SPECTROPHOTOMETER

Purpose of Spectrophotometer
• Spectrophotometers are instruments that send electromagnetic
radiation into a target and measure the resulting interaction of the
energy and the target
• Spectrophotometer optically determines the absorbance or
transmission of characteristic wavelengths of radiant energy (light) by
a chemical properties in solution
• Each molecule absorbs light at certain wavelengths in a unique
spectral pattern because of the number and arrangement of its
characteristic functional groups
Terminology and result interpretation
UV/VIS Spectrophotometer

• UV/Vis or UV-Vis (Ultraviolet and Visible) spectrophotometer offers

the maximum flexibility and is suitable for applications in the
wavelength range 190 to 1100 nm.
• In UV/Visible spectroscopy the UV region is considered to be any
wavelength less than 340 nm.
Single Wavelength Measurement

Also known as Fixed Wavelength. The simplest application of a

spectrophotometer and allows for measurement of the Absorbance or
Transmittance (%T) at a specified wavelength.

Multi Wavelength Measurement

This is an extension of single wavelength measurements where sample

absorbance is recorded at multiple wavelengths. Mathematical
manipulations of this data can often reveal details about the sample's
composition or purity.
Single Beam
• These are the simplest and most economical instruments that all of
the light passes through the sample holder.
• Measurements are made by placing a reference in the sample holder,
which is measured to standardize the instrument.

Double Beam
• In a double beam spectrophotometer the beam from the light source
is split in two.
• One beam illuminates the reference cell holder and the other
illuminates the sample.
• Commonly used when the sample and reference change over time
e.g. kinetics measurements.
Spectral Bandwidth

Also known as spectral bandwidth or bandpass, this relates to the

physical size of the slit from which the light passes out from the
monochromator. Bandwidth is classically measured using the full width
half height method but for instruments with bandwidths <4 nm this
can also be done using the peak and valley ratio of a toluene in hexane
scan

IEC 61010
Safety requirements for electrical equipment for measurement, control,
and laboratory use.
Visible Light Spectrum
Colors and wavelength
Department Involved
• Molecular Biology
• Biochemistry
• Physics
• Material Science
• Chemistry
Parameter of Spectrophotometer
Absorbance = A  Common wavelength for Calibrator
Transmittance =%T filter:
Color and Wavelength (Nm) • 440.0nm
• 465.0nm
• Violet = 400-450nm
• 546.1nm
• Blue = 450-500nm
• 635.0nm
• Green = 500-570nm
• Yellow = 570-590nm
• Orange = 590-610nm
• Red = 610-700nm
Procedure / Protocol of Spectrophotometer
• Select the correct type of cuvette for analysis, quartz cuvettes should be
used for readings in the UV range (200-350nm).Disposable (plastic)
cuvettes can be used for readings in the visible range (350-750 nm).
• Avoid handling the cells by the polished surfaces.
• Cuvettes need to be cleaned properly both before and after use. The
cleaning procedure for glass or quartz cuvettes is dependent on the sample
that was measured, so cleaning is sample specific.
• Before use, the fluorescence characteristics of a dilute solution of the
detergent should be measured to make quite sure that the fluorescence is
minimal at the chosen analytical wavelengths.
• The instrument must have been warm for at least 15 min. prior to use. The
power switch doubles as the zeroing control.
Procedure / Protocol of Spectrophotometer
• Use the wavelength knob to set the desired wavelength. Extreme
wavelengths, in the ultraviolet or infrared ranges, require special filters,
light sources, and/or sample holders (cuvettes).
• With the sample cover closed, use the zero control to adjust the meter
needle to "0" on the % transmittance scale (with no sample in the
instrument the light path is blocked, so the photometer reads no light at
all).
• Wipe the tube containing the reference solution with a lab wipe and place
it into the sample holder. Close the cover and use the light control knob to
set the meter needle to "0" on the absorbance scale.
• Remove the reference tube, wipe off the first sample or standard tube,
insert it and close the cover. Read and record the absorbance, not the
transmittance.
• Remove the sample tube, readjust the zero, and recalibrate if necessary
before checking the next sample .
Good Practice on Handling Spectrophotometer
• For optimum performance a calibration routine should be carried out at
the beginning and end of every sample batch.
• To ensure accurate results are obtained the sample area lid should be kept
in the closed position during measurement.
• Plastic cuvettes are not suitable for use with organic solvents.
• Glass cuvettes should be thoroughly cleaned after use. Discard when
scratches become evident in polished surfaces.
• Chemical reagents should, wherever possible, be of high grade quality.
Contamination can cause problems, even at very low levels. Diluents (i.e;
water or solvents) must be free from impurities.
• Samples and standards can “outgas” when left in the cuvette. Bubbles
formed on the cuvette walls will cause reading errors.
General design of Spectrophotometer

