Addis Ababa University

Course Name: Biomedical Instrumentation and Processing

Course Code: BMED 4241
Instructor: Fikadu M.
Chapter 6: Medical Instruments and Devices
 Hematology Analyzer

03-Jun-18 By Fikadu Mulugeta 1

 Hematology
• From a Greek word “Haemalogos”…Haeme refers to blood and logos refers to study
• Hence it’s the science of blood
• It is concerned with the study of the cause, prognosis, treatment, and prevention of
diseases related to blood
• It includes problems with the:
• Red blood cells, white blood cells and platelets,
• Blood vessels and bone marrow,
• Lymph nodes, spleen, and
• Proteins involved in bleeding and clotting (hemostasis and thrombosis)
• Often, hematology tests are done by taking a sample of a whole blood cell
 Whole blood
Blood collection
• Blood collected in a blood tube is venous blood
• Using needle and syringe
• The tube may contain an anti-coagulant to prevent the blood from
clotting
 Whole blood
Plasma & Serum

without anticoagulant

With anti-coagulant

plasma serum
 Whole Blood Compartment
• After whole blood is centrifuged, two main compartments can be
observed; plasma and blood cell.
• Whole blood = plasma +blood cell
• Blood cell = red blood cell(RBC)+ white blood cell(WBC)+ platelets
• RBC(erythrocytes)
• WBC(leukocytes)
• Platelets(thrombocytes)
 …whole blood…
• Plasma :
o Straw (pale yelolow) colored fluid portion of blood mixed with an anticoagulant to
prevent fibrin formation
o Comprises 55% of the Blood
o 92% of the Plasma is Water and 8% Proteins & Electrolytes
• Buffy Coat
• Thin whitish colored layer of cells between Plasma and Red Blood Cells
• Comprised of White Blood Cells (Leukocytes) and Platelets Plasma

• Red Blood Cells

• Reddish colored, bottom layer of cells
• Consists of Red Blood Cells Buffy Coat
(White Blood Cells
Blood Volume and Platelets)

o 7-8% of the body weight Red Blood Cells

o Adult male with 80kg: ~ 6 liters
o Adult female with 62kg: ~ 4 liters
o Newborns: ~ 300ml
Blood cell
 Cell maturation
• Red Blood Cells, White Blood Cells, and platelets are formed in the bone
marrow by a process known as Hematopoiesis
• Blood Cells begin as undifferentiated cells called Stem Cells
• Under instructions from the rest of the body, they begin to specialize and
mature
 Major functions of blood cell
• As cells mature, their shape and contents change so that they can be most efficient at
the functions they perform.
• Blood performs two major functions:
i. Transport of substances thru the body
• Oxygen and carbon dioxide
• Food molecules (glucose, lipids, amino acids)
• Ions (e.g., Na+, Ca2+, HCO3−)
• Wastes (e.g., urea)
• Hormones
• Heat
ii. Defense of the body against infections and other foreign materials
• All the WBCs participate in these defenses
 … functions of blood…
• In a normal blood sample, all of the white blood cells keep their nuclei

• The red cells, however, lose their nuclei and fill up with the special oxygen-carrying
molecule called hemoglobin

• Platelets participate in clotting of blood

WBC
RBC
 RBC (Erythrocytes)
• Red Blood Cells (erythrocytes) are primarily used to carry oxygen to organs and
tissues.
• Each individual has 20 -25 trillion RBCs
• They contain hemoglobin and have a limited life span of around 100 to 120 days
• Appearance:
• Usually appear round or oval, and are reddish color from the protein
hemoglobin
• Stain darker at the edges than in the middle reflecting their biconcave shape
Normal RBCs
Abnormal RBCs

Nucleated RBC (NRBC)

