Centrifuge and Tachometer PIPETS

• Centrifuge – Separate serum Types according to design

• Tachometer – to calibrate it
1. To contain – holds a particular volume but does
o If centrifuge does not work use
not dispense the exact volume.
tachometer to calibrate it.
2. To deliver – will dispense the exact volume
o If machine is from a company, the
indicated.
company will calibrate it. Or of there is
no reagents needed the company will Types according to drainage characteristics
calibrate it.
1. Blowout – last drop of the liquid should be
• 2 ways to calibrate:
expelled into the receiving vessel.
o Tachometer
2. Self-draining – allow the contents of the pipet
o High and low control for calibration
to drain by gravity. (Ex: Biuret)
(reagents)
Types according to purpose – Measuring or Graduated
CENTRIFUGE
1. Mohr pipet – does not have graduations to the
• Used to separate substances of different mass
tip. It is a self-draining pipet but the tip should
or density.
not be allowed to touch the vessel while the
• RPM (revolutions per minute) vs. RCF (relative
pipet is draining. (Numbering starts near the tip)
centrifugal force)
2. Serologic pipette – has a graduation mark to
o RPM is commonly used because it does
the tip and is generally a blowout pipet. Used in
not need conversion, meanwhile RCF
laboratory and research. (Numbering starts at
needs conversion.
the top)
• RCF = 𝑅𝑃𝑀2 𝑥 𝑟 𝑥 1.12𝑥10−5
3. Micropipette – is a pipet with a total holding
• RPM of the centrifuge is calibrated using volume of less than 1mL.
tachometer.
• Automatic pipette – commonly used pipette in
TYPES OF CENTRIFUGE the laboratory has an air displacement
principle.
1. Horizontal head centrifuge / swinging bucket
type – centrifuge tubes as held in vertical Types according to purpose – Transfer pipets
position when not moving but are horizontal
1. Ostwald-Folin – have a bulb-like enlargement of
when the centrifuge is fully in motion.
the pipet stem.
2. Angle head – has a fixed angle head (25-52
2. Volumetric – is designed to dispense or transfer
degrees) at which the tubes are held during
aqueous solution and is always self-draining.
centrifugation.
3. Ultracentrifuge – generates the highest speed; Type or Name Function Drainage
centrifuge head is held at a fixed angle but Push button Deliver a Blow out
generates tight sediment buttons due to the (Automatic) or variable or fixed
high speed generates. Used to achieve sediment Micropipet volume
buttons. Serological, Deliver a Blow out
4. Vertical tube – even at motion or at rest it is in standard type variable volume
vertical position. Kolmer Deliver a Self-drainage
Serological variable volume
Mohr Deliver a Self-drainage
variable volume
Capillary Contain a fixed Wash out
volume
Lambda (two Contain a fixed Wash out
types) volume Blow out
Deliver a fixed • Wavelength – refers to distance between the
volume peaks of a light wave.
Ostwald-Folin Deliver a fixed Blow out • Wavelength is inversely proportional to amount
volume of energy.
Volumetric, Deliver a fixed Self-drainage
standard type volume
• Push button pipets can be divided into two
types that differ in their mechanics: air
displacement and positive displacement.
o Air displacement pipets – routine
measurements
o Positive displacement pipets – greater
accuracy and precision, and preferred
for samples having high viscosity,
surface tension, density or vapor
pressure. Spectrophotometry – to know concentration
• Automatic pipet – 2 stops (1st stop: gather fluid;
2nd stop: dispense fluid) Distance of cuvette – 1 cm (standard)
• MSM (Mohr, Serologic, Micropipette) – to • What the machine detects is its complementary
deliver o Yellow serum – detects 400 – 450nm
o Blood – detects 450 – 560nm

BEER’S LAW

• Since path length (b) and absorptivity

coefficient (a) are constants, we say that
absorbance (A) is directly proportional to the
concentration (c).

