★ Must-know
Borosilicate glass tolerates prolonged heating and sterilization but may cloud in strong alkali and scratch, whereas aluminosilicate glass is six times stronger and more resistant to scratching and alkali attack.
Volumetric transfer pipettes have one calibration mark and are used for nonviscous samples and standards, whereas graduated pipettes have subdivisions for measuring and delivering fractions of their capacity.
Air-displacement micropipettes separate piston and sample with an air cushion, whereas positive-displacement micropipettes place the piston in direct contact with the liquid and are recommended for viscous, dense, or problematic samples.
Further detail
Flint glass is inexpensive soda-lime glass made from silicon, calcium, and sodium oxides, and released alkali can cause errors in some determinations.
Low-actinic amber-colored or coated glass reduces light transmission and is used to contain photosensitive substances.
Centrifuge speed and temperature are checked with a tachometer or strobe light and thermometer, respectively, and centrifuge quality control is performed quarterly.
Air-displacement pipettes cushion the sample with air; positive-displacement pistons touch the sample.
★ Must-know
📌 ACS analytical-grade reagents are suitable for most analytical procedures, while ultrapure spectrograde or nanograde reagents are used when maximum purity and minimal interference are required.
Type I reagent water is the purest and is used for standards and procedures requiring high accuracy and precision; Type II suits most laboratory procedures, and Type III is used for some qualitative tests, glassware washing, and as feed water for Type I or II.
For a body-fluid spill, apply 5.25% sodium hypochlorite or 10% bleach diluted 1:10 and maintain at least 20 minutes of contact time.
Class II biological safety cabinets use two HEPA filters and protect both the worker and work; Class III cabinets are gas-tight negative-pressure enclosures with glove ports for extremely hazardous organisms.
Further detail
A HEPA filter is 99.97% efficient at 0.3 microns, while a ULPA filter is 99.99% efficient at 0.12 microns.
A threshold limit value is the allowable exposure during an 8-hour shift and is inversely proportional to toxicity.
📐 Formula - Molarity is moles of solute per liter of solution, expressed as .
📐 Formula - Normality is equivalents of solute per liter of solution, with equivalent weight given by .
📐 Formula - Diluted-solution calculations use .
📌 To convert a conventional unit to an SI unit, multiply by the listed conversion factor; to convert SI to conventional units, divide by that factor.
📐 Formula - Coefficient of variation is calculated as , and a lower CV indicates greater precision.
★ Must-know
📌 Accuracy is closeness to the true value, whereas precision is the ability to produce results that agree closely with one another.
📌 Random error affects precision unpredictably, whereas systematic error consistently biases observations in one direction and affects accuracy.
Further detail
📐 Formula - The standard deviation index is calculated as , and an SDI greater than 2.0 indicates poor performance.
Systematic error persists in one direction; random error scatters results and reduces precision.
★ Must-know
The tourniquet should be applied for less than one minute, and a blood-pressure cuff used for venipuncture should be inflated to 40–60 mm Hg.
The evacuated-tube order of draw is:
Hemolysis can increase intracellular analytes including potassium, magnesium, phosphate, iron, LD, AST, ALT, ALP, and CK, while decreasing lipase and glucose measured by hexokinase.
Further detail
EDTA chelates calcium and is used in lavender hematology and pink crossmatch tubes; it can falsely increase sodium and potassium and decrease calcium, magnesium, iron, phosphate, CK, ALP, and amylase results.
Sodium fluoride prevents glycolysis and is used primarily for glucose testing, but it can falsely decrease BUN and uric acid by inhibiting urease and uricase.
For arterial puncture, the modified Allen test checks that the ulnar artery can supply the hand before the radial artery is used.
ETS draw order: culture, light blue, red, SST, PST, green, lavender, gray.
★ Must-know
📌 A blank subtracts absorbance not due to the analyte and sets zero absorbance, a standard has known purity and concentration for calibration or quantitation, and a control has target values for monitoring assay performance.
📌 Turbidimetry measures light blocked by a particle suspension, whereas nephelometry measures light scattered by it.
Further detail
📐 Formula - Absorbance and percent transmittance are related by .
A double-beam spectrophotometer splits light between reference and sample cuvets to compensate for light-source intensity variation.
Fluorometry measures light emitted by excited molecules and is about a thousand times more sensitive than spectrophotometry, but fluorescence can be quenched by prolonged UV exposure, increased temperature, pH changes, contaminants, or very high analyte concentration.
★ Must-know
📐 Formula - Diagnostic sensitivity is , and diagnostic specificity is .