 …erythrocytes…

Decrease in size

Erythroblast (NRBC) loss of nucleus Reticulocyte loss reticulin RBC

• Reticulocyte count reflects the bone marrow’s capacity of producing new RBCs
• Anemia with low retic count: BM disease; Chemotherapy, radiation
 Hemoglobin
• It is an iron containing protein found within the RBC
• Its main function is to bind to and transport oxygen
• Values are expressed as grams (gm) per deciliter (dl) of whole blood
• Normal ranges for hemoglobin depend on the age and sex of the person
• It is detected by spectrophotometry principle
 RBC Indices
• RBC Indices are measurements that describe the size and hemoglobin content of RBCs
• They are used to help in the differential diagnosis of Anemia
Anemia:
• A condition characterized by abnormally low level of RBCs or Hemoglobin and/or
Hematocrit
– Hematocrit is the ratio of the volume of red blood cells to the total volume of blood
• Anemia can be caused by loss of blood or defective RBC production
• It is the most common disorder of the blood
• can be classified according to the size of the red blood cell:
– Decreased (microcytic), normal (normocytic) or enlarged (macrocytic or megaloblastic)
• Most common type of anemia is iron deficiency, which is most often microcytic
• In microcytic/hypochromic Anemia:
– RBCs are smaller than normal and have an increased zone of central
pallor (less hemoglobin in each RBC)
 Hematocrit
• The hematocrit is the proportion, by volume; volume of the blood that consists of
Red Blood Cells
• The hematocrit (hct)is expressed as a percentage
• The hematocrit reflects both the number of red cells and their volume (Mean Cell
Volume). If the size of the red cell decreases, so will the hematocrit and vice versa

• The normal ranges for hematocrit are dependent on age and, after adolescence, the
sex of the individual
 Cont’d
• Hct represents the proportion of whole blood that is occupied by red cells
• It can be calculated by the machine as;
– HCT= (RBC x MCV)/10
 RBC indices
• The RBC indices aid in defining the characteristics of the RBCs.
• The RBC Indices include the following:
– Mean Cell Volume (MCV)
– Mean Cell Hemoglobin (MCH)
– Mean Cell Hemoglobin Concentration (MCHC)
– Red Blood Cell Distribution Width (RDW)
Mean cell volume(MCV)
• Is average volume of red cells
• Determined from the Red Blood Cell size distribution
– MCV (fL) = Hct / RBC
• Impedance analyzers measure the MCV by averaging the amplitude of the
pulses created as the cells pass the aperture of the counter
 …MCV
• The instrument also "channelizes" the impulses, segregating them into
channels representing ranges of cell size.
• This data is then assembled into a cell size histogram.

•Normal range is 86 - 98 femtoliters

•Red cell populations with an MCV of:
• Above reference range are termed to be Macrocytic
• Below reference range are termed to be Microcytic
 Mean cell hemoglobin(MCH)
• It represents the absolute amount of hemoglobin in the average red cell in a sample
• It is calculated from the Hemoglobin and RBC results
(HGB / RBC)*10 = MCH
• Expressed in picograms (pg) per cell
• Normal range is 27 - 32 picograms
– In hematology, the terms Normochromic, Hypochromic and Hyperchromic refer to
the intensity of the color of Red Blood Cells
– This is primarily an indication of the concentration of hemoglobin in the Red
Blood Cell
– Low levels can indicate anemia
 Mean Cell Hemoglobin Concentration (MCHC)
Mean Cell Hemoglobin Concentration (MCHC):
• Indicates the hemoglobin concentration in the RBC mass
• Is calculated from the Hemoglobin, RBC and MCV values
• Expressed in percent grams per deciliter (g/dL)
• Normal value for MCHC is about 33%
• Calculation:
• The MCHC is calculated using the following formula:
(HGB / (MCV * RBC))*100 = MCHC
 Red Cell (Random) Distribution Width (RDW)
Red Cell Distribution Width (RDW):
• Is an index of the variation in cell volume within the red cell population
• The more variation in size, the higher the RDW value
• It’s expressed in percent
• Normal range is 11 – 15

