Chemistry Procedures

Spectrophotometer: Measures the absorption of monochromatic light produced by a grating monochromator
Photomultiplier Tube: (PMT) Photosensitive materials in cathodes release electrons when exposed to light energy. Amplification factor may be as high as 10^6.
Flame photometer: 
Measures light emitted by single atoms burned in a flame
Atomic absorption photometer: Measures light absorbed by atoms dissociated by heat
Fluorometer: measures the light of a specific wavelength emitted by a molecule after it has been excited by electromagnetic radiation of a given energy

Beer-Lambert Law:
 concentration of a substance is proportional to the amount of light absorbed or inversely proportional to the log of transmitted light.
Followed if:
· radiation monochromatic
· solvent absorbance insignificance
· solute concentrations within linearity limits
· no chemical reaction between solute or solvent

Atomic emission spectrophotometry [emission flame photometry]: Metallic salt is burned in a flame heat energy drives an electron out of orbit. When it returns to ground they emit electromagnetic radiation. Each metal emits at its own emission spectrum. Variations in sample aspiration rate, atomization rate, and solution viscosity are compensated for by use of an internal standard.
Ion selective elctrodes used for routine Na, K, Li but atomic emission photometers more reliable for urine Na, K, especially in low concentrations.

Atomic absorption spectrophotometer: Atom is dissociated from its chemical bonds, and it absorbs light at a specific wavelength in its ground state.

Flourometry [molecular emission spectrophotometry]: Certain compounds absorb electromagnetic radiation, become excited, and return to an energy level higher than or equal to their original level. Wavelength of emitted light is longer than that of exciting light. Light emitted from a singlet excited state is called flouresence. From a triplet excited state is phosphoresence. Flouresence is 100-1000x more sensitive than absorption measurements.

Particles too large for absorption spectroscopy. Measurement of light scattered by particles in solution. If the wavelength of light 0.1 l > d diameter of particle then scatter is symmetric around particle. If 10 l < d light scatters forward. Measures antigen-antibody interactions in EIA.

Turbidimetry: Measurement in the reduction of light trasmission caused by particle formation. Microbiology analyzers to determine bacterial growth, coagulation analyzers, quantitate protein concentrations in urine & CSF.

Refractometry: Measurement of refraction of light as it passes from one medium to another. Depends on wavelength, temperature, nature of liquid medium, solute. Used for total serum protein concentrations, specific gravity of urine,

Osmometry: Measurement of osmolality. As ­ osmolality : ­ osmotic pressure, ­ boiling point, ¯ vapor pressure, ¯ freezing point. Freezing point depression most commonly used method.

Flow Cytometry: Transmitted light consists of scattered light (forward and 90°) and flourescent light. Simultaneous analysis of forward and right angle light scatter allows for separation of granulocytes, monocytes, and lymphocytes based on size and granularity. Cells of interest are identified by electronic gating and given an electrical charge. Used for CBC, RBC, WBC, plts, 5 part differential.

Electrical Impedance:
 Change in electrical resistence across an aperture when a particle in conductive liquid passes through this aperture. Used in hematology to enumerate leukocytes, erythrocytes and platelets (Coulter). Voltage pulse proportional to cell size. 2-20 fL platelets, >36 fL erythrocytes. Lytic reagent >35 fL WBC.

Electrochemistry: Measurement of current or voltage generated by the activity of specific ion species. Used for measuring electrolytes and blood gases.
Potentiometry: constant-voltage source is needed as the reference potential.
Nernst equation: E = E° – (0.059/z) log (Cred/Cox)
cell potential at 25° = standard reduction potential – (0.059/# electrons) log (Molar concentration of reduced form/Molar cencentration of oxidized form)
Electrodes = saturated calomel electrdes but unstable above 80°C, so use silver-silver chloride for high temps
pH Electrodes = glass used for measuring [H+]
pCO2 Electrode = pH electrode with plastic jacket with gas-permeable membrane and NaHCO3 buffer
Ion selective Electrode = electrochemical transducer capable of responding to one given ion.
Coulometry: electrochemical titration in which the titrant is electrochemically generated and the endpoint is detected by amperometry. Used for measuring chloride ion concentration. Constant current is applied accross two silver electrodes, liberating silver ions at a constant rate to produce insoluble silver chloride.
pO2 gas-sensing electrode = use an amperonetric or current-sensing electrolytic cell as the indicator system.

