|
Incision (Skin puncture) |
<2.0mm (infants and children) 2-3mm (adults) |
|
1.5-2.4mm |
Distance from the skin surface
to bone or cartilage (middle finger) |
|
Arterialized capillary blood |
Earlobe: Preferred site Lateral plantar heel surface:
most commonly used site |
|
Flea |
Minute metal filling which may
be inserted into the capillary tube before collecting blood to help mix the
specimen while the blood is entering the tube |
|
Indwelling umbilical artery |
Best site for blood gas
analysis (newborns) |
|
1000-3000 RCF for 10 mins |
Centrifugation requirement |
|
Hemolysis |
Increased: “KLA6MP ITC2” -K+ -LDH (150x) -ACP -ALP -Aldolase -ALT -AST -Albumin -Mg2+ -Phosphorus -Iron -Total protein Affects bilirubin levels Inhibits lipase |
|
Refrigeration/Chilling (Low temp) |
Required for: “ABCGLRP2” Ammonia Blood gases Catecholamines Gastrin Lactic acid Renin PTH Pyruvate Decreased: LD 4 and 5 Increased: ALP |
|
Photosensitive analytes |
Bilirubin Beta-carotene Folate Porphyrins Vitamins A and B6 |
|
Oxalate |
Insoluble salt 1-2 mg/mL blood |
|
Citrate |
Non-ionized form 3.2-3.8 g/dL (1:9 ratio) |
|
EDTA |
Chelation 1-2 mg/mL blood Versene: disodium salt Sequestrene: Dipotassium salt |
|
Fluoride |
Weakly dissociated calcium
component 2 mg/mL blood: anti-glycolytic 10 mg/mL blood: anticoagulant |
|
Heparin |
A.k.a. Mucoitin polysulfuric
acid Universal anticoagulant Antithrombin 0.2 mg/mL blood |
|
Lithium heparin |
For glucose, BUN, ionized
calcium, electrolyte studies (K+: best) and creatinine |
|
Orange top tube |
Additive: Thrombin |
|
Royal blue top tube |
Additives: None; Na2EDTA Sodium heparin |
|
Brown top tube |
Lead testing |
|
Tan top tube |
Lead testing |
|
Black top tube |
Additive: Buffered sodium
citrate For ESR |
|
Respinning gel tubes |
Increases potassium |
|
Thixotropic gel |
Gel separator (SG: 1.04) Serum: (SG: 1.03) RBC: (SG: 1.05) |
|
Laboratory
Mathematics |
|
|
% w/v |
Grams of solute = % solution
desired x total volume desired
100 |
|
% v/v |
mL of solute = % solution
desired x total volume desired 100 |
|
% w/w |
Grams of solute = % solution
desired x grams of the total solution
100 |
|
Molarity |
M = _grams of solute_______ GMW x volume of solution |
|
Moles |
Mol = weight (grams) GMW |
|
To prepare a molar solution |
Grams of solute = Molarity x
GMW of the solute x Volume (L) desired |
|
To convert % w/v to Molarity |
M = % w/v Ÿ 10 GMW |
|
Normality |
N = _Grams of solute_ EW x volume (L) |
|
Equivalent weight (EW) |
EW = __MW___ valence |
|
To prepare a normal solution of solids |
Grams of solute = Normality x
EW x Volume (L) |
|
To convert % w/v to Normality |
N = w/v Ÿ 10 EW |
|
Normality |
N = Molarity x Valence |
|
Molarity |
M = Normality valence |
|
Molality |
m = Grams of solute__ MW x kg of solvent |
|
Milliequivalents |
mEq/L = mg/dL Ÿ 10 Ÿ valence MW |
|
Millimoles |
mmol/L = mg/dL Ÿ 10 MW |
|
Ratio |
Ratio = _Volume of solute_ Volume of solvent |
|
Dilution |
Dilution = __Volume of
solute__ Volume of solution |
|
0.179 |
Conversion factor for iron
(mg/dL à μmol/L) |
|
0.01 |
Conversion factor for
phospholipid (g/dL to g/L) |
|
2.27 |
Conversion factor for folate |
|
Analytical reagent (AR) grade |
For qualitative and
quantitative analyses For accuracy Established by American
Chemical Society (ACS) Uses: Trace metal analysis and
preparation of standard solutions |
|
Ultrapure reagents |
Additional purification steps Ex: Spectrograde, nanograde,
HPLC grade Uses: Chromatography, atomic
absorption, immunoassays |
|
Chemically Pure (CP) or Pure Grade |
Indicates that the impurity
limitations are not stated Purity is delivered by meas. of
melting point or boiling point |
|
Technical/Commercial grade |
In manufacturing Never used in clin. lab.
testing |
|
United States Pharmacopoeia (USP) and
National Formulery (NF) |
For human consumption Not applicable for lab.