Normal Abnormal
 White blood cell(WBC)
• Cells of the immune system involved in protecting the body against both infectious
disease and foreign invaders
• Produced and derived from multipotent cells in the bone marrow (hematopoietic stem
cells)
• The WBC number is often an indicator of disease, and its count is an important subset
of the complete blood count
• The normal white cell count is usually between 4 × 109 /L and 1.1 × 1010 /L
• Types of leukocytes
– By structure (granulocytes or agranulocytes)
– By cell lineage (myeloid cells or lymphoid cells)
– By their physical and functional characteristics (neutrophils, eosinophils, basophils,
lymphocytes, and monocytes)
 WBC types
Neutrophils, (1.5 -7.5 x 109 /L) Lymphocytes (1.0 - 4.5 x 109 /L )
• Mainly fight bacterial infections • Fight viral infections
• Usually the first cell type to respond • Primary cell type involved in immune
when infections occur response
• Elevated neutrophil count and – T Lymphocytes
percentage can indicate abnormality in – B Lymphocytes
the body • Plasma cells are the antibody producing
lymphocytes
• Lymphocytes form the memory of the
immune system
 …WBC types…
Eosinophils (0.0 - 0.4 x 109 /L )
Monocytes (0.0 - 0.8 x 109 /L )
• its granules contain basic proteins.
• Largest of all WBC`s
• Eosinophils enter inflammatory areas and
• Have a phagocytic role
have particular functions in:
• Respond to severe infection
(a) Allergic responses
(b) Defense against parasites
Basophils (0.0 - 0.15 x 109 /L )
• Associated with allergic reactions.
• Functions are not completely understood
 Differential WBC count
Automated or semi-automated hematology analyzers
• Give WBC count
• Provide either 3-part or full 5-part differential count
5-diff
 Platelets(PLT)
• Platelets are blood cells that help control bleeding
• When a blood vessel is damaged, platelets collect at the site of the
injury and temporarily repair the tear.
• Platelets then activate substances in plasma which form a clot and
allow the wound to heal.
 Normal Ranges

• Increased PLT is called thrombocytosis

• Decreased PLT is called thrombocytopenia or thrombopenia
Common causes of thrombocytosis
– Infection
– Inflammatory diseases
– After bleeding
– Iron deficiency
 Complete Blood Count
A Complete Blood Count (CBC) report includes information on three main
types of blood cells discussed above
1. White Blood Cells (WBC)
• WBC Differential*
– 3 Part Differential (Lymphocytes, Mid-Cells and Granulocytes)
– 5 differential

2. Red Blood Cells (RBC)

• RBC Indices
• Hemoglobin Blood Sample
• Hematocrit

3. Platelets (Plt)

*Note style of differential varies per type of analyzer used

Hematology Analyzer
 Hematology Analyzer

• Also known as complete blood • Basically there are two measurement

count(CBC) machine or full blood principles
count(FBC) machine – Impedance technology method
Primarily measures: – Flow cytometry technology method
– Hemoglobin concentration and • In either case, the whole blood can be
hematocrit counted
– RBC count and red cell indices
– WBC count
– PLT count
– WBC differential count
 1. Impedance Technology Method
• In impedance technology, the measurement is done by applying resistive
property of the blood
• Impedance measurements are carried out by monitoring the voltage required to
send a constant current through an aperture
• A cell or particle passing through an aperture impedes current flow and the
voltage must increase in order to maintain a constant current
• The voltage first rising above and then falling below a certain threshold level,
indicates that a blood cell or particle has passed through the sensing zone
• This technique is based on the principle that cells are poor conductors of
electricity
…impedance…
 …impedance…
• The number of cells or particles that pass through the aperture can be determined by
counting voltage pulses that occur during a measurement interval
• The volume of each cell can be determined by measuring the amplitude of the
corresponding pulse
• Change in impedance on the aperture is proportional to individual volume of cell
• The impedance transducer assembly performs the impedance measurement of WBCs,
RBCs and PLTs
• Using this method 3-diff of WBC also can be detected or measured
• Each pulse is amplified and compared to internal reference voltage that is relative to
the size of that cell
• The internal reference voltage is called threshold voltage
…impedance method…
Impedance Method - WBC count
 2. Flow cytometry
• Flow cytometry is a process in which individual cell or other biological
particles in a single file produced by a fluid stream are passed through a beam
of light.
• A sensor measures the loss or scattering of light, which indicates the physical
size or chemical characteristics of the cells or particles
• Flow Cytometry uses a Laser beam to intersect cells flowing through the flow
cell in single file
• When the beam intersects the cells, light scatters at different angles
• The System detects and measures the light scatter, which results in data about
the cells
…flow cytometry…
 Hydrodynamic focusing
• A delivery syringe injects the diluted sample through an injection tube
• Simultaneously, 9psi of pressure forces sheath fluid (diluent) into the assembly.
• The sheath converges on the sample in the cone, and forces it into a small stream
approximately 30 microns in diameter.
• Due to the characteristics of the fluid flow, the sample and sheath streams are
“laminar," (do not mix with one another).
• This overall principle is known as hydrodynamic focusing
• Because the sample stream is now very small, cells pass through the laser beam
in single file, allowing them to be processed one at a time
 Hydrodynamic focusing
Various angle of
scatter light