📌 Positive predictive value is the probability that a positive result indicates disease, whereas negative predictive value is the probability that a negative result indicates absence of disease.
Further detail
Scintillation counting detects flashes of light with a photomultiplier tube; gamma counters detect iodine-125 or iodine-131 labels, while beta counters detect tritium or carbon-14 labels.
Continuous-flow automation pumps liquids through continuous tubing, where air bubbles separate and clean samples but the system can cause carry-over and waste reagents.
★ Must-know
📌 Microparticle capture enzyme immunoassay (MEIA) is heterogeneous, uses microparticles, an enzyme label and a fluorogenic substrate, requires separation, and is applied to high-molecular-weight substances; fluorescence polarization immunoassay (FPIA) is homogeneous, uses polarized light to excite a fluorescent label, requires no separation, and is applied to low-molecular-weight substances.
📌 Continuous-flow analysis pumps liquids through tubing and risks carryover and reagent waste; centrifugal analysis transfers liquids into separate cuvettes and supports batch analysis but has low throughput; discrete analysis uses separate containers and supports batch, random-access, and STAT testing.
Further detail
Ion counting detects scintillations as electrical impulses using a photomultiplier tube; crystal scintillation is used for gamma counting with iodine-125 or iodine-131 labels, while liquid scintillation is used for beta counting with hydrogen-3 or carbon-14 labels.
Point-of-care testing reduces turnaround time and supports electronic documentation through connectivity to a laboratory information system; glucose monitors are the most commonly used POCT devices, and quality control is recommended once per day of use.
MEIA separates; FPIA does not.
★ Must-know
Insulin, produced by pancreatic islet beta cells, is the only hypoglycemic hormone; it promotes cellular glucose uptake, glycolysis, glycogenesis, and lipogenesis, and inhibits glycogenolysis.
Glucagon, produced by pancreatic islet alpha cells, is a major hyperglycemic hormone that promotes gluconeogenesis and glycogenolysis and inhibits glycolysis.
Hypoglycemia is a plasma glucose level below 50 mg/dL, and its panic value is below 40 mg/dL; symptoms may appear at 50–55 mg/dL and include neurogenic symptoms such as tremulousness and hunger or neuroglycopenic symptoms such as blurred vision and seizures.
📌 Whipple’s triad requires symptoms of hypoglycemia, low plasma glucose, and relief of symptoms after glucose correction; insulinoma diagnosis additionally involves symptoms, plasma glucose below 50 mg/dL, increased insulin, proinsulin, and C-peptide, and decreased β-hydroxybutyrate.
Further detail
📌 Fasting or post-absorptive hypoglycemia occurs after 10 hours without food and may result from hyperinsulinism, hormonal deficiencies, genetic disorders, autoimmunity, or drugs; reactive or post-prandial hypoglycemia usually occurs within 4 hours after a meal.
Insulin lowers glucose; glucagon raises it.
★ Must-know
📌 Type 1 diabetes results from autoimmune or idiopathic beta-cell destruction causing absolute insulin deficiency, whereas type 2 diabetes involves insulin resistance with progressive insulin deficiency; gestational diabetes is glucose intolerance during pregnancy that may resolve postpartum but converts to type 2 diabetes in 30–40% of cases within 10 years.
📌 Diabetes diagnostic thresholds are random plasma glucose above 200 mg/dL with symptoms, fasting plasma glucose at least 126 mg/dL, 2-hour oral glucose tolerance test glucose above 200 mg/dL, or HbA1c above 6.5%; elevated results require confirmation as specified for the test.
Prediabetes ranges are fasting glucose 100–125 mg/dL, 2-hour glucose 140–149 mg/dL, and HbA1c 5.7–6.4%; fasting testing requires at least 8 hours without food, and the OGTT uses a 75 g glucose load after 8–14 hours of fasting.
Gestational diabetes screening uses a 50 g, 1-hour OGTT; diagnosis uses a 100 g, 3-hour OGTT and requires at least two values meeting or exceeding fasting 95 mg/dL, 1-hour 180 mg/dL, 2-hour 155 mg/dL, or 3-hour 140 mg/dL.
Further detail
📌 HbA1c reflects average glucose over the preceding 2–3 months but is unreliable in hemolytic disorders and hemoglobinopathies; fructosamine represents glycosylated albumin and reflects the preceding 2–3 weeks when HbA1c is unreliable, but is unreliable when serum albumin is below 3 g/dL.
★ Must-know
📌 Copper-reduction glucose assays are nonspecific because any reducing agent can produce a falsely increased result, while enzymatic methods are more sensitive and specific; hexokinase is the reference method and is not subject to interferences described for the other methods.
