GLUCOSE –standard
spx venous plasma glucose
-fasting
glucose in WB 15% lower than serum or plasma
-venous
blood 7mg/dl lower than capillary blood; peritoneal fluid gluc same with plasma
-RT (20-25O)-
glucose ↓7mg/dl/hr -Ref
temp(4OC)- glucose ↓2mg/dl/hr
-Frozen Temp
(-20OC)- almost 0mg/dl/hr decrease
I. CHEMICAL METHOD
a. Oxidation
Reduction Method
1. Alkaline Copper
Reduction Method-
-Reduction of cupric ions to cuprous ions
forming cuprous oxide in hot alkaline sol. by glucose
*Alkaline Copper Tartrate
---glucose, heat-à Cuprous ions
a. Folin Wu Method- Cuprous ions + Phosphomolybdate↓
Phosphomolybdic acid or
Phosphomolybdenum blue
b. Nelson Somogyi- Cuprous ions + Arsenomolybdate ↓
Arenomolybdic acid or
Aresnomolybdenum blue
c. Neocuproine Method (2,9
Dimethyl 1,10 Phenantroline Hydrochloride)
Cuprous
ion + Neocuproine↓
Cuprous-Neocuproine Comples (yellow or Yellow orange)
d. Benedict’s Method
(Modification of Folin-Wu)- detect and quanti reducing subs in body fluid
-use citrate or tartrate as
stabilizing agent
2. Alkaline
Ferric Reduction Method (Hagedorn Jensen)-
reduction of yellow ferricyanide to
colorless ferrocyanide by glucose (Colorimetry)
b. Condensation
Method
Ortho-toluidine (Dubowski Method)
Glucose + Aromatic amines ---Glacial
HAC, heatà
glycosylamine + schiff’s base
II. ENZYMATIC
METHOD- act on glucose but not on other sugard and not on other reducing
subs
1. Glucose
oxidase Method- measure B-D glucose
a.
Colorimetruc Glucose Oxidase Method (Saifer
Gernstenfield Method)
Glucose
+ O2 ---glucose oxidase à Gluconic Acid + H2O2
H2O2
+Chromogenic subs ---peroxidaseà oxidized chromogenic
subs + H2O
b.
Polarographic Glucose Oxidase- meas rate of O2 consumotions, prop to gluc conc.
-conversion glucose quantified by consumption of O2 on oxygen-sensing
electrode
-H2O2 prevented from re-forming O2 by
adding molybdate, iodide, catalase and ethanol
Glucose
+O2 ---glucose oxidaseà Gluconic Acid + H2O2
H2O2
+ C2H5OH ---catalaseà CH3CHO + 2H2O
H2O2
+ 2H + 2 I ---molybdateà I2 + 2H2O
2. Hexokinase Method- most specific
glucose method; reference method;
-plasma -- heparin, EDTA, fluoride,
oxalate, or citrate; other sample urine, CSF, serous fluid
Glucose + ATP ---Hexokinaseà Glucose-6-Phosphate +
ADP
G-6-P +NADP---G-6-PDà 6Phosphogluconolactone
+NADPH-reduced coenzyme meas.
3.
Glucose Dehydrogenase Method- gluc red to prod Chrmophore meas by spectrophoto/
electric.
-Mutarotase- shorten time to reach
equilibrium; endcolor blue
4.
Dextrostics (cellular strip)- imp in establishing correct insulin amount for
next dose
5. Interstitial
Glucose Measuring Device- for continuous monitoring of glucose level in
diabetic px
6.
Glycosylated Hgh (HbA1c) or Glycated Hgb- monitoring
-represents a weighted average of glucose
level, w/ youngest RBC contributing greater extent
-spx EDTA whole blood; Method
electrophoresis, immunoassay, HPLC, Affinity Chromatography
-5.7-6.4% inc risk for diabetes; every 1% =
35mg/dl added to PG
CHOLESTEROL DETERMINATION-
can be measure w/o fasting; measured as a whole
-Px prep-usual
diet for 2 weeks, neither gaining nor losing weight
I. CHEMICAL METHOD- Principle: Dehydration and
oxidation of cholesterol to form a colored compound
a. Lieberman
Burchardt Reaction (Colorimetric) Endproduct= Cholestradienyl monosulfonic
Acid (green color)
Color
Developer Micture (LB rgt)’
-Glacial
acetic acid -con.