Sample stream
Focused laser
beam

Sample feed
Sheath stream
needle
 Fluidics
• A major disadvantage of whole blood measurement relative to electronic particle
counting is the high concentration of cells in whole blood
• Two or more cells in the sensing zone are detected as a single cell and result in a
counting error
• This problem is easily solved by controlled dilution
1. Diluent (Dilution):
– Acts as the diluent for WBCs, RBCs, PLTs, and HGB
– Maintains the stable cell volume of the red cells and platelets during counting and sizing
– Provides a conductive medium for impedance counting
– Provides acceptable background counts
– Makes ease of washes
 …fluidics….
2. Detergent:
– Provides an optically clear solution that is needed to obtain the zero reference
read during the HGB measurement cycle
– Provides proper meniscus formation in the WBC and RBC/PLT metering tubes
– Rinses the WBC counting chamber, the WBC metering tube, RBC/PLT counting
chamber, the RBC/PLT metering tube, and the HGB flow cell with minimal
bubble formation
– Provides an acceptable background count
3. Lyse:
– Rapidly lyses the RBCs
– Releases the hemoglobin from the RBC
– The Lyse permeates the WBC cell membrane causing the cytoplasm to diffuse
through the cell membrane which shrinks around the nuclear material - nucleus
and, when present, granules
– Provides an acceptable background count.
 RBC Interference
• Sometimes, RBCs interfere with the WBC count
• There are many more RBCs than WBCs.
– Need to remove (lyse) or make the RBCs invisible
– When all RBCs removed, WBC is accurate
• When RBCs resistant to lyses are present, they will not be removed and
counted as WBCs
 Coincidence Correction
• A phenomenon known as coincidence can affect cell counting
accuracy.
• Coincidence occurs when two or more blood cells pass through
the sensing zone at the same time.
• When coincidence occurs during impedance measurements, one
large voltage pulse results instead of two or more smaller ones,
causing a lower overall count of cells
• Because coincidence is a predictable occurrence, statistics can be
used to compute a correction factor
 Major components in typical hematology
analyzer
•There are many mechanical and electrical components found in hematology
analyzers.
Flow Panel Components:
Sample Aspiration Probe
–Aspirates the blood sample from an open VACUTAINER tube.
–Delivers the sample to the Pre-Mixing Cup for the first dilution process.
–Aspirates the diluted solution from the Pre-Mixing Cup and delivers the solution to
the RBC/PLT Mixing Chamber for the second dilution process.
Wash Block
–The Wash Block rinses the outside of the sample aspiration probe with diluents
–Excess diluents is routed to the waste container
 …components…
Start Switch
– Located behind the Sample Aspiration Probe
– Pressing the touch plate on the front cover initiates the start switch to start the selected
run cycle
Pre-Mix Cup
–Sample is dispensed into the cup of Diluent where it is bubble mixed
–This dilution is referred to as the pre-mix sample
–Some of the diluted solution are aspirated by the Sample Probe and delivered to the
Mixing Chamber in the RBC/PLT Transducer for the second dilution process.
–The remainder of the diluted sample is transferred to the Mixing Chamber in the WBC
Transducer using vacuum.
 …components…
Transducers
–Two Transducers reside on the flow panel:
• RBC/PLT Transducer
• WBC Transducer
–Use electrical impedance technology to count blood cells
Electrodes
• Two non-corrosive, electrically conductive plates.
• One positively charged and one negatively charged.
• One electrode is located in each transducer chamber.
• Conduct a constant current flow through the aperture during the
measurement portion of each cycle
Aperture Plate
• Inserted into a slot between the two transducer chambers.
 …components...
1. Vacuum & Pressure
– The fluid power supply contains the vacuum and pressure pumps, accumulators, waste
bottles, and associated solenoids and hardware used to move fluids through the system
2. Pressure Gauge and Regulator
3. Solenoids & Motors
– Solenoids are used to control the movement of fluid, vacuum and pressure throughout
the system.
– Motors are used to supply samples and reagents to the system
