Unit 1

Introduction to Hematology

Blood Specimen Collection

Importance of Blood Collection

Safety on Blood Collection

OSHA
All specimens must be
treated as potentially
infectious
Hepatitis, syphilis,
malaria, and HIV
Accidental injury
Indirect transmission

Hand washing
Gloves
Contaminated sharps

Phlebotomist

Physiologic Factors Affecting Test

Results

Three General Procedures for

Obtaining Blood
Venipuncture
Venous blood
Skin puncture
Peripheral or

capillary blood
Arterial puncture
Arterial blood

Precautions Common to all

Blood Collection Procedures
1. The correct patient
identification is critical.
2. The correct specimen
identification is equally
as important.
3. Gloves must be worn at
all times while
performing phlebotomy
techniques.

Precautions Common to all

Blood Collection Procedures
4. The puncture site must
be cleansed by rubbing
vigorously with a gauze
pad or cotton
thoroughly moistened
with 70% isopropyl
alcohol
5. All sharp objects must be
disposed of in a special
puncture-resistant
container labeled as
biohazard.

Venipuncture
Primary source of specimen
Methods
Syringe method
Evacuated tube method

Venipuncture
Patient interaction
Assemble supplies and equipment
Venipuncture
Specimen preparation

Patient Interaction
Identify the patient
Note patient isolation

restrictions
Note patient dietary
restrictions
Reassure patient
Verify paper works
Position patient

Additives in Collection Tubes

Collection Equipment for

Venipuncture
Syringe method
Syringe with needle
Plain tubes or tubes with anticoagulant
Evacuated tube method
Adaptor
Two-way needle
Evacuated tubes
Tourniquet
Cotton or gauze
Solutions for skin preparation
Winged infusion sets (butterflies)

Solutions for Sterilization

1.
2.

70% isopropyl alcohol

Povidone-iodine or 1-2% tincture iodine
- blood culture

3.

Benzalkonium chloride (Zephiran chloride)

- legal blood alcohol level

Equipment for Evacuated Tube

Method

Parts of the Needle and

Syringe
Bevel
Shaft
Hub

Barrel
Plunger

Venipuncture Procedure

Selecting a vein
Routine venipuncture

procedure

Sites of Venipuncture
Antecubital fossa
Median cubital
Cephalic
Basilic
External jugular vein
Femoral vessel
Subclavian vein
Superior longitudinal sinus
Long saphenous vein
Dorsum of the hand

How to Make the Veins

Prominent
Opening and closing of fist
Massaging
Applying hot towel
Slight slapping of area
Tightening the tourniquet
Applying 70% alcohol

Routine Venipuncture
Procedure
1.

Check the request form.

2.Identify the patient.
3.Check diet restrictions, if any.
4.Prepare supplies. Put on gloves.
5.Reassure and prepare the patient.
6.Select the venipuncture site.

Routine Venipuncture
Procedure
7. Cleanse the venipuncture site.
Dry with sterile gauze or cotton.
Otherwise air dry.
8. Apply the tourniquet 2-3 inches
above the venipuncture site not
longer than 1 minute.

Routine Venipuncture
Procedure
Inspect the needle and
equipment.
10. Perform the venipuncture. Anchor
the vein with the thumb 1-2
inches below the site. Insert the
needle with the bevel up, with a
15-degree angle.
9.

Routine Venipuncture
Procedure
11. Collect blood using the correct
order of draw, inverting each tube
immediately.
Order of draw:
- blood culture
- red
- light blue
- other additives (gold, green,
lavender, gray)

Routine Venipuncture
Procedure
12. Release tourniquet. Instruct

patient to open hand.

13. Place gauze or cotton lightly over
the venipuncture site.
14. After the last tube has been
released, remove the needle.
15. Apply direct pressure to the site.

Routine Venipuncture
Procedure
16. Ensure that bleeding has stopped

then bandage the site.

17. Dispose the puncture equipment
and other biological hazards.
18. Label tubes properly.
- patients full name
- ID number
- date of collection
- time of collection
- collectors initials

Specimen Preparation
If syringe is used, fill appropriate

tubes
Discard needle
Label specimens
Transport specimen promptly and
properly

Advantages of Venipuncture
Easiest and most convenient method

of obtaining blood suited for different

tests
Accurate clotting time
Fastest method for sample collection
from many patients

Advantages of Venipuncture
Reduces the number and variety of

apparatus carried during collection

Allows test to be repeated on the
same blood sample
No dilution with tissue juices
No variation of results obtained
from different veins

Disadvantages of
Venipuncture
Stasis due to prolonged application

of tourniquet can lead to pH

changes and errors in clotting time
Cannot be used for peripheral blood
film preparation
WBC count and ESR should be
done within two hours
Prothrombin time should be done at
once or within two hours if
refrigerated and separated from the
red cells

Complications of Venipuncture
Immediate local complication
Hemoconcentration

- Continuous

bleeding
Syncope
Ecchymosis/bruises
Hematoma
- Petechiae
Failure to draw blood - Edema
Hemolysis
- Seizures, tremors
Vomiting, choking
- Allergies