CLEANING OF GLASSWARES

• Presoaking glassware in soapy water is

recommended.
• Cleaning solutions: Potassium dichromate in • The amount of absorbed light is proportional to
Sulfuric acid (H2SO4) or Nitric acid (HNO3) solution concentration.
• Final rinses: Type 1 or Type 2 water • Absorbance is inversely proportional to
• Glassware are sterilized using Dry oven @ 160- transmittance
180C for 1 ½ hours. • Increased concentration = Increase absorbance
o Autoclave – for agar = Decrease transmittance (because the
o Dry oven – for glassware concentration absorbs more)

PHOTOMETRY

Principle:

• Light – a form of electromagnetic energy that

travels in waves.
CALCULATIONS IN SPECTROPHOTOMETRIC ASSAY 5. Readout system
• Measures the magnitude of the current
What is the concentration of a glucose sample that has
generated by the detector
an absorbance of 0.25, if a 100 mg/dL glucose standard
• Galvanometer, ammeter
has an absorbance of 0.50?

Abs = conc
VARIATIONS OF PHOTOMETRY
0.25 ÷ 0.50 = 𝑋 ÷ 100
Fluorometry
𝑋 = 0.25 ÷ 0.50 𝑥 100
Things to remember about fluorometry:
𝑋 = 0.5 𝑥 100
1. 2 monochromators
𝑋 = 50𝑚𝑔/𝑑𝐿 2. Set at right angle (90 degrees)
3. UV light
4. More sensitive/specific
COMPONENTS OF SPECTROPHOTOMETER

Light source → Collimator (lens) → Monochromator

(prisms or grating) → wavelength selector (slit) → LABORATORY MATH
sample solution (cuvette) → detector (photocell) →
digital display or meter Conversion:

1. Light source 1. Convert 3.5 grams into mg

• Visible – infrared range: tungsten 1000𝑚𝑔
3.5𝑔 × = 3500𝑚𝑔
halogen (iodide) lamp 1𝑔
• Ultraviolet range: mercury arc lamp,
xenon lamp, deuterium discharge lamp 2. Convert 444mm into meter
2. Monochromator 1000𝑚
444𝑚𝑚 × = 0.444 𝑚𝑒𝑡𝑒𝑟𝑠
• Colored glass filters, prisms, 1𝑚𝑚
interference filter, diffraction gratings Molarity:
o Prisms – most common
3. Sample cuvette 1. Determine the molarity given the following
data:
• Holds the sample solution
Mass of NaOH: 120g
• Can be plastic or glass
MW: 40
o Plastic – much better
Volume of solution: 750mL
o Cylindrical cuvette – most used
𝑀 = 𝑔 ÷ 𝑀𝑊 ÷ 𝐿
4. Detector
• Convert the transmitted radiant energy 𝑀 = 120𝑔 ÷ 40 ÷ 0.75
into an equivalent amount of electrical
𝑀 = 4.0 𝑀𝑜𝑙𝑎𝑟𝑠
energy.
• Photocell, photomultiplier tube, photo
diode, barrier layer cell
Normality: Serial dilution:

1. Determine the normality given the following

data:
Mass of NaOH: 120g
MW: 40
Volume: 750mL

𝑁 = 𝑀 × 𝑛𝑜. 𝑜𝑓 ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛
𝑁 =4 ×1
𝑁 = 4𝑁

Normality into Molarity:

1. What is the equivalent molarity of 0.5N solution
H2SO4? 1. What is the dilution in tube number 5, if the
𝑁 undiluted sample from tube number 1 is
𝑀 =
𝑛𝑜. 𝑜𝑓 ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛 subjected into a two-fold dilution?
1 1 1 1 1
× × × = 𝑜𝑟 1: 16
0.5 2 2 2 2 16
𝑀=
2

𝑀 = 0.25𝑀 2. What is the dilution in tube number 4 if the

undiluted sample from tube number 1 is
subjected into a five-fold dilution?
Concentration and Volume: 1 1 1 1 1
× × = 𝑜𝑟
1. How much 25% alcohol is needed to prepare 2 5 5 5 125 25
liters of 50% alcohol?

