CLINICAL CHEMISTRY LABORATORY

BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

LABORATORY SAFETY AND BASIC

PRINCIPLES OF CLINICAL CHEMISTRY TRANSMISSION OF BIOLOGICAL
HAZARDS
BIOLOGICAL HAZARDS  Direct Contact – Physical contact
 Refers to a biological substance that with an infected person or
poses a threat to the health of living contaminated surface
organism, primarily humans.  Airborne Transmission –
 Bacteria, viruses, fungi, prions and Inhalation of airborne pathogens
other organisms and their toxins. (droplets)
 Chain of infection (transmission)  Vector – Borne Transmission –
 Chain of infection – necessary to Spread through vectors
prevent infection (mosquitoes, ticks, fleas)
 Requires continuous link between  Food and Water - Ingestion of
three elements (source, mode of contaminated food or water
transmission, susceptible host) TRANSMISSION PREVENTION
 Source – refers to the location of GUIDELINES
potentially harmful microorganisms  Wear appropriate PPE
(person, contaminated of object)  Change gloves between patients
 MOT – direct contact, inhalation,  Wash hands after removing of
ingestion, gloves
 Host – individual who is at risk of  Dispose of biohazardous material in
infection designated containers
 Properly dispose of sharps in
TYPES OF BIOLOGICAL HAZARDS puncture-resistant containers
 Pathogenic Microorganisms –  Do not recap needles
Bacteria, viruses, fungi, prions  Do not activate needle safety device
 Bioactive Substances – Toxins using both hands
produced by microorganisms, plants,  Follow institutional protocol
and animals governing working during personal
 Allergic Agents – Substances that illness
can cause allergic reactions  Maintain personal immunizations
SOURCES OF BIOLOGICAL HAZARDS  Decontaminate work areas and
 Medical and Clinical Settings – equipment
Hospitals, laboratories, and clinics  Do not centrifuge uncapped tubes
where infectious agents are handled  Do not eat, drink, smoke, or apply
 Natural Environment – Soil, water cosmetics in the work are
and air that may contain pathogenic BIOLOGICAL WASTE DISPOSAL
organisms  Equipment and supplies that are
 Biotechnology and Research – contaminated with blood and other
facilities involved in genetic body fluids must be disposed
engineering, pharmaceutical properly accordingly to its respective
development, and biological trash bins (color-coded)
research  Black - non-infectious dry

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 CLINICAL CHEMISTRY LABORATORY
BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

 Green - non-infectious wet includes any items that can puncture

 Yellow - infectious pathologic or cut the skin
 Yellow with black band -  Causing injuries and transmitting
chemical waste and heavy infections
metals  Needles and syringes
 Orange - radioactive waste  Scalpel blades
 Red – Sharps  Lancets
 White or clear plastics - soiled  Broken Glass
linens  Razors and Scissors
 Light Blue or Transparent w/ Hazards associated with sharps
blue inscription – autoclaving  Needlestick injuries – can cause
 For blood and other body fluid spill in puncture wounds (bloodborne
the working area must be pathogens)
disinfected.  Cuts and Lacerations – can lead to
 The most common disinfectant is exposure to infectious materials and
1:10 dilution of Sodium hypochlorite increase the risk of infection
(household bleach) prepared weekly  All sharps must be disposed in
and stored in a plastic (not a glass) puncture-resistant, leak-proof
METHODS OF DISPOSAL containers labelled with the
1. Autoclaving – (sterilization) uses biohazard symbol
high-pressure steam to sterilize waste, CHEMICAL HAZARDS
killing all pathogens (commonly used  Refers to the risks posed by
for laboratory waste) chemicals that can cause harm to
2. Incineration – (Burning waste) human health or the environment
involves burning biological waste at  Toxic chemicals: Acute (immediate
high temperatures, reducing it to ash harm upon exposure) ; cyanide,
3. Chemical Disinfection – Often used carbon monoxide ; Chronic (long-
for liquid waste term health effects) ; asbestos,
4. Landfill Disposal – (Burial) requires benzene
careful regulation to avoid  Flammable chemicals – easily
contamination ignite and cause fires (gasoline,
5. Biological Treatment – (Composting) ethanol, acetone)
microorganisms break down the waste  Corrosive chemicals – can cause
into harmless byproducts; more severe damage to tissues and
applicable to agriculture and food materials (sulfuric acid, hydrochloric
industry waste acid) No part of this material may be
distributed and reproduced. The file
is for your own use only.
 Reactive chemicals – can undergo
violent reactions under certain
SHARP HAZARDS conditions (sodium, potassium,
 Sharps are a specific category of explosives)
medical and laboratory waste that

