SPECIMEN COLLECTION

1.1 Introduction to Specimen Collection

Specimen collection is the first major step in the laboratory testing process. The quality of the laboratory result depends not only on the analytical method but also on the quality of the specimen submitted for examination.

The laboratory testing process can broadly be divided into:

Pre-analytical phase → Analytical phase → Post-analytical phase

Specimen collection is part of the pre-analytical phase.

Pre-analytical phase includes:

• Patient identification
• Test/request verification
• Patient preparation
• Selection of appropriate specimen
• Specimen collection
• Correct container and anticoagulant
• Labeling
• Handling
• Transportation
• Storage/preservation

An incorrectly collected or poorly handled specimen can produce an inaccurate result even when the laboratory instrument and analytical procedure are functioning correctly.

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1.2 General Principles of Blood Collection

Blood collection must be performed carefully to:

• Obtain a representative specimen
• Prevent contamination
• Prevent hemolysis
• Prevent clot formation when anticoagulated blood is required
• Maintain the correct blood-to-anticoagulant ratio
• Protect the patient and laboratory worker from infection
• Prevent needle-stick injuries
• Maintain specimen identity and traceability

Basic safety precautions

Laboratory personnel should:

• Perform hand hygiene before and after the procedure.
• Wear appropriate personal protective equipment (PPE), particularly gloves.
• Use sterile, appropriate needles and collection devices.
• Use clean, dry collection containers.
• Avoid unnecessary handling of blood.
• Dispose of needles and lancets immediately in approved sharps containers.
• Never recap a used needle.
• Treat all blood specimens as potentially infectious.
• Clean and disinfect contaminated surfaces appropriately.

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1.3 Patient Identification Before Collection

Correct patient identification is one of the most important steps in specimen collection.

Before collecting blood, the collector should:

1. Check the laboratory request form.
2. Confirm the patient's identity using appropriate identifiers.
3. Match the patient with the request form and specimen labels.
4. Confirm the requested investigations.
5. Determine the required specimen type and volume.
6. Check whether special preparation is required.
7. Ask about previous problems with blood collection, such as fainting or excessive bleeding.
8. Explain the procedure to the patient.

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1.4 Types of Blood Specimens

For hematological investigations, blood may be obtained as:

1. Capillary blood

Obtained by skin puncture.

2. Venous blood

Obtained by venepuncture.

3. Arterial blood

Obtained from an artery and used mainly for investigations such as blood gases rather than routine hematology.

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1.5 Capillary Blood Collection

Definition

Capillary blood is obtained by puncturing the skin and collecting blood from the small vessels in the capillary bed.

It is particularly useful when:

• Only a small volume of blood is required.
• The patient is an infant or young child.
• Venepuncture is difficult.
• Point-of-care or selected small-volume tests are required.

Examples include selected:

• Hemoglobin testing
• Some hematocrit/PCV measurements
• Blood film preparation
• Malaria parasite examination

Capillary blood is not suitable for every hematological investigation. 

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1.6 Sites for Capillary Blood Collection

Adults and children

The preferred site is generally the middle or ring finger.
The:

• Thumb should generally be avoided.
• Index finger should generally be avoided.

These fingers are more sensitive and may be subject to greater discomfort or frequent use.

Infants

The heel is used.
The puncture should be performed on an appropriate area of the medial or lateral plantar surface of the heel. The heel should not be punctured too deeply because of the risk of injury to underlying structures.

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1.7 Technique of Capillary Blood Collection

Step 1: Prepare the patient

Explain the procedure and position the patient comfortably.

Step 2: Warm the puncture site

A warm site promotes blood flow and reduces the need for excessive squeezing.

Step 3: Clean the site

Clean the area with an appropriate antiseptic, such as 70% alcohol, and allow it to dry.

Step 4: Perform the puncture

Use a sterile, single-use lancet. The puncture should be sufficiently deep to allow free-flowing blood.

Step 5: Remove the first drop

Wipe away the first drop with clean, dry gauze/cotton according to the laboratory procedure. The subsequent drops are used for testing.

Step 6: Collect the specimen

Collect the required amount without excessive squeezing.

Step 7: Stop bleeding

Apply clean, dry gauze to the puncture site until bleeding stops.

