Quick Compendium of CP: Chemistry Enzymes, Serum Proteins, Acid-Base and Electrolytes ZW 7/7/08

Enzymes: Basics Michaelis-Menton kinetics: The rate of enzyme activity varies linearly with substrate concentration up to the point that the enzyme is fully saturated with substrate At this point, enzyme working as fast as it can (Vmax) Rate of reaction at this point varies only with enzyme concentration

Michaelis-Menton Kinetics

Enzymes Can measure enzyme concentration by: Using excess of substrate Or, measure whether a reaction has taken place Measure reaction products NAD to NADH to NAD (NADH absorbs light at 340nm, NAD does not)

Coupled Enzyme Assay (NAD/NADH) Aspartate (Asp) + a-ketoglutarate  oxaloacetate (OAA) + glutamate Does not utilize NADH Aspartate (Asp) + a-ketoglutarate  oxaloacetate (OAA) + glutamate + NADH malate + NAD Add excess NADH and aKG, along with catalysts AST and MD The disappearance of NADH (absorbance at 340nm) can be used as a reflection of AST (enzyme at first arrow)

Measurement of Enzyme Antigen The quantity of enzyme determined by immunoassay corresponds to the enzyme ACTIVITY Discordance between concentration and ACTIVITY usually takes the form of the immuno assay overestimating the activity May be due to serum enzyme inhibitors Deficiency in necessary cofactor Defective enzyme Proteolytically inactivated enzymes

Cofactors, Coenzymes Cofactors: Coenzymes: Substances that bind to an enzyme and enhance activity Include inorgantic cofactors like zinc, calcium, magnesium, iron Organic: also called coenzymes Coenzymes: Organic cofactors, include NAD, protein S, pyridoxine (vit B6)

Macroenzymes Ordinary enzymes bound to antibodies Has 2 effects: Makes it incapable of functioning Prevents it from being cleared from blood

Competitive INHIBITION

Non-competitive Inhibition

Uncompetitive Inhibition

Enzyme Units International Unit (IU): Katal The amount of enzyme that catalyzes the conversion of 1 micromole of substrate per minute Katal 1 katal= the amount of enzyme that catalyzes the conversion of 1 Mole of substrate per second 1IU = 16.7 katals

Hepatic Enzymes Liver transaminases AST and ALT AST ALT Cardiac muscle, liver, skeletal muscle, kidney, brain, lung, pancreas (in descending order) Found within cytoplasm (20%)and mitochondria (80%) ALT MORE SPECIFIC FOR LIVER, confined to liver and kidney Found entirely within cytoplasm

Hepatic Enzymes In children AST activity is slightly higher than ALT Pattern reverses at age 20 In adults, AST activity a little lower than ALT May reverse with old age Both AST/ALT activities higher in adult males over females, and in African-Americans Hemolysis raises AST/ALT

Hepatic Enzymes Intra-individual variation more significant for ALT than AST Marked diurnal variation (highest in afternoon) and day-to-day variation up to 30% Both AST/ALT elevated in heparin therapy to around 3X baseline In renal failure, both significantly lower than in healthy individuals

Hepatic Enzymes Lactate dehydrogenase (LDH): Present in numerous tissues, traditionally separated into 5 isoenzymes by electrophoresis Fastest moving are LD1 and LD2 Found in heart, RBC, kidney Slower moving are LD4 and LD5 LIVER and skeletal muscle LD3 in lung, spleen, lymphocytes, and pancreas LD6- “sixth” LD is sometimes seen migrating cathodal to LD5 PRESENCE THOUGHT TO BE A DIRE FINDING (hepatic insufficiency in setting of cardiovascular collapse)

LDH Concentrations: LD2>LD1>LD3>LD4>LD5 LD 1 elevation (with flipped LD ratio LD1>LD2): Acute MI Hemolysis Renal infarction Elevated LD4 and LD5: LIVER DAMAGE or skeletal insult Elevated LD1 and LD5: Acute MI with liver congestion Chronic alcoholism

Alkaline phosphatase Two types of phosphatases: Alkaline (optimum pH is 9): Bone, bile ducts, intestine, placenta Separate reference ranges for women and children Acid (optimum pH is 5): Found in prostate, RBC, and bone RBC acid phosphatase is susceptible to inhibition by 2% formaldehyde and resistance to inhibition by tartrate (this is also seen in hairy cell leukemia)

Alkaline phosphatase 4 isoenzymes by electrophoresis: Each displays characteristic degrees of inactivation by heating, urea incubation, and l-phenylalanine Heating produces significant inactivation of bone alk phos (bone burns), 50% inactivation of biliary alk phos, and NO inactivation of placental alk phos.