analysis Purpose: For drug manufacturing |
|
Preparation of reagent grade water |
Filtration (1st) à Distillation, Ion exchange,
Reverse Osmosis |
|
Type I Rgt Water |
Min. interference Max. water purity Used immediately For ultramicrochemical
analyses, measurements of nanogram or subnanogram concentrations, tissue or
cell methods (microscopy) and preparation of standard solutions Uses: FEP, AAS, blood gases and
pH, enzyme studies, electrolyte testing, HPLC, trace metal and iron studies |
|
Type II Rgt Water |
For clinical laboratory use
(hematology, microbiology, immunology, chemistry) For prep. of rgts and QC
materials |
|
Type III |
For washing glasswares For urinalysis, parasitology
and histology |
|
Distilled water |
Purified to remove almost all
organic materials |
|
Deionized water |
Free from mineral salts;
removed by ion exchange processes Organic material may still be
present |
|
Occupational Safety and Health Act (OSHA) |
Req. manuf. to indicate lot
no., physical or biological health hazard of the chem.. rgts, and precautions
for safe use and storage |
|
College of American Pathologists (CAP) |
Recommends that a lab. document
culture growth, pH and specific water resistance on reagent grade water |
|
Tests for water purity |
Microbiological content pH Resistivity Chemical oxygen demand Ammonia Ions Metals |
|
Detergent-contaminated water |
Alkaline pH |
|
Hard water |
Contains calcium, iron and
other dissolved elements |
|
NCCLS |
Now: Clinical and Laboratory
Standards Institute (CLSI) |
|
Dilute solution |
Relatively little solute |
|
Concentrated solution |
Large quantity of solute in
solution |
|
Saturated solution |
Excess of undissolved solute
particles |
|
Super saturated solution |
Greater concentration of
undissolved solute particles than does a saturated solution of the same
substance |
|
Primary standard (IUPAC) |
Highly purified Measured directly to produce a
substance of exact known concentration |
|
Secondary standard |
Low purity Concentration is determined by
comparison w/ a primary standard |
|
Laboratory
Safety |
|
|
National Fire Protection Association (NFPA)
Classification of Fires |
|
|
Class A fire |
Ordinary combustibles: paper,
cloth, rubbish, plastics, wood Extinguisher: Water (A), Dry
chemical (ABC), loaded steam |
|
Class B fire |
Flammable liquids: grease,
gasoline, paints, oil Extinguisher: Dry chemical
(ABC), carbon dioxide (BC), halon foam (BC) |
|
Class C fire |
Electrical equipment and motor
switches Extinguisher: Dry chemical
(ABC), Carbon dioxide (BC), halon (BC) |
|
Class D fire |
Flammable metals: mercury,
magnesium, sodium, lithium Extinguisher: Metal X Fought be fire fighters only |
|
Class E fire |
Detonation (Arsenal fire) Allowed to burn out and nearby
materials protected |
|
Standard Hazards Identification System
(Diamond-shaped color coded symbol) |
|
|
Blue quadrant |
Health hazard |
|
Red quadrant |
Flammable hazard |
|
Yellow quadrant |
Reactivity/Stability hazard |
|
White quadrant |
Other special information |
|
Chemical spills |
1st step:
assist/evacuate personnel |
|
1:10 dilution of chlorine bleach (10%) |
To disinfect and clean bench
tops In contact with the area for at
least 20 minutes HBV: 10 minutes HIV: 2 minutes |
|
Poisonous vapors |
Chloroform Methanol Carbon tetrachloride Bromide Ammonia Formaldehyde Mercury |
|
Flammable and combustible solvents |
Acetone Ethanol Toluene Methanol Xylene Benzene Isopropanol Heptane |
|
Flammable liquids |
Flash point below 37.8OC |
|
Combustible liquids |
Flash point at or above 37.8OC |
|
Strong acids or bases |
Neutralized before disposal Water should NEVER be added to
concentrated acid |
|
Ether |
Deteriorate over time à hazardous Forms explosive peroxides |
|
Benzidine |
Known carcinogen |
|
Fumehoods |
Ventilation: velocity of
100-120 ft/min |
|
Safety showers |
Deliver 30-50 gal/min of H2O
at 20-50 psi |
|
Carbohydrates |
|
|
Glycol aldehyde |
The simplest carbohydrate |
|
Sucrose |
Most common nunreducing sugar |
|
Pancreas |
Exocrine: Enzymes (AMS, LPS) Endocrine: Hormones (Insulin,
glucagon, somatostatin) |
|
Hyperglycemic Hormones |
“GAG CHET” Glucagon ACTH GH Cortisol Human Placental Lactogen Epinephrine Thyroxine |
|
Hyperglycemia (≥126 mg/dL) |
Electrolyte Imbalance: Decreased: Sodium, Bicarbonate Increased: Potassium |
|
Hypoglycemia |
50-55 mg/dL = Symptoms ≤50 mg/dL = Diagnostic |
|
Whipple’s triad (Hypoglycemia) |
Low blood glucose concentration Typical symptoms Symptoms alleviated by glucose
administration |
|
6:1 |
Ratio of BHA to AA in severe DM (Normal = 1:1) |
|
Type 1 DM |
IDDM Juvenile Onset Brittle Ketosis-prone 80-90% reduction of beta-cells à Symptomatic Type 1 DM HLA-DR3 and DR4 (+) Glutamic acid decarboxylase
(GAD65) (+) Insulin autoantibodies (+) Microalbuminuria: 50-200
mg/24 hours = Diabetic nephropathy (-) C-peptide |
|
Complications of Type I DM |
Microvascular disorders: Nephropathy Neuropathy Retinopathy |
|
Type 2 |
NIDDM Adult type/Maturity Onset Stable Ketosis-resistant Receptor-deficient Insulin resistance: relative
insulin deficiency Strong genetic predisposition Geneticist’s nightmare If untreated à glucose: >500 mg/dL à nonketotic hyperosmolar coma |
|
Gestational DM |
Screening: 1hr GCT (50g) – bet.