Further detail
Whole-blood glucose is 10–15% lower than plasma glucose, capillary glucose is 2–5 mg/dL higher than venous glucose, and ADA glucose goals are 70–130 mg/dL before meals and below 180 mg/dL after meals.
When prompt analysis is not possible, sodium fluoride or a serum separator tube can limit glycolysis; glucose otherwise falls by 7 mg/dL/hour at room temperature or 2 mg/dL/hour at 4°C, and 10% contamination with 5% dextrose raises plasma glucose by 500 mg/dL.
Glucose oxidase acts specifically on β-D-glucose, which constitutes 65% of plasma glucose, while α-D-glucose constitutes 35% and is converted to the beta form by mutarotase; peroxidase-coupled detection can be affected by interfering agents.
Galactosemia results from GALT deficiency, can cause mental retardation, failure to thrive, and galactosuria, and is included in newborn screening; galactose produces a positive Clinitest because it is a reducing sugar.
★ Must-know
📌 Chylomicrons transport dietary triglycerides from the intestine and contain 86% triglyceride, whereas VLDL transports liver-produced endogenous triglycerides and contains 55% triglyceride; LDL carries cholesterol to peripheral tissues, while HDL transports excess tissue cholesterol toward catabolic sites.
Further detail
Lipoproteins have a surface of apolipoproteins, phospholipids, and non-esterified cholesterol and a hydrophobic core containing triglycerides and cholesteryl esters; LCAT catalyzes cholesterol esterification, which removes cholesterol’s polarity.
Chylomicrons float to form a creamy plasma layer after standing, VLDL makes plasma turbid, and LDL does not alter plasma clarity; IDL is a transient VLDL remnant, β-VLDL occurs in type 3 hyperlipoproteinemia, Lp(a) is associated with cardiovascular disorders, and LpX is associated with obstructive biliary disease and LCAT deficiency.
Abetalipoproteinemia is an autosomal recessive disorder involving absence of Apo B-containing lipoproteins and mutations in MTTP on chromosome 4, whereas hypobetalipoproteinemia is autosomal dominant and involves mutation of the Apo-B gene; Tangier disease is autosomal recessive, results from ABCA1 mutation, and causes absence of HDL, Apo A-I, and Apo A-II.
LDL carries cholesterol to tissues; HDL returns it toward catabolic sites.
★ Must-know
Lipid profiles require 12–14 hours of fasting, but cholesterol testing does not require fasting; HDL-C is routinely measured after precipitating Apo B-containing lipoproteins, and LDL-C is calculated by the Friedewald equation when triglyceride levels permit.
Desirable total cholesterol is below 200 mg/dL, borderline high is 200–239 mg/dL, and high is above 240 mg/dL; normal triglycerides are below 150 mg/dL, borderline high is 150–199 mg/dL, high is 200–499 mg/dL, and very high is above 500 mg/dL.
Further detail
📌 The Friedewald LDL-C equation uses a triglyceride divisor of 5 for mg/dL or 2.175 for mmol/L and is unreliable above 400 mg/dL triglycerides; direct LDL measurement is used when calculation is unreliable, and the source also gives the De Long divisor as 6.5 for mg/dL or 2.825 for mmol/L.
Chemical cholesterol methods include saponification with KOH, lipid extraction with Bloor’s reagent, cholesterol precipitation with digitonin, and color development; current cholesterol reference measurement is GC-MS, while IDMS is described as the definitive gold-standard method.
Enzymatic cholesterol measurement uses cholesterol esterase and cholesterol oxidase followed by a peroxidase-coupled chromogen reaction; the oxidized quinoneimine product is red-purple and its absorbance at 500 nm is proportional to cholesterol concentration.
Reduced GFR → creatinine accumulates in plasma.
★ Must-know
📌 Urea is freely filtered and 40–60% is reabsorbed, whereas creatinine is filtered, not reabsorbed, and minimally secreted; plasma creatinine varies with muscle mass and is inversely related to GFR.
Creatinine clearance estimates GFR using when body surface area correction is needed; its reference intervals are 85–125 mL/min in males and 75–115 mL/min in females, and collection of 24-hour urine is required.
Protein structure is described at four levels: primary structure is the number and types of amino acids in a specific sequence; secondary structure includes α-helices and β-pleated sheets stabilized by hydrogen bonds; tertiary structure is the molecule’s three-dimensional conformation; and quaternary structure results from interactions among multiple protein subunits held by noncovalent forces.