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 CLINICAL CHEMISTRY LABORATORY
BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

 Carcinogens – known to cause ELECTRICAL HAZARDS

cancer (formaldehyde, benzene)  The amount of radioactivity present
 Mutagens and Teratogens – in most medical situations is very
mutagens (cause genetic mutations) small and represents little danger
; ethidium bromide ; teratogens  The effects of radiation are related to
(cause developmental abnormalities) the length of exposure and are
; thalidomide cumulative
*ADD ACID TO WATER  Exposure to radiation is dependent
 When skin or eye contact occurs , on the combination in time, distance,
the best first aid is to flush the area and shielding
immediately with water for at least  Exposure to radiation during
15 minutes and then seek medical pregnancy presents danger to the
attention fetus
 Refer to dangerous conditions where
ROUTES OF EXPOSURE a person can encounter energized
• Inhalation equipment or conductors (electric
• Ingestion shock, burns)
• Skin contact  Electrical equipment is closely
• Injection monitored by designated hospital
personnel
 Equipment that has become wet
should be unplugged and allow to
dry completely before reusing
 Equipment should be unplugged
before cleaning
TYPES OF ELECTRICAL HAZARDS
• Electric shock
• Electrical burns
RADIOACTIVE HAZARD • Arc flash
 The amount of radioactivity present • Arc blast
in most medical situations is very • Fire hazard
small and represents little danger • Static electricit
 The effects of radiation are related to
the length of exposure and are
cumulative
 Exposure to radiation is dependent
on the combination in time, distance,
and shielding
 Exposure to radiation during
pregnancy presents danger to the
fetus

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 CLINICAL CHEMISTRY LABORATORY
BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

FIRE/EXLOSIVE HAZARDS
 Initial steps to follow when a fire is
discovered are identified by the code
word RACE
o Rescue – rescue anyone in
immediate danger
o Alarm – activate the
institutional fire alarm system
o Contain – close all doors to
potentially affected areas
o Extinguish/Evacuate –
extinguish the fire, if possible, General precautions that should be
or evacuate, closing the door observed are the following:
NATIONAL FIRE PROTECTION  Avoid running in rooms and hallways
ASSOCIATION (NFPA)  Be alert for wet floors
 Bend the knees when lifting heavy
objects or patients
 Keep long hair tied back and remove
dangling jewelry to avoid contact
with equipment and patients
 Wear comfortable, closed-toe shoes
with non-skid soles that provide
maximum support
 Maintain a clean, organized work
area
BASIC PRINCIPLES
Water Specifications
 Critical reagent in clinical chemistry
laboratories (solutions, reagents,
diluent)
 Quality of water directly impacts the
accuracy and reliability of clinical
tests
 Specific standards and
specifications for laboratory water
are essential to ensure consistent
and reliable results
 Water – most frequently used
PHYSICAL HAZARDS reagent in the laboratory
 Environmental factors that can  Distilled water – purified by
cause harm to human body without distillation
necessarily touching it  Deionized water – purified by ion
 Injuries, health issues, accidents exchange

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 CLINICAL CHEMISTRY LABORATORY
BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