24 and 28 weeks of gestation Confirmatory: 3-hr GTT (100g) Infants: at risk for respiratory
distress syndrome, hypocalcemia, hyperbilirubinemia After giving birth, evaluate
6-12 weeks postpartum Converts to DM w/in 10 years
in 30-40% of cases |
|
OGTT (GDM) |
FBS = ≥95 mg/dL 1-Hr = ≥ 180 mg/dL 2-Hr = ≥ 155 mg/dL 3-Hr = ≥ 140 mg/dL GDM = 2 plasma values of the
above glucose levels are exceeded |
|
Impaired fasting glucose (Pre-diabetes) |
FBS = 100-125 mg/dL |
|
Impaired glucose tolerance |
FBS = <126 mg/dL 2-Hr OGTT = 140-199 mg/dL |
|
FBS |
WB = 15% lower than in serum or
plasma VB = 7 mg/dL lower than capillary
and arterial blood |
|
CSF glucose |
60-70% of the plasma glucose |
|
Peritoneal fluid glucose |
Same with plasma glucose |
|
Plasma glucose increases w/ age |
Fasting: 2 mg/dL/decade Postprandial: 4 mg/dL/decade Glucose challenge: 8-13
mg/dL/decade |
|
w/in 1 hour (Preferably w/in 30 mins) |
Separate serum/plasma from the
cells |
|
5-7%/hr |
Glycolysis at room temperature |
|
1-2 mg%/hr |
Glycolysis at refrigerated
temperature |
|
Copper reduction methods |
Cupric à Cuprous à Cuprous oxide |
|
Folin Wu |
Cuprous ions + phosphomolybdate
à phosphomolybdenum blue |
|
Nelson-Somogyi |
Cuprous ions + arsenomolybdate à arsenomolybdenum blue |
|
Neocuproine method |
Cuprous ions + neocuproine à Cuprous-neocuproine complex
(yellow) |
|
Benedict’s method |
Reducing substances in blood
and urine |
|
Alkaline Ferric Reduction method
(Hagedorn-Jensen) |
Ferricyanide ---(Glucose)-->
Ferrocyanide (Yellow)
(Colorless) |
|
Ortho-toluidine (Dubowski method) |
Schiff’s base |
|
Glucose oxidase |
Measures beta-D-glucose (65%) |
|
Mutarotase |
Converts alpha-D-glucose (35%)
to beta-D-glucose (65%) |
|
NADH/NADPH |
Absorbance at 340nm |
|
Polarographic glucose oxidase |
Consumption of oxygen on an
oxygen-sensing electrode O2 consumption α glucose concentration |
|
Hexokinase method |
Most specific method Reference method Uses G-6-PD |
|
G-6-PD |
Most specific enzyme rgt for
glucose testing |
|
Interfering substances (Glucose oxidase) |
False-decreased Bilirubin Uric acid Ascorbate |
|
Hemolysis (>0.5 g/dL Hgb) |
Major interfering substance in
hexokinase method (false-decreased) |
|
Dextrostics |
Cellular strip Strip w/ glucose oxidase,
peroxidase and chromogen |
|
OGTT |
Janney-Isaacson method (Single
dose) = most common Exton Rose (Double dose) Drink the glucose load within 5
mins |
|
IVGTT |
For patients with gastrointestinal
disorders (malabsorption) Glucose: 0.5 g/kg body weight Given w/in 3 mins 1st blood
collection: after 5 mins of IV glucose |
|
Requirements for OGTT |
Ambulatory Fasting: 8-14 hours Unrestricted diet of 150g
CHO/day for 3 days Do not smoke or drink alcohol |
|
Glucose load |
75 g = adult (WHO std) 100 g = pregnant 1.75 g glucose/kg BW = children |
|
HbA1c |
2-3 months Glucose = beta-chain of HbA1 1% increase in HbA1c
= 35 mg/dL increase in plasma glucose 18-20% = prolonged
hyperglycemia 7% = cutoff Specimen: EDTA whole blood Test: Affinity chromatography
(preferred) |
|
IDA and older RBCs |
High HbA1c |
|
RBC lifespan disorders |
Low HbA1c |
|
Fructosamine (Glycosylated albumin/ plasma protein
ketoamine) |
2-3 weeks Useful for patients w/
hemolytic anemias and Hgb variants Not used in cases of low
albumin Specimen: Serum |
|
Galactosemia |
Congenital deficiency of 1 of 3
enzymes in galactose metabolism Galactose-1-phosphate uridyl
transferase (most common) Galactokinase Uridine diphosphate
galactose-4-epimerase |
|
Essential fructosuria |
Autosomal recessive Fructokinase deficiency |
|
Hereditary fructose intolerance |
Defective
fructose-1,6-biphosphate aldolase B activity |
|
Fructose-1,6-biphosphate deficiency |
Failure of hepatic glucose
generation by gluconeogenic precursors such as lactate and glycerol |
|
Glycogen Storage Disease |
Autosomal recessive Defective glycogen metabolism Test: IVGTT (Type I GSD) |
|
Ia = Von Gierke |
Glucose-6-Phosphatase
deficiency (most common worldwide) |
|
II = Pompe |
Alpha-1,4-glucosidase
deficiency (most common in the Philippines) |
|
III = Cori Forbes |
Debrancher enzyme deficiency |
|
IV = Andersen |
Brancher enzyme deficiency |
|
V = McArdle |
Muscle phosphorylase deficiency |
|
VI = Hers |
Liver phosphorylase deficiency |
|
VII = Tarui |
Phosphofructokinase deficiency |
|
XII = Fanconi-Bickel |
Glucose transporter 2
deficiency |
|
CSF glucose |
Collect blood glucose at least
60 mins (to 2 hrs) before the lumbar puncture (Because of the lag in CSF
