UNIVERSITY OF SANTO TOMAS | MEDICAL TECHNOLOGY

HEMATOLOGY | HEMATOLOGICAL PROCEDURES

CELL COUNT

 THE COUNTING CHAMBER

 Neubauer Counting Chamber

o see improved neubauer
o central secondary square divided into 16 tertiary squares
 Improved Neubauer Counting Chamber
o On the middle third of the chamber there are 3 parallel platforms extending across the
slide and separated by moats.
o Central Platform is 0.1 mm lower than the lateral platforms. It is subdivided by a
transverse groove into halves each wider than the 2 platforms
 Each half consists of a primary square measuring 9mm sq. and is subdivided into
9 secondary squares each measuring 1 x 1 mm. Each secondary squares are
divided into 16 tertiary squares.
 The four corner secondary squares are used for WBC counting.
 The central secondary square is divided into 25 tertiary squares (each measuring
0.2 mm x 0.2 mm).
 Each of the tertiary squares are divided into 16 smaller squares (Total of 800
smaller squares inside the central secondary square)
 RBCs are counted on four corner tertiary squares and central tertiary square of
the central secondary square (Five Smaller Squares = 80 smaller squares)
o Thick-Coverslip must be used to achieve the 0.1 mm space between the slip and ruled
platform
 Fuchs-Rosenthal Counting Chamber
o Larger than Neubauer
o for low cell counts such as Eosinophil Count, Spinal Fluid Count, and leukopenic blood
count
o Ruled Area measures 4x4 mm
o Depth is 0.2 mm
o Central Ruling us divided into 16 smaller squares
 Speirs-Levy Counting Chamber
o 4 sections; two on each side
o Each ruled area consists of 10 squares each measuring 1x1 mm with total area of 10
mm2
o Squares arranged in two horizontal rows of five squares which are further subdivided into
16 squares
o Depth is 0.2 mm

One section of Speirs-Levy

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 GENERAL FORMULA FOR CELL COUNTS

 RBC COUNTING
 THOMA RBC-DILUTING PIPETTE (0.5, 1, 101 mark)
o Bulb (100 units of volume)
o Red Bead
o Smaller Bore
 Make 1/200 Dilution
o 0.5 mark – RBC
o 101 mark - diluent
 Count in 5 smaller squares of the central tertiary square (Area = 1/5 mm2)
 Short Formula : Cells Counted x 10 000
 Normal Values
o Males: 4.5 to 6.0 M cells / cu. mm or 4.5 – 6.0 x 1012 cells / L
o Females: 4.0 to 5.5 M cells / cu. mm
 Diluting Fluids (Isotonic)
o Dacie’s Fluid / Formol Citrate – best rbc diluting fluid
 40% formaldehyde
o Hayem’s Fluid
 Allows growth of yeasts and produces clumping (cirrhosis px)
 Non corrosive but contains mercuric chloride
o Gower’s Fluid
 Prevents rouleaux; ppt protein in cases of hyperglobulinemia and
hemoglobinemia
o Toisson’s
 High sp. gravity
 Supports growth of fungi
 Stains WBCs
o Bethell’s
o NSS
 Used in cases of ER
 Particularly in excessive rouleaux, and autoagglutinated cells
 Stable and serves as preservative
 0.85 g NaCl on 100 mL distilled water
o 3.8% sodium citrate
 Discard 5-6 drops then charge on chamber
 Stand counting chamber for 5-10 mins
 Count on HPO
 Cell difference between squares is 20 or less
 WBC COUNTING
 THOMA-WBC DILUTING PIPETTE (up to 11 mark)
o Bulb = 10 units
o White Bead
o Larger Bore
 Make 1/20 dilution
 Shortcut : cells counted x 50
 Normal Value = 5000-10 000 cells / cu. mm or 5 – 10 x 10 9 cells / L
 Discard first 2-3 drops
 Charge on chamber. Stand 5-10 mins
 Count using LPO
 Cell difference between squares must not exceed 12
 Diluting Fluids (Hypotonic to lyse RBCs)
o 1 – 3% Acetic acid
o 1% HCl
o Turk’s Diluting Fluid
 In cases of Leukocytosis, use an RBC pipette (use 1:100 dilution)
 In cases of severe Leukopenia, smear the buffy coat
 Corrected WBC Count if there nRBC is greater than or equal to 5 (10 for pediatric patients)
𝑊𝑊𝑊𝑊𝐶𝐶 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐 𝑥𝑥 100
o cWBC =
100+𝑛𝑛𝑛𝑛𝑛𝑛𝑛𝑛 𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐𝑐
 PLATELET COUNTING
 Direct Methods
 Rees-Ecker
• Count platelets in counting chamber using all squares of the central secondary
square (Area = 1 mm2)
• Composition ( solution stable for 30 mins only )
o Brilliant Cresyl Blue – stain
o 40% Formalin – preservative
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

