Hematologic Procedures

Hematologic Procedures
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Topic Outline
• Routine Complete Blood Count
• Cell counts
• RBC Count
• Platelet Count
• WBC Count
• Differential Count
• Hemoglobin
• Hematocrit
• Red Cell Indices
• Special Hematologic Tests
• Automation
Complete Blood Count
• Cell counts
• RBC Count
• Platelet Count
• WBC Count
• Differential Count
• Hemoglobin
• Hematocrit
• Red Cell Indices
Manual Cell Count
• Red Blood Cell Count
• Platelet Count
• White Blood Cell Count
Materials Needed
• Hemacytometer
• Thoma Pipet
• Suction device
• Thick coverslip
• Cell Counter
• Diluting fluids
Hemacytometer
Image source: Rodaks Hematology 6th Edition

Calculations
N-RBC
Falsely counted as WBC
Not lysed by WBC diluting fluids

N-RBCs
CORRECT THE WBC COUNT:
• Uncorrected WBC count x 100
• numbers of nRBC + 100
Indirect Platelet Count
Platelet estimate of Report platelet estimate as
0 – 49,000/uL
50,000 – 99,000/uL
100,000 – 149,000/uL
150,000 – 199,000 /uL
200,000 – 400,000 /uL
401,000 – 599,000/uL
600,000 – 800,000/uL
Above 800,000/uL
10
Disposable Blood Cell Count Dilution Systems
• Capillary pipette and diluent reservoir systems
• WBC count and platelet count
• 1.98mL of 1% ammonium oxalate

• Blood: 20uL
Differential Count
• One hundred WBCs are counted and classified through the use of
push-down button counters
• Results are reported as percentages
• Principle: A stained smear is examined to determine the percentage

of each type of leukocyte present and assess the erythrocyte and
platelet morphology.
Image source: Rodak’s Hematology
Differential Count
• WBC abnormalities are also reported
• Reporting:
• Reactive Lymphocytes:
• separate %, as a % of total lymphocytes
• Semi-quantitatively: occasional to many
• Toxic granulation:
• Present
• Semi-quantitatively: slight to marked / 1+ to 3+
• Specimen
• Peripheral blood
• Bone marrow
• Body fluid sediments
Image source: Clinical Hematology by Turgeon 5th ed.
Absolute count
• More accurate than the relative count
• Actual number of specific WBC in a liter of blood
• Absolute count = percentage x WBC Count

Special
• EOSINOPHIL COUNT
• 50-350x10 /L 6
• Diluting Fluids
Hematology
•Tests
Diluting Fluids Composition • Pilot’s
• Phloxine/eosin/neutral red: stain • Manner’s
• Propylene glycol • Randolphs’s
• Na2CO3 • Hinkleman
• Heparin • Tannen’s
Hemoglobin
Hemoglobin
HIGHER IN THE MORNING AND SCREEN FOR ANEMIA AND MAY HIGHER IN HIGH ALTITUDES INCREASE IN STRENUOUS
LOWER IN THE EVENING DETECT RBC BREAKDOWN/ MUSCULAR ACTIVITY
HEMOLYTIC ANEMIA
Hematocrit
• “Packed Cell Volume”

• Volume of packed RBCs that occupies a given volume of whole
blood
• Reported: percentage(%) or L/L
Rule of Three
• Applicable if red cells are normocytic-normochromic
• The value of the hematocrit should be three times the value of the
hemoglobin plus or minus 3
Red Cell Indices
• Used to define the size and hemoglobin content of the red blood cell
• Aids in diagnosing and differentiating anemia
Blood
• MCV Indices
• MCH
• MCHC
Mean Cell Volume
• Average volume of red blood cells expressed in femtoliters (fL)
• NV: 80-100 fL
Mean Cell Hemoglobin (MCH)
• Average weight of hemoglobin in a red blood cell expressed in
picograms (pg)
• NV: 26-32 pg
Mean Cell Hemoglobin Concentration (MCHC)
• Average concentration of hemoglobin in each individual red blood cell
expressed in grams per deciliter (g/dL)
• NV: 32-36 g/dL