glucose equilibrium time) |
|
< 0.5 |
Normal CSF : serum glucose
ratio |
|
C-peptide |
Formed during conversion of
pro-insulin to insulin |
|
5:1 to 15:1 |
Normal C-peptide : insulin
ratio |
|
D-xylose absorption test |
Differentiate pancreatic
insufficiency from malabsorption (low blood or urine xylose) |
|
Gerhardt’s ferric chloride test |
Acetoacetate |
|
Nitroprusside test |
10x more sensitive to
acetoacetate than to acetone |
|
Acetest tablets |
Acetoacetate and acetone |
|
Ketostix |
Detects acetoacetate better
than acetone |
|
KetoSite assay |
Detects beta-hydroxybutyrate
but not widely used |
|
Normal Values (Carbohydrates) |
RBS = <140 mg/dL FBS = 70-100 mg/dL HbA1c = 3-6% Fructosamine = 205-285 μmol/L 2-Hr PPBS = <140 mg/dL GTT: 30 mins = 30-60 mg/dL above
fasting 1-Hr = 20-50 mg/dL above
fasting 2-Hr = 5-15 mg/dL above fasting 3-Hr = fasting level or below |
|
Lipids |
|
|
Phospholipids |
Most abundant lipid Amphipathic: polar (hydrophilic
head) and nonpolar (hydrophobic side chain) |
|
Sphingomyelin |
Reference material during 3rd
trimester of pregnancy Concentration is constant as
opposed to lecithin Not derived from glycerol but
from sphingosine (amino alcohol) |
|
Forms of phospholipids |
70% Lecithin/Phosphatidyl
choline 20% Sphingomyelin 10% Cephalin |
|
TLC + Densitometric quantitation |
Method for L/S ratio |
|
Microviscosity |
Measured by fluorescence
polarization |
|
Cholesterol |
Not a source of fuel Not affected by fasting 70% Cholesterol ester
(plasma/serum) 30% Free cholesterol
(plasma/serum and RBC) |
|
LCAT |
Esterification of cholesterol |
|
Apo A-1 |
Activator of LCAT |
|
Cholesterol increases after the age of 50 |
2 mg/dL/year between 50 and 60
years old |
|
Liebermann Burchardt |
Cholestadienyl Monosulfonic
acid Green end color |
|
Salkowski |
Cholestadienyl Disulfonic acid Red end color |
|
Color developer mixture (Cholesterol) |
Glacial acetic acid Acetic anhydride Conc. H2SO4 |
|
One-step method |
Colorimetry (Pearson, Stern and
Mac Gavack) |
|
Two-step method |
Color. + Extraction (Bloor’s) |
|
Three-step method |
Color. + Extract. +
Saponification (Abell-Kendall) |
|
Four-step method |
Color. +Extract. + Sapon. +
Precipitation (Schaenheimer Sperry, Parekh
and Jung) |
|
Abell, Levy and Brodie mtd (Chemical method) |
CDC reference method for
cholesterol: -Hydrolysis/saponification
(Alc. KOH) -Hexane extraction -Colorimetry
(Liebermann-Burchardt) |
|
Triglycerides |
Most insoluble lipid Main storage lipid in man
(adipose tissue) – 95% Fasting: 12 hours |
|
Triglyceride increases after the age of 50 |
2 mg/dL/year between 50 and 60
years old |
|
Van Handel & Zilversmith (Colorimetric) |
Chromotropic acid (+) Blue color compound |
|
Hantzsch Condensation (Fluorometric) |
Diacetyl acetone (+) Diacetyl lutidine compound |
|
Modified Van Handel and Zilversmith (Chemical method) |
CDC reference method for
triglycerides: -Alkaline hydrolysis -Chloroform extraction à extract treated w/ silicic
acid -Color reaction w/ chromotropic
acid – meas. HCHO (+) Pink colored |
|
Fatty acids |
Short chain = 4-6 C atoms Medium chain = 8-12 C atoms Long chain = >12 C atoms Saturated = w/o double bonds Unsaturated = w/ double bonds Substrate for gluconeogenesis Most is bound to albumin |
|
Palmitic acid |
16:0 |
|
Stearic acid |
18:0 |
|
Oleic acid |
18:1 |
|
Linoleic acid |
18:2 |
|
Arachidonic acid |
20:4 |
|
Lipoprotein lipase (Lipemia clearing factor) |
Hydrolyzes TAG in lipoproteins,
releasing fatty acid and glycerol |
|
Hepatic lipase |
Hydrolyzes TAG and
phospholipids from HDL Hydrolyzes lipids on VLDL and
IDL |
|
Endothelial lipase |
Hydrolyzes phospholipids and
TAG in HDL |
|
Apolipoprotein |
Protein component of
lipoprotein Amphipathic helix – ability of
proteins to bind to lipids |
|
Chylomicrons |
Largest and least dense Produced by the intestine SG: <0.95 80-95% TAG (exogenous) Apo B-48 (Major) EP: Origin |
|
VLDL |
Secreted by the liver SG: 0.95-1.006 65% TAG (endogenous) Apo B-100 (Major) EP: pre-beta |
|
LDL |
Synthesized by the liver SG: 1.006-1.063 50% CE Apo B-100 (Major) EP: beta Cholesterol transport: LiveràTissues Target of cholesterol lowering
therapy Better marker for CHD risk |
|
HDL |
Smallest but dense SG: 1.063-1.21 45-55% protein 26-32% phospholipid Apo A-1 (Major) EP: alpha Produced by the liver and
intestine Reverse cholesterol transport:
TissueàLiver |
|
IDL |
Product of VLDL catabolism Seen in Type 3
hyperlipoproteinemia (Apo E-III def.; beta-VLDL) SG: 1.006-1.019 |
