Professional Documents
Culture Documents
(Prelims)
1. Gravimetric Method
Concentration of analytes in terms of W (g)
clinical microscopy
total lipid in 24 hour fecal sample
diagnosis of steatorrhea
high amount of fat in the stool
inability of human to digest fat
from food eaten
inadequate bile salts supply to
the small intestine
causes:
obstruction of biliary passage
to small intestine
gallstone; biliary stone
2. Volumetric Method (titrimetric method)
determine volume of desired analyte from
clinical specimen
known volume of clinical specimen is reacted
with standard solution until end point is
reached
o (change color of indicator)
clinical microscopy
total gastric acidity gastric juice
strongly acidic
pH of 1-2
to determine the [H+] concentration in gastric
juice, titrate std. base and gastric juice
Zollinger Ellison Syndrome
[H+] concentration
3. Instrumental Method
a. Colorimeter
concentration of analyte is determined by
basing it from the intensity of the colored
solution.
conc. analyte = color intensity
most common method
yellow flame
violet flame
(isolated by
400mm filter)
red flame
(may use EFP)
thick atom require high energy
Mg+2
Colorimeter
1. Duboscq
color of the unknown solution is compared
with a white light
Beers Law
conc. of light is
directly proportional to the absorbance
inversely proportional to the logarithm of
transmittance
if reading if (ABS) Absorbance:
Autoanalyzer
performs automated analysis of the clinical
specimen
Parts of Autoanalyzer
1. Automatic Sampler
pipets approximate volume of the Sx
Parts
1. Light Source
a. tungsten lamp
b. mercury lamp
2. Monochromator
to separate the incident light to 7 colors of the
rainbow
quartz prism
diffraction grating
3. Cuvet
holder of unknown colored solution
4. Photocell
coverts the color observed to an electric
current
5. Combined galvanometer and potentiometer
to read photocells
reading :
(ABS) Absorbance
% transmittance
2. Dialyzer
removes protein substances from the Sx
o causes turbidity that will mask
the color of the unknown
solution
o causes bubbles / foam
3. Peristaltic pump or proportioning pump
dispenses color reagents
4. Heating bath
promotes color formation of the end product
5. Reading devices
a. Spectrophotometer - records intensity of
color of the unknown solution
b. (EFP) Emission Flame Photometry intensity
of the colored flame
c. (AAS) Atomic Absorption Spectrophotomer
amount of radiation absorbed by the unknown
d. Nephelometer amount of light scattered by
the immunoglobulin
e. Fluorometer amt. or degree of fluorescence
6. Recorder
computes the test values
7, Printer
types the test results of the analysis
2 Categories of Autoanalyzer
1. Chromatography
isolate each amino acid in a mixture
solvent front
Distance travelled by
the Sx from the origin
2. Discrete Analyzer
each specimen has its own dedication reaction
tube/chamber
analysis is done simultaneously
short time to finish
Designs of Autoanalyzer
developing solvent
1. Sequential Analyzer
1 analysis from 1 specimen at a time
2. Batch Analyzer
1 kind of test from many specimens at one
time.
3. Parallel Analyzer
many kinds of test from only 1 serum
4. Random Access Analyzer
modify order or sequence of analysis
prioritize
a. Wet Chem Analyzer
o reagents are in liquid form
b. Dry Chem Analyzer
o reagents are dry
Electrophoresis
separate different protein substances in only
one solution
means of electric current
o
Four Layers
Layer 1 spreader
Layer 2 scavenger
o
o
Layer 3 reagent
Layer 4 support
o
o
o
(-) anions
at high pH
migrate anode
(+) cations
at low pH
migrate cathode
at isoelectric point neutral or zwitterions
isoelectric point of serum protein
pH 3.5 5.0
does not migrate in electric field
rate migration dependent
albumin MW 40,000 (fastest)
gamma globulin MW 150,000
pH used in sepa serum proteins = pH 8.6
at this pH, the protein in anionic
(+)
anode
covered by solid:
1. cellulose acetate
2. agarose
3. polyacrylamide gel
(PAGE) polyacrylamide gel electrophoresis
low MW = separated into 5
Spray dyes
1. Ponceau s.
2. Bromphenol blue
3. Silver stain
4. Coomasie brilliant blue
5. Amido black
Electrophoregram
the result is scanned by a densitometer
o measures
intensity of
color or band
o darkest band =
albumin
(most
abundant)
CARBOHYDRATES (CHO)
Blood Glucose Level
always present C , H , O
always absent N
1. Normal
normolycemia 65-100 mg %
3 Classes of Carbohydrates
2. High
1. Monosaccharide
1 saccharide unit
a. glucose (dextrose)
rotate polarized light to the right
b. fructose (levulose)
rotate polarized light to the left
c. galactose
cannot rotate light
2. Disaccharide
2 saccharide units
a. lactose : 1 mole glucose + 1 mole galactose
a. lactase
b. maltose : 1 mole glucose + 1 mole glucose
a. maltase
c. sucrose : 1 mole glucose + 1 mole fructose
a. sucrose
Disaccharases enzymes whose substrates are
disaccharides
(LT) Lactose intolerance
cannot digest lactose in milk
milk is not allowed
instead, give soya drink
3. Polysaccharide
3 or more saccharide units
a. starch
found in saliva
amylase enzyme to digest starch
o S-form amylase saliva
o P-form amylase - pancreas
b. cellulose
fount in plants (fruits/vegetables)
no nutritional value
no enzyme that can degrade
for normal functioning of the intestines
c. glycogen
found in liver of humans/animals
quick energy
o easily converted to glucose as
energy source
stored energy
o storage form of carbohydrates in
the body
hyperglycemia - >100 mg %
hypoglycaemia - < 65 mg %
3. Low
4. renal threshold
140 160 mg %
highest value of blood glucose afterwhich
glucose appears in the urine
glucosuria - > 160 mm %
5.panic value
blood glucose reaches 35 mg %
irreversible brain damage
RMT should inform the physician and the
nurse immediately
500 mg% organ failure occurs
Carbohydrate Processes
1. glycolysis in the muscles
breakdown of glucose into lactate + pyruvate
finally: CO2 + H20 + energy
2. glycogenesis in the liver
synthesis of glycogen from the glucose
3. glycogenolysis in the liver
breakdown of glycogen into glucose
4. gluconeogenesis in the liver
formation of glucose from non-carbohydrate
sources
examples: amino acid, fatty acids, glycerol
Hormonal Control
HORMONES
SOURCE
EFFECT GLUCOSE
insulin
lower
glucagon
increase
glycolysis
(insulin: glucose to the muscle)
glycogenesis
(insulin: glucose to liver cells)
glycogenolysis
adrenal cortex
increase
gluconeogenesis
delta cells
maintain proper
balance of
insulin/glucagon
cortisol
somatostatin
Insulinoma
tumor in pancreas
no. of beta cells = insulin = glucose
lab finding: low blood glucose
(DM) Diabetes Mellitus
Pancreatic damage
Slow production of insulin by the liver
Blood insulin deficient
Glucose is not utilized as the main source of
energy
lab finding: glucose level
Patterns of Blood Glucose Level
a. 30 minutes after meal
fastest increase of glucose level
b. 1 hour after meal
peak glucose level in the blood stream
c. after 1 hour of meal
glucose level of blood starts to go down
d. after 2 hours of meal
blood glucose returns to original level (prior
to the meal)
MECHANISM
substances
with sugar-like
characteristics
glucose
lower value
true blood glucose
3 SYMPTOMS (P-Triad)
1. Polyuria
excessive urine excretion
3L volume of urine
2. Polydipsia
excessive thirst
3. Polyphagia
excessive hunger
SCREEN TEST
very sensitive thirst
to see if (+) or (-) to DM
measures minute concentration of glucose
always yield a (+) result for presence of
diabetes
a. (FBG) fasting blood glucose
fasting for 8 hours (overnight fasting)
b. (2Pp) 2 hours post prandial
collect specimen 2 hours after a meal
1. FBS or 2Pp
140 mg%
CONFIRMATORY TESTS
candidate: >100 mg% glucose
MONITORING TESTS
medicine prescribed Dos and Donts
a. (HbA1C) glycated Hb
GUIDELINES OF OGTT
a) patients should have 3 days preparation
(CHO) carbohydrates intake daily
should have an average of 150g/day
b) overnight fasting a night prior to the test
c) no physical exertion allowed
2. OGTT
2 values out of 3
200 mg% = (+) DM
glycosylated Hb
(unstable)
hemolysate
glycated Hb
(stable complex)
(irreversible complex)
b. (FS) fructosamine
(HbA1C) glycated Hb
stable complex of Hb + glucose
once in 3 months
(FS) fructosamine
stable complex of albumin + glucose
once a week
albumin halflife of 21 days
specimen hemolysate
subject to column chromatography
Gestational Diabetes
manifested in pregnant women
unclassified
early warning
3. Enzymatic
GLUCOSE METHOD
best specimen blood collected
glucose + O2
H2O2 + o-dianisidine
H2O + O2
glucose
Cu+2
Cu+
(cupric)
(cuprous)
O2 + o-dianisidine
(colorless)
Journal -
oxy-orthodianisidine
(orange-brown)
-glucose oxidized
-glucose not oxidized
b. Hexokinase
hexokinase
glucose + ATP
G6P + ADP
G6PD
G6P + NAD
PGA
+ NADH
(phosphoglutonic
acid)
(colored)
measured!