H2SO4
-acetoc
anhydride
b. Salkowsk’s
Reaction Endproduct= Cholestadieny Disulfonic Acid (red color)
>General Methods:
One Step
Method- Colorimetry (Pearson, Stern and Mac Gavack)
Two Step
Method- Extraction + Colorimetry (Bloors)
Three Step
Method- Saponification + Extraction + Colorimetry (Abell-Kenda)
Four Step
Method- Saponification+ Extraction + Colorimetry + Ppt. (Schoenheimer
Sperry + Parekh + Jung)
II. ENZYMATIC
METHOD- don’t required preliminary extraction step
Cholesterol Oxidase Reaction: commonly
used/ routine test
Cholesterol
Ester + H2O --chol esteraseà cholesterol + fatty
acid
Cholesterol
+ O2—cholesterol oxidaseà cholest-4-en-3-one +
H2O2
H2O2
+ phenol + 4-aminoantipyrine –peroxidaseà quinoneimine dye
CDC reference
method (Abell, Levy and Brodle Method)- 1-Saponification, 2-extration,
3-colorimetry
-use hexane extraction after
hydrolysis w/ alcoholic KOH by rxn w/ Lieberman-Burchardt color reagent
TRIGLYCERIDE MEASUREMENT
–hydrolyze all fatty acid esters of TAG to produce glycerol
I. CHEMICAL METHOD
a. Colorimetric
Method (Van Handel & Zilversmith)
TAG—alcoholic KOHàGlycerol + Fatty Acid
Glycerol
Oxidized by Periodic Acidà
Formaldehyde (HCHO)
HCHO
+ Chromotropic Acidà(+)Blue color compound
b. Fluorometric
Method (Hanstzch Condensation)
TAG –alc. KOHà Glycerol +FA
Glycerol
Oxidized by Periodic Acidà
Formaldehyde (HCHO)
HCHO
+ Diacetyl Acetone + NHà
Diacetyl Lutidine Compound
II. ENZYMATIC
METHOD
a. Glycerol Kinase Method- hydrolysis TAG to free
FA and glycerol, then phosphorylation of glycerol to glyceropho
*CDC reference
method (Modified Van Handel and Zilversmith)-colorimetric pink end color
Saponification- KOH (alkaline
hydrolysis) Adsorption-
Silicic acid Chrom (isolate TAG)
Extraction-
Chloroform Color
Reaction Chromotropic acid +TAG= PINK
Lipoprotein Methodologies:
-differentiated based on
electrophoresis and buoyant density (ultracentrifugation) -TC-HDL-C=non HLD-C
1.
Ultracentrifugation (density gradient)- reference method for quantitationof
lipoproteins (LPPs)
-based on
protein and TAG contents of lipoproteins; epressed in svedsverg(s) units
-Lipid
density 1.0g/mL while Protein density 1.4mg/L
-Reagent:
Potassium bromide solution w/ 1.063 density
2. Electrophoresis-
Pattern: HDL, VLDL, LDL, Chylomicrons
-preferred
supporting medium: Agarose-gel-speed; sensitive; resolves LLPs classes
-Lipid
staining dyes: Oil Red O, Fat Red 7B or Sudan Black B
-VLDL
migrates w/ a-2 globulin (preB); Chylomicrons if present remain at origin
3. Chemical
Precipitation- uses polyanions ) and divalent cations such as Mg, Ca,
manganese.
a. HDL- uses dextran sulphate
(syntheric heparin) w/ magnesium (precipitants)
CDC reference 3-step: ultracentrifugation, heparin manganese precipitation and
Abell-Kendall assay
b. LDL- EDTA plasma ..
