Test Reference Range

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Test Reference Range

·  Usually defined as the values for the central 95% of a “normal” population distribution. That means that 5% of normal people will give an abnormal test result.

·  If multiple tests are performed on a normal person, the probability of a person having an abnormal test result are 1 – (0.95)n where “n” is the number of tests.

Probability of Abnormal Results

·  If 20 tests are performed, there is about a 65% probability of obtaining at least one abnormal value for a normal subject.

ACCURACY AND PRECISION

Case 1

·  A 21-year-old male is admitted to the Burn Unit with second- and third-degree burns over 70% of his body. These injuries resulted from an explosion and fire when the patient lit a cigarette while unloading gasoline.

·  I.V. fluids were started in the ER approximately one hour earlier. SPE was as follows:

Serum Protein Electrophoresis

·  All are decreased except beta and gamma (only slight)

·  SPE pattern interpretation? General Protein Loss

·  Large protein loss into interstitial tissue, albumin also decreased because of negative acute phase reactant protein

·  Fluid also lost as due to rapid shift of body fluids into the interstitial compartments (increased interstitial osmotic pressure due to dead cells releasing contents).

Burns

·  Rapid shift of fluids may result in hypovolemic shock, therefore treat with IV fluids fast (dilution).

·  Seen with burns to 20% of the body.

·  Sepsis is a big problem.

o  A burn situation is "acute stress".

·  Why aren't the alpha globulins increased?

o  Loss of proteins and dilution effect of IV fluids

·  Which protein(s) loss accounts for the low total protein? Why?

o  Albumin - major protein

·  List 3 other conditions in which a similar pattern may be seen

o  Starvation/malnutrition

o  Malabsorption

o  Hemorrhage

Inflammation

·  A complex reaction to injurious agents such as microbes and damaged, usually necrotic, cells that consists of vascular responses, migration and activation leukocytes, and systemic reactions

·  Acute Inflammation:

o  A rapid response to an injurious agent that serves to deliver mediators of host defense (leukocytes and plasma proteins).

o  Three Major Components:

§  Microvascular leakage and edema

§  Vasodilation leading to increased blood flow

§  Leukocyte emigration

Case 2

·  On the second hospital day a serum protein electrophoresis pattern is ordered on a 49-year-old male with an MI.

·  General interpretation for pattern?

o  Acute inflammation / stress

·  What is the pathophysiological basis for the changes in serum proteins?

o  Albumin is a negative acute phase reactant while alpha 1 and 2 are APR.

·  Acute inflammation/Stress:

o  Decreased Albumin (negative acute phase protein):

§  loss into extravascular space, decreased synthesis (TNFa and IL-1 and 6)

·  Increased a globulins

o  AAT, increased synthesis due to cytokines (IL-6) inhibits proteases released in areas of inflammation or necrosis

o  Hp, bacteriostatic, antioxidant, control of lymphocyte and neutrophil function

·  Why isn't there an increase in the beta globulin region?

o  Fibrinogen, C3 and C4 go up and transferrin goes down

·  If you had drawn the sample in a heparin or EDTA tube, would that have affected the pattern?

o  Serum – does not contain clotting factors (e.g. fibrinogen) as blood is allowed to clot prior to centrifugation

o  Plasma – contains clotting factors, fibrinogen would be visible in the beta region

Chronic Inflammation:

·  Inflammation of prolonged duration (weeks or months) in which active inflammation, tissue destruction, and attempts at repair are proceeding at the same time.

·  Three Major Components:

o  Infiltration of mononuclear cells

o  Tissue destruction

o  Attempts at healing, angiogenesis, fibrosis

Polyclonal Gammopathy

·  Normal response to infections

·  Individual immunoglobulins generally will provide little information

Wilson’s Disease

·  Incidence = 1:30,000

·  Typically presents after ~8 years of age - teens.

·  If untreated patient will die by age 30

·  Diagnosis commonly made by Kayser-Fleischer Rings as well as decreased serum ceruloplasmin, DNA (also increased hepatic copper content, and increased urinary copper excretion)

·  genetic defect in the Cu-binding ATPase protein

·  defective liver-specific copper transport

·  defective copper excretion into the bile

·  accumulation of copper in the liver (cytosol of hepatocytes) - hepatic overload

·  toxic copper accumulation in other tissues:

o  brain (basal ganglia and thalamus)

o  renal (proximal renal tubules)

o  cornea (Kayser-Fleischer rings)