Complications of Venipuncture
Late local complication
Thrombosis
Thrombophlebitis
Late general complication
Hepatitis
AIDS

Causes of Hematoma
Failure of the needle to penetrate the vein

completely or through and through

puncture
Failure to release the tourniquet before
withdrawing the needle
Severing the vein
Failure to apply pressure to the site of
puncture
Repeated puncture on the same vein

Causes of Hematoma

Sources of Error in
Venipuncture
Errors in venipuncture preparation
Errors in venipuncture procedure
Errors after venipuncture

completion

Error in Venipuncture
Preparation
Improper patient identification
Failure to check adherence to

dietary restrictions
Failure to calm patient prior to blood
collection
Use of improper equipment and
supplies
Inappropriate method of blood
collection

Error in Venipuncture
Procedure
Failure to dry the site completely after

cleansing with alcohol

Inserting needle bevel side down
Use of needle that is too small, causing
hemolysis of specimen
Venipuncture in unacceptable area

Arm with IV catheter

Burned, damaged, scarred, and occluded

veins

Error in Venipuncture
Procedure
Prolonged tourniquet application
Wrong order of draw tube
Failure to mix blood collected in additive-

containing tubes immediately

Pulling back on syringe plunger too
forcefully
Failure to release tourniquet prior to needle
withdrawal

Error in Venipuncture
Completion
Failure to apply pressure immediately to

venipuncture site
Vigorous shaking of anticoagulated
blood specimens
Forcing blood through a syringe needle
into tube
Mislabeling of tubes

Error in Venipuncture
Completion
Failure to label appropriate specimens

with infectious disease precaution

Failure to put date, time, and initials on
requisition
Slow transport of specimens to
laboratory

Special Considerations

Pediatric venipuncture
22 to 23 gauge needle
Infectious disease precautions
Adverse patient reaction
Dizziness, fainting, or nausea

Quality Assurance in Specimen

Collection
Proper patient preparation
Adequately trained phlebotomist
Proper specimen handling
Selection of appropriate tube

Check on expiration date

Tubes with additives must be completely

mixed by gentle inversion

Correct amount of blood

Correct specimen transport and storage

Reasons for Specimen

Rejection
Request form and tube identification do not

match
Unlabeled or mislabeled tubes
Hemolysed specimen
Wrong time collection
Specimen in wrong tube
Clotted blood (if whole blood is needed)
Contaminated blood
Lipemic blood

Skin Puncture
Method of choice in pediatric

patients
Useful in adults with

Extreme obesity
Severe burns
Thrombotic tendencies

Geriatric patients
Fragile veins

Sites of Skin Puncture

Distal portion of 3rd or 4th finger
Site of choice due to accessibility
Earlobe
Best site due less pain because of
presence of less nerve endings
Heel or big toe
Infants and children

Puncturing Devices
Disposable lancet
Automatic pricker
Needle
Blade
Glass capillary pricker
Depth of wound 2-3 mm

Devices for Blood Collection from

Skin Puncture
Capillary tubes
Heparinized or non-heparinized
Microtainer tubes
Anticoagulated or plain
Unopettes
Varied dilutions and diluents

Sites to be Avoided
Edematous and congested
Cold and cyanotic
Inflamed and pale

Disadvantages of Skin
Puncture
Small amount of blood samples are

collected
Repeated tests cannot be performed
without repeated puncture
Possibility of obtaining hemolyzed
blood sample

Disadvantages of Skin
Puncture

Results obtained are different from those

obtained from venous blood

WBC count higher by 15-20%
RBC count and Hgb higher by 5%
Platelet count and RBC fragility lower

Skin Puncture Procedure

1.
2.
3.
4.
5.

Examine request form.

Assemble equipment and supplies.
Greet and identify the patient (and
parents)
Position the patient with the help of
parents or designated holder.
Put gloves on.

Skin Puncture Procedure

6.Select the puncture site.
7.Warm puncture site. Cleanse the

area with 70% isopropyl alcohol.

Allow to air dry.
8.Perform the puncture. The depth of
the wound should be 2-3 mm.
9.Wipe off the first drop of blood.

Skin Puncture Procedure

10.Collect the specimen.
11.After blood has been collected,

apply pressure until bleeding stops.

12.Label the specimen.
13.Dispose of all puncture equipment.