𝐶1𝑉1 = 𝐶2𝑉2 QUALITY ASSURANCE

(25%)(𝑥) = (50%)(2𝐿)
25𝑥 = 100 • Quality assurance – is a complete system of
25𝑥 100 creating and following procedures and policies
=
25 25 to aim for providing the most reliable patient
𝑥 = 4𝐿 laboratory results and to minimize errors in the
pre-analytical, analytical, and post-analytical
Dilution: phases. It is now known as Quality Assessment.
Ratio = solute:solvent o Aircon temperature – 24-26C
Dilution = solute:solution • Quality control – is an aspect of quality
assessment that is used to assess the analytical
1. What is the dilution if 4mL of sample is mixed phase of patient testing. (Ex: at 12mn the high
with 16mL of diluent? and low control is done)
Answer: 4:20 or 1:5 • Accreditation – process by which an agency or
an organization evaluated and recognizes a
program of study or an institution as meeting
2. How much diluent would be needed to prepare certain predetermined qualifications or
1:10 dilution using a sample volume of 2mL? standards; applied only to institutions and
1: 10 × 2 = 2: 20 programs.
20 − 2 = 𝟏𝟖𝒎𝑳
PHASES OF ANALYSIS WESTGARD QC RULES

Pre-analytical Phase 12 𝑠 One control observation exceeding the mean

+/-2s. A warning rule that initiates testing of
• Patient preparation control data by other rules.
• Time of collection 13 𝑠 One control observation exceeding the mean
• Specimen collection order +/-3s. Allows high sensitivity to random error.
• Quality of specimen collected 22 𝑠 Two control observations consecutively
• Specimen processing, storage and preservation exceeding the same +2s or -2s. Allows high
sensitivity to systemic error.
Analytical Phase 𝑅4 𝑠 One control exceeding the +2s and another
exceeding +1s or -1s. This allows the detection
• Maintenance for equipment and instruments
of random error
• Calibration of equipment, verification of
41 𝑠 Four consecutive control observations
instrument linearity exceeding +1s or -1s. This allows detection of
• Precision, accuracy and overall reliability check systemic error
through the use of standard materials, quality 10x Ten consecutive control observations falling on
control samples, procedures, and QC rules. one side or the other of the mean (no
• Accuracy – the nearness or closeness of the requirement for SD size). This allows the
assayed value to the true or target value. detection of systemic error.
• Precision – the nearness or closeness of the • Quality assurance – uses Westgard rules and
assayed value to a repeated value. Levey Jennings chart except gaussian curve,
• Repeatability – closeness of agreement mean, median, and mode
between results of successive instruments • 13s and R4s – only random error
carried out under the same conditions. (Ex: • 12s – warning only
research)
o Same mechanism same test
• Reproducibility – closeness of agreement Post-Analytical Phase
between results of measurement performed
• DELTA CHECK – checking the current results of a
under changed conditions of measurements.
patient with his or her previous results.
o Different machine same test.
• Alarms and flags
• Reliability – refers to the ability of the analytical
method to maintain accuracy and precision over • Recording and reporting of results
an extended period of time. CLINICAL CHEMISTRY CRITICAL VALUES
• Practicability – the degree to which a method is
easily repeated. Bilirubin >18mg/dL (newborn)
• Systematic errors – errors that occur Glucose <40mg/dL
>500mg/dL
predictably once a pattern of recognition is
Potassium <2.5mEq/L
established; predictable errors of the same sign
>6.5mEq/L
and magnitude.
Bicarbonate <10mEq/L
o Inaccuracy; could be machine error >40mEq/L
• Random errors – errors that occur Arterial or capillary pH <7.2
unpredictably; affects precision and is the basis >7.6
for varying differences between repeated Phosphate <1mg/dL
measurements. Calcium <6mg/dL
o Imprecision; fluctuate of results >13mg/dL
Sodium <120mEq/L
>160mEq/L
Arterial or capillary pO2 <40mmHg
Arterial or capillary pCO2 <20mmHg
>70mmHg
CLINICAL CHEMISTRY CONVERSION FACTORS • Sucrose – most common non-reducing sugar
• Non-reducing sugar do not contain an active
Analyte Conventional SI unit Conversion
ketone or aldehyde group. (Ex: Trehalose)
Unit factor
Albumin g/dL g/L 10 • The brain is completely dependent on blood
AST U/L (mU/mL) ukat/L 0.0167 glucose for energy production – 2/3 of glucose
Ammonia ug/dL umol/L 0.587 utilization in resting adults occurs in the CNS.
(NH3) • Glucose – grape sugar, dextrose
Bicarbonate mEq/L mmol/L 10 • Maltose – brain sugar
(HCO3) • Fructose – sweetest sugar
Bilirubin mg/dL umol/L 17.1 • Reducing sugar is False positive – in benedict’s
BUN BUN mg/dL 0.357 test
Calcium mg/dL mmol/L 0.25
Chloride mEq/L mmol/L 1.0 Glycolysis Metabolism of glucose to lactate
Cholesterol mg/dL mmol/L 0.026 or pyruvate for production of
Cortisol ug/dL umol/L 0.0276 energy
Creatinine mg/dL umol/L 88.4 Gluconeogenesis Formation of glucose 6 phosphate
Creatinine mL/min mL/s 0.0167 from non carbohydrate source
clearance Glycogenolysis Breakdown of glycogen to glucose
Folic acid ng/mL nmol/L 2.27 for use as energy
Glucose mg/dL mg/dL 0.0555 Glycogenesis Conversion of glucose to glycogen
Sodium mEq/L mmol/L 1.0 for storage
Thyroxine ug/dL nmol/L 12.9 Lipogenesis Conversion of carbohydrates to
(T4) fatty acids
Total g/dL g/L 10 Lipolysis Decomposition of fat
protein
Triglycerides mg/dL mmol/L 0.0113 REGULATION OF GLUCOSE METABOLISM
Uric acid mg/dL mmol/L 0.0595
Vitamin B12 ng/mL pmol/L 0.0738 • Insulin
PCO2 mmHg kPa 0.133 • Glucagon
PO2 mmHg kPa 0.133 • Epinephrine
• Cortisol
TYPES OF CARBOHYDRATES • Growth hormone
• Thyroxine
Monosaccharides Disaccharide Polysaccharide • Somatostatin
Glucose Sucrose Glycogen
(Glucose + (stored in liver)
Fructose) (animal)
Fructose Lactose Starch (storage
(Fructose + in plants)
Galactose)
Galactose Maltose Cellulose
(Glucose +
Glucose)
Chitin