Red Cell Distribution Width
• It reflects the degree of red cell variation in size
• RDW = S.D./mean MCV x 100
• NV: 9.0-14.5
• Microcytic/macrocytic – normal RDW + dec or inc MCV
• Anisocyte with size w/in ref range= inc RDW + N MCV
• Anisocyte with size below or above the normal range= abn. MCV and
RDW
Representative Critical Values
• Hemoglobin: less than 5.0 g/dL

• Hematocrit: less than 15%
• Platelet count: less than 30,000 per microliter
• WBC count: less than 2,500 per microliter and
greater than 30,000 per microliter
Reticulocyte Count
• An indicator of the rate of erythrocyte production
• Use of supravital stains
• The count is expressed as a percentage of total erythrocytes

FORMULA
Example: There are 15 reticulocytes counted in 8 months old infant.

Retics count (%) = 15 reticulocytes / 1,000 = 0.015 x 100 = 1.5%
Interpretation: NORMAL
Reference range
• Adult: 0.5-1.5%
• Newborn: 2.0-6.0%
Miller Disc
• Designed to reduce the labor-intensive process of counting
reticulocytes
• Disc is inserted into the eyepiece of the microscope
• Minimum of 112 cells should be counted in the small square

Rodak’s Hematology 5th Edition
Absolute Reticulocyte Count (ARC)
• the actual number of reticulocytes in 1 liter (L) or 1 microliter (mL) of
blood
Corrected Reticulocyte Count (CRC)
• In specimens with a low hematocrit, the percentage of reticulocytes
may be falsely elevated because the whole blood contains fewer red
blood cells.
• A correction factor is used, with the average normal hematocrit

considered to be 45%.
Rodak’s Hematology 5th Edition
0.03
Hematology by Turgeon 5th Edition

Reticulocyte Production Index (RPI)
• Measures erythropoietic activity when stress reticulocytes are
present
• What are “shift reticulocytes”?

Automated Reticulocyte Count
• Analyzers evaluate reticulocytes using optical scatter or fluorescence

after the red blood cells are treated with fluorescent dyes or nucleic
acid stains to stain residual RNA in the reticulocytes.
Erythrocyte Sedimentation Rate (ESR)
• Non-specific measurement used to detect and monitor an
inflammatory response to tissue injury
• Distance in millimeters at which the RBCs fall in 1 hour

Stages of ESR
• Initial rouleaux formation
• Rapid settling of RBCs
• Final sedimentation of RBCs

Osmotic Fragility Test (OFT)
• A measure of the ability of red cells to take up fluid without lysing
• Employed to help diagnose different types of anemia, in which the

physical properties of red cells are altered.
Osmotic Fragility Test
• Increased OFT (decrease resistance): found in hemolytic anemias and
hereditary spherocytosis and whenever spherocytes are found
• Decreased OFT (increase resistance): occurs following splenectomy

and in liver disease, sickle cella nemia IDA and thalassemia
Point of Care Tests
• Hematocrit
• Centrifuge-based device
• Conductivity Method
• Hemoglobin
• measured by modified hemoglobinometers or by oximeters integrated with a
blood gas analyzer
• Cell Counts
• Traditional cell counting methods
• employs a buffy coat analysis method
Automation
• Two General Principles
• Electronic resistance ( impedance)
• Optical Scatter
Electronic Impedance
• Utilizes non-conductive properties of blood cells
• as blood cell passes through orifice of aperture it
displaces its own volume
• RBCs and Platelets counted together, separated by
pulse heights
• Hydrodynamic focusing forces cells to pass single
file through sensing zone

• Platelets: 2-20fL
• MPV: 6.8– 10.2fL
• Red Cells: 36fL
• Lymphocytes: 35-90fL
• Mononuclear cells: 90-160fL
• Granulocytes: 160-450fL
Factors Affecting Volume Measurement
• Aperture diameter
• Red cell/platelet
• White cell
• Protein build-up: decreases diameter
• Cell carryover
• Coincident passage loss
• Orientation of the cell in the center of the aperture
• Deformability of the RBC
• Recirculation of cells back into the sensing zone
Optical Scatter
• A hydrodynamically focused sample stream is directed through a
quartz flow cell past a focused light source
• Cells counted as passed through focused beam of light (LASER)
• Sum of diffraction, refraction and reflection
• Multi angle polarized scatter separation (M.A.P.S.S)
• Forward-angle light scatter ( 00 )
• Forward low-angle light scatter ( 20 to 30).
• Forward high-angle 50 to 150.
• Orthogonal light scatter (900)
Radiofrequency
• Used in conjunction with electrical impedance
• Cell interior density is proportional to pulse height or change in the RF
signal