Alkaline phosphatase Biliary alk phos: Bone alk phos: Most sensitive marker of hepatic metastases Bone alk phos: Produced by osteoBLASTS and reflects bone reforming activity Highest levels seen in Paget’s disease of bone A specific immunoassay for bone alk phos available

Regan Isoenzyme Observed in about 5% of individuals with carcinoma Appears identical to placental alkaline phosphatase

Intestinal Alk Phos Elevation Can be factitious in non-fasting individuals, particularly in Lewis positive type B or O pts Ingesting a meal can elevate alk phos by 30% in 2-12 hours Repeat fasting alk phos

Alk Phos Minor elevations are a common clinical problem Usually higher in men than women Higher in African-Americans Threshold of 1.5 times normal limit for further investigation (repeat in 6 months if borderline) Causes: Pregnancy, CHF, hyperthyroidism, drugs

Alkaline phosphatase Sensitive indicator for hepatic metastases In women, investigation should include assay for anti-mitochondrial antibodies

Gamma-glutamyl transferase (GGT) best test to confirm if elevate alk phos if of biliary tree origin Found in biliary epithelial cell, particularly those of the small interlobular bile ducts and ductules Exquisitely sensitive to biliary injury Also elevated in: Steatosis, diabetes, hyperthyroidism, RA, acute MI, COPD

GGT Present within the smooth endoplasmic reticulum of hepatocytes Whenever there is induction due to excess toxin, GGT levels increase This includes warfarin, barbiturates, dilantin, valproic acid, methotrexate, EtOH 2-3X normal limit in heavy drinkers Returns to normal after 3 weeks abstinence and can be followed as marker for alcohol consumption

5’ Nucleotidase Main source is biliary epithelium Levels highest in cholestatic conditions Another test to confirm if elevated alk phos is due to hepatobiliary disease Low sensitivity, best as confirmatory test, utility less than GGT

Ammonia Hyperammonemia nearly always due to liver failure In children, it should raise the suspicion for an INBORN ERROR IN METABOLISM Sources of ammonia: Skeletal muscle and gut Bacteria in GI tract produce ammonia Normally functioning liver removes this ammonia and discards it in the form of urea which is excreted in urine

Ammonia Blood ammonia can become disastrously high when: Too much collateral circulation Excess protein in gut (excess hemoglobin from variceal bleed) SIGNIFICANT HEPATOCYTE DISFUNCTION In cirrhotic patients, these conditions are often met, and neurotoxicity can result

AMMONIA Measurement requires a FRESH specimen which has been CHILLED during transport and has undergone NO hemolysis Smoking patients must abstain for several hours before draw Anyone care to comment on the current state of the ammonia level as it can or cannot be ordered?

Bilirubin Unconjugated (indirect) bilirubin: Water-insoluble form produced by breakdown of heme Taken to liver tightly bound to albumin where it under goes glucuronidation to produce water-soluble (as in bile) conjugated (direct) bilirubin Conjugated bili excreted in bile where intestinal bacteria convert to urobilinogen

Bilirubin Urobilinogen ends up in feces, some of which is reabsorbed and excreted in urine Some urobilinogen is converted by colonic bacteria into brown pigments (complete biliary obstruction leads to yellow-white stool- the Silver Stool of Thompson)

Bilirubin Unconjugated bilirubin, even when it is quite high, does NOT appear in urine Thus, bilirubinuria indicates CONJUGATED hyperbilirubinemia 2 test methods Diazo-colorimetric methods: Rely on formation of colored dye through reaction of bili with diazo compound Without the addition of an accelerator (alcohol), only conjugated bilirubin is measured Addition of accelerators measures combined unconj and conjugated (total) bilirubin Direct spectrophotometry Bilirubin concentration measured by absorbance (455nm)

Causes of Hyperbilirubinemia Unconjugated: Hemolysis (extravascular) Blood shunting (cirrhosis) Right heart failure Gilbert syndrome Drugs: rifampin Crigler-Najjar syndrome Hypothyroidism

