Clinical Chemistry - Exam #1

Ch 1-4, 6-7, 21

Standard deviation & SDI: Definition & Difference between value & mean

• describes distribution of all data points around the mean; the variance represents the average distance from the mean and every value in the data set

• difference between measured value & mean expressed as a # of SDs; may be pos or neg values

Standard Deviation & SDI: SDI = 0 & SDI formula

• SDI = 0 → value is accurate/100% agreement

• σ = √[ Σ(xᵢ - μ)² / N ]

Normally distributed curve: Total area under curve, % of values found within (±) 1, 2, & 3σ

• distribution is symmetric; total area under the curve is 1.0/100%

• 68.3% under the curve = (±) 1σ

• 95.4% under the curve = (±) 2σ

• 99.7% under the curve = (±) 3σ

Coefficient of variation (CV): Definition, Simplifies comparison of, CV highly precise, CV acceptable range, CV formula

• compare SDs with different units & reflects the SDs in %

• simplifies comparison of SDs of test results expressed in different units & concentrations

• CV of highly precise analyzers can be lower than 1%; acceptable range can be as high as 50%

• CV% = (SD/x-bar)100

Reagent grade water by resistivity/conductivity: Type II (high resistivity, water with more ions, resistivity & conductivity, general applications)

• High resistivity means low conductivity; water with more ions is less pure and will conduct more electrical current

• resistivity of >1, conductivity of <1, <50 ppb of TOCs

  • general applications like making buffers, pH solutions, microbio culture prep, cell culture incubators, feed instruments and analyzers, electrochemistry, sample dilution, radioimmunoassay, etc.

Analytical Sensitivity (define, LOD) vs Specificity (define)

1. how little, or how little of a change in an analyte’s concentration can reliably be measured—what is its detection limit

   • determines the lower limit of detection (LOD) for a given analyte (lowest amount of analyte reported)

2. Can a compound be measured without interfering compounds?; ability to only detect the desired analyte with other substances

Diagnostic Sensitivity vs specificity

1. Ability of a test to detect a given disease or condition

   • DSen = TP/(TP + FN)

   • requires a golden standard; diminished by a high rate of false-negative results

   • unaffected by the rate of false-positive results can tolerate a high rate of them

2. proportion of individuals without a condition who have a negative test for that condition

   • DSpe = TN/(TN + FP)

   • diminished by high rate of false-postive results;

   • false postive % = “false alarm”; rate is the inverse of specificity

3. EX: sensitivity and specificity of 100% = test detects every patient with disease and the test is negative for every patient without the disease

Diagnostic Specificity

Receiver-operator characteristic curve (ROC): Purpose and AUC

• aids in selecting cutoff point that maximizes test sensitivity and/or specificity or optimizes both

• AUC is useful estimate for evaluating the overall efficacy of a diagnostic test

Receiver-operator characteristic curve (ROC): False alarm & Determine diagnostic sensitivity and specificity

• 2D graph plots true postive rate against false alarm rate (100-specificity)

  • EX: which cut off point to best discriminate bombers (+) from geese [(-); false alarm]

• Which is more important?—determine wither the cut off point that maximizes test sensitivity, specificity, or both

  • EX: sensitivity more important (high false alarm rate of air-raid sirens) or specificity more important (consequences of missing enemy planes)

ROC analysis

• every point on ROC curve represents a different cut off level

• for every cutoff level, a sensitivity and specificity value is plotted

• “perfect” test hugs the Y-axis completely and makes sharp turn on x-axis

• cutoff points are selected to max sensitivity, specificity, or both

• complete diagonal = uninformative test

Predictive values

• highly dependent upon: population analyzed & prior probability of disease in an individual patient

  • highly variable and subject to sampling error

• (+) PV: probability of an individual having the disease if the result is abnormal (positive for the condition)

  • formula: TP/(TP + FP)

• (-) PV: probability that the patient does not have a disease if a result is within the reference range (negative for the disease)

  • formula: TN/(TN + FN)

• EX: D-dimer levels in serum

Example for predictive values

• D-dimer levels in serum that are ≤ 0.5 μg/mL have a high negative PV helpful in ruling out the presence of a DVT or PE.

