TOPPERS PSC

CORNER RAILWAY STATION ROAD, KARUNAGAPPALLY,

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BLOOD OF PHLEBOTONY

 Hematology
Haem = Blood, Logos = Study
 Hematology – study of blood
Study of different constituents of blood their formation structure and function
BLOOD
 Blood is a fluid connective tissue
 7-8% of body weight
 Blood volume : Adult – Male: 5-6 Liters, Female: 4-5 Liters
 PH of Blood: 7.35-7.45
 Specific gravity-1.052-1.062
 Blood is a complex fluid which circulate throughout the body in closed channels
called blood vessels [Arteries, Veins & capillaries ]

Fluid part – Plasma (55%) – 90% water

Blood
Solid part – Cellular elements (45%)
 Fluid part of anti-coagulated blood – Plasma
 Fluid part of the clotted /coagulated blood, Defibrinated plasma is called- Serum
 Serum= Plasma – (Fibrinogen & prothrombin)
(Fluid part of coagulated blood, clotted bloods)
 Plasma: Coagulation studies , glucose, D-Dimer, FDP( Fibrin Degradation Product),
Plasma glucose
 Serum: Most biochemistry test, protein electrophoresis, urea, creatinine, albumin,
cholesterol
 Buffy coat- WBC+ platelets ( <1%)
 Colour of plasma- pale yellow – deep yellow
 Jaundice- icteric plasma- dark yellow – greenish yellow
 Red plasma- hemolyis, hemoglobinemia
 White plasma-Lipemic

Methods of Blood Collection

Capillary Arterial Verious / Venipuncture

 Method of blood collection selected depends on the amount of blood required for
investigation
Capillary Blood Collection
 This method is adopted when the amount of blood required is very small
 Capillaries are small vessels connecting arterioles and venules
 The peripheral blood sample is obtained by skin picture using an instrument called
blood lancet
Blood lancet
 Flat body with pointed

 Ideal length of lancet tip is 3mm

 Maximum puncture depth is 3mm. (Allows penetration of skin to a maximum depth of
3mm)
 Collected for rapid testing ( point of care testing) & children
 Sites commonly selected.
 Adult – Finger tip / ear lobe (Index or ring finger is used)
 Infants – Heel or big toe
 Steps –
 Area is cleaned with an alcoholic pad
 Allow dry
 Prick using a sterile lancet
 Blood should flow freely without applying any pressure
 Wipe of the first 2 drops ( May contain tissue fluid).
 Collect succeeding drops for investigation
Use: Hb estimation, Differential leukocyte count, total WBC,Platelets

Venous Blood Collection

 Method adopted when there is large requisite of blood

 Sample is collected with a sterile needle and syringe
 Polypropylene syringes are used
 Size of syringe is selected according to the amount of blood required for
investigation
 21 & 22 G needle – Adult
 22 Guage with black hub is used for blood collection in adults
 Bevel length- Adult- 20mm, children-15mm
 23 Gauge in children
 Guage size increases, diameter decrreases
 Hub colour and guage size

Homolink is a device which is used to collected blood without needles

 Venous collection can also be done using evacuated blood collection tubes, venues
and vacutainers

Venipuncture
Adult: (18-20, 21, 22)guage
Children 23 guage
For blood collection in blood bank 15 – 16 guage
(16-18 guage)

 Sites :- most preferred site – Anticubical veins in upper arms

ADULTS
 Median cephalic vein
 Basilic vein
 Median cubital
 Most preferred- Median cubital
 Infants – Femoral vein
 Steps
 Patient seated comfortably
 Arm extended to table and palm facing up ward
 Apply torniquet above elbow
 Torniquet – Make veins prominent and avoid blind probing ( ideal length 30-40 cm)
 Select the vein for puncture and clean the are using 70% alcohol spirit ( 70 %
isopropanol or 0.5%chlorhexidine)
Prolonged application of torniguet
 Fluid pass to tissue spaces
 False elevation in triglyceride , cholesterol, protein
  Needle is inserted at an angle of and bevel facing

upward
 Entrance in to vein denoted by appearance of blood in the hub
 Release the tourniquet and withdraw blood till required amount of blood fills
the barrel
 Place cotton such at the site of puncture and withdraw needle in a single
movement
 Blood is transferred to suitable containers
 USE: Blood for ESR, PCV, Biochemical investigations