Ceruloplasmin

·  a2-globulin that contains ~ 95% of total serum copper

·  Each molecule binds 6-8 copper molecules

·  Main functions are in plasma redox reactions and copper transport

Characteristics of useful markers for tissue injury

·  Changed by pathological process

o  Synthesis decreased (hereditary deficiencies)

o  Activity inhibited (Pseudocholinesterase)

o  Released from damaged tissues (e.g. LDH, AST, ALT, CK, amylase, troponins)

o  Synthesis induced (alk. phos., GGT, ACE)

o  Tissue/plasma concentration gradient

o  Intracellular location

o  Half-life of elimination of activity from plasma

o  Distribution of total activity from organ to organ

·  Isoenzymes

o  Isoenzymes I

§  True isoenzymes are due to the existence of more than one gene locus coding for the structure of the enzyme protein (~1/3 of human enzymes)

§  Enzymes that are oligomeric or made up of subunits from distinct structural genes are included within the definition of isoenzymes

o  Isoenzymes II

§  Due to the different environment of various cell types the distribution of isoenzymes is not uniform throughout the body

§  Tissue-specific differences in the distributions of some isoenzymes can form the basis for organ specific diagnosis through isoenzyme measurement (e.g. CKMB)

o  Isoenzymes III

§  Each creatine kinase molecule has two subunits

§  There are two different subunit genes --M and B

·  There are three possible combinations of subunits: MM, MB, and BB

Myocardial Infarction

·  Sequence of events:

o  Coronary artery occlusion

o  Myocardial ischemia

o  Anoxia

§  Depletion of ATP, Accumulation of metabolites, e.g. lactate

o  Reversible damage

§  Leakage of ions and water, Cellular swelling

o  Irreversible damage Breakdown of membranes

§  Leakage of intracellular proteins

o  Cell death and tissue necrosis

§  Inflammatory response, local response to injury

o  Injury may be reversible for up to 2 hours of ischemia

·  Reperfusion of ischemic heart muscle needs to occur within several hours to reduce injury to heart tissue.

·  Treatment could include thrombolytic therapy or angioplasty to physically remove coronary obstructions with a catheter.

The Roles of Serum Markers

·  Diagnose the 400,000 cases of MI not detected by ECG

·  Rule out MI in the 4,100,000 patients sent home and 400,000 patients admitted but who do not have an MI.

·  Patients with non-diagnostic ECG are observed for 6-8 hours after onset of chest pain and checked to see that serum markers for MI are normal.

Early Markers of Myocardial Infarction (4-12 hours post-MI):

·  Myoglobin (Increased by cardiac injury, minor skeletal muscle injury, or renal failure)

Intermediate markers for myocardial infarction (8-48 hours post-MI)

·  Creatine kinase (CK) Mostly MM isoenzyme. Not specific for cardiac injury—abundant in all muscle.

·  Creatine kinase-MB isoenzyme (Includes both isoforms. Increased by MI, by major skeletal muscle injury, by chronic muscle regeneration as in chronic muscle disease or extreme muscle training, or by renal failure)

Intermediate/Late markers for myocardial infarction (8 hours – 7 days post-MI)

·  Cardiac Troponin I or Troponin T:

o  Highly sensitive due to low normal serum concentration.

o  Specific for cardiac injury. Does not occur in other tissues.

o  Not elevated by renal failure

Late markers for myocardial injury (48-168 hours post-MI)

·  Troponin I or T (Remains elevated for about 1 week after myocardial injury).

Case 3

·  A 49-year-old laborer is brought to the emergency room complaining of an aching chest pain that had begun 2 hours earlier, after he ate four chili dogs with onions at lunch. He has a history of poorly controlled high blood pressure and smokes two packs a day. He had a recent physical exam where his doctor said his heart sounded fine. His cholesterol was checked and it was normal – 190 mg/dL. His father died at age 55 of an acute myocardial infarction and a brother had a nonfatal myocardial infarction within the last year. On physical examination, the patient is found to be nauseous and is mildly dyspneic. The EKG showed no ST segment elevation or Q-waves.

·  Typical MI changes include pathological Q waves (transmural), ST-segment and T-wave changes (nontransmural).

·  Differential diagnosis for this patient includes dyspepsia (heartburn), gastric ulcer, esophageal reflux and MI.

·  Does the lack of EKG abnormalities indicate that this patient is unlikely to have an MI?

o  No

·  Is EKG a sensitive and specific test for MI?

o  Not really

·  Diagnostic sensitivity?

o  Troponin, is highly sensitive, CKMB isn’t too bad of a marker, but the rest are basically non specific

·  WHO criteria for AMI?

o  High stakes for accurate diagnosis.