Causes of Hyperbilirubinemia Conjugated: Dubin-Johnson syndrome Hepatitis Endotoxin (sepsis) Pregnancy (estrogen) Drugs: estrogen, cyclosporine Mechanical obstruction: PBC, PSC, tumor, stricture, stone

Additional Hepatic Function Tests PT: Factor VII has half life of 12 hours (ON RISE EXAM) Sensitive marker for impaired hepatic synthetic function Impaired bile secretion can lead to Vit K deficiency (bile salts required for absorption) How do you distinguish between a prolonged PT because of cholestasis/impaired Vit K absorption and hepatocyte injury? Add parental vit K Gammaglobulins Serum gammaglobulins elevated in liver injury, especially autoimmune

Neonatal Jaundice Most cases of neonatal jaundice are entirely benign (“physiologic jaundice”) Hepatic enzymes not yet at full capacity leading to build-up of unconjugated bilirubin Usually noted between days 2-3 of neonatal life Usually peaks at 4-5 days; rarely exceeds 5-6 mg/dL

Severe hyperbilirubinemia in neonates Most common causes: Hemolytic disease of the newborn (HDN) Sepsis Poorly developed blood-brain barrier causes unconjugated bili to pass to CNS and cause damage (kernicterus)

When to worry about neonatal jaundice? Appearance in first 24 hours of life Rising bili beyond 1 week Persistance of jaundice past 10 days Total bili that exceeds 12 mg/dL Single-day increase of >5 mg/dL Conjugated bili (direct) that exceeds 2 mg/dL

Therapy for neonatal jaundice Phototherapy: Consider when bili exceeds 10 mg/dL before 12 hours of age; 12 mg/dL before 18 hours of age; 14 mg/dL before 24 hours of age Phototherapy converts unconj bili to a molecule that can be excreted WITHOUT conj Not useful for conj hyperbili Exchange transfusion: When bili exceeds 20 mg/dL

DDX of neonate hyperbili Jaundice in 1st 24 hours: Erythroblastosis fetalis Concealed hemorrhage Sepsis TORCH infection Jaundice between 3rd and 7th day: Bacterial sepsis (usually UTI origin) Arising after 1st week: Breast milk jaundice, sepsis, extrahepatic biliary atresia, cystic fibrosis, congenital paucity of bile ducts (Alagille syndrome), neonatal hepatitis, glactosemia, inherited hemolytic anemia (PK def, hered. Spherocytosis, G6PD def)

Lab Eval of Acute Liver Injury May be symptomatic, Jaundice, Elevated transaminases May be due to viral hepatitis (HAV, HBV, HCV), autoimmune hep, toxin, drug, ischemia, or Wilson disease Labs: Hepatitis serologies, ANA, ceruloplasmin, clinical history (new drugs usually cause damage within 4 months of starting)

Acute liver injury labs Acute viral hepatitis due to HAV, HBV most often leads to complete recovery Acute HCV goes to chronic HCV in >80% of cases Serologic testing for HAV, HBV are very dependable for diagnosing acute infx (IgM anti-HAV, IgM anti-HBc, HBsAg) Anti-HCV test only about 60% sensitive for acute infx HCV RNA testing 90-95% sensitive

Transaminases Acute hepatic injury due to ischemic or toxic injury produce PROFOUND elevations in transaminases- often >100X upper limit of normal (RARE in acute hepatitis) AST > 3,000 U/L = toxin in 90% of cases AST 10X upper limit of normal in acute viral hepatitis, but reaches this level RARELY in alcoholic hepatitis AST:ALT ratio is over 2 in 80% of pts with toxic, ischemic, and EtOH hepatitis (<1 in viral hep) Amount of transam elevation poorly correlates with LEVEL of injury

PT/BILIRUBIN PT (protime)- BILI Probably the best indicator of prognosis in acute hepatic injury >4.0 secs indicates severe injury/unfav prog BILI Jaundice in 70% of pts with EtOH, HAV Jaundice in <20% of pts with HBV, HCV Jaundice rare in kids with acute viral hep, rare in toxic or ischemic injury >15 mg/dL indicates severe liver injury, bad prognosis

Pancreatic Enzymes AMYLASE: Serum amylase = salivary and pancreatic isoenzymes On electrophoresis 6 bands result 1st three are salivary Slower 3 are pancreatic Can be separated by inhibition as well Salivary amylase sensitive to inhibition by wheat germ lectin (treticum vulgaris) Assays based on monoclonal Abs directed against specific isoenzymes are very accurate