Electrophoresis: Separation of charged compounds based on their electrical charge. High voltage (50 – 200 V DC). A dilute buffer causes heat to be generated in the cell and a high ionic strength does not allow goos separation of the fractions. Dye to identify fractions. Densitometry performed. Used for serum proteins, hemoglobins, isoenzymes of CK, LD and Alk Phos.

Isoelectric Focusing: At a specific pH protein will have a net charge of zero. That pH is the protein’s isoelectric point or pI. pH gradient created by adding acid to the anode and base to the cathode. A solution of ampholytes (small amphoteric ions with different pIs) is placed between the elctrodes. Each ampholyte migrates to the area of its isoelectric point creating a stable pH zone for slowly migrating proteins. Advantage: resolve mixtures of proteins. Used for serum acid phosphatase isoenzymes, oligoglonal bands in CSF, isoenzymes of CK and alk phos in serum.

 Absorbance measurement. Built-in integrator to find area under curve.

Chromatography: Different interactions of the specimen compunds with the mobile phase and with the stationary phase as the compounds travel through a support medium. Retention time (tR) is the time it takes a compound to elute. Concentration is calculated from peak height.
Gas Chromatography: Naturally volatile compounds or can be readily converted to volatile form. Used for many drugs. Retention time determined by vapor pressure and volatility. Samples introduced with a hypodermic syringe and the port is heated to high temperatures. Samples are vaporized and swept onto column. If substance is not volatile enough for direct injection it may need to be derivatized into a more volative form: done by silylation, alkylation, acylation.
Capillary columns – higher efficiency and better detection limits. Packed columns – larger specimen or sample capacity.
Flame ionization detectors: detect almost all organic compounds. Measure ions produced by the compounds when burned in a hydrogen-air flame.
Liquid Chromatography: Many organic compounds are too unstable or are insufficiently volatile to be assayed by gas chromatography. Liquid uses lower temperatures and it allows better sepeartion of thermolabile compounds. It is easier to recover a sample in liquid chromatography.
HPLC = high performance liquid chromatography: small, rigid supports and special mechanical pumps producing high pressure to pass the mobile phase through the column.

Mass Spectrometry: 
Based on frangmentation and ionization of molecules using a suitable source of energy. Usually ned to use GC first to isolate compound.. Source of energy can be beam of electrons, or beam of atoms (argon). Sorts the parent molecular ions and their fragment ions according to mass-to-charge ratio.

Scintillation Counters: Flashes of light that occur when gamma rays or charged particles interact with matter. Detects scintillations using a PMT tube and counts the electrical impulses produced by the scintillations.
Crystal Scintillation: detects gamma radiation. NaI crystal. with 1% thallium. Gamma rays strike iodide atoms and raises electrons to a higher energy state. UV radiation released when they drop back to ground. UV absorbed by thallium and emitted as photons. A pulse analyzer sorts pulse signals from PMT according to pulse height.
Liquid Scintillation: Beta particles from radioactive sample ionize the solvent. A secondary scintillator absorbs the photons and emits them at a longer wavelength.

Capillary Electrophoresis: Sample molecules are separated by electroosmotic flow through a fused silica capillary. Advantages over conventional electropheresis and HPLC are short analytical time, resolving power and micro sample volumes. Uszed for separation of serum proteins and hemoglobin variants.

Sequential testing: multiple tests analyzed one after another on a given specimen.
Batch testing: all samples are loaded at the same time and a single test is conducted on each sample.
Parallel testing: more than one test is analyzed concurrently on a given specimen.
Random access testing: any test can be performed on any sample in any sequence

Quality Management
Random error causes dispersion.
Systematic error causes a trend or a shift.

Use 1:2s rule to trigger inspection of control data: occurance of control values outside ±2 SD.
When there are no control observations in a run that exceeds a 2s limit, the run can be accepted w/o further inspection.
If any one rule indicates a rejection – the run should be rejected.
Westgard’s Rules: (Henry p. 129)
· 1:3s – Reject when one observation exceeds the mean ±3 SD.
· 2:2s – Reject when two consecutive observations exceed the same mean +2 SD or the same mean – 2 SD.
· R:4s – reject when one control observation in the run exceeds its mean + 2 SD, and another exceeds its mean – 2 SD.
· 4:1s – Reject when four consecutive control observations exceed the same mean + 1 SD or the same mean – 1 SD.
· 10:mean – Reject when 10 consecutive control observations fall on one side of the mean.