·  Do serum markers have a role when there is a diagnostic ECG?

o  Yes

o  Markers like troponin have a way of telling you how bad an MI is

§  Higher the troponin, the worse the infarct

·  Does a normal cardiac marker level rule out an MI?

o  No. Serial measurements are the key.

·  Does the normal cholesterol level in this patient mean that MI is unlikely?

o  No. Only about 50% of MI’s can be linked to elevated cholesterol.

·  What is the current recommended cholesterol screening strategy?

o  Total cholestorol, triglyceride, HDL, and LDL

·  What other factors may contribute to increased risk of MI?

o  Non modifiable risk factors

§  Age, family history, male

o  Modifiable risk factors

§  Hypertension

§  Diet

§  Smoking

·  What are the pro’s and con’s of the cardiac markers used in this case?

o  Major limitation is lack of specificity, however this can be overcome to a large extent by using the CK-MB/CK ratio

o  Shorter time window for elevation compared to troponins

o  May be useful in:

§  Detecting reinfarction

§  confirming Troponin elevations

·  Troponins

o  Highly sensitive and specific for myocardial damage

o  Limitations

§  Heterophile antibodies continue to be a problem (uncommon)

§  Must be interpreted with respect to time from onset of MI

§  Standardization is poor for cTnI

·  Heterophilie antibodies

o  False increase: HA binds to both the capture and detection antibody simulating the presence of antigen (Note: many/most HA are not species specific)

o  False decrease: HA may bind to capture antibody in such a way as to block the antigen-binding site

·  Myoglobin

o  May improve diagnostic sensitivity when used in combination with other markers (esp. CK-MB)

o  May help with detection of successful reperfusion

o  Limitations

§  Poor specificity limits the usefulness of myoglobin in diagnosis of AMI

§  Time period of usefulness is 2-4 hours post AMI and main value is rule out

§  Not all studies demonstrate improved sensitivity at early time points

·  How would your assessment of this patient change if his Troponin I was 12 mg/L?

o  Rise and fall pattern

o  Cardiac injury

Liver Disease

·  Laboratory studies are relatively useful in helping to detect liver disease and to help distinguish whether there is active liver cell necrosis (hepatitis) or an obstructive process

·  Laboratory tests are often called liver function tests (LFTs) although most measure damage rather than function

·  Pre-hepatic bilirubin is unconjugated

·  Bilirubin is conjugated in the liver

·  Bile is secreted into duodenum

Biliary obstruction

·  Gallstones--usually cholesterol stones, occasionally pigment stones if chronic hemolysis

·  Tumors obstructing biliary tract

·  Biliary cirrhosis

·  Biliary atresia

·  Markers

o  Alkaline Phosphatase:

§  cell membrane associated enzyme

§  increased synthesis in response to extrahepatic obstruction of the biliary tree

§  up to 10-12 fold increase

o  Bilirubin:

§  Heme breakdown product that is metabolized in the liver

§  Conjugation of bilirubin to glucuronic acid in the liver precedes its secretion into the bile

o  Gamma glutamyl transferase (GGT):

§  Induced by cholestasis, alcohol and other drugs such as Phenobarbital

·  Diagnosis

o  Jaundice due to increased conjugated bilirubin (direct bilirubin)

o  Increased alkaline phosphatase and gamma glutamyl transferase due to induction of enzyme synthesis

o  Mild increase in ALT and AST (little cellular necrosis)

o  Identify obstruction by ultrasound or other technique

Case 4

·  A 45-year-old woman is seen in the Emergency Room because of fever, chills, nausea and vomiting, and pain in the right upper quadrant of the abdomen. She has had repeated attacks of these symptoms but this one had progressed over a three day period. Examination noted a very obese woman with jaundice. There was tenderness and board-like spasm in the right upper quadrant of her abdomen.

·  What are the most likely diagnoses?

o  Gallbladder attack – cholecystitis

o  MI

o  Pancreatitis

o  Pregnancy

o  Hepatitis

o  Stones most common reason for attacks (95%)

o  Common >10% of adult population with women more predisposed.

o  Most gallstones remain silent and are never treated

·  Lesions in what other organ results in increased serum alkaline phosphatase activity?

o  Bone

o  Placenta

o  Kidney

o  Small intestine

·  Why is alkaline phosphatase increased in hepatobiliary obstruction?

o  The livers response to biliary tree obstruction is increased synthesis of ALP.

o  Due to enzyme induction in the hepatocytes adjacent to the biliary canaliculi.

o  Some of this newly synthesized enzyme enters the circulation to raise levels

o  Typically highest response is from a complete outside-the-liver block (>3 fold and up to 10-12 fold increase) while only a 2-3 fold increase from an inside-the-liver block.