Cuvette
(for sample)
Light Source

Lens Diffraction
Photocell Calibrated
grating galvanometer
(variable
wavelength
selector)
Types of Spectrophotometer
• Vis Spectrophotometer
• UV/Vis Spectrophotometer
• IR Spectrophotometer
Single Beam Spectrophotometer Component
• Light source
The light source is usually a hydrogen or deuterium lamp for UV measurements
and a tungsten lamp for visible measurements.
• Monochromator
The wavelengths of these continuous light sources are selected with a
wavelength separator such as a prism or grating monochromator.
• Sample holder – Cuvette
• This device holds the sample(s) to be analysed.
• Various sample holder types to accommodate different spectrophotometer
models and sample volumes such as cuvettes, microcells, microplates, test
tubes and continuous flow cells.
Single Beam Spectrophotometer Component
• Cuvette
• Cuvette is a straight-sided, optically clear container for holding liquid samples
in a spectrophotometer or other instrument
• In conventional spectrophotometers, cuvette used is rectangular shape.
• Type of cuvette:
• Standard cuvette
• Semi –Micro Cuvette
• Precision Cell (Lightpath 5-50mm)
• Precision Cell (Lightpath 5-40mm)
Single Beam Spectrophotometer Component
• Detector System
The detection system can be designed with photocells, phototubes,
photodiodes or photomultipliers. This depends on the ranges of wavelength,
the sensitivity and the required speed of response. The detection system
receives light from the sample and converts it into an electrical signal
proportional to the energy received. This electrical signal can be processed
and amplified to be interpreted by the reading system
• Readout System
The signal which leaves the detector goes through various transformations. It
is amplified and transformed until its intensity becomes a proportional
transmittance/absorbance percentage. There are analogous reading systems
(displaying results on a reading scale) or digital ones (showing results on a
screen).
Transmittance and Absorbing
• The Absorbance (or optical density) and Transmission
(or Transmittance) of light through a sample can be
calculated by measuring light intensity entering and
exiting the sample concentration 'C'
• Some light energy is absorbed by the sample
• The amount of light energy exiting the sample has
Intensity 'I'
 Beer-Lambert Law

Io = Light Intensity entering a sample

I = Light Intensity exiting a sample
C = The concentration of analyte in sample
L = The length of the light path in glass sample cuvette
K = a constant for a particular solution and wave length

• Therefore:
Light Absorbance (A) = log (Io / I)= KCL
Light Transmission (T) = I/Io = 10-KCL

• As Concentration (C) increases, light Absorption (A) increases, linearly.

Concentration and absorbance
System Operation
• Light from an electric bulb or other source is focused by the lens and
filtered by the colored glass, diffraction grating device or prism before
passing through a cuvette of sample solution

• Filtering produces monochromatic light

• The color of light selected is the one most strongly absorbed by the
substance in solution

• Any unabsorbed light that is transmitted through the solution is picked up

by the photocell or photoelectric cell, which converts the light to a small
electric current
System Operation

• The strength of the current is directly proportional to the intensity of

the light

• The amount of light transmitted through the solution of unknown

concentration is compared with the light transmitted through
solutions of known concentration of the same substance
Reagent, calibrator and QC
• Regulations for quality control such as ISO 9001, Good Laboratory
Practice (GLP), Good Manufacturing Practice(GMP) or standard
operating procedures require a regular performance testing of the
UV/Vis spectrophotometers used.
• These checks include the testing of the resolution, the wavelength
accuracy as well as the testing of stray light and photometric accuracy
• Calibration standards(or calibrator or calibration filter) are calibrated
using a high performance UV/Vis/NIR spectrophotometer
• This instrument is used exclusively for calibrating purposes and is
tested at regular intervals for its accuracy
Calibration standards
• Also known as calibrator or calibration filter
• Have 2 types :
• Liquid filter
• Wavelength accuracy
• Photometric accuracy
• Stray light behavior
• Resolution
• Solid filter
• Photometric accuracy (in the visible spectral region)
• Wavelength accuracy
• Traceability of the calibration and conformity to the regulations is
a must
Carry Out Preventive Maintenance According
To Manufacturer Checklist (HEPPM)
CHECKLIST NO:
KEMENTERIAN KESIHATAN MALAYSIA 15083-001
BEMS Planned Preventive Maintenance Checklist
BER SEKUTU

BER TAM BAH

M UTU
Spectrophotometers, Ultraviolet / Visible PPM YTD : ( 1 / 1 )
TYPE CODE : 15-083

PART 1 ASSET DETAILS

WORK ORDER NO ASSET NO

MANUFACTURER MODEL

FREQUENCY 3 MONTHLY ( ) 6 MONTHLY ( √ ) 12 MONTHLY ( ) PPM HOURS

PART 2 SPECIAL PRECAUTION

If there is evidence of body fluid contamination, submit the device for cleaning and decontamination before inspecting it.
Wear appropriate Personnel Protection Equipment (PPE) during work.
Wear grounded electrostatic wristband when handling PCB or electronic components.
Refer to the safety procedure for additional precautions and guidance as per manufacturer guidelines.
Make sure the test equipment used are duly calibrated.
Carry Out Preventive Maintenance According
To Manufacturer Checklist (HEPPM)
PART 3 TEST APPARATUS

Tick ( √ ) where appropriate

DESCRIPTION ASSET NO / SERIAL NO CALIBRATION DUE ON

ELECTRICAL SAFETY ANALYZER

FILTER KIT (P/N 80-2107-18)

Qualitative Task
PART 4 QUALITATIVE TASKS

Tick ( √ ) where appropriate

PASS FAIL NA PASS FAIL NA
1. Chassis - verify physical integrity,
( ) ( ) ( ) 8. Calibration check ( ) ( ) ( )
cleanliness and condition.