 RO water – reverse osmosis; pumps  Reducing agents – donate

water across semipermeable electrons
membrane  Oxidizing agents – accept
PROCESS OF WATER PURIFICATION electrons
1. Distillation CONDUCTIVITY
2. Deionization • Conductivity – how electricity passes
3. Reverse Osmosis through a solution
4. Ultrafiltration • Resistivity – resistance to the passage of
5. Ultraviolet electrical current
REAGENT GRADE WATER
 Type I (ultrapure) – requiring
minimum interference; for
preparation of standard solutions,
buffer, and in analytical techniques
(HPLC and mass spectrometry)
 Type II – preparation of reagents
and solutions, general laboratory
procedures
 Type III – glassware
washingVirology- Virologist
SOLUTION PROPERTIES
• Solute – dissolved in liquid
• Analytes – biologic solutes
• Solvent – liquid in which solute is
dissolved
• Solution– solute + solvent
COLLIGATIVE PROPERTIES
 Osmotic pressure – the pressure
required to stop the flow of solvent
into the solution through a
semipermeable membrane
 Vapor pressure – liquid solvent is in
equilibrium with the water vapor
 Freezing point – temperature at
which a vapor pressures of a solid
and liquid phases are the same
 Boiling point – temperature ate
which the vapor pressure of the
solvent reaches one atmosphere
REDOX POTENTIAL
 Oxidation – reduction potential –
measure of the ability of a solution to
accept or donate electrons

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 CLINICAL CHEMISTRY LABORATORY
BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

CLINICAL LABORATORY APPARATUS GLASSWARE

AND SUPPLIES Categories:
THERMOMETERS/TEMPERATURE • Kimax/Pyrex
 Celsius ℃ - Predominant • Corex
 Fahrenheit and Kelvin – also used • High silica
• Low actinic
• Flint
PRECISION AND ACCURACY
Class A: stamped with letter “A”
High-precision laboratory
applications
 All analytic reactions occur at an Class B: twice the tolerance limits of Class
optimal temperature A suitable for educational and
 Reactions that are temperature general laboratory purposes
dependent use some type of VOLUME DESIGNATIONS
heating/cooling cell, heating/cooling To Contain (TC):
block, water/ice bath to contain a specified volume of liquid
 Laboratory refrigerator temperatures When liquid is transferred out, it does not
are often critical and need periodic deliver the exact contained volume due to
verification residual liquid To Deliver (TD) :
2 TYPES OF THERMOMETERS to deliver the specified volume of liquid
1. Liquid-in-glass thermometers accurate delivery of the contained volume,
 use a colored liquid (red or other considering the liquid that remains in the
colored material) vessel
 encased in plastic or glass PLASTICWARE
 20C – 400C • Replace glassware
 continuous line of liquid; free from • Unique high resistance to corrosion and
separation or bubbles breakage
• Inexpensive
 calibrated against an NIST –
• Disposable
certified or NIST – traceable
• Polystyerene
thermometer for critical laboratory
• Polyethylene
applications
• Polypropylene
2. Electronic thermometer or
• Tygon
thermistor
• Teflon
 temperature-sensitive resistors
• Polycarbonate
 can be calibrated against SRM • Polyvinyl chloride
thermometers provided by NIST LABORATORY VESSELS
 fast-reading and accurate  Flasks, beakers, and graduated
 ideal for applications requiring cylinders
immediate temperature adjustments  Volumetric and Erlenmeyer flasks
– containers used in the clinical
laboratory

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 CLINICAL CHEMISTRY LABORATORY
BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

 Volumetric flask – calibrated; hold  Blow-out pipettes – any residual

1 exact volume of liquid (TC) liquid remaining in the tip after
 Erlenmeyer flasks and Griffin dispensing should be expelled
beakers – hold different volumes  Usually indicated by an etched or
rather than one exact amount colored ring near the top of the
 Graduated cylinders- long, pipette
cylindrical tubes usually held upright  1 to 50 ml
by an octagonal or circular base  Mohr pipette - have graduation
PIPETTES markings that stop before the tip
• Glass or plastic  They are calibrated to deliver the
• Used to transfer liquids volume between two specified points
• Reusable or disposable (zero;final)
• 20ml or less  1 to 25 ml
• Larger volumes – automated pipetting  Does not require blowing out
devices BACTERIOLOGIC PIPETTE
PIPETTE CLASSIFICATION  Precise handling and transfer of
microbial cultures and samples
 They are designed to maintain
sterility and facilitate accurate
handling of microbial materials
MICROPIPETTE
 ul to 1000 ul (1ml)
 Adjustable
 Air-displacement micropipettes and
Positive-displacement micropipettes
 Attach disposable tip
 1st stop (aspiration)
 2nd stop (dispense)