o Sodium Citrate – prevents clumping

• Use RBC pipette
• Employ 1:200 dilution
• Shortcut : Platelet Counted x 2000
 Guy and Leake’s
• Composition
o Sodium Oxalate
o Formaldehyde
o Crystal Violet
 Brecher-Cronkite
• Reference method
• Composition
o 1% Ammonium oxalate
o No stain is added, platelets will appear as refractile bodies
• Employs Phase-Contrast Microscopy
• 1:100 dilution
• Count in 25 tertiary squares of the central secondary square (Area = 1 mm2)
• Shortcut :Platelet counted x 1000
• Adapted by the Unopette System
 Nygard’s
 Indirect Methods
 Dameshek / Wet Method
• Capillary blood is used
• 1:5 dilution
• Uses the Rees-Ecker diluting fluid
• Prepare a thin film from the mixture.
• Use of siliconized dropper would prevent blood from adhering to the dropper
• Normal value : 500-900 x10^9 / L
 Fonio’s / Dry Method
• Capillary blood is used
• Diluting fluid placed at site of puncture
• Composition
o 14% MgSO4
o Giemsa / Wright’s Stain
• Make a thin film
• Count platelets at OIO until you reach 1000 RBCs along the process of counting
the platelets
 Olef’s
• Best indirect method
• < 1 / OIF : Thrombocytopenia
• 5 – 20 / OIF : Normal Platelet Count
• < 25 / OIF : Thrombocytosis
 SPECIMEN CONSIDERATIONS
 Inadequate mixing and poor collection causes plt to clump
 Skin puncture is less desirable-the tendency of aggregation or clumping
 If fewer than 50 plt are counted on each side, repeat using 1: 20 DIL
 If more than 500, use 1:200 dilution.
 If normal plt count, rbc squares may be used
 Platelet may occur when EDTA anticoagulant is used.
 To correct platelet sattelitosis, use sodium citrate as anticoagulant.
 Multiply platelet count by 1.1 to correct for the dilution in the citrate evacuated tubes.
Parameters RBC Count WBC Count Platelet Count
Dilution Factor 200 20 100
Area 1/5 mm2 4 mm2 1 mm2
Units per cu. mm | X106 / uL per cu. mm | X103 / uL per cu. mm | X103 / uL
X1012 / L X109 / L X109 / L
Normal Value M : 4.20 - 6.00 x 1012 / L
3.6 – 10.6 x 109 / L 150 – 450 x 109 / L
F : 3.80 - 5.20 x 1012 / L

 RETICULOCYTE COUNT
 index of bone marrow activity
 visualized by using supravital stains such as New Methylene Blue and Brilliant Cresyl Blue
 The mixture contains the dye, sodium chloride for isotonicity, and a preservative (sodium
oxalate in NMB, sodium citrate in BCB)
 Equal Amounts of Blood and Supravital Stain are allowed to mix for 3-10 mins at RT.
 2 Thin films are prepared.
 Platelets are counted under OIO in 1000 RBCs
 Formula : # Retics observed per 1000 RBCs x 100
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