2. AC Plug - verify integrity ( ) ( ) ( ) 9. Self test ( ) ( ) ( )

3. Power Cord - verify proper insulation

( ) ( ) ( ) 10. Lamp check (hour meter) ( ) ( ) ( )
and integrity

4. Strain Relief - verify physical integrity at

( ) ( ) ( )
both ends of line cord
5. Fittings/ Connectors - check all
( ) ( ) ( )
fittings/connectors

6. Controls/Switches - verify proper

( ) ( ) ( )
operation of controls

7. Indicators/ Displays - verify proper ( ) ( ) ( )

illumination and operation
Preventive Maintenance Task

PART 5 PREVENTIVE MAINTENANCE TASKS

Tick ( √ ) where appropriate

DONE NOT DONE ** NA DONE NOT DONE ** NA
1. Clean the Exterior 3. Check and clean exhaust fan
( ) ( ) ( ) ( ) ( ) ( )

2. Inspect/ Clean Interior of unit 4. Clean the optics

( ) ( ) ( ) ( ) ( ) ( )

Notes:
*For all parts, NA is defined as NOT APPLICABLE
**If you have ticked 'NOT DONE', then justify in Part 8
***Choose whichever applicable
Quantitative Task
PART 6 QUANTITATIVE TASKS
Tick ( √ ) where appropriate
Units / Set Measured
Description Limit/Tolerance PASS FAIL NA
UOM Values Values

1 Run QC Test ( ) ( ) ( )

2 Absorbance accuracy check ( ) ( ) ( )

3 Wavelength accuracy check ( ) ( ) ( )

4 Stray light check ( ) ( ) ( )

Familiarization use of Wavelength Calibrator
and Electrical Safety Test

PART 7 ELECTRICAL SAFETY TEST

ELECTRICAL SAFETY TEST, (attach report)

(In accordance to IEC 61010)

PASS FAIL NA
ISE ANALYZER
Purpose of ISE Analyzer
• ISE (Ion Selective Electrodes) analyzers measure electrolyte levels in
the human body to detect metabolic imbalances and measure renal
and cardiac function. The electrolytes measured include sodium
(Na+), potassium (K+), chloride (Cl-) and bicarbonate (HCO3- or CO2)
• ISE analyzers are used in hospital and reference laboratories, and also
point of care settings.
• Most electrolyte analyzers use blood plasma, serum, or urine
samples; some analyzers can use whole blood (for faster turnaround
time) and cerebrospinal fluid (CSF)
 Terminology and result interpretation
Typical
Reasons for
Reference
Analyte Description Increased and
Intervals for
Decreased Values
Healthy Adults
Sodium (Na+) Major extracellular cation responsible 136-145 mmol/L ↑Dehydration, Cushing's syndrome,
for maintaining fluid balance in diabetes insipidus
circulation. Blood levels are controlled
through excretion and resorption by the ↓GI loss (diarrhea and vomiting),
kidneys Addison’s disease, renal disease
Potassium (K+) Major intracellular cation responsible for 3.5-5.1 mmol/L ↑Shock, circulatory failure, renal
muscle contraction and maintenance of disease
normal heart rate
↓GI loss (vomiting and diarrhea),
diuretic use, some cancers
 Typical
Reasons for
Reference
Analyte Description Increased and
Intervals for
Decreased Values
Healthy Adults
Chloride (Cl-) Major extracellular anion; changes in 98-107 mmol/L ↑Dehydration
concentration typically mirror sodium
concentrations ↓Low blood sodium,
vomiting

Calcium (Ca2+) Mineral required for bone formation 8.6-10.3 mg/dL ↑Hyperparathyroidism,
and for blood clotting and important in 2.15-2.50 mmol/L some cancers, excess
nerve and muscle function often vitamin D intake
measured as a screening test since it is
usually tightly controlled and held in a ↓Hypoparathyroidism,
very narrow concentration range. vitamin D deficiency,
Abnormalities can signal a wide range chronic kidney disease,
of metabolic problems pancreatitis
 Typical
Reasons for
Reference
Analyte Description Increased and
Intervals for
Decreased Values
Healthy Adults
pH Acid /base indicator in blood pH 7.35-7.45 ↑Increased acid production within the
body, consumption of substances that
are metabolized to acids, decreased
acid excretion, increased excretion of
base

↓Prolonged vomiting or
severe dehydration, administration or
consumption of base, hyperventilation

TCO2 Diagnosis, monitoring, and treatment of 23-27 mmol/L ↑Vomiting, breathing disorder,
numerous potentially serious disorders (arterial) Cushing Syndrome
associated with changes in body acid-base 24-29 mmol/L
balance. (venous) ↓Addison disease, ketoacidosis,
kidney disease, metabolic acidosis.
Department Involved
• Molecular Biology
• Biochemistry
• Physics
• Material Science
• Chemistry
Types of Samples
• Blood (whole blood, serum and plasma)
Parameter for ISE Analyzer
• Sodium (Na+)
• Potassium (K+)
• Chloride (Cl-)
• Calcium (Ca2+)
• pH
• Total TCO2
Calibration of ISE Analyzer
2 types of calibration

1. Automatic Calibration
• Automatically calculate slope and intercept value
• Calibrator parameter values/batch number need to be key in

2. Manual Calibration
• Slope value, intercept and calibration factor value need to be key in
Procedure / Protocol
• The mixture aspiration roller pump pulls reference solution from the
bottle, past the reference electrode where measurements are taken,
and then out to waste.
• ISE buffer is delivered to the sample pot by the ISE buffer syringe
• Sample is aspirated by the sample probe and dispensed into the
sample pot where the sample and buffer are mixed.
• The mixture aspiration roller pump draws the sample/buffer mixture
through the flow cell where measurements are taken.
• Excess fluid in the pot is pulled through the bypass tubing by the
mixture aspiration roller pump and sent to waste
Procedure / Protocol
• After a sample is processed, the ISE syringe sends ISE buffer to the
sample pot.
• The ISE buffer is pulled through the flow cell by the mixture aspiration
rolling pump to rinse the flow cell after each sample.
• After each sample is processed, the mid standard roller pumps pulls
mid standard from the bottle and delivers it to the pot.
• The mixture aspiration roller pump draws the mid standard solution
through the flow cell where measurements are taken.
• Excess fluid in the pot is pulled through the bypass tubing by the
mixture aspiration roller pump and sent to waste.
General design of ISE Analyzer
Types of ISE Analyzer
• Stand alone ISE Analyzer