SEROLOGIC AND MOHR PIPETTE VOLUMERIC PIPETTE

 Serological pipette – have a  Measure and transfer a single
graduation marking that extend to accurate volume of liquid
the tip  Deliver a specific volume with high
precision
 To deliver (TD)
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 CLINICAL CHEMISTRY LABORATORY
BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

 Class A Volumetric pipette BALANCES

 Class B Volumetric pipette

ANALYTICAL BALANCE
 Measure small masses with very
PASTEUR PIPETTE high accuracy and precision
 AKA dropper or dropping pipette • o grams (resolution)
Small volumes of liquids  Enclosed in a draft shield
 Louis Pasteur  Single pan
 Simple and quick liquid handling  Frequent calibration and
SYRINGE maintenance

TOP-LOADING BALANCE
 Less precise; sufficient accuracy for
laboratory applications
 grams to 0.001 grams
 Open pan (do not require a draft
shield)
 Routine weighing
 More cost-effective
 Easier to maintain
CENTRIFUGE
 Used to separate components of a
mixture (spinning)
 Utilizes the principles of centrifugal
force
 Density, size and shape
 General Purpose Centrifuges
 Refrigerated Centrifuges
 Ultracentrifuges
COMPONENTS OF CENTRIFUGE
1. Rotor – central component (samples
are placed for spinning) Fixed-angle,
swinging bucket rotors, vertical
rotors
2. Speed control – rotation per minute
(rpm) and duration (time) of the spin

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3. Temperature control – some 3. Glass nonadditive tubes

centrifuge offer temperature control  Tube Stopper Color: Red
features (maintains integrity)  Rationale: Prevents contamination
4. Safety features – lid locks and by additives in other tubes.
imbalance detection 4. Plastic clot activator tubes |
TYPES OF CENTRIFUGE Serum separator tubes (SST)
1. General Purpose Centrifuges –  Tube Stopper Color: Red, Red and
routine laboratory applications gray rubber, Gold plastic
2. Refrigerated Centrifuges –  Rationale: Filled after coagulation
Equipped with cooling mechanisms tests because silica particles
to maintain low temperatures activate clotting and affect
(suitable for samples sensitive to coagulation tests (carry-over of silica
heat) into subsequent tubes can be
3. Ultracentrifuges – Capable of overridden by anticoagulant in
reaching extreme high speeds them).
(separation of very small particles or 5. Plasma separator tubes (PST) |
molecules) Heparin tubes
OPERATION OF CENTRIFUGE  Tube Stopper Color: Green and
1. Loading – samples are loaded into gray rubber, Light-green plastic,
centrifuge tubes or rotor buckets Green
2. Centrifugation – spins rapidly  Rationale: Heparin affects
(heavier particles-settle ate the coagulation tests and interferes in
bottom; lighter particles-move to the collection of serum specimens;
top); based on their density and size causes the least interference in tests
3. Collection – extracted using other than coagulation tests.
pipettes or decanting methods 6. EDTA tubes
ORDER OF THE DRAW  Tube Stopper Color: Lavender,
Pink
 Rationale: Responsible for more
carry-over problems than any other
Order of Draw | Tube Stopper Color | additive; elevates Na+ and K+
Rationale for Collection Order levels, chelates and decreases
1. Blood cultures (sterile calcium and iron levels, elevates PT
collections) and PTT results.
 Tube Stopper Color: Yellow SPS, 7. Plasma preparation tubes (PPT) |
Sterile media bottle Oxalate/fluoride tubes
 Rationale: Minimizes chance of  Tube Stopper Color: Gray
microbial contamination.  Rationale: Sodium fluoride and
2. Coagulation tubes potassium oxalate elevate sodium
 Tube Stopper Color: Light blue and potassium levels, respectively,
 Rationale: The first additive tube in after hematology top tubes because
the order because all other additives oxalate damages cell membranes
affect coagulation tests. and causes abnormal RBC