|
Lp(a) |
Sinking pre-beta lipoprotein SG: 1.045-1.080 Apo B-100 EP: pre-beta (VLDL) UC: like LDL Independent risk factor for
atherosclerosis |
|
LpX |
Found in obstructive jaundice
(cholestasis) and LCAT deficiency 90% FC and PL Apo C and albumin |
|
Beta-VLDL |
Floating beta-lipoprotein SG: <1.006 EP: beta (LDL) UC: like VLDL Found in type 3
hyperlipoproteinemia (Apo E-III def; IDL) Rich in cholesterol content
than VLDL |
|
Lipoprotein methodologies |
Specimen: sample from serum
separator tubes (preferred) EDTA plasma: choice for
research studies of LPP fractions Fasting state: TAG à VLDL Nonfasting state: TAG à CM |
|
Ultracentrifugation |
Reference method for LPP
quantitation Reagent: Potassium bromide (SG:
1.063) Ultracentrifugation of plasma
for 24 hours Expressed in Svedberg units |
|
Electrophoresis |
Electrophoretic pattern: (+) HDL ßVLDL ß LDL ß CM (Origin) (-) Agarose gel: sensitive medium VLDL: migrates w/ alpha2-globulin
(pre-beta) |
|
Chemical precipitation |
Uses polyanions (heparin and
divalent cations) and polyethylene glycol Dextran sulfate-Mg2+ Heparin-Mn2+ |
|
3-step procedure: Ultracentrifugation Precipitation Abell-Kendall assay |
CDC Reference method for HDL |
|
Beta quantification + Ultracentrifugation +
Chemical precipitation |
Method for LDL Sample: EDTA plasma |
|
Immunoturbidimetric assay |
Measures Lipoprotein (a) |
|
LDL Cholesterol |
Total Cholesterol – HDL – VLDL |
|
Friedewald method |
Most commonly used VLDL = TAG/2.175 (mmol/L) VLDL = TAG/5 (mg/dL) Not applicable if TAG is
>400 mg/dL |
|
De Long method |
VLDL = TAG/2.825 (mmol/L) VDL = TAG/6.5 (mg/dL) |
|
Apo A-1 |
Activates LCAT |
|
Apo B-100 |
LDL à LDL receptor |
|
Apo B-48 |
CM (major) Not recognized by LDL receptor |
|
Apo C-II |
Activates LPL |
|
Apo D |
Activates LCAT |
|
Apo E |
Apo E-4: associated w/ high
LDL, higher risk of CHD and Alzheimer’s disease |
|
Apo(a) |
Lp(a) Homologous to plasminogen |
|
Abetalipoproteinemia (Basses-Kornzweig syn.) |
Autosomal recessive Defective apo B synthesis Deficient fat soluble vitamins |
|
Niemann-Pick disease |
Sphingomyelinase deficiency |
|
Tangier’s disease |
Deficiency of HDL (1-2 mg/dL) Defects in the gene for the
ABCA1 transporter |
|
LPL deficiency (Chylomicronemia) |
TAG = 10,000 mg/dL Do not develop premature coronary
disease (CM are not atherogenic) Abdominal pain and pancreatitis |
|
LCAT deficiency |
Fish-eye disease Low HDL |
|
Tay-Sachs disease |
Hexosaminidase A deficiency |
|
Fredrickson Classification |
|
|
Type 1 |
LPL deficiency
(Chylomicronemia) Increased: CM (TAG) |
|
Type 2a |
Familial hypercholesterolemia Increased: LDL
(cholesterol) |
|
Type 2b |
Combined hyperlipidemia (most
common primary hyperlipidemia) Increased: LDL (cholesterol), VLDL (TAG) |
|
Type 3 |
Dysbetalipoproteinemia Increased: IDL, (+) beta-VLDL (+) Apo E-II (+) Eruptive and palmar xanthomas |
|
Type 4 |
Hypertriglyceridemia Increased: VLDL (TAG) |
|
Type 5 |
Increased: VLDL (Endo.TAG), CM (Exo.TAG) |
|
Normal Values (Lipids) |
Cholesterol: Desirable = <200 mg/dL Borderline high = 200-239 mg/dL High = >240 mg/dL Triglycerides: Desirable = <150 mg/dL Borderline high = 150-199 mg/dL High = 200-499 Very high = >500 mg/dL HDL: Low = <40 mg/dL (Cutoff) High = >60 mg/dL LDL: Optimal = <100 mg/dL Near/above optimal = 100-129
mg/dL Borderline high = 130-159 mg/dL High = 160-189 mg/dL Very high = >190 mg/dL |
|
Proteins |
|
|
Proteis |
First rank of importance |
|
Proteins |
Amphoteric: positive and
negative charges Effective blood buffers Synthesized by the liver except
immunoglobulins (plasma cells) Provide 12-20% of total daily
body energy requirement Composed of 50-70% of the
cell’s dry weight |
|
Primary structure |
Amino acid sequence Det. the identity of protein,
molecular structure, function binding capacity, recognition ability |
|
Secondary structure |
Winding of polypeptide chain Specific 3-D conformations:
alpha-helix, beta-pleated sheath, bend form |
|
Tertiary structure |
Actual 3-D configuration Folding pattern Physical and chemical
properties of proteins |
|
Quarternary structure |
Association of 2 or more
polypeptide chains à protein |
|
Albumin |
No quarternary structure |
|
Glucogenic amino acids |
Alanine (pyruvate) Arginine (alpha-ketoglutarate) Aspartate (oxaloacetate) |
|
Ketogenic amino acids |
Degraded to acetyl-CoA Leucine Lysine |
|
Simple proteins |
Hydrolysis à Amino acids Fibrous: fibrinogen, troponins,
collagen Globular: hemoglobin, plasma proteins,
enzymes, peptide hormones |