• Simply Retics counted / 10

 Normal : 0.5 to 1.5% for Adults
 Newborns would have 1.8 to 5.8% on the first 2 weeks
 Miller Disk Method / Optical Disk
 Placed in the ocular of the microscope
 Two Squares
• Large : Retic Counting
• Small : RBC Counting (retics on this square are counted as rbcs)
 Minimum of 112 RBCs must be counted
 Retics % = [Retics Counted / Total RBCs] x [100 / 9 ]
 ABSOLUTE RETICULOCYTE COUNT
 # Retics per Liter of blood
 ARC = [retics % x RBC count] x 10
 Normal : 15 to 75 x 109 Reticulocytes per Liter of Blood
 CORRECTED RETICULOCYTE COUNT
 Reticulocyte Index
 Corrects for the Reticulocyte Count considering the Hematocrit of the patient
 CRC = [retics %] x [Hct / 0.45]
 RETICULOCYTE PRODUCTION INDEX Hematocrit Maturation In Days
 Shift Correction 40 – 45 10
 Indicator of rate of erythropoiesis 35 – 29 15
 RPI = CRC / Maturation Time in Peripheral 25 – 34 20
Blood 15 – 24 25
 Significance < 15 3
• RPI must be > 3  indicates adequacy of BM activity
• RPI < 2  Inadequacy
 ABSOLUTE EOSINOPHIL COUNT
 measure of eosinophils in 1 cubic mm of blood
 Randolph Method
 1:10 dilution
 10-15 mins incubation
 Count on all 9 secondary squares
 Fluid Composition 1
• Acetone – fixative
• Distilled Water – lysis of RBC
• Eosin – stain
 Fluid Composition 2 / Pilot
• Phloxine
• Heparin
• Sodium Carbonate
• Propylene Glycol
 Diluting Fluids
 Phloxine
• 1% Phloxine – Stain
• 10% Sdoium Bicarbonate – Lyse WBC / Accentuator
• Propylene Glycol – Lyse RBC
 Pilot
• Heparin – prevent clumping
• Phloxine Fluid
 Absolute Eo per cu.mm = [total eo] x 100/9
 ABSOLUTE LEUKOCYTE COUNTS
 Derived from Total WBC Count and the % of the specific leukocyte from differential count
 Absolute Count = [%Leukocyte/100] x [Total WBC count]
 LEUKOCYTE DIFFERENTIAL COUNT
 Determine percentile of each type of leukocytes
 Use OIO
 Specimen
 PBS from EDTA whole blood
 Buffy coat if extremely low wbc count
 Alternatives
• Addition of albumin (22%) and NSS allows better cell separation and minimizes
spreading artifact
 Expected Differential Count of Normal Patients
 Neutrophils :
 Lymphocytes :
 Monocytes
 Eosinophils :
 Basophils :
 Methods of Classification
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Schilling hemogram
• all the leukocytes (granulocytes and non–granulocytes) are classified and
grouped according to maturity of the cells into:
o Granulocytes | Neutrophils, Eosinophils, Basophils
o Non–granulocytes | Lymphocytes, Monocyte
• The polymorphonuclear neutrophils are further classified according to maturity of
the cells as:
o Myelocytes
o Metamyelocytes
o Bands or stabs
o Segmenters
• The Schilling hemogram may be represented as:

Basophil 0.25 – 0.5%

Eosinophil 2 – 4%
Myelocyte 0%
Metamyelocyte 0 – 1%
Stab 2 – 6%
Segmenters 55 – 65%
Lymphocytes 25 – 35%
Monocytes 2 – 8%
 Arneth’s classification
• In this method, the polymorphonuclear neutrophils are classified according to the
number of lobes which their nuclei possess. The more lobes, the older the cells:

Class I (with lobe or indented nucleus) – 5%

Class II (with 2 lobes) – 35%

Class III (with 3 lobes) – 41%

Class IV (with 4 lobes) – 17%

Class V (oldest with 5 lobes) – 2%

• Under the traditional unit, the result in differential leukocyte count is reported in
percentage, under the S.I. unit, the proportion of each type of cell is reported as a
decimal fraction and is called the leukocyte type number fraction.
 Haden’s classification
• This method classifies the neutrophils according to the presence of filaments.
These neutrophils whose lobes are connected by thin filaments are classified as
filamented, while those that are not connected by filaments are grouped under
non–filamented cells.

Filamented cells – 60%

Non–filamented cells – 7%

Eosinophils – 3%

Basophils – 1%

Lymphocytes – 21%

Monocytes – 8%

HEMOGLOBIN

 Points to Remember

 Per gram of Hb = 1.34 mL Oxygen = 3.47 mg Ferrous iron

 34 g/dL within RBC

 Classical Methods

 Copper Sulfate / Specific Gravity Method

 1.053 specific gravity of blood

 Iron Content / Kennedy Wong’s Method

𝐼𝐼𝐼𝐼𝐼𝐼𝐼𝐼 𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶𝐶 𝑖𝑖𝑖𝑖 𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏𝑏 (𝑚𝑚𝑚𝑚/𝑑𝑑𝑑𝑑)
Hb =
3.47 𝑚𝑚𝑚𝑚 𝑖𝑖𝑖𝑖𝑖𝑖𝑖𝑖 /𝑔𝑔 𝐻𝐻𝐻𝐻𝐻𝐻

 Oxygen Combining / Van Slyke / Gasometric Method

𝑂𝑂𝑂𝑂𝑂𝑂𝑂𝑂𝑂𝑂𝑂𝑂 𝑖𝑖𝑖𝑖 𝑚𝑚𝑚𝑚/𝑑𝑑𝑑𝑑 /
Hb =
1.34 𝑚𝑚𝑚𝑚 𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑜𝑒𝑒𝑒𝑒 / 𝑔𝑔 𝐻𝐻𝐻𝐻𝐻𝐻