• Levels ≥ 0.5 μg/mL do not have a high positive value (PPV) for predicting the presence of such a blood clot because:

  • Fibrin turnover as measured by d-dimer can be high (e.g., inflammation) in the absence of a blood clot

  • D-dimer levels are elevated in the presence of a circulating blood clot

  • D-dimer is a sensitive but not specific test for a blood clot

Beer’s Law: Transmittance of light and %T

• concentration of substance is directly proportional to the amount of light absorbed; inversely proportional to the log of transmitted light

• Percent transmittance (%T) and absorbance (A)

• all light absorbed or blocked → %T = 0

• Absorbance (A) is amount of light absorbed & mathematically derived from %T

  • %T = I/I₀ × 100

  • T = I/I₀

  • I₀ = incident light (light entering cuvette)

  • I = transmitted light (light sensed by photodetector)

  • Colored solutions absorb light at an appropriate wavelength: T < 1.0 & %T < 100

Beer-Lambert Law: Absorbance

• Absorbance = -logT

• Absorbance = 2 -log(%T)

• relates absorbance of a colored compound to its concentration in solution

• A = ε * b * c or A = abc

  • ε = molar absorptivity (fraction of specific wavelength of light absorbed by a given type of molecule

  • b = length of light path through the solution

  • c = concentration of absorbing molecules in M

• amount of light absorbed at a particular wavelength depends on molecular and ion types present and may vary with concentration, pH, or temp

  • Thus: absorbance is directly proportional to concentration

  • used to calculate concentrations of analyte in serum sample

Dynamic range/Linearity

• over what range of values from very low to very high can be reliably measured?

• EX: “one touch” glucose meter’s dynamic range: 20-600 mg/dL

Matrix effects

• Unintended and often undesirable influences that the components of an analyte have on the accuracy and precision of your measurement

• can an assay that measures calcium in serum also be reliably used for urine? breast milk?

• QC materials must be the same matrix as the patient’s specimen

Westgard rule violations on Levey-Jennings’ Charts

• 1_2s = 1 control observation exceeding the mean ±2s → warning to test of control data by other rules

• 1_3s = 1 control observation exceeding the mean ±3s → high sensitivity to random error

• 2_2s = 2 control observations exceeding the same ±2s or -2s → high sensitivity to systematic error

• R_4s = 1 control exceeding the ±2s and another exceeding -2s → detection of random error

• 4_1s = 4 consecutive control observations exceeding ±1s or -1s → detection of systematic error

• 10_x = 10 consecutive control observations failing on one side or the other of the mean → detection of systematic error

Amine and Carboxyl group vs N-terminal and C-terminal

1. NH2

2. —COOH

3. NH3+

4. —COO-

5. always written with the N-terminus toward the left

Amino acid side chain classification: uncharged polar side groups

asparagine, glutamine, serine, threonine, tyrosine

Amino acid side chain classification: nonpolar side groups

alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, glycine, cysteine

Amino acid side chain classification: acidic side chains

aspartic acid and glutamic acid

Amino acid side chain classification: basic side chains

lysine, arginine, histidine

Peptide bonds

4 atoms form a rigid planar unit; no rotation around the C—N bond

Methionine, cysteine, and disulfide bonds

• stabilize primary and secondary protein structure

• amino acid backbone with side group: 2 carbon chain, sulfur atom, methyl group (CH2-CH2-S-CH3)

• amino acid backbone with side group: thiol (SH → HS-CH2-CH(NH2)-COOH)

• amino acid backbone with side group: disulfide (S-S)

A-1-antitrypsin deficiency

• A-1 protects lungs against by inhibiting neutrophil proteases → excess breaks down elastin & connective tissue → emphysema

  • SERPIN - inhibitor of neutrophil proteases

• misfolding of A-1 → cannot be secreted by liver cells →accumulate in ER → liver cell apoptosis → injury → fibrosis and cirrhosis → liver cancer

• causes liver and ling disease (juvenile emphysema)

Maple syrup urine disease

• elevated leucine → cerebral edema & intoxication, poor feeding/irritability → lethargy, intermittent apnea, cerebral edema, coma, death

• elevated valine, leucine, isoleucine, and L-alloisoleucine are detected

Phenylketonuria (PKU)