|
Conjugated proteins |
Protein (apoprotein) +
nonprotein moiety (prosthetic group) Metalloproteins: ferritin, ceruloplasmin,
hemoglobin, flavoproteins Lipoproteins: VLDL, HDL, LDL, CM Glycoproteins: haptoglobin, alpha1-antitrypsin
(10-40% CHO) Mucoproteins or proteoglycans: Mucin (CHO > CHON) Nucleoproteins: Chromatin (combined w/
nucleic acids) |
|
Nitrogen balance |
Balance bet. anabolism and
catabolism |
|
Negative nitrogen balance |
Catabolism > anabolism Excessive tissue destruction |
|
Positive nitrogen balance |
Anabolism > catabolism Growth and repair processes |
|
Prealbumin (Transthyretin) |
Transports thyroxine and
retinol (Vit. A) Landmark to confirm that the
specimen is really CSF |
|
Albumin |
Maintains osmotic pressure Negative acute phase reactant |
|
Alpha1-antitrypsin |
Acute phase reactant Major inhibitor of protease
activity 90% of alpha1-globulin
band |
|
Alpha1-fetoprotein |
Gestational marker Tumor marker: hepatic and
gonodal cancers Screening test for fetal
conditions (Spx: maternal serum) Amniotic fluid: confirmatory
test Increased: Hepatoma, spina bifida, neural
tube defects Decreased: Down Syndrome (Trisomy 21) |
|
Alpha1-acid glycoprotein/
orosomucoid |
Low pI (2.7) Negatively charged even in acid
solution |
|
Alpha1-antichymotrypsin |
Acute phase reactant Binds and inactivates PSA Increased: Alzheimer’s disease, AMI,
infection, malignancy, burns |
|
Haptoglobin (alpha2) |
Acute phase reactant Binds free hemoglobin (alpha
chain) |
|
Ceruloplasmin (alpha2) |
Copper binding (6-8 atoms of
copper are attached to it) Has enzymatic activities Decreased: Wilson’s disease (copper à skin, liver, brain, cornea
[Kayser-Fleisher rings]) |
|
Alpha2-macroglobulin |
Larges major nonimmunoglobulin
protein Increased: Nephrotic syndrome (10x) Forms a complex w/ PSA |
|
Group-specific component (Gc)-globulin (bet.
alpha1 and alpha2) |
Affinity w/ vitamin D and actin |
|
Hemopexin (beta) |
Binds free heme |
|
Beta2-microglobulin |
HLA Filtered by glomeruli but
reabsorbed |
|
Transferrin/Siderophilin (beta) |
Negative acute phase reactant Major component of beta2-globulin
fraction Pseudoparaproteinemia in severe
IDA Increased: Hemochromatosis
(bronze-skin), IDA |
|
Complement (beta) |
C3: major |
|
Fibrinogen (bet. beta and gamma) |
Acute phase reactant Between beta and gamma
globulins |
|
CRP (gamma) |
General scavenger molecule Undetectable in healthy
individuals hsCRP: warning test to persons
at risk of CAD |
|
Immunoglobulins (gamma) |
Synthesized by the plasma cells IgG>IgA>IgM>IgD>IgE |
|
Myoglobin |
Marker: Ischemic muscle cells,
chest pain (angina), AMI |
|
Troponins |
Most important marker for AMI |
|
TnT (Tropomyosin-binding subunit) |
Specific for heart muscle Det. unstable angina (angina at
rest) |
|
TnI (Inhibitory subunit or Actin-binding
unit) |
Only found in the myocardium Greater cardiac specificity
than TnT Highly specific for AMI 13x more abundant in the
myocardium than CK-MB Very sensitive indicator of
even minor amount of cardiac necrosis |
|
TnC |
Binds calcium ions and regulate
muscle contractions |
|
Glomerular proteinuria |
Most common and serious type Often called albuminuria |
|
Tubular proteinuria |
Defective reabsorption Slightly increased albumin
excretion |
|
Overload proteinuria |
Hemoglobinuria Myoglobinuria Bence-Jones proteinuria |
|
Postrenal proteinuria |
Urinary tract infection,
bleeding, malignancy |
|
Microalbuminuria |
Type 1 DM Albumin excretion ≥30 mg/g
creatinine (cutoff: DM) but ≤300 mg/g creatinine Microalbuminuria: 2 out of 3
specimens submitted are w/ abnormal findings (w/in 6 months) |
|
CSF Oligoclonal banding |
2 or more IgG bands in the
gamma region: Multiple sclerosis Encephalitis Neurosyphilis Guillain-Barre syndrome Neoplastic disorders |
|
Serum Oligoclonal banding |
Leukemia Lymphoma Viral infections |
|
Alkaptonuria |
Ochronosis (tissue
pigmentation) |
|
Homocystinuria |
Impaired activity of
cystathione beta-synthetase Elevated homocysteine and
methionine in blood and urine Screen: Modified Guthrie test
(Antagonist: L-methionine sulfoximine) |
|
MSUD |
Markedly reduced or absence of
alpha-ketoacid decarboxylase Screen: Modified Guthrie test
(Antagonist: 4-azaleucine) Diagnostic: Amino acid analysis
(HPLC) |
|
PKU |
Deficiency of
tetrahydrobiopterin (BH4) à elevated blood phenylalanine |
|
Normal Values (Proteins) |
Total protein = 6.5-8.3 g/dL Albumin = 3.5-5.0 g/dL Globulin = 2.3-3.5 g/dL |
|
Kidney
Function Tests |
|
|
Tests for GFR |