Serum Amylase Rises within 2-24 hours of onset of acute pancreatitis Returns to normal in 2-3 days Higher levels don’t correlate with severity Higher levels are more specific for acute pancreatitis Persistance in elevation suggests complication like pseudocyst

Urine amylase Nearly all pts have concomitant increase in urine amylase Amylase primarily cleared by glomeruli Renal insufficiency = spurious amylase elevation Fractional excretion of compound (x) = FEx

Amylase Sensitivity of serum amylase for acute pancreatitis is 90-98% Specificity is only around 70-75% Specificity of urine amylase and FEamylase is higher

Additional causes of increased amylase Diabetic ketoacidosis, peptic ulcer dz, acute cholecystitis, ectopic pregnancy, salpingitis, bowel ischemia, intestinal obstruction, macroamylasemia, and renal insufficiency, opioid analgesics (contraction of sphincter of oddi)

Pancreatic Enzymes LIPASE: Unlike amylase, essentially specific for pancreas Rise parallels rise in amylase, but remains elevated for 14 days Less reliant on renal clearance than amylase Often considered superior to amylase in dx of acute pancreatitis

Lab Eval for Acute Pancreatitis Amylase limited in sensitivity and specificity Hypertriglyceridemia (common cause of acute pancreatitis) interferes with amylase assay (false negative) Lipase remains elevated longer giving greater sensitivity Others: trypsinogen-2 and elastase-1 (not used often) but have excellent negative predictive value

Acute Pancreatitis Prognosis Ranson Criteria: Aggressive management ICU admission Parenteral feeding Systemic antibiotics Provides specificity of 90% Cannot be assigned until 48 hours after admission Serum amylase/lipase are poor predictors of outcome

Etiology of Acute Pancreatitis Stones EtOH Viruses Inherited diseases: Mutations in Cationic trypsinogen (PRSS-1) Pancreatic secretory trypsin inhibitor (PSTI) Cystic fibrosis transmembrane conductance regulator (CFTR)

Pancreatic EXOCRINE function Tests include: Secretin-cholecystokinin (secretin CCK, secretin pancreozymin), fecal elastase-1, fecal fat FECAL FAT: Oil-red O stain and 72 hour fecal fat quant Stain has sensitivity of 70% Fecal fat quant quite sensitive for panc. Insuff.

Myocardial Enzymes CK (creatine kinase): Three isoenzymes distinguishable by electrophoresis CK-MM, CK-MB, CK-BB Fastest migrating is BB (CK1), then MB (CK2), then MM (CK3) CK-BB: found primarily in brain, with lesser amounts in bladder, stomach, and prostate. CK-MM (CK3) found in skeletal muscle and cardiac muscle (Skel.muscle: 99% MM, cardiac: 70%MM). In normal subjects, serum CK is 100% MM CK-MB (CK2) found in cardiac and skeletal muscle (card: 30%, skel: 1%), skeletal muscle is source of nearly all MB in circulation in serum

CK Now use immunoassays to measure CK Much faster and more accurate than electrophoresis (particularly in low/clinical range) Total CK enzymatic assay Ratio of CK-MB to total CK (RI: relative index) adds to ability to distinguish MI RI of 2% is usually cut-off

Troponin I Group of enzymes consisting of Troponin T (TnT) Troponin I (TnI) Troponin C (TnC) Involved in mediating the actin-myosin interactions that result in muscle contraction Immunoassays distinguish cardiac troponins (cTnI and cTnT) from skeletal muscle troponins

Troponin Vast majority of cardiac muscle troponin is bound to actin/mysosin Very small amount free in cytoplasm So: Immediate release of cytoplasmic troponin in MI (4-8 hours) And sustained release of bound troponin over next 10-14 days

Cardiac Troponin I Only cTnI is currently widely available for use in clinical diagnosis cTnT marginally less cardiospecific cTnI NOT elevated in skeletal muscle injury cTnI may be elevated in other forms of cardiac muscle injury(contusion, myocarditis)

Myoglobin Mgb is THE MOST SENSITIVE of the cardiac markers Earliest marker of acute MI Myoglobin should be elevated as soon as an infarcting patient present to ED LEAST CARDIOSPECIFIC of cardiac markers!

