CC1 LAB

MODULE 1: QC Statistics

Mean (x̄) = Σxi/n — lab's best estimate of true value

SD = √[Σ(Xn-Mean)²/(n-1)] — quantifies precision; sets control limits (Mean ± 1SD, 2SD, 3SD)

CV% = (SD/Mean) × 100 — lower CV = better precision; used to compare methods/instruments

SDI = (Your Mean – Consensus Mean)/Consensus SD

• 0 = ideal; ±1–1.5 = acceptable/marginal; ±1.5–2.0 = marginal, may need corrective action

CVR = Your CV/Consensus CV

• <1 = better than peers; 1–1.5 = acceptable/marginal; 1.5–2.0 = marginal

Shift = sudden/abrupt change in control values → systematic error (light source failure, reagent lot change, calibration issue, instrument maintenance)

Trend = gradual loss of reliability → systematic error (deteriorating reagents, light source, calibration drift)

Systematic Error = trend/shift away from mean Random Error = random deviation from mean

Number of controls:

• 1 control → accept if within ±2SD

• 2 levels → apply Westgard Multirule

Westgard Rules:

If out of control: STOP testing → identify/correct problem → repeat testing → do NOT report patient results until resolved.

MODULE 2: Amino Acids & Proteins

Total Protein Methods:

Biuret reagent components: KOH (alkaline medium), Na-K tartrate (prevents Cu precipitation), KI (antioxidant)

Specimen: Serum preferred; hemolysis falsely elevates TP; lipemia interferes with Biuret

Albumin methods (Dye-binding): BCG and BCP most common; BCP preferred (more specific, precise); Bilirubin interferes with BCP.

A/G Ratio = Albumin/Globulin; Globulin = TP – Albumin

• ↓A/G: ↑globulin (myeloma, inflammation, RA) or ↓albumin (liver/kidney disease)

Electrophoresis: pH 8.6 buffer; albumin migrates farthest to anode; order: Albumin > α1 > α2 > β > γ

• Isoelectric point (pI): pH > pI → negative charge → migrates to anode

Key patterns:

• Nephrotic syndrome: ↓albumin, ↑α2/β

• Cirrhosis: ↓albumin, "beta-gamma bridge," ↑gamma

• Multiple myeloma: monoclonal gamma spike

• α1-antitrypsin deficiency: ↓α1 peak

• Inflammation (acute phase): ↓albumin, ↑α1/α2/β

Calculations:

• Total Protein (g/dL) = (Abs sample/Abs standard) × 6

• Albumin (g/dL) = (Abs sample/Abs standard) × 3

• If reagent blank given: subtract from BOTH sample and standard absorbance first

MODULE 3: Carbohydrates (Glucose)

Specimen: Serum/plasma preferred, unhemolyzed; separate within 30 min (glycolysis ↓ glucose 5-7%/hr); NaF inhibits glycolysis in whole blood.

Chemical Methods:

• Alkaline Copper Reduction: Folin-Wu, Nelson-Somogyi, Neocuproine, Benedict's

• Alkaline Ferric Reduction: Hagedorn-Jensen

• Condensation: Ortho-toluidine (Dubowski) — green color, 630nm

Enzymatic Methods (preferred):

• Glucose Oxidase (Trinder reaction): Glucose+O2+H2O → gluconic acid + H2O2 (glucose oxidase); H2O2 + chromogen → colored product (peroxidase). Interferences: uric acid, bilirubin, ascorbic acid (false ↓); bleach (false ↑)

• Hexokinase: MOST SPECIFIC/reference method; Glucose+ATP→G6P; G6P+NADP→NADPH (measured at 340nm); less interference

• Glucose Dehydrogenase: NADH = glucose conc; close agreement with hexokinase

Glucose Calculation: C = (Abs sample/Abs standard) × standard conc. (convert mmol/L to mg/dL: divide by 0.055)

Diagnostic Criteria for DM (ADA):