4. Power Supply
– Supply necessary voltage to drive the whole system
Typical counter circuit
 Hemoglobin Measurement
• Hemoglobin is measured by absorption Spectrophotometry
• This process is based on the linear relationship between the amount of light
that a well-mixed, non-flowing sample absorbs at a particular absorption band
and the concentration of the absorbing entity in the sample (Beer’s Law)
• It is based on the fact that substances absorb or emit electromagnetic energy at
different wavelengths
• The concentration of hemoglobin is proportional to the absorbance of the light
by the sample in the green 540 nm wavelength region
 HGB measurement
• The Hemoglobin Flow Cell consists of the following:
– Fully enclosed, transparent glass flow cell —holds the HGB sample dilution
or reagent blank for measurement
– Light source—illuminates the sample dilution or the reagent blank
– Band pass Filter —allows light in the 540-nm region to pass
– Photo detector—measures the amount of light passing through the sample
dilution or reagent blank
HGB Meas’t
HGB Meas’t
 Troubleshooting
• Most problems are related to erroneous reading
• What to do when control value is out of limit?
– Duplicate tests on patient samples
– Subsequent duplicate values should be within these defined limits
– Patient data can also be used to monitor precision in a laboratory performing >100
samples a day
– Day-to-Day variation in MCV, MCH and MCHC should be analyzed using Bull's
algorithm
• This facility is available in the software of many auto analyzers.
– The use of stable controls, however, is the method of choice
• Still out of control?
– Check integrity of material –Troubleshoot –Verify instrumentation
 Specimen-Related Problems
• An instrument problem is differentiated from a specimen- related
problem by running a control
• If the control results are acceptable, the problem is probably specimen-
related
• Check for:
– Clots
– Hemolysis
– Lipemia (the presence of an abnormally high concentration of emulsified fat in
the blood)
 Instrument Problems
• If the control shows similar problems, it indicates an instrument problem.
– Electronic? – Pneumatic / Hydraulic? – Reagent?
• Because it is easiest to detect a problem in the electronic subsystem and
hardest to detect a problem in the reagent subsystem, the subsystems are
usually checked in the following order: electronic, pneumatic / hydraulic,
reagent
 Reagent Troubleshooting
• A reagent problem can be as obvious as precipitate in the reagent tubing
• In the less obvious cases, the most effective way of detecting a problem
is by keeping a log of the lot numbers with the opening and expiration
dates of the reagents in use
– And knowing how each reagent affects the data.
• Referring to the labeling information with your reagents for details will
also help a lot
 Preventive Maintenance
• Refer to your instrument manual to determine which, if any, preventive
maintenance procedures are required for your instrument.
• Most Beckman Coulter instruments do not require routine preventive
maintenance
– But there are cleaning and replacement procedures available for troubleshooting
purposes
• Maintain a log documenting your instrument’s use.
• This will assist you both in the laboratory routine and when you need
service
• Use your log book and your instrument certification documents to keep
your system’s history current
 Calibration
• It is done to compensate for any inaccuracies of the pneumatic, hydraulic
and electric systems.
• Calibration fine tunes your hematology analyzer and provides the most
accurate results possible.
• Automated Hematology analyzers should be calibrated using calibrators
that have traceability to standard reference material or methods.
• Controls are not used for calibration
 Calibration tips
• Never adjust to a specific value for an individual sample
• For best performance, calibrate all the CBC parameters
• The WBC differential is calibrated at the factory
– They do not require calibration in the laboratory

• When to Calibrate?
– At installation,
– After the replacement of any component that involves dilution characteristics or
the primary measurements (such as the apertures), and
– When advised to do so by your service representative
Thank you!