• Built-in ISE Analyzer

 Types of ISE Analyzer components
• Washing block • Aspirating pump • Liquid outlet valve
• Aspirating tube • Lactic acid pump • Gas exhaust valve
• Sample probe • Pressure sensor • Reagent pack
• Positioner • Anti overflow chamber • Waste bottle
• Electrode assembly • Reaction chamber
• Outgoing sample tube • Lactic acid tube
• Liquid distribution valve • Gas tube to pressure
• Standard tube A&B sensor
• Liquid outlet tube • Waste tube
System operational characteristics
• Electrolyte analyzer have use ion selective electrode method to
achieve precise measurement of the testing.
• The apparatus are six electrodes: sodium, potassium and chlorine,
calcium ions, lithium and CST electrode.
• Each electrode has a ion selective film, will be measured and samples
corresponding ion responses, the membrane is a ion exchanger, and
ionic charge reaction and change the membrane potential, it can
detect the liquid, samples and membrane potential between.
• Film on both sides of the two electric potential tested value will
produce the current, samples, reference electrode, reference
electrode liquid form "loop" side, membrane, internal electrodes
liquid, internal electrodes are the other side.
• Internal electrodes fluid and sample the difference between the
concentration of the ions will work on both sides of the film electrode
in create electrochemical voltage, through high voltage of the
conductance of the internal electrodes to lead to the amplifier,
reference electrode also LED to the location of the amplifier.
• Through the test a known precisely the concentration of the ions
standard solution for calibration curve, and the test sample the
concentration of the ions.
Reagent, calibrator and quality control
• Filling solution (Na+, K+, Cl-, Ca2+, pH, TCO2)
• Solution AB Calibrator
• Reagent (Solution A&B)
Performance and Maintenance of ISE
Analyzer
Daily Maintenance
• Wash aspirating needle with 90% alcohol after daily use

Weekly Maintenance
• Check and refill the filling solution if it is less than 2/3 of total volume
• Check and clean salty crystal on electrode
• Check sample aspiration volume is correct
• Run cleaning program in service menu
• Check electrode voltage
Monthly Calibration
• Check tubing system and make sure there is no leakage and blockage
• Check internal electrode for any flaking or stain
• Check electrode voltage for the minimum voltage of 20mV
• If it is lower than 20mV, change the filling solution or reference membrane
• Run calibration if slope are low

Yearly maintenance
• Change reference electrode’s membrane
• Change peristaltic pump pipe
• Wash measuring chamber of TC02
• Use specific PPM Kit
 Qualitative tasks
PART 4 QUALITATIVE TASKS

Tick (  ) where appropriate

PASS FAIL NA PASS FAIL NA
1. Chassis - verify physical integrity, and 8. Sampler, Sensor, Valve and Reagent
( ) ( ) ( ) ( ) ( ) ( )
condition Modules - verify physical integrity

2. Power Cord / AC plug - verify physical 9. Motor/Pump - verify physical integrity &
( ) ( ) ( ) ( ) ( ) ( )
integrity proper operation
10. Indicators/ Displays - verify proper
3. Mount / Fasterners - verify integrity ( ) ( ) ( ) ( ) ( ) ( )
illumination and operation

4. Circuit Breaker / Fuses - verify integrity and

( ) ( ) ( ) 11. Printer Assy - verify operation ( ) ( ) ( )
rating

5. Strain Relief - verify physical integrity at

( ) ( ) ( ) 12. Others _____________________ ( ) ( ) ( )
both ends of line cord

6. Fittings / Connectors - check all

( ) ( ) ( )
connections are leak free

7. Controls / Keypad - verify proper operation

( ) ( ) ( )
of controls
 Preventive maintenance task

PART 5 PREVENTIVE MAINTENANCE TASKS

Tick (  ) where appropriate

DONE NOT DONE ** NA DONE NOT DONE ** NA
5. Perform following tests:-
1. Clean the Exterior ( ) ( ) ( )
a) Pump Test ( ) ( ) ( )
2. Inspect / Clean Interior of Unit ( ) ( ) ( ) b) Valve Test ( ) ( ) ( )
c) Sampler Switches ( ) ( ) ( )
3. Clean Fluid Path with Daily Cleaner ( ) ( ) ( ) d) Reagent Reader ( ) ( ) ( )
e) Fluid Flow Test ( ) ( ) ( )
4. Check / Replace*** f) Bubble Detector Test ( ) ( ) ( )
a) Pump Tubing ( ) ( ) ( ) g) Pump Calibration Test ( ) ( ) ( )
b) Probe Wiper ( ) ( ) ( )
c) Reagent Module ( ) ( ) ( )
d) Printer Paper ( ) ( ) ( ) Notes:
e) Sensor (specify): ________________ *For all parts, NA defined as NOT APPLICABLE
**If you have ticked 'NOT DONE', then input relevant remarks in Part 8
***Choose whichever applicable. Please indicate in Part 8 for any part replaced
Quantitative task
PART 6 QUANTITATIVE TASKS

Tick (  ) where appropriate

Set Measured
Description UOM Limit/Tolerance PASS FAIL NA
Values Values

1 Sensor Status

a) Calibrant A

Na+ mV 100 - 150 ( ) ( ) ( )

K+ mV 100 - 150 ( ) ( ) ( )

Cl- mV 50 - 100 ( ) ( ) ( )

b) Calibrant B

Na+ mV 80 - 130 ( ) ( ) ( )

K+ mV 120 - 170 ( ) ( ) ( )

Cl- mV 70 - 120 ( ) ( ) ( )

c) Slope

Na+ mV/decade 52 - 64 ( ) ( ) ( )

K+ mV/decade 52 - 64 ( ) ( ) ( )