Arterial Puncture
 Used for arterial blood cells analysis
 Often collected from radial artery
 Allen test is employed before collecting blood from radical artery
 This test ensure that ular artery is in good condition, test for collateral circulation
 Heparinized syringes are used for collecting arterical blood
 1000 IV of heparin/ml of blood
 Guage size- 23-26 G
 Needle is inserted at an angle 900, Radial- 45, brachial-60, femoral -90
 For blood gas analysis needle tip is closed immediately after collecting blood
 Arterial blood samples are transported at 2 – 4 degree Celsius

Types of Blood samples

Whole Blood Serum Plasma

Haematologytests Fluid part of coagulated/ Fluid part of anti

cleated blood coagulated blood

ABG Biochemistry , protein Plasma glucose

electrophoresis
Ammonia, lead other Coagulation studies
poison detection

Precautions for Blood collection

To prevent hemolysis
 Make sure syringe and needle are dry
 Plastic disposable apparatus are preferred
 Avoid rough handling of blood at any stage of collection or processing
 Do not eject blood from the syringe through the needle. Remove needle first
 Gently eject blood through the sides of the tube avoiding foaming
 Mix the blood with anticoagulant gently by rotation
 Do not freeze the blood because the cell will hemolysis on thawing

 When taking blood a minimum amount of constriction should to the arm

 The blood should flow slowly and freely into the syringe.
 Avoid squeezing
 The blood collecting contains should be clean and dry
 Excessive amount of anticoagulants should not be used

Preparation of thin blood smear

 Capillary blood collected by skin puncture one various blood collected in EDTA can
be used
 Approximate size of a blood drop used for a thin smear is 2mm in diameter
 Ideal length of a blood smear 3-4cm
 When using venous blood smear should be made with in 1-2 hours
 Ideal blood smear will be tongue shaped
 Head is thick portion followed by body Tail is thin portion
 Body portion preferred for morphological studies
 Feather edge is a region near tail
 It is labeled at head for identification
 Smear is allowed to day before staining
 Preparation of a good smear depends on
 Quality of the glass slide is spreader slide used
 Size of drop of blood
 Angle between spreader and slide
 Spead by which the spreader is moved blood slide

Identification of Blood cells – Leishman’s staining procedure and identification

 In hematology in order to study cellular morphology we use staining techniques
 Most commonly used stains in haematology are Romanowsky stains
 Simplest Romanowsky stain : Leishman – Most commonly used stain for differential
count
 Giemsa’s – Most complex
 Jenner’s – Simplest
 MaY –Grunwald
 Field stain
 Staining of cells are based up on their plt
Romanowsky stains
 Neutral stains both the acidic and basic part of the stains are coloured
 Made up of oxidized methylene blue (azure) as basic group and Eosins as acidic
group
 PH of buffer solutions used for all Romanowsky stain is 6.8 – 7.2
 During staining the nucleus being acidic is stained by basic component of the die.
Methylene blue forming purple colour
 Cytoplasm is stained by acidic part of the stain cosign, forming red coloration
Leishman’s stain
 Commercially available in powder form
 Eosin – Acidic
 Basic – Methylene blue
 Solvent used is methanol to fix the stain
 Methanol used should be acetone free to avoid hemolysis of blood cells
 Blood films are fixed with cretonne free methyl alcohol for 30 – 60 sec
 100ml methyl alcohol + 0.15 gm of Leishman powder
 Keeping the stain in an incubator at for 24 hours

 Keep for a few days for ripening of the stain which increases the staining property
Steps
 Cover the air dried smear using Leishman’s stain
 Keep for 2-3 min.
 Dilute with buffered distilled water of + 6.8 to twice the volume of the stain
 Mix well and keep for 7 – 10 min.
 Wash with buffered distilled water and then with tap water
 Clean the bottom side of the slide
 Dry in coir and examine under the micro scope
Differential Leukocyte count
 Preparation of smear
 Staining of smear
 Microscopic examination
 Strip method, Battlement method
 RBC – Salmon Pink colour
 Platelets – Deep Violet
 Nucleous of leukocyte – Purple
 Eosinophilic granules – Basic – Organic red
 Basophilic granules – Acidic Highly – Dark blue
 Normal values
 Neutrophils – 40 – 60%
 Eosinophils – 2 – 8%
 Basophils – 0 – 1 %
 Lymphocytes – 20 – 40%
 Monocyte – 2 – 6%
 Long term preservation of blood smear requires alcohol fixation
 Simplest Romanowsky stain – Jenner’s
 Most complex – Giemsa’s stain
 pH of buffer recommended for Leishman’s stain 6.8
 PH for schuffner’s dot in material infection – 7.2
 stains made up of combination of acidic and basic dye – Romanowsky
 Hb molecule is stained by Eosin.