dehydrogenase removes H+
G6PD for RBC durability
glucose oxidase:
mutarotase
-glucose
-glucose
Autoanalyzer Method
Fe(CN)6-3 + glucose
(serum)
Fe(CN)6-4
(colorless)
2 x 14
4x1
1 x 12
1 x 16
= 28
=4
= 12
= 16
60 MW
urea 60
BUN 28
albumin
smallest protein
40,000 50,000 MW
IgG MW 150,000 g/n
IgM MW 900,000 g/n
b. NPN - crystal in nature
Proteins colloids
COMPONENTS OF NPN
1.
2.
3.
4.
5.
6.
Urea 45 50%
Amino Acids 20%
Uric Acid 20%
Creatinine 5%
Creatine 1-2%
Ammonia 0.2%
1. Urea
waste product of protein metabolism
excreted through urine
carnivorous rich in protein - urea
90% urea is excreted in bloodstream
2. Creatinine
waste product of muscle metabolism
99% creatinine excreted in bloodstream
excreted through urine
more reliable for KFT
o 99% excreted by kidney
o Not influenced by protein diet
meat = correspondently
increase
urea
but
not
creatinine
o muscle mass origins of retaining
doesnt change abruptly
remains constant
used to evaluate for the completeness of the
24 hour urine sample
RR-male
higher muscle mass
not always true
RR-female
P plasma/serum creatinine
1.73 average surface area
SA surface area of patient
3L polyuria = DM
Measured in lab
1. Urea - KFT
2. Creatinine - KFT
3. Uric Acid - gout
4. Ammonia hepatic Coma
immediately
tests for patient who will die/ seriously
illed
STAT!
avoid delay
NH3 - determination of glutamine
CCr
before
medication
prescribe
anti-hypertensive
if equal = normal
MEASUREMENT OF CREATININE
H2O
creatine
creatinine
dehydration
CCr
after
medication
a. Jaffes Reaction
not specific for creatinine.
may also measure other substances.
o reducing substances (ascorbic acid,
Vitamin C, glucose, uric acid)
creatinine in serum + alkaline picrate creatinine
(color rgt.)
picrate
unstable
(orange-red
compound)
Picric acid + 10% NaOH
NH3
urea
liver
kidney
creatine precursor
(yellow crystals)
3. Uric Acid
end product of purine or nucleic acid
metabolism
after chemotherapy = greatly elevated UA
if elevated
o has the tendency to be deposited at
joints
o fluid dries UA crystals (tophi)
o friction
o inflammation
o pain
2 KINDS OF URIC ACID
a. exogenous
from food (beans, peanuts, mongo, etc.)
b. endogenous
manufactured by body from purine
metabolism
4. (NH3) Ammonia
from bacterial breakdown of urea
lowest concentration of all NPN
liver converts all NH3 to urea which is
excreted through urine
measured to monitor hepatic coma
liver not functioning
NH3
o special test
o unscheduled
o for seriously ill patients
normal NH3 normal liver
elevated NH3 defective liver
creatinine
anhydride of
creatine
H2O
urine
5. Amino Acids
not measured in chemistry
measured in clinical microscopy
Proteins
separated by electrophoresis
most common
hormone
serum
proteins in nature
enzyme
UREA DETERMINATION
1. Direct Method
measures urea
Rosenthal Method
Fearon Method
DAM (diacetyl monoxide) Method
urea + DAM
yellow product
2. Indirect Method
measures BUN by Kjeldahl-Nessler Method
a. digest N
NH4+
b. Neisslerization
Neisslers Reagent K2HgI4
[OH-]
NH4
K2HgI4
NH3
NH2Hg2I3
(yellow)
(diamino mercuric iodide)
urea in serum
CO2 + NH3
measured using
Berthelots Rxn
blue indophenol
Na nitroprusside
measured by spectrophotometry
Interferent - NH3
Disadvantage urease is inactivated by NaF
Urea output
not correction due to muscle mass
muscle mass is not related to urea output
Creatinine Output
correction due to muscle mass
muscle mass = creatinine output