Clearance: -Inulin clearance -Creatinine clearance -Urea clearance Phenolsulfonphthalein dye test Cystatin C |
|
Tests for Renal Blood Flow |
BUN Creatinine Uric acid |
|
Tests Measuring Tubular Function |
Excretion: -Para-amino hippurate test
(Diodrast test) -Phenolsulfonphthalein dye test Concentration: -Specific gravity -Osmolality |
|
GFR |
Decreases by 1.0 mL/min/year
after age 20-30 years 150 L of glomerular filtrate is
produced daily |
|
Inulin clearance |
Reference method |
|
Creatinine clearance |
Best alternative method Measure of the completeness of
a 24-hour urine collection Excretion: 1.2-1.5 g
creatinine/day |
|
Urea clearance |
Demonstrate progression of
renal disease or response to therapy |
|
Cystatin C |
Low MW protease inhibitor FilteredàNot secretedàCompletely reabsorbed (PCT) Indirect estimates of GFR Its presence in urine denotes
damage to PCT |
|
BUN |
Synthesized from Ornithine or
Kreb’s Henseleit cycle First metabolite to elevate in
kidney diseases Better indicator of nitrogen
intake and state of hydration |
|
2.14 |
BUN à Urea (mg/dL) |
|
Fluoride or citrate |
Inhibit urease |
|
Thiosemicarbazide Ferric ions |
Enhance color development (BUN
mtd) |
|
Diacetyl monoxime method |
Yellow diazine derivative |
|
Urease method |
Routinely used Urease: prepared from jack
beans Urea ---(Urease)--> NH4
+ Berthelot reagent (Measure ammonia) |
|
Coupled urease |
Glutamate dehydrogenase method UV enzymatic method |
|
Isotope dilution mass spectrometry |
Reference method For research purposes |
|
NPN |
45% Urea 20% Amino acid 20% Uric acid 5% Creatinine 1-2% Creatine 0.2% Ammonia |
|
Creatinine |
Derived from alpha-methyl
guanidoacetic acid (creatine) Produced by 3 amino acids
(methionine, arginine, lysine) Most commonly used to monitor
renal function |
|
Enzymatic methods (Creatinine) |
Creatinine Aminohydrolase – CK
method Creatinase-Hydrogen Peroxide
method – benzoquinonemine dye (red) Creatininase (a.k.a. creatinine
aminohydrolase) |
|
Direct Jaffe method |
Formation of red tautomer of
creatinine picrate |
|
Interferences (Direct Jaffe) |
Falsely increased: Ascorbate Glucose Uric acid Alpha-keto acids |
|
Folin Wu Method |
(+) Red orange tautomer |
|
Lloyd’s or Fuller’s Earth method |
True measure of creatinine Sensitive and specific Uses adsorbent to remove
interferences (UA, Hgb, Bili) |
|
Lloyd’s reagent |
Sodium aluminum silicate |
|
Fuller’s earth reagent |
Aluminum magnesium silicate |
|
Jaffe reagent (Alk. picrate) |
Satd. picric acid + 10% NaOH |
|
Kinetic Jaffe method |
Popular, inexpensive, rapid and
easy to perform Requires automated equipment |
|
Azotemia |
Elevated urea and creatinine in
blood |
|
Pre-renal azotemia |
Decreased GFR but normal renal
function Dehydration, shock, CHF Increased: BUN Normal: Creatinine |
|
Renal azotemia |
True renal disease Decreased GFR Striking BUN level but slowly
rising creatinine value BUN = >100 mg/dL Creatinine = >20 mg/dL Uric acid = >12 mg/dL |
|
Post renal azotemia |
Urinary tract obstruction Decreased GFR Nephrolithiasis, cancer or
tumors of GUT Creatinine = normal or slightly
increased |
|
Uremia |
Marked elevation of urea,
accompanied by acidemia and electrolyte imbalance (K+ elevation)
of renal failure Normocytic, normochromic anemia Uremic frost (dirty skin) Edema Foul breath Urine-like sweat |
|
Uric acid |
From purine (adenine and
guanine) catabolism Excretion: 1g/day |
|
Hyperuricemia |
-Gout -Increased nuclear metabolism
(leukemia, lymphoma, MM, polycythemia, hemolytic and megaloblastic anemia) –
Tx: Allopurinol -Chronic renal disease -Lesch-Nyhan syndrome (HGPRT
deficiency) |
|
Hypouricemia |
Fanconi’s syndrome Wilson’s disease Hodgkin’s disease |
|
Methods (Uric acid) |
Stable for 3 days Potassium oxalate cannot be
used Major interferences: Ascorbate
and bilirubin |
|
Phosphotungstic acid mtd |
Uric acid + Phosphotungstic
acid ---(NaCN/NaCO3)--> Tungsten blue + Allantoin |
|
NaCN |
Folin Newton Brown Benedict |
|
NaCO3 |
Archibald Henry Caraway |
|
Lagphase |
Incubation period after the
addition of an alkali to inactivate non-uric acid reactants |
|
Uricase method |
Simplest and most specific
method Candidate reference method Uric acid (Absorbance at 293nm)
---[Uricase]--> Allantoin (No absorbance) Decrease in absorbance α uric acid
concentration |
|
Para-amino hippurate test |
Measures renal plasma flow Reference method for tubular
function |
|
Phenolsulfonphthalein dye test |
Measures excretion of dye