4. Chromatographic method- utilizes Gel chromatography or
affinity chromatography
5. Immunochemical methods0 uses AB specific to epitopes o
apolipoproteins
6. Immunoassay or Immunonephelometry- Apolipoprotein assay
-meas
turbidity created by apolipoprotein-Ab complexes
Friedewald Method
(Indirect Method)
Formula for LDL-Cholesterol (LDL-C)= Total
Cholesterol –HDL-VLDL
VLDL (mmol/L)= Plasma
TAG/2.175 VLDL(mg/dL)=
Plasma TAG/5
DeLong Method (Indirect Method)
VLDL(mmol/L)=
Plasma TAG/2.825 VLDL(mg/dL)=
Plasma TAG/6.5
KINDEY FUNCTION TESTS
1. BUN
Determination: Fluorid, citrate inhibit urease; thriosemicarbazide and
ferric ions enhance color
I. Chemical Method (Direct Method)- Diacetyl Monoxime Method Urea+DMà
Yellow diazine derivative
II. Enzymatic Method (Indirect Method)
a. Hydrolysis of Urea by Urease (Routine metho) Urea+Urease à
NH3 + CO2
-ammonia
then treated with Berthelot rgt
b. Coupled Urease/ Glutamate Dehydrogenase (GLD) method-
UV enzymatic method
Urea
+ Urease à
NH4 + CO2 NH4+2-oxoglutarate + NADH—GLDà
Glutamat+NAD+H2)
c. Isotope
Dilution Mass Spectromoetry (IDMS)- Reference method
2. Creatinine
I. Chemical
Method- Direct Jaffe Method: Red-orange
tautomer of creatinine picrate is formed when creatinine
is mexed with alkaline picrate rgt
a. Folin Wu
method- sensitive but nonspecific method
b. Lloyd or Fuller’s earth Method-
sensitive and specific
Adsorbent:
Lloyd’s reagent (Sodium aluminium silicate) Adsorbent:
removes interference present
Fuller’s earth rgt (Aluminum Mg silicate) in spx and elution done
to separate crea
Jaffe Reagent
(Alkaline Picrate): Saturated Picric Acid; 10% NaOH
II. Kinetic Jaffe Method- serum mixed with alkaline
picrate sol, rate of change in absorbance measured bet 2points
III. Enzymatic Method- routine method; specific than
jaffe
a. Creatinine
Aminohydrolase- CK method –require large vol of pre-incubated sample: not
widely used
b. Creatinase
Hydrogen Peroxide Method- Creatinase aka creatinine aminohydrolase
Creatinine
+ H2O—creatinaseà Creatine + H2O—creatinaseàsarcosine
+ urea
Sarcosine
+ H2O + O2—sacrosine oxidaseàglycine + HCHO+ H2O2
H2O2+
phenol + 4-aminophenazone –peroxidaseà benzoquinonemine dye (Red)
IV. Isotope Dilution Mass Spectrometry (IDMS)- reference
method
3. Blood Uric Acid-
I. Chemical Methods: Reduction-Oxidation (Redox) Reaction
Uric acid + Phosphotungstic Acid
–NaCN/NaCO3àTungsten Blue + Allantoin + CO2
*Sodium
cyanide—Folin NewtonBenedict àBrown
*Sodium carbonate –Archibald Henryà Caraway
II. Enzymatic Method
Uricase Method-
routine, specific method
Uric
acid has absorbance of 293nm, allantoin do not
Uric
acid + O2 –uricaseà allantoim + CO2 + H2O
III. Isotope Dilution Mass Spectroscopy (IDMS)- reference
method
Osmolality
a. Direct Method: Freezing point osmometry*; Vapor
pressure osmometry (Seebeck effect) ↑Osmo↓FP,VP
b. Indirect Method: -use glocuse or urea in osmolality
Serum
Osmolality= 1.86X Na = Gluc(mg/dL)/18 + BUN(mg/dL)/2.8
*Osmolal gap-
difference between measured amd calculated plasma osmolality
LIVER FUNCTION TEST
I. Test Measuring
Hepatic Synthetic Function- quantitate severity of hepatic dysfunction
(Albumin, Vit-K dep CF)
A. Total Protein- RV: 6.5-8.3g/dL
a. Kjeldahl
Method- measure Nitrogen(15.1-16.8%) content of CHON; referenc method; 1gN= 6.45g CHON
Serum
+ tungstic
acidà
Protein-free filtrate (PFF)
-reagent:
H2SO4 (digesting agent) -Endproduct:
Ammonia
b. Biuret Method-