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 CLINICAL CHEMISTRY LABORATORY
BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

morphology. Oxalate interferes in  Sample holder - usually a cuvette

enzyme reactions. (placed in the path of the isolated
EVACUATED TUBES light beam)
Cap Color | Inversions  Quartz, glass, plastic (transparent)
 Light Blue: 3-4x  Detector – photodiode or
 Light Green/PST: 8x photomultiplier tube
 Green: 8x  Measures the intensity of the
 Lavender: 8x transmitted light (inversely related to
 Pink: 8x the amount of light absorbed by the
 Grey: 8x sample) No part of this material may
 Red (Plastic with clot activator): 5x be distributed and reproduced. The
 Red (Glass tubes): 0x file is for your own use only.
 Gold: 5x  Readout and Data Analysis –
 Tan: 8x instrument converts the detected
 Yellow: 8x light intensity into a digital signal,
 Orange: 8x displaying the absorbance (or
transmittance) on a readout screen
 Royal Blue (no additive glass
serum): 8x  Beer – Lambert Law
BEER-LAMBER LAW
SPECTROPHOMETER  AKA Beer’s Law (fundamental
 Analytical instrument used to principle in spectrometry)
measure the intensity of light as a  The concentration of a substance is
function of its wavelength directly proportional to the amount of
 For quantitative analysis of light absorbed or inversely
substances proportional to the logarithm of the
transmitted light
 Provides highly accurate and precise
measurements of light absorbance
 Concentration of substances and
identifying molecular structures
PRINCIPLE OF OPERATION
 Light source – emits a broad
spectrum of light, typically ranging
from UV to Vis and sometimes into
the IR region
 Deuterium lamps (UV); Tungsten or
Halogen lamps (Vis) TYPES OF SPECTROPHOTOMETER
 Monochromator– isolates specific  UV – Visible – Measures light
wavelengths of light (prism or absorbance in UV and Vis regions
diffraction gratings) (190-1100nm) ; used for analyzing
 Allows only the desired wavelength nucleic acids, proteins, biological
to pass through to the sample molecules
 IR Spectrophotometer – Measures
absorbance in the IR region

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(NIR:700 – 2500nm; MIR: 2500-

25,000nm; FIR: 25,000nm – 1mm)
 Atomic Absorption
Spectrophotometer (AAS) –
Measures the concentration of metal
ions in solutions by detecting the
absorption of light by free atoms

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BSMT | MED 224 TRANSES | 3RD YEAR – 1ST TERM

QUALITY MANAGEMENT 2. Personnel or operator errors –

QUALITY ASSURANCE usually random errors that usually
 Ensuring the accuracy, reliability, affect only a few analyses
and consistency of laboratory test  Mislabeling the specimen
results  Analytical result is assigned to a
 Critical for patient diagnosis, wrong specimen
treatment, and monitoring  Wrong number entry on test result
 Includes the pre – analytic, analytic, ACCURACY AND PRECISION
and post – analytic phases 1. Accuracy
• Pre-analytic phase –  Nearness to the true value
involves all the steps  Method is reflected by its ability to
taken before the actual reproduce the values of reference
analysis of the samples of unknown concentration
specimen 2. Precision (expressed in terms of
• Analytic phase – SD)
involves the actual  Reproducibility of a laboratory
testing of the specimen determination when it is run
• Post-analytic phase – repeatedly under identical conditions
involves all the steps STANDARD DEVIATION
taken after the analysis Measure of the amount of variation or
to ensure the accurate dispersion in a set of values
reporting and
interpretation of results
QUALITY CONTROL
 Part of quality assurance
 Focusing on monitoring and
maintaining the accuracy and
precision of laboratory tests
 Help ensure that test results are
reliable and valid 2 major type:
1. External QC – monitors primarily
the accuracy of laboratory tests
2. Internal QC – monitors the day-to-
day performance of laboratory tests,
namely precision
ERRORS
1. Analytical errors – usually
systematic errors
 Erroneously calibrated pipettor
 Deteriorating reagent
 Improperly calibrated instrument
Sample data set of tests scores: 85, 90, 78,
92, 88