• elevated phenylketones in urine

• deficiency of phenylalanine hydroxylase activity or in the synthesis or recycling of its biopterin cofactor

• treatment is dietary restriction of phenylalanine with supplement of tyrosine

• if untreated during infancy and childhood → intellectual disability, seizures, “mousy” odor, fair skin/hair, and eczema

5 classical bands of SPE and proteins found

• Albumin: albumin

• A-1: antitrypsin

• A-2: haptoglobin

• Beta: transferrin, LDL, C3

• Gamma: Immunoglobulins (IgA, IgM, IgG)

Where synthesized: (1) albumin, (2) antitrypsin, (3) haptoglobin, (4) transferrin, (5) LDL, (6) C3, (7) immunoglobulins

• 1-6: liver

• 7: secreted by B-lymphocytes/plasma cells

SPE methodology

• proteins in an alkaline buffer at pH of 8.7 migrate in an agarose gel placed in an electrical field, toward (+) charged electrode/anode

• rate of migration depends on magnitude of (-) charge and charge/mass ratio

• the gel is dried → stained with protein dye → cleared and canned in densitometer to enable quantification of each classic bands

Physiologic functions of albumin

• responsible for maintaining water in vascular compartment (oncotic pressure)

• transports insoluble ions (Ca2+) and insoluble compounds (bilirubin, free fatty acids, many hormones, many acid (-) charged and neutral drugs)

• negative acute phase reactant (creases during acute phase reaction)

Clinical consequences of circulating albumin below its normal range

• hypoalbuminemia

• common finding in liver failure

• causes edema or anasarca (widely disseminated edema)

• decreases total but not free calcium level

Causes of decreased serum albumin and key clinical signs of that

• liver disease → leads to edema

• kidney disease → leads to edema and albumin present in urine

SPEP patterns: acute phase reaction/inflammation and myeloma

• increase in Ig and A-1 fraction proteins, complement consumption

• elevated TP & Ca2+, “spike” in gamma region, monoclonal (M protein)

SPEP patterns: cirrhosis, nephrosis, polyclonal gammopathy

• beta-gamma bridging pattern

• decreased albumin and UPEP pattern mimicking SPEP

• immune system overactivated → increased immunoglobulins

Diagnostic criteria for myeloma

• increased immunoglobulin fraction

• spike in gamma region of SPEP is indicative of myeloma or MGUS

• elevated TP & Ca2+

• CRAB: high calcium levels, renal insufficiency, anemia, bone lesions

What type of lymphocyte represents the neoplastic (malignant) clone in myeloma?

plasma cells; accumulate in bone marrow → produce useless antibody (M protein)

Tumor marker in myeloma

monoclonal (M) protein; spike in gamma region of SPEP is indicative of monoclonal gammopathy of undetermined significance (MGUS)

Clinical utility of immunofixation electrophoresis

• characterize monoclonal proteins in serum, urine, or cerebrospinal fluid (CSF)

• sample is placed in all six lanes of an agarose gel → electrophoresed to separate the proteins → Cellulose acetate is saturated with an Ab reagent → applied to one lane of the separated protein

• Ab reagent recognizes the protein → insoluble complex is formed → staining and drying of the agarose film → interpretation is based on the migration and appearance of bands

• Monoclonal proteins present will appear as a discrete band

• Polyclonal proteins will appear as a diffuse band

Total Protein determination and RR

• Biuret: form violet-colored chelate by cupric ions in complex groups; reagent contains sodium potassium tartrate

• Dye binding: binds to protein causing spectral shift in absorbance max; dyes include bromophenol blue, amido black, lissamine green, etc.