widely used recommended by Int’l federation of clinical chemistry (IFCC) expert
panel
-required at
least 2 peptide bonds and an alkaline medium
-Principle:
Cupric ions complex groups involved In peptide bond forming violet-colored chelate, proportional to
number of peptide bonds present and represent total protein level @454nm
-Reagents:
Alkaline Copper Sulfate, Rochelle Salt (NaK Tartrate), NaOH and K iodide
c. Folin-Clocateu
(lowry) Method- highest analytical sensitivity
-Priciple:
Oxidation of phenolic compounds such as tyrosine, tryptophan, histidine àdeep blue color
-Reagent: Phosphotungstic-molybdic acid or
phenol reagent; Biuret rgt (color enhancer)
d. Ultraviolet Absorption Method- absorbance of CHON
@210nm due to abs. of peptide bonds @speci. waveL
e. Serum Protein Electrophoresis (SPE)- migration of
charged paticle in an electric field
-important in
ID or monoclonal spike of Ig and differentiating them from polyclonal
hypergammaglobulinemia
Normal SPE pattern:
Albumin
(1st band)- fastest band 53-65%
Alpha
1-Globulin (2nd fastest)- glycoproteins, AAT, AAG, TBG; inc in
nonspecific response to inflammation Alpha
2-globulin (3rd fastest)- haptoglobin, AMG, ceruloplasmin 7-13%
Beta-globulin
(4th band)- transferrin, beta-lipoprotein, hemopexin, complement
(C3, C4) 8-14%
Gamma-globulin
(5th band)- immunoglobulin and CRP 12-22%
Abnormal Serum
Electrophoretic Patterns:
i. Gamma spike- multiple myeloma iv. a1-globulin flat curve0
juvenile cirrhosis (AAT deficien.)
ii. beta-gamma bridging- hepatic cirrhosis v. Spikes of a1,
a2, B globulin bands- inflammation
iii. a2-globulin band spike- nephrotic syndrome
f. Refractometry- alternative test to chem analysis of
serum total protein; refractive index of solutes in serum
g. Turbidimetric and Nephelometric Method- utilizes SSA
and TCA
h. Salt Fractionation- globulins can be separated from
albumin by salting-out procedures using Na salts
-Rgt: Sodium
Sulfate Salts
B. Prothrombin
Time (Vit K Response Test)- diff intrahepatic disorder (prolong PT) from
extrahepatic obs (NO PT)
Albumin/Globulin Ratio- validate if globulin is higher
than albumin
-if
globulin > than alb= inverted A/G ratio: cirrhosis, multiple myeloma,
Walderstrom’s macroglobulinemia
-RV:
1.3:1 to 3:1
II. Test Measuring
Conjugation and Excretion Function *bili
(mg/dL) x17.1 (mmol/L)
A. Bilirubin Assay –unconjugated bilirubin
reacts slowly, accelerants (Caffeine and Methanol) meas Total Bili
-deletion of accelerants
allow determination of direct-reacting or conjugated bilibun J
-Accelerators
allow indirect bilirubin to react (solubilize) w/ the color reagent; read after
15min
Principle: Van den Berg Reaction- diazotization of
bilirubin to produce azobilirubin
a. Evelyn and Malloy Method: Coupling
Accelerator Methanol *PINK
to PURPLE azobilirubin
b. Jendrassik
and Grof*: Coupling Accelerator Caffeine
Sodium Benzoate Buffer: Sodium Acetate
*PINK to BLUE azobilirubin
B. Bromsulfonthalein
(BSP) Dye Extraction Test –test for hepatocellular func and potency of bile
duct
a. Rosenthal
White (Double Collection)- dose 2mg/kg BW; collect after 5min(50%) and 30min(0% dye retention)
b. Mac Donald
Method (Single Collection)- dose 5mg/kg; collect spx after 45min; NO=
+/- 5% dye retention
III. Test for
Detoxification Function- involves enzymes and ammonia tests
A. Enzyme Test-assess
extent of liver damage & diff. hepatocellular (functional) from obstruct
(mechan) disease.
