Complete Blood Count - Student's

COMPLETE BLOOD COUNT:
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AUTOMATED, MANUAL
METHODS
By: BROMARIE M. OLARTE, RMT, MLS (ASCPi)

AUTOMATIONS IN
HEMATOLOGY
CBC PARAMETERS
MEASUREMENTS GENERATED BY AUTOMATED HEMATOLOGY PROFILING
INSTRUMENTS
RBC RBC count, Hemoglobin, Hematocrit, MCV, MCH, MCHC,

PARAMETERS RDW, Retic
WBC WBC count, Relative and absolute count of Segmenters,

Band, Lymphocytes, Monocytes, Eosinophils and
PARAMETERS Basophils
PLATELET Platelet count, MPV

PARAMETERS
BASIC COMPONENTS OF HEMA ANALYZERS
1. HYDRAULICS
– aspirating unit, dispensers, dilutors, mixing chambers, aperture baths and /or
flow cells and hemoglobinometers
2. PNEUMATICS
– vacuums and pressures for operating valves and moving the sample through
the system
3. ELECTRICAL SYSTEMS
– electronic analyzers and computing circuitry for processing data
BASIC CELL COUNTING PRINCIPLES
1. ELECTRICAL IMPEDANCE
– a.k.a Electronic Resistance or low – voltage direct current (DC) resistance or
“Coulter Principle”
– Most common methodology used
– Principle: Isotonic solutions conduct electricity better than cells do
– Cell counting and sizing is based on the detection and measurement of changes
in electrical impedance (resistance) produced by a particle as it passes through
a small aperture.
Ohm’s Law: Voltage = Current x Resistance
ELECTRICAL IMPEDANCE – CONT.
Procedure:
1. Aspirated blood is divided into two separate volumes for measurements
2. One volume is mixed with diluent and is delivered to the cell bath, where erythrocyte
and platelet counts are performed. As a cell passes through the aperture partially
occluding it, the electrical impedance increases producing a voltage pulse.
– Size of pulse: related to the cell size
– Number of pulses: related to the number of cells
3. Particles measuring between 2 – 20 fl are counted as platelets, whereas those

measuring >36 fl are counted as RBCs.
ELECTRICAL IMPEDANCE – CONT.
4. The other blood volume is mixed with diluents and a cytochemical lytic agent that
lyses only RBCs. A leukocyte count and cyanmethemoglobin test is performed on
this mixture.
5. Particles >35 fl are recorded as leukocytes.

 35 – 90 femtoliters: lymphocytes (small)
 90 – 160 femtoliters: monocytes (medium)
 160 – 450 femtoliters: granulocytes
6. The analyzer derives the MCV and RDW from the red cell histogram, whereas the
MPV and PDW are derived from the platelet histogram. The hematocrit, MCH and
MCHC are calculated from measured and derived values.
HISTOGRAM
TYPES OF ERRORS THAT MAY BE ENCOUNTERED
INSTRUMENTAL ERRORS
ERROR RESULT
Aperture plugs*most common POSITIVE ERROR
Extraneous electrical pulses POSITIVE ERROR
Bubbles POSITIVE ERROR
Improper setting of aperture current or threshold EITHER
Excessive lysing of RBCs NEGATIVE ERROR
ERRORS INHERENT TO THE SAMPLE
Giant platelets may be counted as RBCs or WBCs
Fragments of leukocytes may be counted as RBCs or platelets
Increased number of schistocytes decreased RBCs, increased platelets
Agglutinated red cells or platelets increased WBCs
Platelet satellitism decreased platelets
Sickle cells, hypochromic cells, target cells (resist lysis) increased WBC
BASIC CELL COUNTING PRINCIPLES
2. LIGHT (LASER) SCATTER
– a light sensitive – detector measures light scattering of cells when they pass through a laser.
Forward (low) angle scatter – cell size
Side (high) angle scatter – cell granularity, complexity, lobularity
Number of pulses – cell number
3. FLOW CYTOMETRY
– Similar as light scatter but is also capable of measuring fluorescent emission from
particles labelled with fluorescent tags.
– Primarily used for the diagnosis, classification, and management of hematologic
malignancies.
SCATTERGRAM / SCATTERPLOT
MANUAL METHODS IN
HEMATOLOGY
HEMOCYTOMETRY
• Numerical evaluation of the formed elements

of blood or the estimation of the number of
blood cells in a known blood volume.
• “heart of manual cell counting”
Methods:
1. Turbidimetric
2. Microscopic
 Improved Neubauer Counting Chamber – most
commonly used
3. Automated – optical, electrical
HEMOCYTOMETER RULING
• Total area = 9 mm2

