Topic:

Different Method of CBC

submitted to
Dr.Hafiz Iftikhar Hussain
Group No:4
Farrukh siyyer
Shahzaib
Hammad Hassan
Habiba Faraz
Muhammad Rehan Asghar
Introduction:
• Blood consist of plasma and blood cells
• Plasma is fluid portion
• Major blood cells are WBCs, RBCs, Platelets
• Produce in bone marrow and release into
blood stream
• CBC is A complete blood count Also known as
Full blood count, full blood exam
 Parameters measure:

• RBCs
• Hemoglobin (Hb)
• Hematocrit (Hct)
• Mean corpuscular volume (MCV)
• Mean corpuscular Hemoglobin (MCH)
• Mean corpuscular hemoglobin concentration (MCHC)
• Red cells distribution width (RDW)
• WBCs
• Platelets
• Mean platelet volume (MPV)
• Reticulocytes
Functions:
RBCs :Transport hemoglobin that carry oxygen
Hemoglobin: A protein which carry in blood
Hct: proportion of blood cells in plasma
MCV: Average volume of RBC. It measures
Average size or RBC
MCH: content of Hb of average red cell
MCHC: Average concentration of Hb in given volume of cells
RDW: measure variation in RBCs size
Platelets: involve in prevention of bleeding
WBCs: involve in immunity.
MPV: Measure average size of platelets
 WBCs:
• In WBCs we count
• Neutrophils ( 40-60 %)
• Eosinophils ( less than 1%)
• Basophils (1-4%)
• Monocytes (2-8% )
• Lymphocytes (20-40%)
Normal Ranges:
Why we perform CBC:
• to detect various disease
• For routine health examination
• General screening by doctor
• Anemia is suspected
• Leukemia is suspected
• Evaluate abnormal bleeding
• Bone marrow problems
• Nutritional deficiency
• Genetic Hb structural or functional
Problems
Method of CBC:
• There are 2 methods of CBC
Automated method
Manual method
Blood collection:
Whole blood
Vein puncture ( anticubital Fossa)
( Back side of hand)
Take in EDTA tube
CBC analyzer machine
CBC analyzer machine:
• 2 types on base of DLC
 3-part diffenrtial cell count
 5-part differential cell count
 7 part is also used.
Principle of working:
• Aspirate the required quantity of blood
• Quantifies
• Classifies
• Describe cell population using electrical and optical
Techniques
3 modern technologies used
 photometric method for Hb
 Electrical impedance
 Flow cytometery
Photometric method for Hb:
• RBCs lyses and Hb release
• Monochromatic light pass through it
• Absorbance of solution measured by photo sensor
• Calculate Hb concentration
Electeical impedance:
• Whole blood pass between 2 electrode through narrow
Appeature.
• 1 cell pass at a time.
• It measure cell volume
Flow cytometery:
• Tells about morphology of cells
• Determines granulatiy
• Fluorescence flow cytometery:
• Measure specific population of cells
• Fluorescence dye reveal-plasma ratio
Parts of CBC analyzer:
• Display and graphic screen
Menu and functions displayed
• Keyboard panel
numerous key, navigation keys
Shortcut keys.
• Probe
• Start button
• Trap chamber
• Waste chamber
Procedure:
• Take whole EDTA blood
• Turn the machine ON from power supply.
• Check the background of machine
• Gave command to machine
• Mix the blood sample in blood mixing
machine
Sysmex xp 300 automated
• Set the mixed sample to sample probe and press hematology analyzer.
start switch
• Wait for sometime
• The result will display on the screen of machine
• Print that result.
Video link :
https://www.youtube.com/watch?v=bFsute5CEos&feature=youtu.be

Advantages:
Accurate and precise
Large samples
Reduction of labour
Accurate determination of RBC incides.
Disadvantages:
• Flagged sample need reviews
• Platelets clump count as single cell
• Expensive
• Sample review by smear formation
Manual method of CBC:
• Hematocrit or PCV:
Procedure:
1. Anticoagulated blood
2. Filled capillary tube or
wintrobe tube Upto 100 mark.
3. Centrifuge it with speed of 3000 rpm for 30 mints
4. RBCs will settle at the bottom than a buffy coat layer
5.Plasma remain at top.
Video link:
https://www.youtube.com/watch?v=DfG6C0gKjhU
Hb identification:
Procedure:
• N/10 Hcl is taken in Hb tube upto 12 mark.
• Anticoagulated blood is taken 20 microliter
In Hb pippete.
• Dispense the blood in N/10 Hcl in Hb tube and mix
it Properly.
• Place for 10 mnt at room temperature
• Place tube in sahli’s comparator box.
• Dilute it with distelled water drop wise and mix it with help of strierr
• Did it until the color of blood change from dark brown to standard color
 Continue:
• If color match to standard
• Lift the strrir up and note down reading
• Read tube by taking lower minscus