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• Calculate the mean: find the sample mean  Normalized measure of the
dispersion of the probability
= 85+90+78+92+88/5 distribution
= 433/5  Ratio of the standard deviation to the
= 86.6 mean
• Calculate each deviation from the mean
and square it Find the deviation of each
score from the mean and square the result
(85-86.6)2 = (-1.6)2 = 2.56  The sample mean = 86.6
(90-86.6)2 = (3.4)2 = 11.56  The sample standard deviation =
(78-86.6)2 = (-8.6)2 = 73.96 5.46
(92-86.6)2 = (5.4)2 = 29.16  Calculate CV
(88-86.6)2 = (1.4)2 = 1.96 CV = 5.46/86.6 x 100% = 6.30%
DESCRIPTIVE STATISTICS: MEASURES
 Sum the squared deviations Sum OF CENTERS,SPREAD, AND SHAPE
these squared deviations
2.56+11.56+73.96+29.16+1.96 = 119.2
 Divide by the number of data points
minus one (n-1)
For a sample, we divide by n-1,
where n is the number of data
points: 119.2/5-1 = 119.2/4 = 29.8
 Take the square root Finally, take MEASURES OF CENTER
the square root of this value to Three most used descriptions of the center
obtain the sample SD: s= √29.8 of a date set:
= 5.46  Mean
VARIANCE  Median
 Calculate the Mean (86.6)  Mode
 Calculate each deviation from the 1. Mean – commonly used and often
mean and square it called the average
 (85-86.6)2 = (-1.6)2 = 2.56 2. Median – middles point and often
used with skewed data (calculation
 (90-86.6)2 = (3.4)2 = 11.56
is not significantly affected by
 (78-86.6)2 = (-8.6)2 = 73.96
outliers)
 (92-86.6)2 = (5.4)2 = 29.16 3. Mode – rarely used as a measure of
 (88-86.6)2 = (1.4)2 = 1.96 data’s center (often used to describe
 Sum the squared deviations data that seem to have 2 centers
2.56+11.56+73.96+29.16+1.96 = MEAN
119.2  Data set is calculated by summing
 Divide by the number of data points all the values and then dividing by
minus one (n-1) 119.2/5-1 =119.2/4 the number of values
= 29.8  Formula for the mean is different
COEFFIECIENT OF VARIATION (CV) depending on whether you are

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dealing with a population or a *the median is the average of the

sample second and third values in the
ordered list: 85+90/2 = 175/2 = 87.5
MODE
• Value that appears most frequently in
data set • Data set may have one mode,
more than one mode, or no mode at all
Types of Mode:
• Unimodal – data set with one mode
• Bimodal – data set with two mode
• Multimodal – data set with more than two
modes
• No mode – data set where no value
Example calculation: repeats
Using the previous data set: Steps to find the mode
85, 90, 78, 92, 88 85+90+78+92+88/5 1. Count the Frequency – determine
=433/5 =86.6 the frequency of each value in the
MEDIAN data set
 Middle value of data set when it is 2. Identify the most frequent value –
arranged in ascending or the value with the highest frequency
descending order is the mode
 If the data set has an odd number of Example Calculation:
observations, the median is the 85,90,78,92,88,90,85,90 Count the
middle number frequency
Example calculation: 85 – 2 times
Previous data set: 85, 90, 78, 90 – 3 times
92, 88 78 – 1 time
Arrange the data: 78, 85, 88, 92 – 1 time
90, 92 88 – 1 time
Determine the middle Identify the most frequent value:
position: 5 85 – 2 times
observations (5+1)/2 = 3 90 – 3 times
*the median is the 3rd value in the 78 – 1 time
ordered list, which is 88 92 – 1 time
 If the data set has an even number 88 – 1 time
of observations, the median is the Example with no mode: 85,90,78,92,88
average of the two middle numbers Each value appears only once
Example calculation: Example with multiple modes:
Data set: 78,85,90,92 85,90,78,92,88,85,90
Arrange the data: 78,85,90,92 85 – 2 times
Determine the middle position: 4 90 – 2 times
observation (4/2 = 2 ; (4/2)+1 = 3) 78 – 1 time
92 – 1 time
88 – 1 time