proportional to renal tubular mass 6 mg of PSP is administered IV |
|
Concentration tests |
Collecting tubules and loops of
Henle Specimen: 1st
morning urine |
|
Specific gravity |
Affected by solute number and
mass SG >1.050: X-ray dye and
mannitol 1.010 = SG of ultrafiltrate in
Bowman’s space |
|
Osmolality |
Total number solute particles
present/kg of solvent (moles/kg solvent) Affectted only by number of
solutes present Urine osmolality = due to urea Serum osmolality = due to
sodium and chloride Det. by Colligative
properties: Freezing point (incr. osm. =
decr. FP) Vapor pressure (incr. osm. =
decr. VP) Osmotic pressure (incr. osm. =
incr. OP) Boiling point (incr. osm. =
incr. BP) |
|
Direct methods (Osmolality) |
Freezing point osmometry =
popular method Vapor pressure osmometry
(Seebeck effect) |
|
Incr. plasma osmolality |
Incr. vasopressin (H2O
reabsorption) à decr. plasma osmolality |
|
Tubular failure |
Increased: BUN, creatinine, calcium Decreased: Phosphate |
|
Osmolal gap |
Difference between measured and
calculated osmolality Sensitive indicator of alcohol
or drug overdose |
|
Osmolal gap: >12 mOsm/kg |
DKA Drug overdose Renal failure |
|
Normal Values (Kidney Function Tests) |
Creatinine Clearance: Male = 85-125 mL/min Female = 75-112 mL/min BUN = 8-23 mg/dL Creatinine = 0.5-1.5 mg/dL Uric acid: Male = 3.5-7.2 mg/dL Female = 2.6-6.0 mg/dL Renal plasma flow (PAH) = 600-700 mL/min Renal blood flow (PSP) = 1200 mL/min SG = 1.005-1.030 Osmolality: Serum = 275-295 mOsm/kg Urine (24-hr) = 300-900 mOsm/kg [<290 mOsm/kg = kidney
damage] Urine osmolality: Serum
osmolality
= 1:1 to 3:1 [>1:1 = Glomerular disease] [1.2:1 = loss of renal
concentrating ability] [<1:1 = Diabetes Insipidus] |
|
Liver
Function Tests |
|
|
Liver |
Receives 15 mL of blood per
minute Lobule: anatomic unit |
|
Synthetic function |
Proteins, CHO, lipids, LPP,
clotting factors, ketone bodies, enzymes Albumin: 12g/day |
|
Conjugation function |
Bilirubin metabolism Bilirubin: 200mg/day |
|
Detoxification and Drug metabolism |
Drugs Ammonia à Urea à Excreted |
|
Excretory and Secretory functions |
Bile acids: cholic acid and
chenodeoxycholic acid Bile salts: bile acids + amino
acids (glycine and taurine) |
|
Storage function |
Vitamins Glycogen |
|
Test measuring the Hepatic Synthetic Ability |
Total Protein Determination: -Kjeldahl method -Biuret method -Folin-Ciocalteu (Lowry) method -UV absorption method -Electrophoresis -Refractometry -Turbidimetric and
Nephelometric methods -Salt fractionation Prothrombin Time (Vitamin K Response
Test) |
|
Test measuring Conjugation/Excretion Function |
Bilirubin Assay: -Evelyn and Malloy method -Jendrassik and Grof Bromsulfonphthalein (BSP) Dye
Excretion test |
|
Test for Detoxification Function |
Enzyme tests: ALP, AST, ALT, 5’NT, GGT, OCT,
LAP, LDH Ammonia: -Kjeldahl (Digestion) method -Nesslerization reaction -Berthelot reaction |
|
Plasma protein |
0.2-0.4 g/dL higher than serum
due to fibrinogen |
|
Kjeldahl (Digestion) mtd |
Standard reference method Measurement of nitrogen content Serum + Tungstic acid à PFF 1g N2 = 6.54g
protein 15.1-16.8% = N2
content of proteins Rgt: H2SO4 End product: NH3 |
|
Biuret method |
Most widely used method (IFCC
recommended) Req. at least 2 peptide bonds
and an alkaline medium Rgts: Alkaline CuSO4 Rochelle salt (NaK Tartrate) NaOH KI End product: Violet color (545nm) |
|
Folin-Ciocalteu (Lowry) method |
Highest analytical sensitivity Oxidation of phenolic compounds
(tyrosine, tryptophan, histidine) Rgts: Phenol (or
phosphotungstic-molybdic acid) Biuret (color enhancer) End product: Blue color |
|
Electrophoresis |
MI: elevated APRs (AAT, HPG, a1-x) |
|
Gamma-spike |
Monoclonal gammopathy (multiple
myeloma) |
|
Beta-gamma bridging |
In serum: Hepatic cirrhosis
(IgA) In plasma: normal (fibrinogen) |
|
Alpha2-globulin band spike |
Nephrotic syndrome |
|
Alpha1-globulin flat curve |
Juvenile cirrhosis (AAT
deficiency) |
|
Alpha1, alpha2,
beta-globulin band spikes |
Inflammation |
|
Polyclonal gammopathy |
Chronic inflammation (RA,
malignancy) |
|
Small spikes in beta region |
IDA (transferrin) |
|
Free hemoglobin |
“Blip” in the late alpha2
or early beta region |
|
Refractometry |
Refractive index |
|
Turbidimetric and nephelometric methods |
SSA TCA |
|
Salt fractionation |
Salt: Sodium sulfate |
|
Albumin |
Soluble: Water Moderately concentrated salt solution Concentrated salt solution Insoluble: Hydrocarbon solvents Highly concentrated salt
solution Saturated salt solution |