MUST KNOW
• Except somatostatin and insulin, which does not
• Glycol aldehyde – simplest carbohydrate (CHO) increase blood glucose instead they decrease
• Glucose, maltose, fructose, lactose and blood glucose.
galactose – reducing substances/sugars • Somatostatin – inhibit insulin and glucagon
SPECIMEN CONSIDERATIONS AND PREPARATIONS NEOCUPREINE METHOD

• Whole blood, plasma, serum, CSF, urine, serous 𝐶𝑢1+ + 𝑁𝑒𝑜𝑐𝑢𝑝𝑟𝑒𝑖𝑛𝑒 → 𝑦𝑒𝑙𝑙𝑜𝑤 − 𝑦𝑒𝑙𝑙𝑜𝑤 𝑜𝑟𝑎𝑛𝑔𝑒
fluid, synovial fluid can be used as sample.
• Standard clinical specimen in hospital is serum
but in board exam it is fasting venous plasma BENEDICT’S METHOD
• Fasting blood sugar should be obtained after 10
• For reducing sugars
– 12 hours of fasting.
• Fasting for lipids is 12-14 hours 𝑔𝑙𝑢𝑐𝑜𝑠𝑒
𝐶𝑢𝑆𝑂4
• Fasting blood glucose with lipid is 10-12 hours 𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑠𝑢𝑔𝑎𝑟𝑠
• Whole blood gives 10-15% lower than the → 𝑔𝑟𝑒𝑒𝑛, 𝑦𝑒𝑙𝑙𝑜𝑤, 𝑜𝑟𝑎𝑛𝑔𝑒, 𝑏𝑟𝑖𝑐𝑘 𝑟𝑒𝑑 𝑝𝑟𝑒𝑐𝑖𝑝𝑖𝑡𝑎𝑡𝑒
glucose levels than serum or plasma.
• Serum is appropriate for glucose analysis if it is
separated from the cells within 30-60 minutes.
• Glucose is metabolized at room temperature at Chemical methods:
a rate of 7mg/dL/hr.
• At 4 degree Celsius, glucose decreases by • Ferric reduction
approximately 2mg/dL/hr • Condensation method
• 2mg of sodium fluoride per mL of whole blood
prevents glycolysis for up o 48 hours (2 days).
• Fluoride binds magnesium which causes FERRIC REDUCTION
inhibition of the enzyme enolase. 𝑔𝑙𝑢𝑐𝑜𝑠𝑒
• CSF glucose concentration is approximately 60 - 𝐹𝑒 3+ → 𝐹𝑒 2+
𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑠𝑢𝑔𝑎𝑟𝑠
70% that of plasma concentration.
• CSF glucose should be obtained 1-2 hours
before spinal tap. CONDENSATION METHOD
∆100𝐶
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂𝑟𝑡ℎ𝑜𝑡𝑜𝑙𝑜𝑢𝑖𝑑𝑖𝑛𝑒 𝑚𝑒𝑡ℎ𝑜𝑑
METHODOLOGIES FOR GLUCOSE ASSAY 𝐻𝐴𝐶
→ 𝑏𝑙𝑢𝑖𝑠ℎ 𝑔𝑟𝑒𝑒𝑛 𝑐𝑜𝑙𝑜𝑟