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Why should excessive squeezing be avoided?

Excessive pressure can cause tissue fluid to enter the specimen and dilute the blood.
This may produce:

Tissue-fluid contamination → altered cellular/chemical composition → inaccurate result

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1.8 Advantages of Capillary Blood

• Requires a small volume.
• Useful in infants and young children.
• Relatively simple procedure.
• Useful for selected point-of-care investigations.
• Particularly useful for preparing blood films in some settings.
• Useful when venous access is difficult.

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1.9 Limitations of Capillary Blood

Capillary collection has several disadvantages:

• Limited specimen volume.
• Excessive squeezing may cause tissue-fluid contamination.
• Blood may clot rapidly.
• Repeat testing may not be possible because of limited volume.
• It is unsuitable for many tests requiring larger volumes.
• Platelet distribution/clumping may affect some film-based assessments.

Capillary blood should be selected because it is appropriate for the test—not simply because it is easier to collect.

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1.10 Venous Blood Collection

Definition

Venous blood is blood obtained from a vein by venepuncture.
Venous blood is the preferred specimen for many hematological investigations because:

• A larger volume can be obtained.
• Multiple tests can be performed.
• Repeat testing may be possible.
• Appropriate anticoagulant tubes can be used.
• Standardized laboratory methods generally use venous blood.

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1.11 Common Venepuncture Sites

The preferred site is usually the antecubital region of the upper limb.
Common veins include:

• Median cubital vein
• Cephalic vein
• Basilic vein

The median cubital vein is commonly preferred because it is often relatively large and stable.
Other sites, such as veins on the dorsum of the hand, may be considered when appropriate.

Avoid:

• Sites with infection
• Significant hematoma
• Severe edema
• Burns
• Areas with vascular complications
• An arm with an unsuitable intravenous infusion, unless an approved institutional procedure specifically permits collection

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1.12 Basic Technique of Venepuncture

Step 1: Verify the request

Confirm:

• Patient identity
• Tests requested
• Specimen type
• Required volume
• Appropriate collection tube

Step 2: Position the patient

The patient should be comfortably seated or positioned appropriately. This reduces the risk of injury if the patient becomes faint.

Step 3: Apply the tourniquet

Apply the tourniquet sufficiently to make the vein prominent. Avoid prolonged venous stasis.

Step 4: Select the vein

Palpate the vein and select an appropriate vessel.

Step 5: Disinfect the site

Clean the puncture site using the approved antiseptic and allow it to dry. Do not touch the cleaned site unnecessarily.

Step 6: Perform venepuncture

Insert the needle with the bevel facing upward and obtain the required volume of blood.
Avoid:

• Excessive suction
• Excessive probing
• Repeated movement of the needle

Step 7: Release the tourniquet

Release the tourniquet at the appropriate stage, according to the collection procedure.

Step 8: Remove the needle

Remove the needle safely and apply pressure to the puncture site.

Step 9: Transfer the specimen

If using a syringe, blood should be transferred carefully into the appropriate labeled containers. Do not force blood through the needle into the tube, because this can contribute to hemolysis.

Step 10: Mix anticoagulated specimens

Anticoagulant-containing tubes should be mixed gently according to the laboratory's validated procedure.

Step 11: Label immediately

The specimen should be labeled correctly and promptly.

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1.13 Evacuated Blood Collection System

An evacuated tube system uses a tube containing a predetermined vacuum.
The vacuum draws blood into the tube when the needle enters the vein and the tube is connected to the collection system.

Advantages

• Standardized blood volume
• Appropriate blood-to-additive ratio
• Reduced blood exposure
• Convenient multiple-tube collection
• Improved specimen handling

Different tubes contain different additives depending on the requested test.

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1.14 Capillary vs Venous Blood

Feature	Capillary blood	Venous blood
Collection method	Skin puncture	Venepuncture
Volume	Small	Larger
Common use	Infants, small-volume tests	Most routine laboratory tests
Multiple tests	Limited	Usually suitable
Repeat testing	Limited	More feasible
Sampling error	Relatively greater	Generally lower when properly collected
Common site	Finger/heel	Antecubital veins
Major limitation	Small volume	Requires venepuncture

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1.15 Anticoagulants in Hematology

An anticoagulant is a substance that prevents blood from clotting.
The choice of anticoagulant depends on the investigation.
The major anticoagulants relevant to hematology are:

• EDTA
• Sodium citrate

Heparin is used for selected laboratory investigations but is generally not the routine anticoagulant for standard hematology testing.