-enzymes secreted by liver: ALP,
aminotransferases, 5’nucleotidase, GGT, OCT, LAP, LD
B. Ammonia-
from deamination of AA, converted by liver to uea RV: 19-60ug/dL (11-35mmol/L)
↑cirrhosis,
hepatitis, Reye’s syndrome, chronic renal disease and acetaminophen poisoning
ENYMES
^Alkaline
Phosphatase^
1. Electrophoresis-
Liver, Bone most Anodal (neuraminidase,wheat germ lectin-separ.); intestinal
ALP least anodal
- High-resolution
electrophoresis using polyacrylamide gel and isoelectric focusing resolve bands
of ALP
2. Heat
Fractination/Stability Test- performed @ 56OC for 10-15min
-placental
ALP (most heat stable); bone ALP most heat labile; Order decresing: Placenta,
Intestinal, Liver, B
3. Chemical
Inhibition Test- use diff conc of phenylalanine, synthetic urea and
levamisole
-P and I inhibited by phenylalanine rgt
; 3M urea inhibit Bone; Levamisole
inhibit L and B ALP
4. Bowers and Mc
Comb (Szasz modification)-most specific method IFCC recommended
-continous-monitoring
technique requiring a pH 10.15 and read @405nm
p-nitrophenylphosphate
←ALP→p-nitrophenol + phosphate ion
|
Method |
Substrate |
End Product |
|
1. Bodansky |
|
|
|
2. Shinowara |
Beta-glyceroPO4 |
InorganicPO4 + Glyverol |
|
3. Jones |
|
|
|
4. Reinhart |
|
|
|
5. King and
Armstrong |
Phenylphosphate |
Phenol |
|
6. Bessy Lowry
& Brock |
p-nitro phenyl PO4 (PNPP) |
p-nitrophenol or yellow
nitrophenozide ion |
|
7. Bowers and
McComb |
PNPP |
p-nitrophenol or yellow
nitrophenozide ion |
|
8. Huggins and Talalay |
Phenolphthalein PO4 |
ALpa-naphtol |
|
9. Moss |
Alpha naphthol PO4 |
Alpha- naphthol |
|
10. Klein, Babson and Read |
Buffered phenolphthalein PO4 |
Free phenolphthalein |
-Refrigeration leads to inc ALP; ALP inhibited by
phosphorous; Zinc component of ALP
^Acid
Phosphatase^ Summary of ACP
Methods
|
Method |
Substrate |
End Product |
|
1. Gutman and
Gutman |
Phenyl PO4 |
Inorganic PO4 |
|
2. Shinowara |
PNPP |
p-nitrophenol |
|
3. Babson, Read and Phillips |
Alpha naphthyl PO4-continuous mon |
Alpha-naphtol |
|
4. Roy and
Hillman |
Thymolphthalein
MonoPO4* |
Free
thymolphthalein |
*Tartrate-resistant
Acid Phosphatase (TRAP)- present in certain chronic leukemia, esp Hairy Cell Leukemia
*Postatic ACP
used together w/ Prostate Specific Anitgen (PSA) to monitor recurrence of
prostate cancer
^Aspartate
Aminotransferase^
Karmen Method- pH 7.5; 340nm- use
malate dehydrogenase (MD), monitor change in absorbance at 340nm
Aspartate + a-ketoglutarate ←AST→ Oxaloacetate + Glumate
Oxaloacetate + NADH + H ←MD→ Malate + NAD
^Alanine
Aminotransferase^ -present in plasma, bile, CSF, saliva; require
pyridoxal phosphate (vitB6) as coenzym
Coupled Enzymatic Reaction: using pH 7.5
@340nm
Alanine + a-ketoglutarate ←ALT→ Pyruvate + Glutamate
Pyruvate + NADH + H ←LD→ Lactate + NAD
|
|
AST/ SGOT |
ALT/ SGPT |
|
Major Organ Affected |