Ischemia-modified albumin (IMA) When albumin circulates through harsh environments (acidosis, hypoxemia, free radicals, altered calcium- all seen in ischemia), its ability to rapidly and tightly bind cobalt is altered Altered Cobalt Binding (ACB) assay: Add cobalt, measure unbound amount Measurement reflects ischemia modified albumin

B-type Natriuretic Peptide (BNP) Natriuretic peptides: Cause vasodilation and sodium excretion A-type: stored in granules in atrial myocytes, affected by atrial filling pressure, ventrilar wall tension B-type: synthesized in ventricular myocytes Correlates directly with ventricular wall tension N-terminal peptide fragment (N-terminal pro-BNP) is cleaved from pro-BNP to make active hormone BNP N-terminal pro-BNP is more stable, provides more longitudinal info Elevated in heart failure Distinguishes between cardiac/non-cardiac dyspnea Provides prognostic info for pts with CHF and acute coronary syndrome (ACS)

ACS Acute coronary syndrome: Encompasses many clinical situations with myocardial ischemic damage Stable angina, unstable angina, acute MI, sudden cardiac death Lab assays good at diagnosing MI, bad at diagnosing the remainder Usual biomarkers for necrosis (MB, myoglobin, troponin) are overall poor markers for non-AMI ACS BNP is predictive for both recurrence and higher likelihood of sudden cardiac death in non-AMI ACS C-reactive protein good predictor of development of ACS in healthy individuals

ACUTE MI Typical rise and fall of CK-MB or troponin with ischemic symptoms ECG changes Or interventionally demonstrated coronary artery abnormality TROPONINS: single positive troponin is highly specific for AMI Single low troponin has low sensitivity for negative predictor CK-MB: increase detectable within 3-6 hours of AMI, peaks at 20-24 hours, returns to normal in 72 hours; sensitivity using serial measurements approaches 100% Myoglobin: rapidly released from damaged muscle, early indicator, negative predictive value 2 hours post symptoms approaches 100%

SERUM PROTEINS Protein quantitation: Nitrogen count (Kjeldahl technique) is gold standard; involves acid digestion of protein to release ammonium ions which are quantified Assumption is serum proteins are 16% nitrogen by mass Colorimetry (Biuret technique) is recommended routine method for measuring total protein Absorbance from chelate with copper at 540 nm is proportional to total protein

Protein Separation Use PRECIPITATION Electrophoresis: Movement of proteins due to electrical potential Charge applied across a medium composed of solid support (gel) and fluid buffer. Charge creates electromotive force Solid support has slight neg charge and is drawn towards the anode (+ pole), but being solid, cannot move Compensatory flow of fluid buffer towards negative pole (cathode) This flow is called endosmosis- has capacity to carry substances suspended in medium

Protein Separation If proteins added, two forces are on the proteins: Electromotive force Endosmotic force Most proteins have negative charge Electromotive force pulls towards anode Endosmosis pulls towards cathode Gammaglobulins= weak net negative charge Electromotive force exceeds endosmotic force Move to variable extent towards anode

Serum Protein Electrophoresis SPEP: When electrophoresis is carried out on serum at pH 8.6 on agarose gel, then fixed and stained: Five distinct bands can be seen Fastest moving band is albumin Next fastest- two a bands (a1 and a2) Then the b band Then the g band (move very slowly)

Serum Protein Electrophoresis

Protein Electrophoresis Increasing endosmosis is used to separate gamma globulins into oligoclonal bands in CSF electrophorsis Capillary electrophoresis can also be used if there is Small sample size Needed automation Need for speed

Immunofixation Electrophoresis IFE Method for characterizing a suspected monoclonal band observed in SPEP or UPEP Much simpler to interpret than IEP Place pt serum into six wells in agarose gel Five different monospecific antisera are applied Anti-IgG, IgA, IgM, Kappa, and lambda Entire gel is stained IEP (immunoelectrophoresis)- not commonly used anymore

IFE

Serum Proteins Albumin Most abundant protein in human plasma 2/3 of total plasma protein Many functions: Maintains serum osmotic pressure, carries multiple substances Congenital absence NOT a serious problem (mild anemia and hyperlipidemia) Several allotypes: Most common is Albumin A When variant is present, might get 2 peaks