• Protein electrophoresis: migration of proteins based on density and charge under influence of electric field; reagent is buffer

• TP reference range: 6.0-8.3 g/dL

Albumin determination and RR

• dye-binding procedures using bromocresol green (BCG) or bromocresol purple (BCP)

• pH of solution adjusted so albumin is (+) charged → bind to dye → concentration calculated by absorbance of albumin-dye complex

• Albumin reference range: 3.5-5.5 g/dL

3 filtration barriers in the glomerulus

1. fenestrated endothelial cells: prevent passage of cells into filtrate and highly permeable barrier

2. basement membrane (GBM) beneath endothelium: enriched with non-linear type 4 collagen

3. epithelial cell projections (foot processes): wrap around GBM and form filtration slits; enriched in transmembrane protein (nephrin) mesh

5 characteristics of an ideal renal filtration marker

1. be freely filterable (no protein-bound like calcium)

2. not be metabolized (an end-product)

3. be produced at a steady state level

4. not be reabsorbed by tubules

5. not be secreted by tubules

Urea: source & clinical significance of increased circulating levels

• produced by urea cycle in liver to detox ammonia (produced by gut flora) and from amino acid catabolism (RR: 7-20 mg/dL)

• increased BUN levels > 20:1 caused by prerenal azotemia (hypoperfusion caused by hemorrhage, shock, volume depletion, CHF, renal arterial stenosis) and by increased protein catabolism

Creatine: source, methodologies, clinical significance of increased values

• synthesized in the liver from arginine, glycine, and methionine

• PCr synthesized and stored in skeletal muscle using ATP

• Increases in circulating creatinine seen <50% of functional nephrons or nephron activity lost

• methodologies: (1) ancient dye-binding method & (2) enzymatic creatinine assay

(1) ancient dye-binding method & (2) enzymatic creatinine assay for creatinine

1. alkaline solution of picrate; many interferences (glucose/protein); kinetic analysis is less subject to interference compared to end-point

2. 3-enzyme method; peroxide generation coupled to oxidation of a chromogen; new reference ranges needed due to lack of interferences; many variations on this theme

Creatinine clearance (CrCl) and RR

• RR: (male) 97-137 mL/min; (females) 88-128 mL/min

• measure of the amount of creatinine eliminated from the blood by the kidneys, and GFR are used to gauge renal function

• CrCl = U_cr(V_u)/(P_crt)

  • U_cr = urine creatinine concentration

  • P_cr = plasma creatinine concentration

Variables used in estimating GFR & normal values

• volume of plasma filtered (V) by the glomerulus per unit of time (t)

• GFR = UV/P*1440

  • U = urinary creatinine level in mg/dL

  • V = 24hr urine volume in mL per 24hrs

  • P = plasma creatinine in mg/dL

  • 1440 minutes = 24 hours

• Normal values:

  • plasma creatinine: 1.0 mg/dL

  • 24hr urine volume: 1000 mL

  • Urinary creatinine: 120 mg/dL

• Creatinine, age, sex, race, cystatin C (cysteine protease inhibitor)

Arginine vasopressin (Antidiuretic hormone; ADH)

• angiotensin II induces secretion of ADH from the posterior pituitary

• recruits water channels (aquaporin-2) to the apical membrane of connecting tubules and collecting ducts in distal nephron

• aquaproins allow for free passage of water from the filtrate back into circulation

Central vs Nephrogenic Diabetes insipidus (DI)

• etiology: head injury, brain trauma, radiation therapy, severe illness

  • not enough ADH secreted → H2O is lost via inability of kidney conservation

  • hypernatremia, copious and dilute urine, decreased urine osmolality

• ADH resistance: inherited or acquired defects in vasopressin type-2 receptor or aquaporin-2 genes causing ADH sensitivity

Syndrome of Inappropriate Antidiuretic Hormone Secretion (SIADH)

• etiology: Paraneoplastic syndromes, for example ectopic ADH secretion from small cell lung cancer, epilepsy, meningoencephalitis, medications (anticholinergics, carbamazepine), pulmonary disease

• excessive ADH secretion; too much water moves into vascular space

Serum/Urine osmolality and Na levels in Diabetes Insipidus & SIADH

• water is lost via inability of kidney conservation; decrease in urine osmolality and increase in serum osmolality

• too much water moves into vascular space; increased urine osmolality and dilutional hyponatremia

Major cation: extracellular and intracellular

• extra: Na+

  • important role in generating AP in excitable cells

  • responsible for: water balance (where NaCl goes, H2O follows), important player in neuromascular function