In protein electrophoresis at pH 8.6, serum proteins carry a net negative charge and migrate from sample wells near the cathode toward the positive anode.
📌 A protein has a negative charge when pH is above its pI, a positive charge when pH is below its pI, and zero net charge when pH equals its pI.
Further detail
📌 Inulin clearance is the gold standard for GFR but requires intravenous infusion and timed urine collections; cystatin C is measured in plasma without urine collection, is inversely related to GFR, and is unaffected by diet, gender, race, age, or muscle mass, whereas β-trace protein is unaffected by glucocorticoids.
Uric acid is produced by purine catabolism, is freely filtered and 98–100% reabsorbed, and can deposit in joints and tissues above 6.8 mg/dL; reference intervals are 3.5–7.2 mg/dL in males and 2.6–6.0 mg/dL in females.
Ammonia is a neurotoxic product of amino-acid catabolism metabolized in the liver through the urea cycle; arterial blood is the preferred specimen, samples should be kept in ice water until cell separation, and the reference interval is 19–60 µg/dL.
Glutamine is the most abundant amino acid in the body, is synthesized from glutamic acid, and helps transport ammonia to the liver for metabolism.
Support media and stains include:
High-resolution protein electrophoresis uses higher voltage, a cooling system, and more concentrated buffer to separate proteins into 12 or more bands and detect minor or unusual monoclonal bands.
Primary → secondary → tertiary → quaternary structure.
★ Must-know
Decreased albumin lowers plasma oncotic pressure and can cause edema and transudative effusion.
α1-antitrypsin is the most abundant protein in the α1 band, is a positive acute-phase reactant, and inhibits proteolytic enzymes including chymotrypsin, plasmin, thrombin, collagenase, and elastase.
α2-macroglobulin is the largest non-immunoglobulin protein and a protease inhibitor; its concentration rises ten-fold in nephrotic syndrome and other protein-losing conditions.
Haptoglobin is a positive acute-phase reactant that binds free hemoglobin and preserves iron; its concentration decreases in intravascular hemolysis as the haptoglobin–hemoglobin complex is removed by the liver.
Transferrin transports ferric iron, is a negative acute-phase reactant, and increases in iron-deficiency anemia and pregnancy while decreasing in infections, liver disease, and nephrotic syndrome.
C-reactive protein is the most sensitive positive acute-phase reactant, enhances phagocytosis, and can increase up to 1000 times during inflammation.
Further detail
Pre-albumin is a better indicator of malnutrition than albumin and transferrin and forms a distinct band in CSF electrophoresis, where it is the second most abundant protein.
Elevated serum albumin reflects dehydration and is a relative increase that returns to normal when fluids are administered; the course identifies no physiological disease that increases liver production of albumin.
Albumin may decrease with:
α1-antitrypsin deficiency produces an almost flat α1 band and is associated with pulmonary emphysema and severe juvenile hepatic disorders that may result in cirrhosis.
α1-acid glycoprotein (orosomucoid) is a positive acute-phase reactant that binds progesterone and some basic drugs, remains negatively charged even at acid pH, and has high carbohydrate content.
Ceruloplasmin is a copper-binding protein with oxidase activity and is a positive acute-phase reactant; it decreases in Wilson’s disease, malnutrition, malabsorption, and severe liver disease.
📐 Formula - Transferrin in mg/dL is calculated as , where TIBC is in mg/dL.
Albumin and transferrin fall in inflammation; positive acute-phase proteins rise.
★ Must-know
📌 Monoclonal gammopathy causes an absolute increase in one immunoglobulin, usually IgG or IgM, producing a sharp M spike; multiple myeloma is associated with increased IgG and Waldenström macroglobulinemia with increased IgM.
Urine total protein is quantified using a 24-hour urine specimen; its reference range is 1–14 mg/dL and less than 100 mg/day.
Urine albumin reference is less than 30 mg/day; microalbuminuria is increased urinary albumin not detected by dipstick and can precede diabetic nephropathy.
Further detail
Protein loss, including nephrotic syndrome, protein-losing enteropathy, blood loss, or extensive burns, is associated with decreased albumin and gamma globulins and increased α2 globulins, including a ten-fold rise in α2-macroglobulin.
Dehydration causes relative hyperproteinemia that returns to normal when the patient is hydrated.
CSF is a plasma ultrafiltrate formed in the brain ventricles, and its protein reference range is 15–45 mg/dL.
Protein loss lowers albumin but can raise α2-macroglobulin.
★ Must-know
📌 Myoglobin is a heme-containing oxygen-carrying protein in skeletal and cardiac muscle that rises earlier than troponin after myocardial infarction but lacks cardiac specificity and is more useful for ruling out than establishing myocardial infarction.