Albumin Clinical utility: Nutritional status (half-life is 17 days) Hepatic synthetic function (ESLD) Diabetic control In normal pts, up to 8% of albumin is glycosylated In diabetics with poor control, up to 25% may be glycosylated Negative acute phase reactant (decreases in inflammatory conditions)

Prealbumin Fastest migrating protein on SPEP Sparse, not normally seen on traditional SPEP Binds T4 and T3 Binds and carries retinal-binding protein:vitamin A complex Also, prealbumin is the precursor protein in senile cardiac amyloidosis Short ½ life of 48 hours True elevations seen in chronic EtOH, steroids Negative acute phase reactant

a1 antitrypsin (AAT) Major component of the a1 band Main function is to inactivate various proteases like tyrpsin and elastase SPEP can be used to screen for AAT deficiency Markedly positive acute phase reactant

a1-acid glycoprotein (orosomucoid) Briskly positive acute phase reactant Minor component of a1 band Major component of increased a1 band seen in inflammation May be used to monitor chronic inflammatory conditions like ulcerative colitis

a2 macroglobulin Protease inhibitor Serum concentration elevated in liver and renal disease Large size prevents its loss in nephrotic syndrome, leading to a 10-fold increase in concentration

Ceruloplasmin a2 protein Functions in copper transport Decreased serum ceruloplasmin important marker for Wilson disease, ddx includes: Hepatic failure malnutrition Menke syndrome Acute phase reactant: Elevated in inflammation and pregnancy

Haptoglobin Third major component of a2 Binds free hemoglobin Decreased or absent in acute intravascular hemolysis Very sensitive marker for hemolysis Haplotypes I and II: Phenotypes 1-1, 1-2, 2-2 2-2 phenotype is independent risk factor for CAD in diabetes Acute phase reactant

Transferrin Major b globulin Functions to transport ferric iron (Fe3); normally 30% saturated Marked increase in Fe def.; abnormally masquerades as an M-protein Increased in pregnancy and estrogen tx Decreases in acute phase; but rises is inflammation persists Blood brain barrier transports transferrin to CSF in a modified form (Tau-protein) Carbohydrate-deficient transferrin: superior to GGT as marker for EtOH abuse

Fibrinogen Also called b globulin In normal course of events, no fibrinogen in serum as it is consumed by formation of clot If specimen clots incompletely (heparinized pt) fibrinogen may be seen Can straddle the b-g interface When present in serum, may be misinterpreted as an M-protein May be seen in serum in: Dysfibrinogenemia, APL syndrome, liver dz, vitamin K def, or heparin

C-reactive protein (CRP) Produced in liver Predictive value of low level elevations (>2-3 mg/L) for cardiac events High sensitivity CRP (hsCRP) now available with sensitivity of <0.5 mg/L Distribution NOT Gaussian curve- Curve skewed significantly with dense cluster in very lowest CRP levels and long tail extending into >10 range Half of population has CRP >2

CRP Three categories based on CRP: Low level elevation may indicate: Normal CRP <3 mg/L High CRP >10 mg/L (active inflammation) Low-level elevations 3-10 mg/L (cellular stress) Low level elevation may indicate: Minor disease states Genetic factors Demographic variables Behavioral patters Individuals normal set point is inherited

CRP Low-level CRP elevation predicts poor outcome following cardiovascular events Also correlates with mortality in non-cardiac dzs

SPEP Patterns Normal serum: Bisalbuminemia: Invisible prealbumin band Very large albumin band Then, small peaked a1, a2, a bimodal B, and a broad gamma band Bisalbuminemia: Seen in heterozygotes for albumin allotypes Double albumin spike No clinical consequence

SPEP Patterns a1-antitrypsin (AAT) deficiency: Nephrotic syndrome: AAT is the major component of a1 band Genotype PiZZ individuals have a visibly and quantitatively decreased band Nephrotic syndrome: Massive loss of small proteins, particularly albumin Minimal change disease: especially high loss of albumin With other forms of nephrotic syndrome, gamma globulins also lost LARGE PROTEIN MOLECULES RETAINED Result: dimming of all electrophoretic bands, with exception of a2 which contains a2-MACROglobulin