• intra: K+

  • determine resting membrane potential

  • important component of AP in excitable cells

  • crucial role in cardiac conduction and function

Dietary sources of potassium

veggies and fruits

Reserve of potassium in the body

• inside of our cells

• can be donated to plasma in times of depletion within limit

• intracellular levels = 150 mM

Artifactual or physiological hemolysis of RBC/WBC

• hemolysis increases K+ levels in serum

• 35x higher inside cells than extracellular fluid

• animals move K+ in and Na+ out of cells against concentration gradient (requires ATP)

Renin-angiotensin-aldosterone axis

• decreased NaCl delivery drives macula densa (MD) to release prostaglandins → dilate afferent arteriole → increase renin release → aldosterone secretion from renal cortex → increase Na+ and H2O retention and BP

  • decreased BP increases renin secretion

• increased NaCl delivery to MD → vasoconstriction of afferent arteriole → decreasing GFR and renin secretion

  • net effect: decrease Na+ and water retention and BP

• aldosterone: increases rate of K+ excretion and Na+ retention by distal tubule

ACE-inhibitor and Angiotensin II receptor (ARB) blocker

• ACE inhibitors block ACE which prevents the conversion of angiotensin I to angiotensin II (which constricts blood vessels)

  • ex: lisinopril

• ARB: angiotensin II constricts blood vessels and salt and water retention; ARBs blocks it → vessels relax → decrease in BP

  • ex: losartan (cozaar)

Aldosterone: function & increased vs decreased secretion

• secreted in response to hyperkalemia (help excrete K+) and hyponatremia (via renin)

  • important in rapid conservation of Na+ and excretion of K+

• Oversecretion: Primary hyperaldosteronism; Conn’s syndrome; adrenal ademona; hypokalemia

• Deficient secretion: primary adrenal insufficiency; Addison’s disease; hyperkalemia and hyponatremia

• Urine and serum osmolality (total solute) measurments are helpful when plasma Na+ is pertubed

Conn’s syndrome and Pseudohyperaldosteronism

• adrenal adenoma (tumor) that produces excess hormones: hypokalemia

• seen in individuals that consume a high amount of real licorice or may be dosed on other medications for heart failure or Abiraterone

  • compounds are potent inhibitors of an enzyme → 11-ß hydroxysteroid dehydrogenase type 2 (11-ß-HSD2) converts cortisol (has aldosterone-like activity) → to cortisone (does not have such activity)

  • 11ßHSD2 keeps cortisol from behaving like an unregulated aldosterone in the kidney → lets aldosterone take over salt, water and H+ balance at the level of the tubules without interference from glucocorticoid cortisol

  • overingestion of licorice root’s glycyrrhizic acid → lethal hypokalemia

Clinical utility of aldosterone to renin ratio (ARR)

• aldosterone can be stimulated by angiotensin II and independently by hyperkalemia

• ARR informative in differentiating primary from secondary hyperaldosteronism (HA)

• high circulating level of aldosterone → low plasma K+ and EKG cardiac conduction abnormalities

• Primary HA caused by adrenal adenoma secreting high levels of aldosterone → hypokalemia; ARR ratio higher

• Secondary HA: decreased arterial perfusion/pressure to kidney in settings like heart failure or congenital or acquired conditions; ARR ratio lower

Hypokalemia

• consequences:

  • cardiac arrhythmias and ECG abnormalities

  • renal abnormalities and glucose intolerance

  • severe muscle weakness or rhadomyolysis

  • donate intracellular reserve of K+ → extracellular fluids → overwhelmed

• etiology:

  • GI loss: lower GI tract (secondary to HCO3)

  • urinary loss: hyperaldosteronism, pseudo hyperaldosteronism, diuretics acting proximal to K+ secretion site

  • licorice intoxication

  • insulin administration (stim Na/K ATPase)

  • excessive sweating, dialysis, plasmapheresis

  • beta adrenergic activity

  • metabolic alkalosis

Hyperkalemia

• consequences: muscle weakness and paralysis, cardiac conduction abnormalities and arrythymia

• etiology:

  • metabolic acidosis (exchangeable with H+)

  • Intravascular hemolysis (also high WBC, PLT)

  • Artifactual hemolysis

  • Insulin deficiency or insensitivity

  • Beta-blockers

  • Adrenal insufficiency (aldosterone)

