Screening Tests

Most are immunoassays Often drug-class specific (e.g. opiates, amphetamines) Qualitative Drug/metabolite must be present above a threshold concentration (cut-off) for a positive result to be reported Suitable cut-off value must be selected by the lab based on the utility of the test

Typical Urine Screening Detection Periods

Amphetamines Cannabinoids Cocaine Opiates Benzodiazepines Barbiturates Methadone LSD Phencyclidine Methaqualone Ethanol
1-2 days 3-7 days or longer 2-3 days About 2 days 3 days or longer 2-20 days About 3 days Several days 7 days or longer 7 days or longer Up to 12-24 hours

Amphetamine Immunoassay
Urine screen able to detect amphetamines and methamphetamines Also detects MDA and MDMA (ecstasy) False positives occur with over the counter and prescription meds
Ephedrine, pseudoephedrine

Confirmatory testing can identify individual agents

Urine Immunoassays
Phencyclidine
Detects PCP and derivatives of PCP Single dose detectable for 7 days Major cause of false positives is due to medications containing dextromethorphan

Dextromethorphan

Benzodiazepines
Urine screen detects for 5-7 days or longer Benzodiazepine parent drugs and metabolites are detected Certain benzodiazepines are not well detected (assay specific)

PCP

Cannabinoids Immunoassay
Screening test highly specific. Detects inactive metabolites of tetrahydrocannabinol in urine (often 11-Nor-9-THC-9-carboxylic acid) Stored in fat, generally detectable in urine for 3-7 days With chronic use can be detectable for much longer periods (weeks) Serum toxicology screens are not typically designed to detect cannabinoids Passive inhalation
Federal drug testing cut-off 100 ng/ml

Cocaine Immunoassay
Short half life in serum (1 hour) limits blood detection Urine test detects cocaine metabolite benzoylecgonine Single dose detectable for 48 hours in urine With typical laboratory cut-offs (300 ng/ml) passive inhalation very unlikely to cause a positive result

Opiates Immunoassay
Urine opiate testing detects heroin, many opiates, codeine Detectable for about 1-4 days post-exposure Naloxone and methadone do NOT cross react in most opiate assays A methadone specific immunoassay is available The poppy seed muffin defense. Poppy seeds contain opiates (morphine and codeine) that may be detected
Federal drug testing cut-off Typical laboratory cut-off 2000 ng/ml 300 ng/ml

Heroin and Codeine Metabolism

Positive opiate screen may be followed by confirmation (often GC/MS) Detection of 6-MAM is regarded as definitive evidence of exposure to heroin 6-MAM is best detected in urine collected within 8-12 hours of heroin use Immunoassays specific for 6-MAM are now available

Thanks!

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