Page 5 of 14
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Colorimetric Methods
 Direct Matching
 Blot & Match with color
 Tallquist & Dare’s Method
 Acid Hematin
 0.1 N HCl
 HbF resists acid elution
 Principle : Acid Elution / Kleihauer-Betke
 Alkaline Hematin
 0.1 N NaOH
 Principle : Precipitation
 Oxyhemoglobin
 Photometric
 0.007 N NH4OH
 Read at 540 nm
 Cyanmethemoglobin
 Photometric
 Most reliable except that it cannot measure sulfhemoglobin
 Uses Drabkin’s Reagent
o Sodium Bicarbonate = lysing agent
o Potassium Ferricyanide = oxidizes ferrous to ferric
o Potassium Cyanide = stabilizes ferric iron = Cyanmethemoglobin
 Read at 540 nm
 Automated Analyzers utilize Sodium Lauryl Sulfate = SLS-MetHb

HEMATOCRIT

 Volume of the packed RBCs occupying a given volume of whole blood

 Packed Cell Volume / Erythrocyte Volume Fraction
 1% Hct = 0.34 g Hgb = 107 000 RBC per cu.mm
 ADAM’S MICROHEMATOCRIT
o Uses a 75 mm long capillary tube (heparinized if from fingerstick, nonheparinized if using EDTA
samples) with a 1.2 mm diameter bore
 Heparinized : Red Band
 Nonheparinized : Blue Band
o Can hold 0.05 mL of blood
o Uses microcentrifuge. 5 mins @ 10,000 RPM
o Layers after centrifugation
 Fatty Layer
 Plasma Layer
 Buffy Coat
 RBC
 Sealing Clay – 4-6 mm at the end of the capillary tube on the ringed portion
o Microhematocrit reader is used. The level of RBC packing is read and the buffy coat is excluded.
 Adjust the top of the sealant to the zero mark of the reader.
 Read from the top of the red cell portion
 Do not include the buffy coat.
o Normal Values
 Male : 40% to 54%
 Female : 35 to 49%
 Newborn : 53% to 65%
 Temporarily low values after blood loss
 Temporarily high values when dehydrated.
o Sources of Error
 Falsely Decreased
• Short draw of EDTA sample / Double Anticoagulant  RBC shrinkage
• Improperly mixed EDTA sample
• Presence of interstitial fluid from capillary collection
 Falsely Increased
• Insufficient centrifugation
• Buffy coat was included in the reading
• Trapped Plasma (seen in sickle cell anemia, macrocytic anemia, hypochromic
anemias, spherocytosis, and thalassemia)  1-3% increase
• Improper flushing of catheter before blood collection
 MACRO METHODS
o WINTROBE METHOD
 Uses Double Oxalate anticoagulant and a Wintrobe Tube that is calibrated from 10 to 0
 HCT % = [height rbc layer / height of whole blood] x 100
o HADEN’S MODIFICATION
 Uses 1.1% Sodium Oxalate and a calibrated tube

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Centrifuge blood for 20 mins at 3000 RPM

 HCT = volume PRBC x 20 [if > 5 mL blood] | use 100 if blood < 5 mL
o VAN ALLEN METHOD
 Uses 1.6% Sodium Oxalate and a bulbed tube with 1-10 cm calibration or 10-100 mm
 Centrifuge for 15-30 mins at 2,500 RPM
 Each unit of division is equivalent to 1% hematocrit
o SANFORD-MAGATH
 Uses 1.3% Sodium oxalate and a 5 inches tube with a funnel-like mouth
 High speed centrifugation at 15 mins
o BRAY’S METHOD
 Uses Heparin and Bray’s Tube which resembles Wintrobe but is calibrated at 10-50 mm
(1 mm marking each) with 5 mL capacity