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*Bimodal 3. Center Line – represents the mean

CONTROL SPECIMEN (target) value of the control measurements
 Standard – reference of the 4. Control Limits – usually set at ±1, ±2,
unknown, to achieve accuracy and ±3 standard deviations (SD) from the
 Control – to check for precision mean
 Blank – to achieve accuracy by ±1 SD: warning limits
setting the reading to zero ±2 SD: Action limits
 Unknown – sample/specimen ±3 SD: Critical action limits
Characteristics of control specimens: Creating a Levy-Jennings QC chart
1. Should behave like the real specimen 1. Collect data: Obtain a series of control
2. Should be available in sufficient quantity measurements over time
to last for a minimum of 1 year 2. Calculate the Mean and SD
3. Should be stable over a period of 1 year 3. Determine Control Limits: ±1, ±2, and
4. Should be available in convenient vial ±3 SD from the mean
volumes 4. Plot the Data: Plot the control
5. Should vary minimally in concentration measurements on the Y-axis against time
and composition from vial to vial or sequence on the X-axis ; Draw the mean
6. Should include clinically normal, high, line and the control limits (±1, ±2, and ±3
and low abnormal ranges SD)
7. Should be preferably lyophilized require Interpretation of the Chart
reconstitution before use  Within ±1 SD – indicates that the
Sources: process is well within control
1. Pooled control sera  Between ±1 SD and ±2 SD –
2. Assayed commercial control indicates a warning, process may
3. Unassayed commercial control need to be monitored more closely
PRECISION MONITORING TECHNIQUES  Between ±2 SD and ±3 SD –
Levey-Jennings QC chart indicates an action level, process
 The control results are plotted on the may require adjustment
ordinate versus time on the abscissa  Beyond ±3 SD – indicates a critical
 Random error shows a wider range action level, process likely needs
of scatter of points on the control immediate attention
chart, while systematic error can be Example Calculation and Plot Control
seen when the points drift or shift on measurement data set:
one side of the central solid line 98,102,100,101,99,103,97,104,96,100
 Solid line indicates the mean of the Calculate the Mean:
control, dotted lines are the control
limits (usually +/- 2SD)
LEVEY-JENNINGS QC CHART
Calculate the SD
Components of L-J QC chart
1. X-axis – represents time or the
sequence of the control measurements
2. Y-axis – represents the measurement
values of the quality control sample

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2. Y-axis – represents the

measurement values of the quality
control sample
3. Center Line – represents the mean
(target) value of the control
measurements
Determine Control Limits 4. Control Limits
 1s Control Limits: ±1 SD from the
mean
 2s Control Limits: ±2 SD from the
mean
 3s Control Limits: ±3 SD from the
mean
Plot the Data  2s Warning Limits: indicates a
warning zone before reaching the 2s
control limits R4s/R6s/R10s Rules:
These are additional rules used to
detect trends or shifts in data points
over time
Rules in Westgard QC chart
1s,2s,3s Control Limits
WESTGARD QC CHART 1. These limits help identify when a
 Developed by James O. Westgard measurement falls outside expected
 Used extensively in clinical variability (1s), indicating a potential
laboratories to monitor the issue
performance of analytical tests and 2. 2s Warning Limits These are
ensure the accuracy and reliability of typically set closer to the mean than
laboratory results the 2s control limits and serve as an
 Designed to detect both random early warning for potential issues
errors and systemic errors  R4s, R6s, R10s Rules These rules
are used to detect systematic shifts
or trends in the QC data
 R4s – one control measurement falls
beyond ±2s
 R6s – 2 consecutive control
measurements fall beyond ±1s
 R10s – 9 consecutive control
measurements fall on the same side
of the mean
Components of Westgard QC chart Example: Suppose we have a series of
1. X-axis – represents time or the daily control measurements for a specific
sequence of measurements analyte in a clinical laboratory
98,102,100,101,99,103,97,104,96,100