Chemical Methods
Copper reduction:
Enzymatic Methods:
• Folin Wu method
• Nelson Somoygi • Glucose oxidase
• Neocupreine method o Polarographic
o Colorimetric
• Hexokinase G6PD – oldest; reference method;
FOLIN WU MTEHOD sensitive and specific
𝑔𝑙𝑢𝑐𝑜𝑠𝑒
𝐶𝑢2+ → 𝐶𝑢1+
𝑅𝑒𝑑𝑢𝑐𝑖𝑛𝑔 𝑠𝑢𝑔𝑎𝑟𝑠
GLUCOSE OXIDASE
𝐶𝑢1+ + 𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑚𝑜𝑙𝑦𝑏𝑑𝑎𝑡𝑒 → 𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑚𝑜𝑙𝑦𝑏𝑑𝑒𝑛𝑢𝑚 𝑏𝑙𝑢𝑒
𝑔𝑙𝑢𝑐𝑜𝑠𝑒 𝑜𝑥𝑖𝑑𝑎𝑠𝑒
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑂𝑥𝑦𝑔𝑒𝑛
→ 𝑔𝑙𝑢𝑐𝑜𝑛𝑖𝑐 𝑎𝑐𝑖𝑑 + ℎ𝑦𝑑𝑟𝑜𝑔𝑒𝑛 𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑒

• Polarographic
NELSON SOMOYGI • Colorimetric
𝐶𝑢1+ + 𝑎𝑟𝑠𝑒𝑛𝑜𝑚𝑜𝑙𝑦𝑏𝑑𝑎𝑡𝑒 → 𝑎𝑟𝑠𝑒𝑛𝑜𝑚𝑜𝑙𝑦𝑏𝑑𝑒𝑛𝑢𝑚 𝑏𝑙𝑢𝑒
𝐻𝑦𝑑𝑟𝑜𝑔𝑒𝑛 𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑒 + 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛
𝑝𝑒𝑟𝑜𝑥𝑖𝑑𝑎𝑠𝑒 • Cholesterol – serves as part of cell membrane,
as parent chain for cholesterol based hormones
→ 𝑊𝑎𝑡𝑒𝑟
such as aldosterone, cortisol and sex hormones.
+ 𝑂𝑥𝑖𝑑𝑖𝑧𝑒𝑑 𝐶ℎ𝑟𝑜𝑚𝑜𝑔𝑒𝑛
o Exists in the body in two different
forms:
o Cholesterol esters – accounts to
HEXOKINASE-G6PD
approximately 70% of total cholesterol
𝐻𝑒𝑥𝑜𝑘𝑖𝑛𝑎𝑠𝑒 of the body, composed of a cholesterol
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝐴𝑇𝑃 → 𝐺6𝑃𝐷 + 𝐴𝐷𝑃
ring with a fatty acid.
𝐺6𝑃𝐷 o Free cholesterol – accounts to
𝐺𝑙𝑢𝑐𝑜𝑠𝑒 + 𝑁𝐴𝐷𝑃 approximately 30% of total cholesterol;
→ 6 𝑝ℎ𝑜𝑠𝑝ℎ𝑜𝑔𝑙𝑢𝑐𝑜𝑛𝑎𝑡𝑒 + 𝑁𝐴𝐷𝑃𝐻 also known as unesterified cholesterol.