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2.16 EDTA

EDTA = Ethylenediaminetetraacetic acid

EDTA prevents coagulation mainly by chelating calcium ions required for the coagulation process.

Common uses

EDTA-anticoagulated blood is commonly used for:

• Hemoglobin measurement

• Complete blood count (CBC)

• RBC count

• WBC count

• Platelet count

• Hematocrit/PCV

• Reticulocyte count

• Peripheral blood film morphology

Why is EDTA preferred?

It generally preserves blood-cell morphology better than many other anticoagulants when used at the correct concentration.

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2.17 Importance of Correct EDTA Concentration

The ratio between blood and EDTA is critical.

Excess EDTA can cause:

• RBC shrinkage

• Cellular degeneration

• Platelet changes

• Alteration of PCV

• Changes in calculated indices

Therefore:

The correct volume of blood must be added to an EDTA tube.

An underfilled EDTA tube may produce an excessive anticoagulant-to-blood ratio and compromise results.

Mixing

Immediately after collection, EDTA blood should be gently mixed according to the tube manufacturer's instructions.

It should not be vigorously shaken.

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2.18 Sodium Citrate

Sodium citrate also acts by binding calcium and is used particularly when preservation of the coagulation system is required.

Important uses

1. ESR

Citrated blood may be used for erythrocyte sedimentation rate testing according to the method employed.

2. Coagulation studies

Sodium citrate is the standard anticoagulant for many coagulation tests.

Important principle

For coagulation testing, the blood-to-citrate ratio must be carefully controlled.

A commonly used ratio is:

9 parts blood : 1 part sodium citrate

Incorrect filling of the tube can significantly affect coagulation results.

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2.19 Heparin

Heparin is an anticoagulant used for selected applications such as:

• Clinical chemistry

• Some blood gas investigations

• Immunophenotyping/flow cytometry in appropriate protocols

It is not generally the preferred anticoagulant for routine hematology morphology because it may cause cellular aggregation and can adversely affect blood-film appearance.

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2.20 Specimen Containers and Additives

Different specimens require different containers.

Specimen	Common additive/container	Major use
EDTA blood	EDTA tube	CBC and routine hematology
Citrated blood	Sodium citrate tube	Coagulation/selected ESR methods
Serum	Plain/clot-activator tube	Serology/chemistry
Heparinized blood	Heparin tube	Selected chemistry/other tests
Bone marrow aspirate	Appropriate anticoagulant when required	Morphology/ancillary studies
Bone marrow biopsy	Fixative	Histopathological examination

Always follow the laboratory's validated SOP and the manufacturer's instructions for the collection tube.

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2.21 Bone Marrow Specimen Collection

Definition

Bone marrow is the specialized tissue responsible for the production of blood cells.

Examination of bone marrow provides direct information about:

• Hematopoietic cell populations
• Cell maturation
• Marrow cellularity
• Abnormal infiltrates
• Certain malignant and non-malignant disorders

Bone marrow examination is particularly important in the investigation of selected hematological disorders, including suspected leukemia and marrow failure.

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2.22 Methods of Bone Marrow Examination

There are two major specimen approaches:

1. Bone marrow aspiration

A needle is used to aspirate liquid marrow.

The aspirated material is used primarily for:

• Cytomorphological examination
• Cytochemistry
• Immunophenotyping
• Cytogenetic studies
• Molecular investigations
• Selected microbiological studies

2. Bone marrow trephine biopsy

A core of bone and marrow is obtained using a biopsy needle.

It provides information about:

• Marrow architecture
• Cell distribution
• Overall cellularity
• Fibrosis
• Infiltration
• Focal lesions

Key difference

Aspiration → individual cell morphology

Trephine biopsy → tissue architecture and spatial distribution

Both may be required because they provide complementary information.

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2.23 Indications for Bone Marrow Examination

Bone marrow examination may be performed for diagnostic, staging, or treatment-response assessment.