• Video link
https://youtu.be/mWAEIvu1mV8
Blood cells incides:
Mean corpuscular volume:
MCV = Hct × 10/RBC
• Value (84-96 fL)
• Mean corpuscular Hb:
MCH = Hb × 10/RBC
value (26-36 pg)
Mean corpuscular Hb concentration:
MCHC= Hb × 10/Hct
value (32-36%)
 WBCs morphology:
• Neutrophils: These are multilobed cells have 3 to 5 lobes.
• Cell diameter is 12- 15micro meter.
Eosinophil: These cells have bilobed nucleus
• cytoplasm is grimson color and cell diameter
• is about 12-15micrometere cytoplasm is pinkish color
Basophils: These are also bilobed cells and cytoplasm is
purplish and cell diameter is about 12-15micrometer.
Lymphocytes: These have no lobes and cytoplasm
is basophilic cell diameter is about 12-16micrometer.
Monocytes: These have no lobes and cytoplasm is blue-gray
and have granules ,cell diameter is about 14-20micrometer.
 WBCs count:
• Manually it is done by smear formation
Procedure:
 Take clean, dry (grease free) slide.
 Transfer a small drop of blood near the edge of the slide.
 Place the spreader slide at an angle of 45 degree . Pull back the spreader until
touches the drop of blood.
 Let the blood run along the edge of the spreader.
 Push the spreader forward to the end of the slide with a smooth movement.
 Dry the blood smear at room temperature.
Adequate drying is essential to preserve quality of the film.
 Continue:
• Fix with methanol by taking drops of methanol
or dip in methanol for 2 minute.
• Place slide on staining rank and dry the slide.
• leishman’s stains drop on slide and spread over it left it for 2 minute
• Dilute it with distilled water wait for 10 minutes.
• wash again with distilled water.
• Allow to air dry.
 Continue:
• Microscopic Examination:
• Now put the slide on stage focus
it with first 10X than move to high power 100X.
• DLC is counted on body tail junction on slide.
• Count and write down there to find out their
number.
 RBCs Morphology:
• The human erythrocytes are discoid (bi-concave)
• about 7–8 μm in diameter
• with a central area of pallor
• It carries hemoglobin
• No nucleus
• No mitochondria
• Life span 120 days
 RBCs count manually:
• Hematocytometer :
Naubers chamber:
Procedure:
Draw blood up to „0.5‟ mark.
 Carefully wipe the excess blood outside
the pipette by using cotton or a gauze.
 Draw diluting fluid up to 101 mark
( haymens fluid)
The pipette in rotated rapidly by keeping it horizontal during mixing.
 Continue
 After two minute, by discarding few drops
ring mixing from the pipette and holding it
slightly inclined small volume of the fluid is
introduced under the cover slip which is
placed on the counting chamber.
 Allow the cells to settle for 2 to 3 minutes.
 Continue:
 Place the counting chamber on the stage of the microscope.
 Switch to low power (10 X) objective.
Adjust light and locate the large square in the center with 25 small squares.
 Now switch to high power (40 X) objective.
The red blood cells in the four corners and central Square of „R‟ are counted.
Counting Rule:
• Leave left lower
• Count right upper
Vice versa.
 Formula:
• Total RBC/mm3 = Number of RBC counted × Dilution factor × Depth factor
No. of chambers counted

no of RBCs in 1 mm3 = n* 1000 per cumm

Reticulocyte Count:
• Immature stage of RBCs
• Non-nucealated
• Ribisomes present
• It is a reflection of the amount of effective
red blood cell production taking place in the bone marrow.
 Reticulocyte count:
Procedure:
 Mix equal amounts of methylene blue and EDTA (two to three drops) on a small
test tube.
 Mix the tube and allow standing at room temperature
 This allows the reticulocytes adequate time to take up the stain.
 Mix blood and stain mixture thoroughly and make two thin wedge smears and
allow to air dry.
 Place the first slide on the microscope stage and, using the low power objective
10X.
 Carefully change to the "oil immersion objective (100x) and further locate an
area.
 Formulas:

• % Reticulocytes = Number of reticulocytes in 1000 RBCs × 100

1000 (RBC's observed)

Corrected reticulocyte count (%)=Patient's hematocrit × Reticulocyte count (%)

Normal Hematocrit (%)
Normal values:
Newborn 2.5 – 6.0 %
Adult 0.5 - 2.0%
 Platelets morphology:
• Platelets are very small non-nucleated cells
with fine granules that derive from
fragmentation of megakaryocytes.
• (1.5-3 μm) diameter
• Life Span is 8-12 days.
 Platelet count:
• Same procedure as RBCs
Draw blood up to „0.5‟ mark.
 Carefully wipe the excess blood outside
the pipette by using cotton or a gauze.
 Draw diluting fluid up to 101 mark (Ammonium oxalate)
The pipette in rotated rapidly by keeping it horizontal during mixing
 After two minute, by discarding few drops
ring mixing from the pipette and holding it
slightly inclined small volume of the fluid is
introduced under the cover slip which is
placed on the counting chamber.
 Allow the cells to settle for 2 to 3 minutes.
 Continue:
Place the counting chamber on the stage of the microscope.
 Switch to low power (10 X) objective.
Adjust light and locate the large square in the center with 25 small squares.
 Now switch to high power (40 X) objective.
The red blood cells in the four corners and central Square of „R‟ are counted.
Counting Rule:
• Leave left lower
• Count right upper
• Formula
Plt count =n x 1000 per cumm
 Diagnosis:
• RBCs normal value:4.7 to 6.1 (cells/mcL)
• women – 4.2 to 5.4 (cells/mcL.)
• If RBCs count High
Polycythemia
Kidney diseases
If Rbcs count low
Anemia
Chronic inflammatory disease
 sickle cell anemia
Iron deficiency
 Conti..
• Hb normal range:
• 13.5 to 17.5 g/dl
• women 12.0 to 15.5 g/dl
Hb high:
Dehydration
Heart failure
Liver cancer
Hb low:
Loss of blood
Iron, folate deficiency
Kidney failure
 Hct:
• Normal ranges:
• Men 45%
• Women 42%
Hct high:
Dehydration
Genetic defect
Less rbcs production
Hct low:
Bleeding
Destruction of rbcs
 Blood cells incides:
• MCV:  80–100 fL.
MCV high:
megaloblastic anemia
MCV low:
 microcytic anemia
MCH: 27.5 and 33.2 (pg)
MHC high:
 macrocytic anemia
MHC low:
Decreased level of
hemoglobin
 MCHC:
• Normal range: 33.4–35.5 (g/dL)
MCHC high:
 macrocytic anemia
Liver diseases
spherocytes
MCHC low:
 anemia due to iron deficiency
Microcytic hypochromic anemia
 WBCs:
• Neutrophil 2.0–7.0 x 109 /l
High:Typoid, malaria, kala azar, enzene chemical, irradiation etc.
Low: Pneumonia, burns, ischemic necrosis, acute hemmorages etc
basophil 0.02–0.1 x 109 /l
High: Allergic infection,hay fever, drug allergy etc
Low: Chronic myeloid leukemia, Polycythaemia vera
Eosinophil 0.02–0.5 x 109 /l
High: Steriod administration, cushing’s syndrome etc
low: Brochial asthma, Hay fever, worms entery etc
 Cont…
Lymphocyte 1.0–3.0 x 109 /l
High: Aplastic anemia, AIDS, Hodgking’s disease etc
Low: Viral hepatits, tuberclosis, leukemia etc
Monocyte 0.2–1.0 x 109 /l
High: Aplastic anemia, leukimeia etc.
low:Malaria, tuberclosis, multiple myloma etc.
Platelet count :
Normal range: 150,000-400,000 per cubic mm.
High: Acute leukemia, aplastic anemia, Liver disease, Metal poisoning ,
Megaloblastic anemia
Low: Polycythemia, Chronic granulocytic anemia, immadietly after surgery
 Refrance:
• Dacie and lewis practical hematology
• Oxford Handbook of Clinical Haematology
• Pics refrance from Google,
Wikipedia Dacie and lewis book
• Youtube videos