• Phospholipid – structurally similar to

SPECIMEN CONSIDERATIONS AND PREPARATIONS triglyceride, except 2 fatty acids and a
phosphate group is attached to the glycerol
• Glucose in the solutions exists either as an alpha
backbone.
glucose or beta glucose. Alpha glucose is
o Constituent of the cell membrane,
approximately 35% of the total glucose; beta
contains polar and non polar end.
glucose is 65%.
• Glucose oxidase is specific to beta.
• Free fatty acids – building blocks of lipids,
• Alpha glucose is converted into beta glucose
hydrocarbon chains with a terminal COO-
using the enzyme mutarotase.
group.
• Hemolyzed and icteric samples cause decreased
glucose with the Hexokinase-G6PD method
• Presence of strong reducing and oxidizing
TRANSPORT PROTEINS FOR LIPOPROTEINS – THE
agents interfere with glucose oxidase method.
LIPOPROTEINS

• Apolipoproteins
LIPIDS
Functions Major
Functions: Source
Apo A1 Major structural protein in Liver
• Storage HDL. Activates LCAT; ligand and
• Cushion for HDL binding intestine
• Integral part of phospholipid Apo A-II Structural protein in HDL; Liver
• Hormone synthesis activates LCAT. Enhances
hepatic triglyceride lipase
activity.
FORMS OF LIPIDS Apo A-IV Component of intestinal Intestine
lipoproteins
• Triglyceride Apo B-48 Primarily structural protein Intestine
o Possess 3 molecules of fatty acids and a in chylomicrons
molecule of glycerol which serves as the Apo B-100 Major structural protein in Liver
backbone. VLDL and LDL. Ligand for
o Serves as the main storage of lipid in the LDL receptor.
man, as insulator or shock absorber, Apo C-I Activates lipoprotein lipase. Liver
Apo C-II Activates lipoprotein lipase; Liver
and as an integral part of the cell
activates LCAT.
membrane.
Apo C-III Inhibits lipoprotein lipase. Liver Step 3:
Inhibits receptor
recognition of Apo E. • Reagent: sodium periodate
Apo E2, 3, Binds to LDL-receptor and Liver • Purpose: converts glycerol to formaldehyde
4 remnant receptor.
Step 4:
Apo (a) Structural protein for Lp (a). Liver
May inhibit plasminogen • Reagent: chromotropic acid dissolve in H2SO4
binding. • Purpose: Color reaction
Lp(a) – abnormal lipoprotein seen in patients with CHD
and AMI.
SPECIMEN CONSIDERATIONS AND PATIENT
PREPARATION
CHEMICAL COMPOSITION OF DIFFERENT LIPOPROTEIN
CLASSES: • Fasting requirement at least 12 hours
• No to hemolyzed sample.
Prot Cholest Cholest Triglycer Phospho • Avoid water contamination
ein erol eryl ides lipid
• Serum or EDTA-plasma may be used
esters
CM 1-2 1-3 2-4 80-95 3-6 • No to icteric specimens.
VL 6-10 4-8 16-22 45-65 15-20 • Serum is preferred; 24 hour urine and serous
DL fluids can also be used.
IDL Intermediate between VLDL and LDL
LD 18- 6-8 45-50 4-8 28-24
ELECTROPHORESIS
L 22
HD 45- 3-5 15-20 2-7 26-32 • Regions are stained using ponceau s, coomasie
L 55 brilliant blue, amido black
• Electrophoretic patterns:
SEPARATING LIPOPROTEINS o Beta-gamma bridging – seen in patients
with liver cirrhosis.
• Electrophoresis o Bence jones protein – seen in cases of
• Ultracentrifugation monoclonal gammopathy.
o Increase a2-macroglobulin, decrease
albumin – seen in patients with
ABNORMAL LIPOPROTEINS nephrotic syndrome.
o Decrease a1 antitrypsin – seen in
• Beta-VLDL patients with emphysema
• Lp(a)

METHODOLOGIES IN PROTEIN DETERMINATION

METHODOLOGIES FOR TRIGLYCERIDES
DETERMINATIONS • Kjeldhal Method – reference method;
quantifies protein by its nitrogen content.
Chemical Method – Van Handel Zilversmith o Assumes average nitrogen content of
Step 1: 16%.
• Biuret Reaction – measures protein by peptide
• Reagent: chloroform bonds.
• Purpose: separate tag and protein carrier o Violet color reaction.
Step 2: • Dyes:
o Bromcresol purple – most sensitive,
• Reagent: Alcoholic potassium hydroxide specific and precise among the dye-
• Purpose: divide tag binding assays.
o Bromcresol green – most commonly
used.
o Other dyes used: methyl orange, HABA
• Salt precipitation (turbidity) test – used for
urine and CSF.