SPEP Patterns Acute inflammation: Acute phase reactants account for increases in a1, a2 bands Albumin slightly increased Prolonged inflammation shows polyclonal gamma globulin increase Beta-gamma bridging (BODOR noted this has been on several Board Exams): Hallmark of CIRRHOSIS Also, hypoalbuminemia, blunted a1 and a2 Beta-gamma bridging mainly due to increase serum IgA

Monoclonal Gammopathy SPEP shows a prominent discrete dark band (M-spike, m-protein) usually within the gamma region (sometimes in B or a2) A monoclonal gammopathy (paraprotein) shows immunochemically homogeneous immunoglobulin (M-protein) in serum May be result of: Multiple myeloma Neoplastic proliferations like solitary plasmactyoma, MGUS, Waldenstroms macroglobulinemia (lymphoplasmacytic lymphoma), and CLL/SLL

Biclonal Gammopathy 2 M-proteins Occurs in 3-4% of cases If biclonal gammopathy has IgA spikes having a single light chain (by IFE): Most likely due to appearance of both monomers and dimers in SPEP Should be considered monoclonal

SPEP Hypogammaglobulinemia 10% of SPEPs When not due to myeloma: May be due to congenital deficiency, lymphoma, nephrotic syndrome, or corticosteroids Pts with myeloma and hypogammaglobulinemia are likely to have free-light chains in urine (Bence-Jones proteins)

IFE Indicated to characterize M-protein on SPEP Even if no m-spike is seen, do IFE if: Strong suspicion of myeloma (lytic bone lesions in 80 year old male) Systemic AL amyloidosis Hypogammaglobulinemia In pts with negative serum screens with high suspicion, do UPEP (for light chain disease) May be losing all of light chain in urine

M-protein Usually an intact immunoglobulin composed of 2 heavy and 2 light chains Sometimes, it is light chain only or very rarely, heavy chain only Systemic amyloidosis may result from monoclonal gammopathy, when M protein produced has unusual (amyloidogenic) properties

After diagnosis: Protein electrophoresis is used to follow disease progression and efficacy of treatment 3 quantitive results are important: M-protein concentration Degree of suppression of other immunoglobulins (IgG, IgA, IgM, whichever is not involved) Quantity of free serum light chain, especially in light chain only myelomas (extremely sensitive to myeloma recurrence after tx)

Hyperviscosity syndrome Normally, viscosity of serum is 1.5-1.8 centipoise (cp) Hyperviscosity syndrome: exceeds 3.0 cp Symptoms: Nasal bleeding, blurred vision, retinal vein dilation, neurologic symptoms

Cryoglobulins May be looked for in pts with M-proteins Precipitate reversibly at low temps Blood is drawn and kept at 37 degrees C until clotted Centrifuged at 37 degrees Remaining serum stored at 4 deg C for at least 3 days, then centrifuged at 4 deg C Any precipitate that is formed is CRYOPRECIPITATE and can be subject to electrophoresis

Cryoglobulins 3 types: Type I: monoclonal immunoglobulins associated with MM or Waldenstroms Type II: mix of monoclonal IgM and polyclonal IgG. IgM has Rheumatoid factor activity (anti-IgG). This is the most common type of cryo Type III: mix of two polyclonal cryoglobulins

Mixed Cryoglobulinemia Types II & III Found in variety of conditions: Lymphoproliferative d/o, chronic infx, chronic liver dz, autoimmune dz (SLE) Most common in women in 4th-5th decades of life 30-50% of cases have underlying HepC virus (most common cause) Clinical manifestation: palpable purpura, arthralgias, hepatoslenomegaly, lymphadenopathy, anemia, sensoineural defects, and glomerulonephritis Tx: corticosteroids, plasmapheresis, a-interferon

UPEP In proteinuria, UPEP can determine source of protein Glomerular proteinuria pattern: Strong albumin, a1, B bands Very large and very small proteins don’t make it into urine, leaving albumin, AAT, and transferrin Tubular proteinuria pattern: Weak albumin band, strong a1 and B bands Impaired tubular reabsorption of LMW proteins normally filtered freely by glomerulus (a2-macroglobulin, b2 microglobulin, light chains) Overflow proteinuria pattern: Monoclonal light chain (Bence Jones) Remember that these back up into SPEP in MYELOMA KIDNEY

CSF Protein electrophoresis Different than serum All proteins in serum, but smaller quantities Prominent prealbumin band and double beta (transferrin) band (transferrin conjugated to Tau-protein on way into CSF)