CBC Parameters
PARAMETER UNIT OF REPORTING COMMON METHOD OF DETERMINATION
WBC X 109/L Impedance count X calibration (cal) factor
RBC X 1012/L Impedance count X calibration factor
HGB g/dL Colorimetric absorbance in proportion to hemoglobin
From RBC histogram,

MCV fL #of RBCs X size of RBCs X cal constant OR Calculated: HCT X 10
RBC
Calculated: RBC X MCV
HCT %
10
Calculated: HGB X 10
MCH Pg
RBC
Calculated: HGB X 100
MCHC g/dL or %
HCT
RDW % Impedance (from histogram)
Platelet X 109/L Impedance count X cal factor
Absolute: X109 /L
WBC Diff Light Scatter , flow cytometry
Percent of WBC : %
CBC Quality Control
• Commercial Controls:
• 3 levels (low, normal, high)
• Values stored in instrument computer
• Levey-Jennings graph generated and stored for each parameter
• Mode to Mode QC:
• Most automated hematology instruments have a primary and secondary mode
of sample aspiration. Controls must be run on BOTH and correlate.
• Primary=Automated or Closed
• Secondary=Manual or Open
• Delta Checks
• When the Laboratory Information System (LIS) and the instrument are
interfaced (connected) delta checks are conducted by the LIS on select
parameters.
Advantages of the automated analyzers
Ørapid ,objective, statistically significant
Ønot subject to the distributional bias of the manual count.
Ømore efficient and cost effective
Ø The precision of the automated differential makes the absolute
leukocyte counts reliable and reproducible.
Disadvantages of the automated analyzers
Øproduce cell counts which are falsely increased or decreased.
ØSome analyzers check only the volume and number of particles .
Ø Platelet clumps may be misclassified as leukocytes or erythrocytes,

and nucleated red blood cells can be misclassified as leukocytes or,
specifically, lymphocytes.
References
• Rodak’s Hematology: Clinical Principles and Applications, 6th Edition
• Clinical Hematology: Theory and Procedures, 5th Edition, Turgeon,
M.L.
• Steininger, Cheryl et al. Clinical Hematology: Principles, Procedures
and Correlations.
Thank you…

Hematologic Procedures

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Hematologic Procedures

Image source: Rodaks Hematology 6th Edition

Falsely counted as WBC

Not lysed by WBC diluting fluids

• 1.98mL of 1% ammonium oxalate

• Results are reported as percentages

• Principle: A stained smear is examined to determine the percentage

• Absolute count = percentage x WBC Count

• “Packed Cell Volume”

• NV: 32-36 g/dL

• RDW = S.D./mean MCV x 100

• Anisocyte with size w/in ref range= inc RDW + N MCV

• Hemoglobin: less than 5.0 g/dL

• Use of supravital stains

• The count is expressed as a percentage of total erythrocytes

Example: There are 15 reticulocytes counted in 8 months old infant.

• Disc is inserted into the eyepiece of the microscope

• Minimum of 112 cells should be counted in the small square

• A correction factor is used, with the average normal hematocrit

Hematology by Turgeon 5th Edition

• What are “shift reticulocytes”?

• Analyzers evaluate reticulocytes using optical scatter or fluorescence

• Distance in millimeters at which the RBCs fall in 1 hour

• Rapid settling of RBCs

• Final sedimentation of RBCs

• Employed to help diagnose different types of anemia, in which the

• Decreased OFT (increase resistance): occurs following splenectomy

• Electronic resistance ( impedance)

Image source: Rodaks Hematology 6th Edition

Image source: Rodaks Hematology 6th Edition

WBC X 109/L Impedance count X calibration (cal) factor

RBC X 1012/L Impedance count X calibration factor

HGB g/dL Colorimetric absorbance in proportion to hemoglobin

From RBC histogram,

Platelet X 109/L Impedance count X cal factor

ØSome analyzers check only the volume and number of particles .

Ø Platelet clumps may be misclassified as leukocytes or erythrocytes,

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