Cardiac troponins are the gold-standard markers for acute coronary syndrome because they are cardiac-specific and remain elevated; cTnI has greater cardiac specificity than cTnT.
BNP is secreted by myocardial ventricles in response to volume expansion and pressure overload, promotes vasodilation and sodium and water loss, and increases in congestive heart failure; its reference range is below 100 pg/mL.
Further detail
After acute myocardial infarction, myoglobin rises in 1–3 hours, peaks in 5–12 hours, and returns to normal in 18–30 hours.
After myocardial infarction, cTnT rises in 3–4 hours, peaks in 10–24 hours, and returns to normal in 10–14 days; cTnI rises in 3–6 hours, peaks in 14–20 hours, and returns to normal in 5–10 days.
Cystatin C is an endogenous serum marker of GFR measured by immunoassay without collecting a 24-hour urine specimen and is useful when creatinine measurement is inappropriate.
Myoglobin rises early but lacks cardiac specificity; troponins are cardiac-specific.
★ Must-know
The Kjeldahl method is the ultimate reference method for protein measurement and estimates protein from nitrogen content, assuming nitrogen is 16% of protein weight and using a factor of 6.25.
The biuret method detects peptide bonds through a violet or purple complex formed when cupric ions bind peptide bonds in alkaline medium; the color intensity is proportional to protein concentration.
The liver stores glycogen, fat-soluble vitamins, and synthesizes or metabolizes lipids, lipoproteins, carbohydrates, proteins, and coagulation factors; it detoxifies drugs, poisons, unconjugated bilirubin, and ammonia and secretes bile containing conjugated bilirubin.
📌 Unconjugated bilirubin is nonpolar, water-insoluble, indirectly reacts with diazo reagent, is not excreted in urine, and can affect the central nervous system; conjugated bilirubin is polar, water-soluble, directly reacts with diazo reagent, and is excreted in urine.
📌 Pre-hepatic hemolysis causes unconjugated hyperbilirubinemia with increased urine urobilinogen and negative urine bilirubin, whereas post-hepatic biliary obstruction causes conjugated hyperbilirubinemia with increased urine bilirubin, decreased urine urobilinogen, and pale stools.
Total bilirubin assays use diazo reagent with an accelerator to measure all fractions, direct bilirubin assays use diazo reagent without an accelerator to measure conjugated and delta bilirubin, and indirect bilirubin is calculated as total minus direct bilirubin.
The enzyme classes are:
In the catalytic mechanism, enzyme and substrate form an enzyme–substrate complex that either produces product and releases the enzyme or dissociates if reaction requirements are not met; the enzyme is not consumed.
📌 First-order kinetics occurs when enzyme concentration exceeds substrate concentration and substrate is rate-limiting; zero-order kinetics occurs when substrate exceeds available enzyme or active sites are saturated, making enzyme concentration rate-limiting.
Further detail
In the Kjeldahl method, sulfuric acid digestion converts protein nitrogen to ammonium ion, which is measured colorimetrically with Nessler’s reagent or distilled as ammonia and titrated.
The Lowry method is more sensitive than the biuret method and produces a deep blue color through oxidation of protein phenolic compounds with phosphotungstomolybdic acid.
UV absorption measures increased absorbance at 280 nm, mainly from tryptophan, tyrosine, and phenylalanine, without adding reagents.
Turbidimetric protein analysis measures decreased light transmission after protein precipitation, using trichloroacetic acid for CSF, sulfosalicylic acid for urine, or benzalkonium chloride for automated methods.
Delta bilirubin is conjugated bilirubin covalently bound to albumin, is not excreted in urine, and has a half-life of 17 days.
Take a 58-question practice quiz on Clinical Protein and Enzyme Analysis, with every answer explained.
1. A laboratory needs glassware for prolonged heating and sterilization, but it may also encounter strong alkali. Which comparison between borosilicate and aluminosilicate glass is accurate?
2. A technician needs to deliver a measured fraction of a pipette’s capacity. Which pipette design is suited to this task?
Memorize the key concepts of Clinical Protein and Enzyme Analysis with 95 flashcards.
What prolonged treatment can borosilicate glass tolerate?
Prolonged heating and sterilization.
How do volumetric transfer pipettes differ from graduated pipettes?
Transfer pipettes have one calibration mark; graduated pipettes have subdivisions for measuring and delivering fractions of their capacity.
How does an air-displacement micropipette separate its piston from the sample?
With an air cushion.
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