Heart |
Liver |
|
Substrate |
Aspartic
a-ketoglutaric acid |
Alanine
a-ketoglutaric acid |
|
End Product |
Glutamic Acid + Oxaloacetic
Acid |
Glutamic Acid + Pyruvic
Acid |
|
Color Developer |
2,4 DNPH |
2,4 DNPH |
|
Color Intensifier |
0.4N NaOH |
0.4N NaOH |
|
Method |
Reitman and
Frankel |
Reitmand and
Frankel |
*De Ritis Ratio
(ALT:AST) > 1.0, seen in acute hepatitis
^Amylase/
Alpa-1-4-Glucohydrolase (AMS)^ Substrate for all methods Starch- polysaccharide, carbohydrate
1. Saccharogenic-
reference method expressed in Somogyi units;
-measure reducing sugars prod by
hydrolysis of starch by the usual glucose method
2. Amyloclastic –measure
amylase activity by following decreases in substrate conc (defradation of
starch)
3. Chromogenic- meas
amylase activity by increase in color intensity of soluble dye-substrate sol
prod in rxn
4. Coupled-enzyme-
measure amylase activity by a continuous-monitring technique
^Lipase/
Triacylglycerol Acylhydrolase (LPS)^
-uses Olive oil as substrate bec other esterases can hydrolyze TAG and
syntheric diglycerides
-Collipase (CHON from
pancreas) and Bile salts- make assay more sensitive and specific
1. Cherry Crandal (reference
method)- substrate 50%olive oil; End product Fatty Acid
-Hydrolysis
of olive oil after incubation for 24hrs @37OC and titration of FA
using NaOH
TAG
(olive oil) +2H2O ←LPS→
2-monoglyceride + 2 Fatty acids
2. Tiets and
Flereck
3. Peroxidase
coupling- most common; doesn’t use 50% olive oil
^Lactate
Dehydrogenase^ -Lactate more specific substrate than pyruvate; LD1
prefer forward; LD5 reverse rnx
1. Wacker Method
(forward/direct reaction)- reaction @ pH 8.8; most common
Lactate
+ NAD –LDà
Pyruvate + NADH @340nm
2. Wrobleuski La
Due (reverse/ indirect reaction)- reaction @ pH7.2; 2x faster; prefer for
dry slide technology
Pyruvate
+ NADH –LDà Lactate + NAD
3. Wrobleuski
Cabaud
4. Berger Broida
^Creatine
Kinase (CK)
1. Tanzer-Gilbarg
Assay (forward/direct method) –pH9.0 @340nm
Creatine + ATP –CKàCreatine
PO4 + ADP + Phosphoenolpyruvate ←PK→
Pyruvate + ATP
Pyruvate
+ NADH ←LD→ Lactate + NAD
2. Oliver-Rosalki
(reverse/indirect method)-most common; faster; pH 6.8 @340nm
Creatine
PO4 + ADP –CPKà Creatine + ATP +
glucose ←hexokinase→ ADP+
glucose-6-PO4
Glucose-6-PO4
+ NADP ←G-6-PD→ 6-phosphogluconaye +
NADPH
3.
Electrophoresis- reference method
*Adenosine monophosphate (AMP)- added to reverse method
to inhibit AK (Adenylate kinase)-it hydrol ADP
*imidazole- buffer; urate and cysteine- potent inhibitor
of CK; CK light and pH sensitive
^Sodium and
Potassium^- use heparinized blood
1. Emission Flame Photometry
2. Ion Selective Electrode (Valinomycin gel)
3. Atomic Absorption Spectrophotometry
4. Colorimetry (Lockhead and Purcell)
^Chloride^
1. Mecurimetric
Titration (Schales and Schales) -Diphenylcarbazone
–indicator; HgCl2 (blue violet)=end
p
2.