Examples include investigation of:

• Acute leukemias
• Aplastic anemia
• Megaloblastic anemia in selected cases
• Unexplained cytopenias
• Plasma-cell disorders such as multiple myeloma
• Myelofibrosis
• Suspected marrow infiltration/metastasis
• Selected storage disorders
• Selected infections
• Assessment of treatment response
• Cytogenetic or molecular investigation of hematological disease

The exact indication should be determined by the treating physician/hematologist.

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2.24 Sites for Bone Marrow Aspiration

The site depends on:

• Age
• Patient anatomy
• Clinical indication
• Disease distribution
• Operator expertise

Common sites

1. Posterior superior iliac crest

This is the major preferred site for many patients.

Advantages include:

• Good marrow yield
• Relatively safe access
• Suitable for aspiration
• Suitable for trephine biopsy
• Can often be used for repeated sampling

2. Sternum

Sternal aspiration may be used in selected adults by appropriately trained specialists.
Because of the proximity of vital structures, this procedure requires strict technique and appropriate equipment.

3. Other sites

Other sites have historically been described, but their use depends on patient age, clinical circumstances, and institutional practice.

Important safety point

Bone marrow aspiration and biopsy are invasive medical procedures and should only be performed by appropriately trained and authorized healthcare professionals.

MLT personnel generally support the procedure by preparing equipment, receiving and processing specimens, preparing slides, and performing laboratory examination according to their scope of practice.

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2.25 Bone Marrow Aspirate: Laboratory Role

The laboratory must be prepared before the procedure.

Typical materials include:

• Clean labeled glass slides
• Appropriate specimen containers
• EDTA tube when required
• Sterile gauze
• Appropriate PPE
• Specimen labels
• Materials for immediate smear preparation
• Containers for ancillary investigations

Critical laboratory principle

Bone marrow aspirate should be processed immediately because it can clot rapidly.

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2.26 Preparation of Bone Marrow Smears

Immediately after obtaining the aspirate:

1. Place an appropriate amount of marrow on a clean slide.

2. Identify marrow particles/fragments.

3. Prepare smears using an appropriate spreading technique.

4. Prepare multiple slides when required.

5. Allow smears to dry appropriately.

6. Label slides correctly.

7. Send specimens for requested ancillary studies.

Why must smears be prepared immediately?

Bone marrow contains a high concentration of cells and may clot rapidly.

Delay → clot formation → poor cellular distribution → compromised morphology

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2.27 Bone Marrow Trephine Biopsy

A trephine biopsy provides a cylindrical core of bone containing marrow.

The specimen is placed in an appropriate fixative and transported to the histopathology laboratory.

Major uses

Trephine biopsy is particularly useful when evaluation of:

• Marrow architecture

• Cellularity

• Fibrosis

• Infiltration

• Focal lesions

is required.

It is also useful when aspiration produces a dry tap.

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2.28 Aspiration vs Trephine Biopsy

Feature	Bone marrow aspiration	Trephine biopsy
Specimen	Liquid marrow	Tissue core
Main information	Individual cell morphology	Marrow architecture
Cell morphology	Excellent	More limited
Cell distribution	Limited	Excellent
Cellularity	Estimated	Better assessed
Fibrosis	Limited assessment	Better assessed
Cytochemistry	Applicable	Usually not primary method
Immunophenotyping	Applicable	Selected applications
Cytogenetics/molecular tests	Applicable	Selected applications
Histology	No	Yes

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2.29 Specimen Handling

Specimen handling begins immediately after collection.

The major principles are:

1. Correct identification

Every specimen must be traceable to the correct patient.

2. Correct container

Use the container appropriate for the requested test.

3. Correct volume

The required volume should be collected.

4. Correct additive

Use the appropriate anticoagulant or preservative.

5. Proper mixing

Anticoagulated specimens should be gently mixed immediately after collection.

6. Avoid contamination

Specimens should not be contaminated with:

• Tissue fluid

• Water

• Disinfectants

• Other specimens

• Incorrect additives

7. Minimize delay

Specimens should reach the laboratory section and be processed within the validated stability period.

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2.30 Specimen Labeling

Specimen labeling is a critical patient-safety procedure.