GDM criteria: Fasting >92mg/dL, 1h >180, 2h >153

HbA1c: reflects 2-3 month average; specimen = whole blood, EDTA; falsely decreased in hemolysis/hemoglobinopathy (shortened RBC lifespan)

Fructosamine: short-term (3-6 wk) control; useful when HbA1c unreliable

MODULE 4: Package Inserts (Key Values)

*Note: possible typo in original (likely 6 g/dL)

Unit conversions: Glucose mg/dL × 0.055 = mmol/L; Protein/Albumin g/dL × 10 = g/L

MODULE 5: Lipids & Lipoproteins

Fasting: TAG & LDL require fasting (12h ideal, 9h per NCEP-ATP III); TC & HDL don't require fasting.

Posture: standing→sitting causes 10% ↓ in TC/HDL/LDL (fluid shift); tourniquet >2min causes hemoconcentration (↑10-15% cholesterol)

Anticoagulants:

• Citrate: falsely low (osmotic effect)

• Heparin: acceptable but alters electrophoretic mobility (avoid for LPP electrophoresis)

• EDTA: MOST preferred — prevents lipid oxidation

Storage: Serum preferred over plasma for lipids (less protein aggregation); Samples for ultracentrifugation must NOT be frozen (destroys chylomicrons)

Cholesterol Methods:

• Chemical: Liebermann-Burchard (bluish-green), Salkowski (red), Abell-Kendall (reference method)

• Enzymatic: Cholesterol esterase → cholesterol oxidase → peroxidase; RED color, 500nm

HDL measurement: Precipitate non-HDL lipoproteins (dextran sulfate/Mg²⁺ most specific) → measure cholesterol in supernatant

Friedewald Equation:

• LDL (mg/dL) = TC – HDL – (TAG/5)

• LDL (mmol/L) = TC – HDL – (TAG/2.175)

• Invalid if TAG >400 mg/dL

MODULE 6: Renal Function

NPN composition of plasma: Urea 45-50%, amino acids 25%, uric acid 10%, creatinine 5%, creatine 1-2%, ammonia 0.2%

GFR: best overall indicator of kidney function; 150L filtrate produced daily; declines 1mL/min/year after age 20-30

Clearance formula: C(mL/min) = (U × V)/P × (1.73/A)

• U = urine conc, P = plasma conc, V = urine volume(mL), 1.73 = avg BSA, A = patient's BSA

Creatinine Clearance (endogenous, most common):

• Reference: Male 85-125 mL/min; Female 75-112 mL/min

• NOT affected by protein diet (unlike urea)

• Increased in: pregnancy, high cardiac output

• Decreased in: impaired kidney function, CHF, shock

Urea Clearance: ~50% of creatinine clearance (variably reabsorbed)

Cystatin C: not affected by muscle mass/age/diet/gender; GFR (mL/min) = 84.69 × Cystatin C(mg/L) × 1.384

Osmolality: Serum 275-295 mOsm/kg; Urine 300-900 mOsm/kg

• Serum Osmolality = 1.86×Na + Glucose/18 + BUN/2.8

Urea (BUN):

• Synthesized in liver from ammonia (via urea cycle)

• Early marker of acute kidney disease (rises before creatinine)

• Avoid Na citrate/fluoride as anticoagulants (inhibit urease)

• Reference: 6-20 mg/dL (adult)

• Conversion: Urea N × 2.14 = Urea (mg/dL)

Methods: Conventional (urease hydrolysis + Nessler's/Berthelot), Kinetic (urease-GLDH, measures NADH disappearance at 340nm — reference method), Chemical (Diacetyl monoxime, o-Phthalaldehyde)

BUN:Creatinine Ratio (normal 10-20:1):

• Low (<10:1): low protein diet, liver disease

• High (>20:1) + normal Cr: dehydration, pre-renal azotemia

• High (>20:1) + increased Cr: renal failure, post-renal azotemia

Creatinine:

• Byproduct of muscle metabolism (creatine/creatine phosphate breakdown)