Spectrophotometric Methods Mercuric
Throcyanate (Whiterhorn Titration Method) =Reddish
complex
Ferric
Perchlorate =colored complex
3. Coulometric
Amperometric Titration –Cotlove Chloridometer- Sweat Chloride (cystic
fibrosis)
4. Ion Selective
Electrode-using ion exchange membrane (tri-n-octylpropylammonium chloride
decanol); common
^Calcium^
-serum
1. Precipitation and
Redoc Titration: Clark Collip ppt- endproduct (purple color)
Ferro Ham
Chloranilic Acid ppt- endproduct: Chloranilic acid (purple color)
2.
Ortho-Cresolpthalein Complexone Dyes (Coloremetric Method) Dye: Arzeno III
3. EDTA Titration
Method (Bachra, Dawer and Sobel)
4. Ion Selective
Electrode (Liquid membrane)
5. Atomic
Absorption Spectrophotometry-reference method
6. Emissio Flame
Photometry
^Inorganic
Phosphorus^- require fasting, high CHO diet dec level; only inorganic
phosphate is measured
-affected by circadian rhythm- high level late morning,
low in evening
1. Fiske Subbarow
Method (Ammonium Molybdate method)- most common
-Reducing
agent: *Pictol (Amino Naphthol Sulforic Acid); elon, senidine
-Endproduct:
Ammonium molybdate complex (unstable); Reduced form blue color det bet 600 to 700nm
^Magnesium^
1. Colorimetric
Methods
a. Calmagite Method= (+) Reddish-violet
complex
b. Formazen Dye Method= (+) Colored
complex
c. Magnesium Thymol Blue Method= (+)
Colored complex
2. Atomic
Absorption Spectrophotometry- reference method
3. Dye-Lake
Method- Titan Yellow Dye (Clayton Yellow or Thiazole Yellow)
^Bicarbonate^
-spx blood anaerobically collected
1. Ion selective
electrode (using pCO2 electrode)
2. Enzymatic (Phosphoenolpyruvate
carboxylase and dehydrogenase)
*Cystic
Fibrosis-
Sweat Inducer:
Pilocarpine]
Diagnostic test:Sweat-teest Coulometry (↑Sodium and Cl)
Reference
Method: Gibson and Cooke Pilocarpine
Iontophoresis
>50mg sweat sample collected w/in
30min
(+)
Result: >65 mmol/L sweat
electrolytes (RV: 5-40mmol/L)
*Iron
1. Colorimetry
(HCl and ferrozine) –(+) Blue Color
2. Anodic
Stripping Voltammetry- 1st separation form transferrin by
acidification,
^Blood Gas^-
spx Arterial Blood; Anticoagulant:
0.05mL heparin/mL blood
1. Gasometer 2.
Electrodes
a. Van Slyke a. pH (potentiometry)
b. Natelson i. Silver-silver chloride electrode- reference
electrode
i. mercury- to produce vacuum ii. Calomel electrode (Hg2CL2)- reference
electrode
ii. caprylic alcohol- anti-foam reagent iii. Gas electrode- most common,
used for pH
iii. Lactic acid b.
pO2 Clark electrode- polarography-amperometry
iv. NaOH and
NaHSO3 c. pCO2
Severinghaus electrode –potentiometry
Thyroid Function
Test:
1. TRH Stimulation
Test (Thryrotropin Releasing Hormone)- measure relationship bet TRH and TSH
secretions
-differentiate euthyroid and
hyperthyroid Px w/ both undetected TSH; detect thyroid hormone resistance synd.