Drugs: Volume of Distribution
Vd = D0/D
D0 = amount of drug administered
D = concentration of drug in serum
If Vd is larger than the expected drug volume the drug is stored in tissues.

Metabolic Derangements
Hypernatremia (134 < Na <146)

Asses Clinically Extracellular Fluid Volume
Urine Na >20 <10 Variable Urine Na > 20
Urea diuresis
GI Losses (Lactulose)
* Central:
Tumor, Histiocytosis, Sarcoid, Trauma
* Nephrogenic: Renal disease Hypercalcemia, Hyperkalemia, Li, Sickle cell, Amyloidosis, Urinary tract obstruction, Demeclocycline
Hypertonic NaHCO3
NaCl Tablets
Hypertonic IVF
Mineralcorticoid: hyperaldosterone, Cushing’s, congenital adrenal hyperplasia
Hypertonic hemodialysis, peritoneal dialysis
Rx: Hypotonic Saline Rx: Water Replacement Rx: Diuretics & Water Replacement

Most common etiology for hypernqatremia in a hospitalized patient is nephrogenic DI secondary to loss of medullary gradient due to poor po intake coupled to dehydration from poor po intake/vomiting.
Urine osm <150 defect in water conservation; >150 osmotic diuresis

Hyponatremia (134 < Na <146)
Sx: GI (N/V), CNS disturnances from edema: obtundation, coma, seizures, death
FENA = (Urine Na/Plasma Na)/(Urine Cr/Plasma Cr)

Serum Osmolality
Normal (280-285)
1. Pseudohyponatremia:
· Hyperlipidemia
· Hyperproteinemia
2. Isotonic infusions: glucose, glycerol, mannitl, sorbitol, glycine, ethanol
Low (<280)

Asses Clinically Extracellular Fluid Volume

High (>285)
1. Hyperglycemia
2. Hypertonic infusions: glucose, glycerol, mannitl, sorbitol, glycine, ethanol
Urine Na >20 <10 or FENA <1% <10 >20 <10 >20
Renal Damage
Partial Obstruction
Adrenal insufficiency
Salt-wasting nephritis
GI – vomit, diarrhea
Skin/Lung Losses
H20 Intoxication RENAL FAILURE
· Idiopathic
· Lung carcinoma
· Hypopituitary
· Drugs: thiazides, chlorpropramide, clofibrate, phenytoin
Adrenal Insufficiency
Rx: Isotonic Saline Rx: Water Restrict

Hyperkalemia (3.5<K<5.5 mEq/L)
Sx: neuromuscular weakness, paralysis, cardiac arrythmias (K>6.0) with prolonged PR, peaked T, lose P, increased QRS then sin wave, V tach.
· Acidosis
· Insulin Deficiency
· Severe renal failure
· Aldosterone insufficiency (lead, ACE inhib, Addison’s, NSAIDS, interstitial renal disease, obstructive uropathy)
· Aldosterone resistance (sickle cell, SLE, amyloid, spironolactone…)
Pseudohyperkalemia: leukocytosis, thrombocytosis – measure plasma K, hemolysis – check serum
Urine K+<30 mEq/L impaired renal secretion.

Hypokalemia (3.5<K<5.5 mEq/L)
Sx: neuromuscular weakness, paralysis, cardiac arrythmias, decreased insulin release w/glucose intolerance, polyuria, polydipsia, ileus
· Insulin
· Epinephrine
· Folic acid/B12 therapy
· Acute alkalemia
· Hypokalemic periodic paralysis: Asians with thyroid disease/catecholamine Rx
· Inadequate dietary intake < 10 mEq/day
· Diarrhea
· Vomiting – ¯ intravascular volume – ­aldosterone – metabolic alkalosis
· Fistula – acidosis
· Discontinued diuretics – alkalosis
· Skin losses – sweating
· Drug induced: diuretics, PCN, Gent, Amphotericin B
· Hypertensive
– Hepereninemic: malig HTN, renovascular, renin secreting tumor, CHF, cirrhosis
– Normoreninemic: normoadosterone (Liddle’s Syndrome)
– Hyporeninemic· Hyperaldosterone (adrenal adenoma/hyperplasia) · Hypoaldosterone (mineralcorticoid ingestion, Ectopic ACTH, Cushings, CAH)
· Renal Tubular: RTA, Bartter’s Syndrome, Diuretics, Laxatives