  • Aldosterone insensitivity

  • Acute or chronic kidney disease

  • Hyperkalemic periodic paralysis

Hyponatremia

• consequences:

  • Nausea & vomiting, Headache & confusion, Loss of energy & fatigue

  • Restlessness & irritability

  • Muscle weakness, spasms or cramps

  • Cerebral edema (brain swelling) & death

  • Seizures & coma

• Etiology:

  • volume depletion: GI losses (vomiting, diarrhea) and renal (thiazide diuretics)

  • reduced cardiac output (hypervolemia)

  • SIADH: inappropriate ADH secretion (water excess)

  • adrenal insufficiency

  • Polydipsia: Psychogenic, Exercise-related, MDMA (Ecstasy) induced

Hyponatremia

• consequences: Thirst, Brain cell shrinkage, Confusion, Muscle twitching & spasms, Seizure & coma (brain shrinkage)

• etiology:

  • unreplaced water loss (sweat, bacterial diarrhea)

  • Central or nephrogenic diabetes insipidus

  • glucosuria-induced osmotic diuresis (solvent drag)

  • salt poisoning/iatrogenic causes

How hyponatremia leads to cerebral edema & how hypernatremia shrinks neurons in the CNS

• decrease serum osmolality → osmotic gradient draws water from blood into cells → cells swell → cerebral edema

• high concentration of Na → hypertonic → draw water out of cells via osmosis → cells shrink

Direct vs Indirect methods for potentiometric measurements of Na

• Direct: measures electrolytes in whole blood

  • WB: 93% H2O / 7% solids

  • electroyltes are present and active in water compartment

  • electrolyte activity is expressed in units of mm/kg of water

• Indirect: measures electrolytes in serum or plasma

  • after dilution: do not take into account the real % of plasma water in the sample if that compartment has been diluted by an abnormally high level of solids

  • Na+ is more affected than K+ in the setting of lipemia or hyperproteinemia.

  • Expert panel: error in sodium measurement of [-17 mM] in hyperproteinemic samples, and [-26 mM] error in lipemic samples

Pseudohyponatremia

• not actual low sodium value in diluted serum or plasma that has been centrifuged and run in an automated instrument.

• Consequence: Na+ is reported as too low when actually in the normal range, or normal to low in a sample that actually has an elevated Na+

• Recommendation: for those who are critically ill, perform sodium assay using a direct ISE

Clinical significance of sweat chloride measurments

• use for diagnosis of cystic fibrosis

• DNA level test are reliable but may miss rare mutations in the CFTR

• patience must be > 48 hours age, sweat collected from arms or legs in duplicate and analyzed by methodology/instrument

• infants up to 6 months Cl- level: < 29 mmol/L = unlikely CF; > 60 mmol/L = likely CF

• Older than 6 months Cl- level: <39 mmol/L = unlikely CF; > 60 mmol/L - likely CF

RR: Na, K, Cl, HCO3 in serum and for serum and urine omolality

• 135-145 mM; immediate action values (IAV) < 120, > 160 mM

• plasma RV: 3.5-5.0 mM; IAV: < 3.0, > 6.0 mM

• RV: 95-105 mM; IAV: <80, >120 mM

• RV: 22-26 mM; IAV: <10, >40 mM

Potentiometry (ISE)

• glass can be manufactured to selectively interact with an individual ion in solution

• Alteration contains variable quantities of: Na2O, CaO, SiO2 and Al2O3 → produces chemically active binding sites in the glass that have hydronium, sodium, or other cation selectivity

• When ISE interacts with solution of that ion, electrical potential difference across surface of glass “membrane” changes in proportion to concentration of ion

  • Ion selective electrodes (ISE): internal solution contains fixed concentration of cation of interest fixing internal electrode surface potential, while external varies

Utility of Nernst Equation

• E = E⁰ - (RT/zF) * lnQ

• permits the calculation of reduction potential of a reaction from the (1) standard electrode potential, (2) absolute temp, (3) number of electrons involved in oxido-reduction reaction, and (4) activities of chemical species undergoing reduction and oxidation

• useful for predicting how cell voltage changes as a reaction progresses and reactant/product concentrations fluctuate