-↑1O
hypothyroidism; ↓hyperthyroidism
2. TSH Test-
most important thyroid function test- best from detecting clinically
significant thyroid dysfunction
-detect 1O
thyroid disorfers; differentiate 1O from 2Ohypothyroidism
↑1O
hypothyroidism, hashimoto’s thyroiditis. TSH Ab; ↓1O
hyperthyroidism, 2O and 3O hypothyroidism
3. Radioactive
Iodine Uptake (RAIU)- measure ability of thyroid gland to trap iodine;
..hyperthyroidism
-high uptake
=metabolically active; high uptake + TSH deficiency= autonomous thyroid
activity
4. Thyroglobulin
(Tg) assay- normally used as postoperative marker thyroid cancer,
↑untreated and
metastatic differentiated thyroid cancer, nodular goiter and hyperthyroidism
↓infants w/
goitorous hypothyroidism and thyrotoxicoxis factitia
Method:
double-Ab RIA, ELISA, IRMA, immunochemiluminescent assay (ICMA)
5. Reverse T3
(rT3)- formed by removal one iodine from inner ring of T4; enproduct of T4
metabolism
-identifies
px w/ euthyroid sick syndrome (↑rT3)
6. Free Thyroxine
Index (FTI or T7)- indirectly assess level of free T4 in
blood;↑hyperthyroidism; ↓hypothyroid
7. Total 3 (TT3),
Free T3 (FT3) and FreeT4 (FT4)- FT4 used to differentiate drug induced TSH
elev and hypothyroid
-TT3 or
FT3 confirm hyperthyroidism; direct/reference method: Equilibrium dialysis
(FT4)
8. T3 Uptake-
measure number available binding sites of thyroxine-binding proteins, a test
for TBG
-reflects
serum level of TBG, inversely related to TBG- ↓T3 uptake ↑TBG, vice versa
↑hyperthydoisim, ↓hypothyroidism
9. Thyroxine
binding globuline (TBG)- confirm results of FT3 and FT4, or abnormalities
in relation to TT4 and THBR
-hyperthydoism (↑T4 + NO TBG); euthyrdoism (↑T4 and TBG);
hypothyroidism ↓TBG
10. Fine-needle
aspiration- most accurate tool in evaluation of thyroid nodules
11. Recombinant
Human TSH- test pc w/ thyroid cancer
12. Tanned
Erythrocyte Hemagglutination- measure antithyroglobulin Ab
13. Serum
Calcitonin- tumor marker for detecting thyroid metastasis in medullary
thyroid carcinoma
14. Pentagastrin
(Pg) Stimulation Test- diagnose MTC
^Pheochromocytoma^:
Pharmacologic Tests:
a. Clonidine Tests- diff. pheochromocytoma (not
suppressed) to neurogenic hypertension (50% ↓catecholamine)
b. Glucagon Stimulation Test- for indiv w/ normal blood
pressure and when catecholamines only modestly elev.
^Neuroblastoma^
Spx: 24hr urine and blood (plasma)
a. Chromatography-
HPLC or GC-MS (VMA and metanephrines)
b.
Radioimmunoassay- sensitive screening test for total plasma catecholamines
>2000pg/mL plasma catecholamines- diagnostic for pheochromocytoma
-Urine preservation: 10mL 6N HCl
^Hormonal Assay^
1.
Whole blood- LH, testosterone
2.
Plasma- EDTA (ACTH, ADH, PTH) and Heparin (Catecholamines, cortisol, dopamine,
FSH)
3.
Serum- aldosterone, androstenedione, DHEA, estrogen, FSH, GH, HCG, progesterone
4.
Urine- for measurement of estriol
-Boric acidin a concentration of 1g/dL
urine elements such as estriol and estrogen for up to 7days
-for catecholamines, VMA, 5-HIAA
collections, 10mL 6N HCl in 3-4L container
-HCl establishes a pH of approximately
<3.0, good for chemical testing
a. Classical Assay
-Bioassay-
-
Competitive Protein Binding (CPB)
b. Immunologic Assays
-Radioimmunoassay (RIA)
-Immunoradiometric (IRMA)
-Enzyme-Linked Immunosorbent Assay
(ELISA)
-Enzyme Multiplied Immunosorbent
Technique (EMIT)-for TDM
-Immunometric- for TSH
c. Fluorescent Technique-
FPIA
d. High Performance Liquid
Chromatography (HPLC)
e. Colorimetry
i. Porter-Silber Method- for 17-OHCS
ii. Zimmerman Reaction- measure those
steroid w. 17-keto structure
iii. Pisano Method- for quantitating metanephrines
and normetanephrines
iv. Kober Reaction- for estrogen (H2SO4 +
hydroquinone = (+) reddish-brown color