Cl- mV/decade 40 - 55 ( ) ( ) ( )

2 Run QC Test and attach results ( ) ( ) ( )

Familiarization to the Calibration, mean, SD
and CV
Calibration

• Calibration is a comparison between measurements – one of known

magnitude or correctness made or set with one device and another
measurement made in as similar a way as possible with a second
device
• Including the process of adjusting the output or indication on a
measurement instrument to agree with value of the applied standard,
within a specified accuracy
• Calibration can be called for:
• with a new instrument
• when a specified time period is elapsed
• when a specified usage (operating hours) has elapsed
• when an instrument has had a shock or vibration which potentially may have
put it out of calibration
• sudden changes in weather
• whenever observations appear questionable
Mean
• In statistics, mean has two related meanings:
• the arithmetic mean.
• the expected value of a random variable, which is also called the population
mean.
• Mean is the sum of the values divided by the number of values
Standard Deviation (SD)

• The length of a distribution curve defines the variance of the variable.

The most common measure of variance is standard deviation (SD or s).
• Standard Deviation can be calculated by the formula:
Coefficient of Variation (CV)

• CV% is a normalized measure of dispersion of a probability

distribution. It is defined as the ratio of the standard deviation to the
mean and expressed as a percentage:
Familiarization use of QC and Electrical Safety

PART 7 ELECTRICAL SAFETY TEST

ELECTRICAL SAFETY TEST, (attach report)

PASS FAIL NA
Familiarization use of QC and Electrical Safety
Quality Control (QC)

• Quality control is designed to detect, reduce, and correct deficiencies in a

laboratory's internal analytical process prior to the release of patient
results, in order to improve the quality of the results reported by the
laboratory.
• Quality control is a measure of precision, or how well the measurement
system reproduces the same result over time and under varying operating
conditions.
• Laboratory quality control material is usually run at the beginning of each
shift, after an instrument is serviced, when reagent lots are changed, after
calibration, and whenever patient results seem inappropriate.
• Quality control material should approximate the same matrix as
patient specimens, taking into account properties such as viscosity,
turbidity, composition, and color.
• It should be stable for long periods of time, and available in large
enough quantities for a single batch to last at least one year
• Interpretation of quality control data involves both graphical and
statistical methods
• Quality control data is most easily visualized using a Levey-Jennings
chart
• Controls are run:

• To periodically monitor the performance of the testing system - see site

procedure/handout for the control policy followed.
• For any test whenever reagents for that test are replaced.
• When troubleshooting an instrument problem or following maintenance
procedures.
Strict adherence to laboratory protocols is vital to prevent erroneous
results caused by:

Pre-analytical variables (sample collection and processing)

• Specimen integrity is critical to accurate coagulation results. Avoidance of

tissue trauma and hemolysis is essential to minimize activation of the
coagulation process prior to analysis.

Analytical variables (reagents, instrumentation, equipment, technique)

• Due to the many variables that may cause instrument or reagent related
errors, it is imperative that commercial control plasmas are used to
monitor the performance of the testing system. Reagent and/or control
problems can arise if manufacturer recommendations for reconstitution
and handling are not followed. The operator must be aware of instrument
linearity limits.
Post-analytical variables (reporting results)

• Patient values should be correlated with clinical information (if

available) before reporting. All laboratories establish critical/panic
values that must be verified and/or action taken per lab policy.
Questionable results must be investigated before reporting, e.g.,
inconsistent values or results that fail a delta check with previous
results.
Hematology Analyzer
Purpose of Hematology Analyzer
• Hematology analyzers are computerized, highly specialized and
automated machines that count the number of different kinds of
white and red blood cells in a blood sample.
• The results they provide are collectively known as complete blood
counts (CBCs) or complete blood count with differentiation of cells
(CBCs with diff).
• The CBC can be used to detect a wide range of pathological states
including anemia, infection and hematological malignancy, as well as
for the monitoring of cancer patients undergoing chemotherapy
Terminology and result interpretation
• CBC /FBC = Complete Blood Count / Full Blood Count
• PCT = Platelet Crit
• MPV = Mean Platelet Volume
• PDW = Platelet Distribution Width
• Automatic sampling = Sample is automatically stirred, diluted and
measured
• Two sampling modes = Close tube and open tube sampling
• Emergency sampling count = Highest priority of sampling
• Pre-diluted blood = Blood diluted by solution
Substance What It Is? Reference Ranges Low Number May High Number May
Mean Mean
White blood cell count Measures the total 4,500-10,000 cells/mcL Autoimmune diseases, Infection,
(WBC) number of white blood immunosuppression, inflammation,
cells, which defend the bone marrow failure, leukemia, intense
body against infection; chemotherapy, viral exercise, stress,
there are several infections corticosteroids
different types of
white blood cells:
lymphocytes,
monocytes,
neutrophils,
eosinophils, and
basophils