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Interpretation
 Points falling within the 1s control
limits indicate normal variation
 Points beyond the 1s control limits
but within the 2s control limits may
indicate a need for closer monitoring
 Points beyond the 2s control limits
indicate a significant deviation that
requires investigation and corrective
action
INDICATORS OF SYSTEMATIC ERROR
1. Trend – because of reagent,
increasing values passing through
mean, reject on the 6th test
2. Shift – because of standard, stable
on one side of the mean, reject on
the 6th test
• Sensitivity – ability to
measure minute
concentration
• Specificity – ability to
react with only one
unknown

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PIPETTING TECHNIQUES  Single- Channel Pipettes

PIPETTE  Multi-Channel Pipettes
 Pipette (pipet) – laboratory  Adjustable-Volume Pipettes
equipment  Fixed-Volume Pipettes
 Pipetting is a fundamental laboratory POSITIVE DISPLACEMENT PIPETTES
technique used for transferring  Used for viscous, volatile, or high-
precise volumes of liquids Based on density liquids (have a direct piston
volume/capacity to dispense mechanism that makes them more
 Micropipettes – dispense between accurate for challenging liquids
1- 1000 ul Aspirating:
 Macropipettes – dispense greater  The piston is in direct contact with
volume the liquid
 When the plunger is depressed, the
PARTS OF PIPETTE piston moves down, displacing an
equivalent volume of liquid into the
capillary tip
Dispensing:
 As the plunger is pressed again, the
piston pushes the liquid out of the
tip, ensuring complete delivery
without the risk of residual liquid or
air bubbles
AIR DISPLACEMENT PIPETTE Types:
 Used for aqueous solutions (include 1. Manual Positive Displacement Pipettes
single-channel and multichannel 2. Electronic Positive Displacement
pipettes) Pipettes
Aspirating: PIPETTES AND COLOR CODING
 When the plunger is pressed to the
first stop, the piston inside the
pipette moves downward, displacing
air from the tip
 Upon releasing the plunger, a
vacuum is created, drawing liquid
into the tip up to the calibrated
volume
Dispensing:
 Pressing the plunger to the first stop
dispenses the aspirated liquid from
the tip
 Pressing the plunger to the second GUIDELINES
stop expels any residual liquid to Select the right pipette and tip:
ensure complete delivery  Use a pipette appropriate for the
Types: volume you intend to transfer

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 Choose the correct tip that fits the

pipette securely to avoid leaks or
inaccuracies
Pre-rinse the pipette tip:
 Aspirate and dispense the liquid 2-3
times to condition the tip, especially
when using new tips
Proper tip attachment:
 Attach the tip firmly by pressing it
onto the pipette
Aspirating technique:
 Press the plunger to the first stop
before immersing the tip in the liquid
 Immerse the tip just below the
surface of the liquid to avoid drawing
air
 Release the plunger slowly and
steadily to aspirate the liquid,
ensuring no air bubbles are drawn
into the tip
Dispersing technique:
 Touch the tip to the side of the
receiving container to prevent
splashing
 Press the plunger smoothly to the
first stop to dispense the liquid
 Press the plunger to the second stop
to expel any remaining liquid in the
tip
MAINTENANCE AND CALIBRATION
1. Regular Calibration
Calibrate pipettes regularly
according to the manufacturer’s
instructions to ensure accuracy
2. Clean Pipettes
Clean pipettes regularly to prevent
contamination (follow manufacturer
guidelines for cleaning procedure)
3. Proper Storage
Store pipettes vertically on a stand
to prevent damage to internal
components and to avoid
contamination

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