Introduction to Clinical
Hematology
Anticoagulants

Anticoagulants

Chemical agents that prevent or

hinder clotting or coagulation of

blood

Characteristics of Anticoagulants
Must not alter the size of RBC
Must not cause hemolysis
Must minimize platelet aggregation
Must minimize disruption of WBC
Must be readily soluble in blood
Must not affect the result of the test

Actions of Anticoagulants

Making Ca++ unavailable

Precipitating Ca++ (oxalate)
Binding Ca++ in a non-ionized form (citrate and

EDTA)
Neutralizing thrombin and thromboplastin

(heparin)

Actions of Anticoagulants

Delaying the activation of factor XII or preventing

platelet adhesion on wet surfaces (siliconized

glasswares)
Removing fibrin as it is formed (defibrination)

Ethylene Diamine Tetra Acetic

Acid (EDTA)
Anticoagulant of choice for hematologic

determinations
Synonyms
Sequestrene
Versene
Sequester-sol

Dispensing 1-2 mg/ml of blood

Action prevents Ca++ from ionizing

(chelation)

Ethylene Diamine Tetra Acetic

Acid (EDTA)

Disodium, dipotassium, or tripotassium

Powder or liquid form
Prevents artifact formation
Excess concentration causes
Shrinkage of RBC, decreased Hct, increased

MCHC, low ESR, degenerative changes in WBC,

swelling and breaking of platelets

Advantages of Using EDTA

WBC count, RBC count, and Hct determination

can be done even after many hours

Preserves RBC and WBC morphology
Prevents clumping of platelets

Advantages of Using EDTA

Can be used in blood transfusion with very little

toxic effect
Samples can be stored overnight without
deterioration (40C)
Prevents formation of artifacts even after
prolonged standing

Citrates
Sodium citrate (buffered or

unbuffered)
Acid citrate dextrose (ACD)
Citrate phosphate dextrose (CPD)
Citrate phosphate dextrose with
adenine
(CPD-A)

Sodium Citrate
Action
Combines with Ca++ to form an insoluble salt
Helps prevent the rapid deterioration of labile and

stable factors (V and VII)

Dispensing
3.2% aqueous solution (0.109 M)
3.8% trisodium or disodium citrate
ESR 1 part anticoagulant + 4 parts blood
Coagulation tests and platelet count 1 part
anticoagulant + 9 parts blood
Use blood transfusion

ACD
Action
Source of nutrient for RBC thus

prolonging their survival

Uses
Blood transfusion

Shelf life
14 days

CPD and CPD-A

CPD

Shelf life 21 days

CPD-A

Shelf life 35 days

Action
Adenine

Source of ATP
Provides energy for RBC

Heparin
Action
Inactivating thromboplastin
Neutralizing thrombin
Inactivating prothrombin
Preventing fibrin formation from fibrinogen

Dispensing
0.1-0.2 mg/ml
15-30 units/ml

Advantages of UsingHeparin
Excellent natural anticoagulant
Absolute minimal hemolysis
Used for electrolytes determination,

osmotic fragility test (OFT), blood gas

analysis, pH assays, Hct
determination, ESR, , and blood
transfusion in open-heart surgery

Disadvantages of UsingHeparin
Expensive
Does not readily mix with blood
Not recommended for WBC count,

platelet count, and blood smear

preparation

Oxalates
Ammonium oxalate and potassium

oxalate
Potassium oxalate

Ammonium Oxalate and

Potassium Oxalate
Synonyms

Dried oxalate
Balanced or double oxalate
Paul Hellers mixture
Wintrobe mixture
Function

Ammonium prevents swelling of RBC

Potassium prevents shrinkage of RBC

Ammonium Oxalate and

Potassium Oxalate
Ratio 3:2
Dispensing 2 mg/ml of blood
Action forms an insoluble salt with

Ca++
Uses

RBC count
ESR
Blood indices

Advantages of Using Double

Oxalate
Cheap
Easy to prepare
Requires no dilution
Produces the least distortion of cell
Provides sample suitable for Hgb

determination, WBC count, RBC

count, and Hct determination

Disadvantages of Using
Double Oxalate
Cannot be used in transfusion
Toxicity of oxalate
Inability of liver to metabolize oxalate
Not recommended for platelet count
Not recommended for BUN, NPN, and K

determination due to NH4

Not recommended for blood smear preparation
Decomposes at high temperature (800C)

Potassium Oxalate
Used for BUN, HCO3, Cl, creatinine,

and glucose determination

Fluorides
Action

Better preservative than anticoagulant

Powerful enzyme poison

Dispensing

Preservative

2 mg/ml
Blood and CSF sugar determination

Anticoagulant
10 mg/ml

Defibrination

Equipment
Erlenmeyer flask
Glass beads or glass rod or sealed tube with 0.5 cm

fine glass rod or capillary tubes fused at one end

Applicator stick with 3-4 paper clips

Defibrination

Procedure
Glass rod is held in the flasks neck by a holed

stopper
Blood is delivered to the flask
Flask is rotated in a figure of eight motion for 5-10
minutes (fibrin will adhere to the beads or end of
the rod)

Defibrination

Uses

Good for WBC and RBC morphology,

buffy coat preparation

Gives high serum yield

Siliconized Glasswares
Prevents or reduces platelet loss
Glassware (clean and dry) is coated with

water-soluble silicon concentrate

Immerse in solution for 5 seconds or longer

Glassware is rinsed with water and dried

Room temperature for 24 hours or 1000C for 10

minutes