Red blood cell count Measures the number Male: 4.7-6.1 Iron, vitamin B12, or Dehydration, renal
(RBC) of red blood cells, million/mcL folate deficiency; bone problems, pulmonary
which pick up oxygen marrow damage; disease, congenital
from the blood and Female: 4.2-5.4 leukemia or heart disease,
deliver it to tissues million/mcL lymphoma; acute or polycythemia vera
throughout the body chronic blood loss; red
blood cell hemolysis
Substance What It Is? Reference Ranges Low Number May High Number May
Mean Mean
Lymphocytes, absolute Measures the number 800-5,000 cells/mcL Immunosuppression, Viral infections,
(LY, abs) or percentage or percentage of (abs) HIV-AIDS, bone leukemia, lymphoma
(LY, %) lymphocytes, which 18-45 (%) marrow failure,
are white blood cells chemotherapy
that include B-cells, T-
cells, and natural killer
cells
Monocytes, absolute Measures the number 400-1,000 cells/mcL Immunosuppression, Chronic infections,
(MO, abs) or or percentage of (abs) bone marrow failure, autoimmune diseases,
percentage (MO, %) monocytes, which are 1-10 (%) chemotherapy leukemia
white blood cells that
move out of the
circulating blood and
into the tissues, where
they mature into
macrophages
Substance What It Is? Reference Ranges Low Number May High Number May
Mean Mean
Granulocytes, absolute Measures the number 1,800-8,300 cells/mcL Immunosuppression, Infection,
(GR, abs) or or percentage of white (abs) bone marrow failure, inflammation,
percentage (GR, %) blood cells with 45-75 (%) chemotherapy leukemia, intense
granules in their exercise, stress,
cytoplasm and two or corticosteroids
more lobes in their
nuclei; an inclusive
term for neutrophils,
basophils, and
eosinophils, although
neutrophils are by far
the most abundant
Neutrophils, absolute Measures the number 1,800-8,300 cells/mcL Immunosuppression, Infection,
(NE, abs) or or percentage of (abs) bone marrow failure, inflammation,
percentage (NE, %) neutrophils, which are 45-75 (%) chemotherapy leukemia, intense
normally the most exercise, stress,
abundant circulating corticosteroids
white blood cells and
respond quickly to
infection
Substance What It Is? Reference Ranges Low Number May High Number May
Mean Mean
Eosinophils, absolute Measures the number 0-800 cells/mcL (abs) Generally not a Parasitic infections
(EOS, abs) or or percentage of 0-7 (%) concern
percentage (EOS, %) eosinophils, which
combat parasitic
infections and are
involved in asthma or
allergy responses

Basophils, absolute Measures the number 0-100 cells/mcL (abs) Generally not a Active allergic
(BAS, abs) or or pecentage of 0-0.5 (%) concern response
percentage (BAS, %) basophils, which are
involved in allergy
responses

Reticulocytes Measures the 0.5-2.0 % Generally not a Anemia, recent blood

(optional in percentage of concern loss, red blood cell
Hematology Analyzer) circulating immature hemolysis
red blood cells
Substance What It Is? Reference Ranges Low Number May High Number May
Mean Mean

Hemoglobin (HgB) Oxygen-carrying Male: 13.8-17.2 g/dL Iron, vitamin B12, or Dehydration, renal
pigment in red blood Female: 12.1-15.1 g/dL folate deficiency; bone problems, pulmonary
cells marrow damage; disease, congenital
leukemia or heart disease,
lymphoma; acute or polycythemia vera
chronic blood loss; red
blood cell hemolysis

Hematocrit (HCT) The percentage of red Male: 40.7%-50.3% Iron, vitamin B12, or Dehydration, renal
blood cells Female: 36.1%-44.3% folate deficiency; bone problems, pulmonary
marrow damage; disease, congenital
leukemia or heart disease,
lymphoma; acute or polycythemia vera
chronic blood loss; red
blood cell hemolysis
Substance What It Is? Reference Ranges Low Number May High Number May
Mean Mean
Measures the number 150-400 Bone marrow failure, Leukemia,
Platelet count (PLT) of platelets, which are Thousand/mcL chemotherapy, viral myeloproliferative
important for blood infections, lupus, disorders (which cause
clotting pernicious anemia blood cells to grow
(due to vitamin B12 abnormally in bone
deficiency), leukemia marrow), inflammatory
or lymphoma, conditions
sequestration in the
spleen, certain
medications

Mean corpuscular Average size of red 80-95 fL Iron deficiency Vitamin B12 or folate
volume (MCV) blood cells deficiency

Mean corpuscular The amount of 23-31 pg Iron deficiency Vitamin B12 or folate
hemoglobin (MCH) hemoglobin per red deficiency
blood cell
Substance What It Is? Reference Ranges Low Number May High Number May
Mean Mean
Mean corpuscular The average 32-36 g/dL Iron deficiency Sickle cell disease,
hemoglobin concentration of hereditary
concentration (MCHC) hemoglobin in a given spherocytosis
volume of red blood
cells

Red cell distribution A measurement of the 11-15% Generally not a Iron deficiency, vitamin
width (RDW) variation in red blood concern B12 or folate
cell size deficiency, recent
blood loss
Department Involved
• Hematology
• Blood Bank
• Health Clinics
• Emergency Department
Types of Samples
• Whole blood
• Pre diluted blood
Parameter in Hematology Analyzer
• Whole WBC (white blood cell) (Analysis principle: DC detection
method) WBC count in 1 µL of whole blood with its component:
• Neutrophil (NE)
• Eosinophil (EO)
• Lymphocyte (LY)
• Monocyte (MO)
• Basophil (BA)
• RBC (red blood cell) (Analysis principle: DC detection method) RBC
count in 1 µL of whole blood
• PLT (platelet)
Parameter in Hematology Analyzer
• HGB (Hemoglobin) (Analysis principle: Non-Cyanide hemoglobin
analysis method) Volume (gram) of hemoglobin in 1 dL of whole
blood
• HCT (Hematocrit value) (Analysis principle: RBC pulse height
detection method) Ratio (%) of whole RBC volume in whole blood
• MCV (Mean RBC volume) Mean RBC volume (fL) in whole blood,
which is calculated by Hct/RBC.
• MCH (Mean RBC hemoglobin) Mean hemoglobin volume (pg) per
RBC, which is calculated by Hgb/RBC.
• MCHC (Mean RBC hemoglobin concentration) Mean hemoglobin
concentration (g/dL), which is calculated by Hgb/Hct
Calibration of Hematology Analyzer
CBC parameters

Calibrator must be used only 3 days after opening and stored at 2 – 8oC

WBC 5 parts differential parameters

Blood sample of different healthy person within 8 hours after collection

and measurement can be done by using Hematology Analyzer or
Hematocrit Centrifuge or Spectrophotometer
Procedure / Protocol

•Let see the video!