ERYTHROCYTE SEDIMENTATION RATE

 Sensitive nonspecific indicator of inflammation

 Rate of RBC aggregation as they settle to the bottom
 Directly proportional to the weight of RBCs and inversely related to blood viscosity (primarily the plasma)
 Events during 1 hr standing
o Initial Period of Aggregation during the first 10 mins of standing
o Period of Fast Settling occurs on the next 40 mins
o Final period of packing on the last 10 mins
 Wintrobe Method
o Oxalate anticoagulated blood on a 100 mm column with 0-10 markings [the left side of the tube,
color red] (if ESR 10-0, located at the right side of the tube color white)
o Now uses EDTA and a short column
 Westergren Method
o Uses 3.8% sodium citrate (black top)
o Westergren : 30 cm tube with 2.5 mm bore
o New : Plastic Tube by Sediplast
 Modified Westergren
o Uses EDTA and then blood from EDTA is diluted to 3.8% sodium citrate or 0.85% NaCl (4:1)
o Uses 200 mm column with 2.55 mm bore
 Sources of Errors
o Falsely Decreased o Fasely Increased
 Increased anticoagulant  Use of oxalate and heparin
concentration causing RBC which promotes RBC
sphering shrinking
 Bubbles in the column  High room temp
 Low room temp  Tilted ESR column
 Narrow ESR column  Vibrations
diameter  Use of refrigerated sample
 Clotted sample  Severe anemia – hence
 Delayed testing (sx not ESR is of little diagnostic
tested within 4 hrs) value in such cases
 Clinical Correlations
o Decreased ESR o Increased ESR
 Increased bile salts,  Increased choleesterol,
phospholipids, albumin, fibrinogen, and gamma
glucose but not DM globulins
 Decreased fibrinogen and  Decreased albumin
gamma globulins  Macrocytosis, Leukemia,
 Acanthocytosis, HgbC, Malignancies, Multiple
Microcytosis, Sickle Cell, Myeloma
Spherocytosis  Heavy metal poisoning
 Polycythemia, Thalassemia,  Acute infections, syphilis,
Leukocytosis Rheumatic Fever
 Cohexia, Congestive Heart  Diabetes Mellitus, End-
Failure stage renal failure
 Newborn Status  Myocardial Infarctionm
 ACTH, Cortisone Collagen Vascular Disease
 Ethambutol, Quinine,  Rheumatoid Arthritis, Gout,
Salicylates Inflammatory conditions
 Pregancy, Menstruation
 Dextran heparin,
procainamide, theophylline,
penicillamine vitamin A

Normal Values Modified Westergren Wintrobe

Adult Male 0 to 10 mm / hr 0 to 9 mm / hr
Adult Female 0 to 15 mm / hr 0 to 20 mm/hr
Page 7 of 14
UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

OSMOTIC FRAGILITY TEST

 Uses heparinized blood

 Determine hereditary spherocytosis or thalassemia
 Main Factor is the shape of RBC
o Shape is dependent on volume, surface area and the function of RBC membrane
o Spherocytes rupture more easily than normocytes, target cells, and sickle cells
 Fragility increases as the rate of hemolysis increases
 Griffin-Sanford Method
o 0.5% NSS and distilled water
o 12 Wasserman Tubes
 Label from 25 to 14 (corresponds to number of NSS dropped)
 Total volume must be 25 drops.
 Deliver 1 drop of blood to each tube
 Incubate at RT for 2 hours
o Expected Results
 Initial Hemolysis at Tube 23
 Final Hemolysis at Tube 17
o Computation of OFT
 % Fragility = Tube # x 0.02
o Normal Values
 Initial Hemolysis = 0.44 +/- 0.02
 Final Hemolysis = 0.32 +/- 0.02
 Clinical Correlations
o Increased OF in Hereditary Spherocytosis
 Initial Hemolysis at 0.65% NaCl
 Final Hemolysis at 045% NaCl
o Decreased OF in Thalassemia and Sickle Cell Anemia
 Initial Hemolysis at 0.35% NaCl

RBC INDICES

THE HEMOGRAM / COMPLETE BLOOD COUNT

 PARAMETERS
o RBC COUNT AND RBC INDICES
o HEMOGLOBIN – HEMATOCRIT
o WBC COUNT AND DIFFERENTIAL COUNT
o PLATELET COUNT AND PLATELET INDICES
 AUTOMATED CBC
o Components in Automation
 Hydraulics – aspirating unit, dilutior, mixing chambers, aperture baths and/or flow
cells, hemoglobinometer
 Pneumatics – vacuums and pressures for operating valves anf moving the sample
 Electrical system – electronic analyzers, computing circuit
o Electrical impedance
 Cell counting principle involving the measurement of the change in resistance produced
by a particle as it passes through an aperture
 Coulter Principle
 Blood cells are poor conductors
o Hydrodynamic Focusing Technology
 Counting of RBCs and PLTs
 Usually used with direct current detection method