Hypercalcemia (4.25 < ionized Ca < 5.25 mg/dL) (8.5 < serum Ca < 10.5 mg/dL)
50% plasma protein bound. Ionized Ca ­ with acidosis, ¯ with alkalosis
Sx: hypercalciuria, renal calculi, osteitis fibrosis cystica, bone cysts, osteopenia, anorexia, weight loss, constipation, n/v, abdominal pain, muscle weakness, shortened QT on EKG.
· Primary Hyperparathyroidism (80-90% adenoma, 10-20% hyperplasia, 1% carcinoma MENI)
· Malignancy: with bone mets (breast ca, myeloma, lymphoma) or without (PTHrP producing – hypernephroma, pancreatic ca, sq cell of lung, cx, esoph)
· Sarcoidosis
· Familial hypocalciuric hypercalcemia – AD
· Vit D intoxication
· Milk-alkali syndrome (hypercalcemia, alkalosis, renal failure)
· Hyperthyroidism
· Thiazide diuretics
· Immobilization
· Paget’s Disease
· Addison’s disease
· Recovery from ARI from rhabdomyolysis

Hypocalcemia (4.25 < ionized Ca < 5.25 mg/dL) (8.5 < serum Ca < 10.5 mg/dL)
Sx: fatigue, weakness, tetany, laryngeal stridor, prolonged QT, Chvostek’s sign (facial nerve), Trousseau’s sign (BP cuff carpal spasm), seizures, muscle/abd cramps. 
 Hypoparathyroidism (+ hyperphosphatemia): post-surgical or idiopathic (assoc w/mucocutaneous candidiasis & adrenal insufficiency)
· Pseudohypoparathyroidism (end organ resistance to PTH) e.g. Albright’s hereditary osteodystrophy, ­PTH levels
· Hypoalbuminemia
· Renal failure
· Malabsorption: tropical sprue, sm intestinal fistula
· Viamin D deficiency
· Acute pancreatitis
· Osteoblastic mets (prostate, breast, lung ca)
· Hypomagnesemia
· Drugs: gentamycin, cisplatin, ethylene glycol, colchicine, EDTA, protamine, dilantin, phenobarbital, mithramicin, citrated blood

Hyperphosphatemia (2.6 < PO4 < 4.6 mg/dL)
Sx: hypocalcemia, hypotension, renal failure
· Renal Failure
· Cell lysis syndromes
· Exogenous PO4 administration – laxatives, enemas, IV
· Hypoparathyroidism
· Tumoral calcinosis
· GH excess – acromegaly
· Hyperthyroidism *
· Sickle cell anemia *
· Cyclosporine/FK 506 *
* Excessive renal absorption

Hypophosphatemia (2.6 < PO4 < 4.6 mg/dL)
Sx: obtundation, coma, ascending paralysis, seizures, hemolytic anemia, rhabdomyolysis, increased bone resorption, decreased cardiac function
· Dietary Deficiency
· Antacids – Bind phosphate in GI tract
· Starvation/Refeeding – increased cell uptake
· Redistribution: glycolysis, resp alkalosis, DKA recovery, sepsis, epinephrine (glucose infusion is the #1 cause in hospitalized patients)
· Hyperparathyroidism
· Renal Tubular Defects – Phosphate wasting: Heavy metal poisoning, multiple myeloma, SLA, Wilson’s, Amyloid, Cystinosis, Fanconi’s syndrome
· Hypophosphatemic Vitamin D Rickets
· Glycosuria

Hypermagnesemia (1.5 < Mg < 3.0 mg/dL)
Sx: >10 mEq/L muscular paralysis, CNS depressant, hypotension, bradycardia, vasodilation
· Iatrogenic: laxatives, antacids, IV MgSO4 given for toxemia
· Renal failure
· Adrenal insufficiency

Hypomagnesemia (1.5 < Mg < 3.0 mg/dL)
Sx: tremor, muscle twitching, weakness, hypokalemia (decreased reabsorption)
70% of Mg absorbed in thick ascending limb
 Inadequate dietary intake
· Malabsorption: diarrhea, laxative abuse
· Redistribution: ETOH withdrawl, Insulin, resp alkalosis, s/p parathyroidectomy for osteitis fibrosa cystica
· Primary tubular disorders: Bartter’s syndrome, RTA, postop diuresis, s/p renal transplant
· Drugs: thiazides, furosemide, cisplatin, gentamicin, amphotericin B
· Hormone: hyperaldosterone, hypoparathyroidism
· Ion-induced: hyperkalemia, hypophosphatemia, ETOH