General design of Hematology Analyzer
Detection and measurement of RBC, WBC and PLT
• Impedance Counter for WBC,RBC,PLT (a.k.a The Coulter Principle)

Detection and measurement of WBC component

• Flow Cytometry

Manipulation of measured value (Calculation Method)

Impedance Counter for WBC,RBC,PLT (a.k.a
The Coulter Principle)

Cell count detector

Electrode Electrode
B
A
C

Blood Aperture
Impedance Counter for WBC,RBC,PLT

80mm for RBC, PLT

100mm for WBC
Flow Cytometry
Calculation Method
Types of Hematology Analyzer
• Low Volume Hematology Analyzer
Throughput: 60 samples per hour
• Mid Volume Hematology Analyzer
Throughput: 100 samples per hour
• High Volume Hematology Analyzer
Throughput: 100 samples + 140 slides prepared per hour
• Ultra High Volume Hematology Analyzer
Throughput: 300 samples + 140 slides prepared per hour
Types of Hematology Analyzer components
• Sub Bath Unit • Table Unit
• CBC Measuring Unit • Display Unit
• Laser Optics Unit • Master Board
• Complex Pump Unit • Slave CBC Board
• Sample Pump Unit • Slave MS Board
• Sheath Pump Unit
• Closed Sampler Unit
• Open Sampler Unit
Reagent, calibrator and quality control

Reagent
• Diluent: An isotonic saline solution
used to dilute whole blood specimens
and to rinse the fluidic system
between measuring procedures.
• Lysing reagent: Used to prepare blood
hemolysate for WBC and HGB
measurement.
• Cleaner/Detergent: Used to perform
cleaning process of the fluidics.
Calibrator
QC solution
• High
• Normal
• Low
System operational characteristics

2 basic systems :

• Impedance Counter for RBC, WBC and PLT

• Optical Counter (Flow Cytometry) for WBC Classification

The Coulter Principle

• Particles moving in an electric field cause measurable disturbances in that field.

• The magnitudes of these disturbances are proportional to the size of the particles
in the field.
• The particles should be suspended in a conducting liquid.
• The electrical field should be physically constricted so that the movement of
particles in the field causes detectable changes in the current.
• The particles should be dilute enough so that only one at a time passes through
the physical constriction, preventing an artifact known as coincidence.
• Coulter Principle can be implemented in a variety of designs, including an
aperture format and a flow cell format
The Coulter Principle
(Aperture Format)
RBC/WBC/PLT
Count Processing
Area
 Optical Counter (Flow cytometry)
• The hematology analyzer uses the light scatter technique to differentiate
WBC into neutrophil, lymphocyte, monocyte, eosinophil and basophil
counts
• The diluted blood sample is injected into the flow cell.
• Blood cells pass through the sensing zone in a single file.
• A laser beam through the sensing zone is scattered by the passing cells
and scattered light is detected
• The angle and intensity of scattered light depend on the volume and
characteristics of the cell.
• From this, WBC can be differentiated into 5 parts
Optical Counter
(Flow cytometry)
Flow Cytometry

Flow-cell
Flow Cytometry

Sample
Flow
Flow Cytometry

Sheath Flow
(around sample)
 Flow Cytometry

Because of a laminar
flow, sample and
sheath liquid are not
mixed.
Flow Cytometry

Laser Unit
 Flow Cytometry

While each cell is

passing through single
laser beam, the
scattering light is
generated.
 Flow Cytometry

Forward Small
angle Scattering
light (FSS)
 Flow Cytometry

Forward Large
angle Scattering
light (FL)
Flow Cytometry

SiDe angle
Scattering light
(SDS)
 Optical Counter for Differential

FS means Size
Y-axis : Forward small angel
Information of
each cell scattering light Intensity
1st X-axis : Forward large angel
FL complexity
scattering light Intensity

2st X-axis : Orthogonal

SD granurarity
scattering light Intensity
 Basic Graph for WBC Characteristic

Size ( FS )

Granularity ( SD )

Complexity ( FL )
 BASIC WBC CLASSIFICATION ALGORITHM

Mo+Ba Ne
Ne+Eo
Forword small angle

Forword small angle

Forword small angle

Eo

Ly
Ba
Mo

Orthogonal angle Forward large angle Orthogonal angle

Lymphocyte
Monocyte
Neutrophil
Eosinophil
Basophil
Performance and Maintenance of
Hematology Analyzer
 Qualitative tasks
PART 4 QUALITATIVE TASKS

Tick (  ) where appropriate

PASS FAIL NA PASS FAIL NA
1. Chassis - verify physical integrity, 11. Controller Board- Verify physical
( ) ( ) ( ) ( ) ( ) ( )
cleanliness and condition. Integrity

12. Plunger & Syringe motor drive -

2. Mount/ Fasteners - verify physical integrity. ( ) ( ) ( ) ( ) ( ) ( )
Verify physical Integrity
13. Vacuum & Pressure Pump - Verify
3. Cables - verify integrity ( ) ( ) ( ) ( ) ( ) ( )
physical integrity

14. Solenoid valve - Verify physical

4. AC Plug - verify integrity ( ) ( ) ( ) ( ) ( ) ( )
integrity
5. Power Cord - verify proper insulation and
( ) ( ) ( ) 15. Printer - verify operation ( ) ( ) ( )
integrity

6. Strain Relief - verify physical integrity at

( ) ( ) ( )
both ends of line cord

7. Fittings/ Connectors - check all ( ) ( ) ( )

fittings/connectors.