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Eliminates potential errors in cell counters such as coincidence, re-circulation, and stress
changes associated with conventional methods
 Employed by Sysmex XN-1000
o Flow Cytometry
 Based on physical, antigenic, and functional prioperties of a particle suspended in a fluid
 Forward Scatter
 Fluoresecence Flow Cytometer (Laser at 633 nm)
• FSC – measures cell size | bigger = stronger
• SSC – measures intracellular structure | more complex = stronger
• SFL – measures type and amount of RNA/DNA = more NA = stronger
o Passes through dichromatic mirror
 Lineage Associated markers analyzed routinely in FC
LINEAGE MARKER
CD34
Immature CD 117
tDNT
CD 13,14,15
Granulocyte-Monocyte
CD33
CD71
Erythroid
Glycophorin A
CD 41, 42
Megakaryocytic
CD 61
CD 19, 20, 22
B-Cell
Light Chains
CD 2, 3, 4 , 5
T-Cell
CD 7, 8
BECKMAN COULTER Abbott CELL-DYN
PARAMETER Sysmex XN Series Siemens ADVIA 2120i
UniCEL DxH 800 Sapphire
Fluorescent staining Light scatter (primary
Impedance volume
Forward scatter count) Light Scatter
WBC Conductivity
Side Fluorescent light Impedance Absorption
5 angle light scatter
detection (secondary)
Low-angle and High-
RBC Impedance Impedance Impedance angle laser light
scatter
Modified Cyanmeth Sodium lauryl sulfate Modified Cyanmeth Modified Cyanmeth
Hgb
(525 nm) (555 nm) sulfolysis (540 nm) (546 nm)
Cumulative RBC
Hct (RBCxMCV) / 10 (RBCxMCV) / 10 (RBCxMCV) / 10
pulse height detection
Mean RBC volume Mean RBC volume Mean RBC volume
MCV [HCT/RBC] x 10
distribution histogram distribution histogram distribution histogram

MCH [HGB/RBC] x 10 [HGB/RBC] x 10 [HGB/RBC] x 10 [HGB/RBC] x 10

MCHC [HGB/HCT] x 100 [HGB/HCT] x 100 [HGB/HCT] x 100 [HGB/HCT] x 100

CV % of RBC
RDW-SD (fL) or Relative value; CV % of RBC
RDW histogram
RDW-CV% equivalent to CV Histogram
or RDW-SD (fL)
Impedance; Light Dual-angle light scatter Low-angle and High-
Impedance volume Scatter Forward analysis
angle laser light
PLT Conductivity Scatter Impedance count for
verification scatter
5 angle light scatter Side Fluorescent light
CD61 monoclonal ab ct Refractive index
detection
Supravital stain Supravital (oxazine
Fluorescent staining Fluorescent staining ;
Impedance volume 750) Low angle and
Forward scatter low-angle scatter and
RETIC Conductivity high angle light
Side fluorescent light fluorescent light
Light scatter scatter and
detection detection
measurement absorbance
Red fluorescent dye
Fluorescent staining
Impedance volume staining; forward Multiangle light scatter
Forward scatter
nRBC Conductivity scatter and measurements in two
Side fluorescent light
5 angle light scatter fluorescent light WBC diff channel
detection
detection
Multiangle polarized Peroxidase stain; light
Fluorescent staining scatter and absorption
Impedance volume scatter separation
Forward scatter For basophils –
DIFF Count Conductivity (MAPSS); three color
Side fluorescent light differential lysis, low-
5 angle light scatter fluorescence angle, high-angle laser
detection
detection light scatter

 Measurement Principles
o Direct Measurement:
 RBC –Impedance, hydrodynamic focusing
 WBC -Impedance, hydrodynamic focusing
 Platelets –Impedance (2-20 fl), hydrodynamic focusing
 Hgb–mod. Cyanmethemoglobin(525 nm)
 MCV –mean RBC volume (histogram)

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

o Indirect Measurement:
 Hct, MCHC, and MCH (calculations)
o RDW and MPV (CV of respective histograms)
o WBC differential
 VCS –volume, conductivity, scatter
 employs differential shrinkage
o Reticulocyte
 Supravitalstain
 Fluorescent detection

HISTOGRAM

 RBC HISTOGRAM AND PLT HISTOGRAM

o Gaussian
o X axis – volume
o Y axis – number of cells
o There are two flexible discriminators LD (25-75 fl) and UD (200-250fl
o Peak of curve should fall within the normal MCV range of 80-100 fl
o Erythrocytes have a size of 80-100 fl and are counted between 25 and 250 fl.
 Platelets have a size between 8 and 12 fl and are counted between 2 and 30 fl.
 PLT counted between 2 fl and 30 fl.
• 1 flexible Discriminator PL 2 to 6 fl.
• 1 flexible Discriminator PU 12-30 fl.
• 1 fixed Discriminator at 12 fl
o MCV is perpendicular line from peak of the curve to base
o The distribution should always start and end on base line and should be located between the two
discriminators

FLAGS ON RBC HISTOGRAM

o RL | Abnormal Height at Lower Discriminator

 Giant Platelets
 Microerythrocytes
 Dysplastic RBCs
 Platelet Clumps
o RU | Abnormal Height at Upper Discriminator
 Cold Agglutinin
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