• NOT reabsorbed, NOT recycled — solely excreted

• Not affected by protein diet

• Jaffe Reaction: creatinine + alkaline picrate → red-orange complex

• Reference method: Isotope dilution mass spectrometry

• Enzymatic: Creatininase-CK method (kinetic, measures NADH decrease at 340nm)

Uric Acid:

• From purine breakdown (guanine, adenine) via xanthine oxidase

• 98-100% reabsorbed in PCT; exists as monosodium urate

• Gouty arthritis = uric acid deposition in joints

• Lesch-Nyhan syndrome: HGPRT deficiency, X-linked, self-mutilation, treated with allopurinol

• Methods: Caraway (tungsten blue), Uricase (293nm), Coupled enzymatic (peroxidase, 545nm)

Ammonia:

• From amino acid metabolism; converted to urea in liver

• Reye Syndrome: viral infection + aspirin → hepatic damage → ammonia accumulation → coma (children)

• Specimen: ice immediately, avoid hemolysis; RBCs have 2-3x more ammonia than plasma

• Method: GLDH enzymatic (most common); Conway method (older, diffusion-based)

MODULE 7: Liver Function

3 Main Functions Tested:

1. Hepatic Synthesis: Total protein, Albumin, Prothrombin time (↑PT = liver not making clotting factors)

2. Conjugation/Excretion: Bilirubin, BSP dye test, Urobilinogen

3. Detoxification: Liver enzymes (ALT, AST, ALP, GGT, LDH), Ammonia

Bilirubin types:

• Unconjugated/Indirect: non-polar, albumin-bound, needs accelerator (diazo reagent)

• Conjugated/Direct: polar, water-soluble, reacts directly without accelerator

• Delta bilirubin: conjugated bilirubin bound to albumin (seen in hepatic obstruction)

Bilirubin Metabolism: RBC breakdown → heme → biliverdin (heme oxygenase) → bilirubin (biliverdin reductase) → bound to albumin → hepatocyte uptake → conjugation (bilirubin glucuronyl transferase) → excreted in bile → gut bacteria → urobilinogen

Methods:

• Malloy-Evelyn: methanol accelerator, 560nm

• Jendrassik-Grof: no accelerator (direct only); with caffeine-benzoate = total bilirubin; 600nm; preferred/candidate reference method

Specimen: protect from light (photo-oxidizes to biliverdin); avoid hemolysis

Reference ranges (adult): Total 0.2-1.0 mg/dL; Direct 0.0-0.2; Indirect 0.2-0.8

AST/ALT: elevated = hepatocellular damage (liver-specific enzyme leakage)

MODULE 8: Trace Elements

Essential in humans: Chromium, Cobalt, Copper, Fluorine, Iodine, Iron, Manganese, Molybdenum, Selenium, Zinc (only Cu, I, Fe, Se, Zn have well-characterized deficiency states)

Methods: AAS (flame for Zn/Cu; graphite furnace for Se/Cd/Pb), ICP-MS (ultratrace, method of choice)

Iron:

• Total body: 3-5g; 2-2.5g in hemoglobin

• Stored as ferritin/hemosiderin (liver, spleen, bone marrow)

• Deficiency: ↓serum iron, ↑TIBC/transferrin, ↓ferritin (most sensitive)

• Overload: Hemochromatosis (tissue damage present) vs. Hemosiderosis (no tissue damage)

• % Saturation = (Serum Iron/TIBC) × 100; normal 20-50%

Copper:

• Component of ceruloplasmin, cytochrome C oxidase, superoxide dismutase

• Deficiency: Menke's syndrome (X-linked, kinky hair)

• Excess: Wilson's disease (Kayser-Fleischer ring, hepatolenticular degeneration)

Zinc: cofactor for 300+ enzymes (ALP, carbonic anhydrase); deficiency → growth retardation, poor wound healing

Others: Cobalt (vit B12 component), Chromium (insulin activator, glucose metabolism), Fluoride (dental caries prevention), Manganese (enzyme activator, protein-bound), Molybdenum (oxidase cofactor), Selenium (glutathione peroxidase cofactor, antioxidant)