• Area of each of the 9 large squares = 1 mm2
• Area of each of the 16 small corner squares
= 0.0625 mm2
• Area of each of the 25 smaller central
squares = 0.04 mm2
• Depth = 0.1 mm2
• Total volume = A x D = 0.9 mm3
BLOOD DILUTING PIPET
Thoma RBC pipet

 Bead color: RED
 Volume of the bulb: 100 uL
 Commonly used dilutions: 1:200
 Bore size: small
Thoma WBC pipet

 Bead color: WHITE
 Volume of the bulb: 10 uL
 Commonly used dilutions: 1:20
 Bore size: large

DILUTING FLUIDS
RBC Diluting fluids
– should be isotonic
 Recommended: Formol – Citrate (Dacie’s fluid)
 Most common: NSS
 Others: Hayem’s, Gower’s, Toisson’s, Bethel’s fluid, 3.8 sodium citrate
WBC Diluting fluids

– contains weak acids capable of lysing erythrocytes without destroying
leukocytes (hypotonic)
 Glacial acetic acid w/ gentian violet (Turk’s)
 Glacial acetic acid w/ methyl violet
 1% hydrochloric acid
DILUTING FLUIDS
Platelet Diluting fluids

– must be able to preserve platelet integrity while inhibiting their aggregation
 Recommended for phase contrast microscopy: Brecher – Conkrite (1%

ammonium oxalate)
 Recommended for light microscopy: Tocantin
 Guy and Leake: Citrate + Formalin + Crystal Violet
FORMULAS
RED BLOOD CELL COUNT
- reported in million/mm3 ; x 1012/L
WHITE BLOOD CELL COUNT
- reported in thousand/mm3 ; x 109/L
Number of cells counted x Dilution Factor
Total Area Counted x Depth Factor
• RBC Long Method: RBC counted x 10 x 200 x 5

• RBC Short Cut Method: RBC counted x 10,000 (or add 4 zeroes)
• WBC Long Method: WBC counted x 10 x 20/ 4
• WBC Short Cut Method: WBC counted x 50
REFERENCE RANGES
RBC COUNTS WBC COUNTS PLATELET COUNTS

 Male
12
4.2 -6.0 x 10 /L
9 9
 Female 4.5 – 11.0 x 10 /L 11.0 x 10 /L
3.5- 5.6 x 1012/L
 Newborn
5.0 – 6.5 x 1012/L
Notes:
The manual _____________ is the most difficult and least accurate of all manual tests.
One RBC omitted = error in count by __________.
One WBC omitted = error in count by __________.
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What are the main components of hematology analyzers?

What are the main components of hematology analyzers?

What are the basic cell counting principles used in automated hematology?

What are the basic cell counting principles used in automated hematology?

COMPLETE BLOOD COUNT:

Click to add text

By: BROMARIE M. OLARTE, RMT, MLS (ASCPi)

RBC RBC count, Hemoglobin, Hematocrit, MCV, MCH, MCHC,

WBC WBC count, Relative and absolute count of Segmenters,

PLATELET Platelet count, MPV

– Number of pulses: related to the number of cells

3. Particles measuring between 2 – 20 fl are counted as platelets, whereas those

5. Particles >35 fl are recorded as leukocytes.

• Numerical evaluation of the formed elements

• Total area = 9 mm2

Thoma RBC pipet

Thoma WBC pipet

 Commonly used dilutions: 1:20

 Bore size: large

WBC Diluting fluids

Platelet Diluting fluids

 Recommended for phase contrast microscopy: Brecher – Conkrite (1%

• RBC Long Method: RBC counted x 10 x 200 x 5

RBC COUNTS WBC COUNTS PLATELET COUNTS

Riverside College, Inc. - RiversideCollegeBcd RiversideCollegeBcd

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