 Agglutination
 Rouleaux
o Multiple Peaks
 Iron deficiency in recovery (therapy)
 Dimorphic picture
 Multiple RBC transfusions
 Extreme leukocytosis(> 600 x 10³/μl)
 Note if RBC, MCV, RDW-SD & RDW-CV are flagged
• Extreme anisocytosis is found. In case of anisocytosis the RBC result is not
affected.
• extreme high numbers of leukocytes may cause high incorrect RBC results

RDW HISOGRAM

 RDW-CV (%) = 100 x s/μ

• μ = L2 + L1 / 2
• s = L2 –L1 / 2
• Normal value: 11 -16 %
 RDW-SD is calculated in 20 % of the total height ofthe distribution curve.
• Normal value: 37 – 46 fl
• Clinical relevant > 60 fl

FLAGS ON PLT HISTOGRAM

o PL
 High blank value
 Cell fragments
 High numbers of bacteria
 Contaminated reagent
 Platelet aggregation
o PU
PLT clumps

• EDTA-incombatibility
• Clotted sample
 Giant Platelets (False low)
 Microerythrocytes(False High)
 Fragmentocytesor dysplastic RBC
o Multiple Peaks
 Platelet anisocytosis
 Recovery after chemotherapy
 Platelet aggregation
 Platelet transfusion
 WBC HISTOGRAM

o LD at the WBC-Histogram seperates the Leukocytes from the Thrombocytes

o The LD is flexible, but can not be lower than 30 fl
 WL FLAG
• PLT Clumps EDTA-Incombatibility / coagulated Sample
• High osmotic resistance (Erythrocytes not lysed)
• Erythroblasts
• Cold agglutinins
 WU FLAG in Leukocytosis

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

TROUBLESHOOTING

 RULE OF THREE – reliability of results ; only when RBC pop is normocytic normochromic

HGB x 3 = HCT +/- 3

 USING MCHC and MCV (from Sysmex XN-1000)

RESULTS CAUSES SUGGESTION
Low MCV, Low MCHC Microcytic hypochromic anemia N/A
Low MCV, Normal MCHC Hemoglobinopathies/Thalassemia N/A
High/N MCV, Low MCHC Hypernatremia, Hyperglyemia Dilute sample 1:7
Hyponatremia
Dilute Sample 1:7
Hemolysis
Low/N MCV, High MCHC Severe Lipemia
Plasma replacement with diluent for
Icterus
severe lipemia or icterus
Abnormal Protein Precipitation
Prewarm sample at 37C for 15-30
mins
RBC Agglutination
High MCV, High MCHC
Rouleaux
Plasma replacement if severe
agglutination is observed.

CONDITIONS CAUSING INTERFERENCE

Condition Parameters Rationale Indicators Action

Cold Agglutinin Low RBC RBC agglutination Dual RBC Warm specimen
High MCV,MCHC population and rerun
Grainy appearance Righ shift
Lipemia, Icterus High Hgb, MCHC Increased Turbidity Rule of Three Plasma
violation, Abnormal replacement
histogram
Hemolysis Low RBC Count RBC not counted Rule of Three Recollection of sx
High Hematocrit violation
Lysis-resistant RBCs High WBC, Hgb Hgb S,C,F may fail Noise-wbc Manual Dilution,
to lyse and are interface increased
counted as WBC interference incubation time
Microcytes/Schistocytes Low RBC Volume below Left Shift on Review blood film
High PLT RBC threshold or Histogram
within PLT Abnormal PLT
threshold histogram
nRBCs, megakaryocyte High WBC (on old Counted as WBCs nRBC flagging cWBC; automatic
fragments, instruments only) correction by
micromegakaryoblasts newer instruments
PLT clumps Low PLT Large plt clumps PLT clump Redraw sx with
High WBC counted as WBCs flagging sodium citrate;
multiply result by
1,1
WBC less than 100K / High Hgb, RBC Turbidity Rule of Three Manual HCT;
uL Incorrect Hct above linearity manual Hgb;
recalculate indices;
dilute for WBC
count if above
linearity
Leukemia Low WBC Fragile WBCs Delta check Review film, phase
High PLT counted as PLT plt count, CD61
count
Old Sx High MCV, MPV PLT/RBCs swollen Abnormal New Sx
Low PLT WBCs degenerate clustering on WBC Establish stability
Differential histogram of sx
Incorrect

PERIPHERAL BLOOD FILM EVALUATION AND CORRELATION WITH CBC

 PREPARATION OF THE BLOOD FILM

o Sample : EDTA whole blood
o Smears must be prepared within 1 hr (optimum) [up to 4 hrs]
o Mostly done by using two slides, a drop of blood is placed on a slide and another slide is used to
spread the blood
o The blood film must cover up to ¾ of the silde (two-thirds is the minimum requirement)