8. Controls/Switches - verify
( ) ( ) ( )
proper operation of controls.
9. Indicators/ Displays - verify proper
( ) ( ) ( )
illumination and operation
10. Sample probe - verify integrity ( ) ( ) ( )
 Preventive maintenance task

PART 5 PREVENTIVE MAINTENANCE TASKS

Tick (  ) where appropriate

DONE NOT DONE ** NA DONE NOT DONE ** NA
1. Clean the Exterior ( ) ( ) ( ) 8. Run Autoclean ( ) ( ) ( )
2. Inspect/ Clean Interior of unit ( ) ( ) ( ) 9. Run daily shutdown ( ) ( ) ( )
3. Replace PPM kits (Refer to Part 8.) ( ) ( ) ( ) 10. Sample asprition Probe- Cleaning ( ) ( ) ( )
4. Power On Self Test (POST) ( ) ( ) ( ) 11. Pinch valve & Lyse pump tubing - ( ) ( ) ( )
5. Fan Filter - Cleaning/replace if necessary ( ) ( ) ( ) clean and replace if necessary
6. Aperture Plates- cleaning ( ) ( ) ( ) 12. Close sample holder - cleaning ( ) ( ) ( )
7. Diluent Syringe, sample syringe, sample ( ) ( ) ( )
aspiration probe- cleaning

Notes:
*For all parts, NA is defined as NOT APPLICABLE
**If you have ticked 'NOT DONE', then justify in Part 8
 Quantitative task

PART 6 QUANTITATIVE TASKS

Tick (  ) where appropriate
Units / Set Measured
Description Limit/Tolerance PASS FAIL NA
UOM Values Values
1 Background Check ( ) ( ) ( )

1.1 WBC 10³/µ 0 < 0.05 ( ) ( ) ( )

1.2 RBC 10/µ 0 < 0.05 ( ) ( ) ( )

1.3 HGB g/µ 0 < 0.02 ( ) ( ) ( )

1.4 PLT 10³/µ 0 < 10.0 ( ) ( ) ( )

2 Count time

2.1 WBC s 5.00 4-6 ( ) ( ) ( )

2.2 RBC s 7.00 6 -8 ( ) ( ) ( )

QC Check- Refer to Calibrator data sheet

3 ( ) ( ) ( )
attached
Familiarization to the Calibration, mean, SD
and CV
Calibration

• Calibration is a comparison between measurements – one of known

magnitude or correctness made or set with one device and another
measurement made in as similar a way as possible with a second
device
• Including the process of adjusting the output or indication on a
measurement instrument to agree with value of the applied standard,
within a specified accuracy
• Calibration can be called for:
• with a new instrument
• when a specified time period is elapsed
• when a specified usage (operating hours) has elapsed
• when an instrument has had a shock or vibration which potentially may have
put it out of calibration
• sudden changes in weather
• whenever observations appear questionable
Mean
• In statistics, mean has two related meanings:
• the arithmetic mean.
• the expected value of a random variable, which is also called the population
mean.
• Mean is the sum of the values divided by the number of values
Standard Deviation (SD)

• The length of a distribution curve defines the variance of the variable.

The most common measure of variance is standard deviation (SD or s).
• Standard Deviation can be calculated by the formula:
Coefficient of Variation (CV)

• CV% is a normalized measure of dispersion of a probability

distribution. It is defined as the ratio of the standard deviation to the
mean and expressed as a percentage:
Familiarization use of QC and Electrical Safety

PART 7 ELECTRICAL SAFETY TEST

ELECTRICAL SAFETY TEST, (attach report)

PASS FAIL NA
Familiarization use of QC and Electrical Safety
Quality Control (QC)

• Quality control is designed to detect, reduce, and correct deficiencies in a

laboratory's internal analytical process prior to the release of patient
results, in order to improve the quality of the results reported by the
laboratory.
• Quality control is a measure of precision, or how well the measurement
system reproduces the same result over time and under varying operating
conditions.
• Laboratory quality control material is usually run at the beginning of each
shift, after an instrument is serviced, when reagent lots are changed, after
calibration, and whenever patient results seem inappropriate.
• Quality control material should approximate the same matrix as
patient specimens, taking into account properties such as viscosity,
turbidity, composition, and color.
• It should be stable for long periods of time, and available in large
enough quantities for a single batch to last at least one year
• Interpretation of quality control data involves both graphical and
statistical methods
• Quality control data is most easily visualized using a Levey-Jennings
chart
• Controls are run:

• To periodically monitor the performance of the testing system - see site

procedure/handout for the control policy followed.
• For any test whenever reagents for that test are replaced.
• When troubleshooting an instrument problem or following maintenance
procedures.
Strict adherence to laboratory protocols is vital to prevent erroneous
results caused by:

Pre-analytical variables (sample collection and processing)

• Specimen integrity is critical to accurate coagulation results. Avoidance of

tissue trauma and hemolysis is essential to minimize activation of the
coagulation process prior to analysis.

Analytical variables (reagents, instrumentation, equipment, technique)

• Due to the many variables that may cause instrument or reagent related
errors, it is imperative that commercial control plasmas are used to
monitor the performance of the testing system. Reagent and/or control
problems can arise if manufacturer recommendations for reconstitution
and handling are not followed. The operator must be aware of instrument
linearity limits.
Post-analytical variables (reporting results)

• Patient values should be correlated with clinical information (if

available) before reporting. All laboratories establish critical/panic
values that must be verified and/or action taken per lab policy.
Questionable results must be investigated before reporting, e.g.,
inconsistent values or results that fail a delta check with previous
results.