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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

o The blood film is shaped like a tongue with a thin feathery end.
o Requires technical skills
 The angle of the spreader is optimum at 30 degrees or lower. The higher the angle, the
shorter the film, and thicker it will be. Hence, if the hematocrit of the patient is high, one
must decrease the angle of the spreader to produce a good quality smear.
 The speed of spreading the blood also affects the length and the thickness of the smear.
Irregular acceleration of spreading would cause uneven spreading of the blood and
ridges. The quicker the movement, the shorter it will apear. The slower the movement,
the longer it will be. Sometimes when the movement is slow, no tongue shape is formed
and the spreading of the blood will appear like it was just wiped through the slide.
 Holes would appear when dirt is present on either of the slides or when one is using
greasy slide (if the slide is greasy, the blood will not adhere to the slide, hence some part
of the smear appears to have been removed/wiped away). Holes are normal in cases of
leukemic and pediatric patients. It is also commonly seen in blood samples with viscosity
less than the normal.
o Other Ways to Prepare the Film
 Coverslip Technique – the only advantage is on the even WBC distribution
 Beacom’s Method : Uses a glass slide and a coverslip
 Ehrlich’s Method – two-coverslip method
 Automated
 Miniprep : semiautomatic
 Centrifugal : uses 0.2 mL blood
o Staining the Film
 Differential Staining
 Romanowsky : polychromatic ; MB / Azure B + Eosin B/Y
o Wright’s
o Giemsa
o May-Grunwald
 Panoptic : Romanowsky + another dye
o Wrights-Giemsa
o Jenner-Giemsa
o May-Grunwald-Giemsa
 Components
 Fixative usually methanol
 Acid Dye
 Buffer pH 6.8
 Basic Dye
 Manual Method usually uses wright’s stain involving flooding of the smear on a tray.
Staining takes around 3-5 mins. The buffer is added after the addition of the stain and
mixing must be ensured.
 Rapid Manual Method – Dipping of the slide in couplin jars containing the solutions
 Fixative : Methanol : 30 sec
 Eosin : 5 sec
 Methylene Blue : 15 - 20 sec
 Buffer | Aged Distilled Water | Phosphate Buffer : 45 sec
 MICROSCOPY
o Methods for Examination
 Four-Field Meander
 The count is made by dividing the smear into four fields and proceeds as in
exaggerated battlement method
 Two-Field Meander
 The count is made by dividing the smear into two fields and proceeds as in
exaggerated battlement method
 Exaggerated Battlement
 The count starts at one edge of the smear and counting all the cells, advancing
inward to 1/3 of the width of the smear, then on the line parallel to the edge, then
out of the edge, then along the edge.
 Strip Differential / Battlement
 All the cells are counted in the longitudinal strip that is, from the head to the tail of
the smear.
o Scan first using LPO. Find the monolayer area of RBCs (RBCs are not too for nor to close to
each other)
 DIFFERENTIAL COUNT
o Already discussed above
o Done whenever immature granulocytes are suspected or flagged in the machine
 WBC ESTIMATE
o 40x HPO | WBCs per field x 2000
o 50x Dry OIO | WBCs per field x 3000
 PLT ESTIMATE
o 100x OIO | count in 10 fields | Average Plt per field x 20,000
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UNIVERSITY OF SANTO TOMAS
FACULTY OF PHARMACY | DEPARTMENT OF MEDICAL TECHNOLOGY

o If there is significant anemia or erythrocytosis

 [Ave Plt per field x Total RBC] / 200 RBCs/field

SUMMARIZING RESULTS

 WBC PARAMETERS
o TOTAL WBC
 Compare WBC histogram and scatterplot to respective cell counts
 Check for nRBCs (corrected automatically by new analyzers)
 Elevated WBCs – leukocytosis – check what specific WBC is increased
 Decreased WBCS -leukopenia
o DIFFERENTIAL COUNT %
 Examine specific cell line affected in abnormal wbc count
o ABSOLUTE DIFFERENTIAL COUNTS
 A specific cell line may be relatively increased or decreased but their absolute count are
within normal range or vice versa.
o WBC MORPHOLOGY
 Note for bands and immature cells
 Left shift – increased immature cells
 WBC precursors in PBS may indicate malignancy
 Check for inclusions.
 RBC PARAMETERS
o RBC Count
o Hgb – check for rule of three ; more sensitive for anemia than hct
o Hct
o MCV – correlate with RBC histogram
o MCH
o MCHC
o RDW
o RBC Morphology Correlate size with MCV, correlate pallor with MCHC
 Normocytic : MCV 80 – 100 fL
 Microcytic : < 80 fL
 Macrocytic : > 100 fL

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