A specimen label should contain information according to institutional policy, commonly including:

• Patient name/identifier

• Unique laboratory or hospital number

• Specimen type where required

• Date and time of collection

• Collector identification where required

Important principle

Label the specimen immediately after collection and before moving to the next patient.

Never rely on memory.

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2.31 Specimen Transportation

Transportation should preserve the integrity of the specimen.

Important factors include:

• Time

• Temperature

• Light exposure where relevant

• Physical protection

• Leakage prevention

• Correct packaging

• Biosafety

General principle

Transport conditions must be appropriate for the specimen and test.

Blood specimens should be protected from unnecessary exposure to:

• Excessive heat

• Freezing

• Direct sunlight

• Excessive agitation

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2.32 Specimen Preservation

Preservation refers to maintaining specimen integrity between collection and analysis.

Different specimens have different stability requirements.

For example:

EDTA whole blood

For hematological testing, testing should generally occur as soon as possible.

If immediate testing is not possible, storage conditions should follow the validated laboratory procedure and analyzer/test manufacturer's requirements.

Citrated blood

Some coagulation tests have relatively short stability periods and should be processed promptly.

Serum

Serum may require separation from cells before storage.

Depending on the investigation, serum may be refrigerated or frozen.

Bone marrow

Bone marrow aspirate smears should be prepared promptly.

Trephine biopsy tissue should be placed promptly in an appropriate fixative.

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2.33 Effect of Storage on Blood Cells

Blood cells undergo progressive changes after collection.

Prolonged storage can produce morphological alterations such as:

• Neutrophil degeneration

• Nuclear changes

• Cytoplasmic vacuolation

• RBC crenation

• Altered RBC morphology

• Platelet deterioration

Therefore:

Longer storage → greater cellular alteration → potentially unreliable morphology

This is particularly important when preparing and interpreting peripheral blood films.

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2.34 Avoiding Hemolysis

Hemolysis is the rupture of RBCs with release of intracellular contents into plasma or serum.

Hemolysis may occur during collection, transportation, or processing.

Common causes

• Excessive suction

• Very rapid blood withdrawal

• Excessive needle movement

• Inappropriate needle size

• Wet collection equipment

• Vigorous shaking

• Forcing blood through a needle

• Rough specimen handling

• Excessive mechanical trauma

Prevention

• Use appropriate collection equipment.

• Ensure equipment is clean and dry.

• Avoid excessive suction.

• Allow blood to flow appropriately.

• Avoid vigorous shaking.

• Mix tubes gently.

• Handle specimens carefully.

• Use appropriate centrifugation procedures.

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2.35 Common Pre-analytical Errors

Error	Possible consequence
Wrong patient identification	Result assigned to wrong patient
Wrong tube	Invalid/inaccurate result
Insufficient blood	Incorrect blood-to-additive ratio
Excess EDTA	Cellular and index alterations
Clotted EDTA specimen	Invalid cell counts
Hemolysis	Analytical interference and altered results
Excessive tourniquet time	Hemoconcentration
Excessive squeezing during capillary collection	Tissue-fluid contamination
Delayed processing	Cellular deterioration
Incorrect storage temperature	Specimen instability
Poor labeling	Identification/traceability failure
Vigorous shaking	Hemolysis/cellular damage
Inadequate mixing	Microclot formation or uneven distribution
Wrong anticoagulant	Test may be invalid
Underfilled citrate tube	Incorrect coagulation results

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2.36 Blood Specimen Rejection

A laboratory may reject a specimen when it does not meet acceptance criteria.

Common reasons include:

• Unlabeled specimen
• Mislabeled specimen
• Insufficient volume
• Wrong container
• Wrong anticoagulant
• Clotted anticoagulated blood
• Gross hemolysis where the test is affected
• Leakage or contamination
• Improper transportation
• Excessive delay
• Inappropriate storage
• Incorrect patient identification

Important principle

Specimen rejection is not merely an administrative decision.

It is a quality and patient-safety measure.

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2.37 Special Considerations for Blood Culture

Although blood culture belongs primarily to microbiology, students should understand the basic collection principle because contamination can significantly affect diagnosis.

Blood culture collection requires:

• Strict aseptic technique
• Appropriate skin antisepsis
• Correct culture bottles
• Appropriate blood volume
• Correct timing when clinically indicated
• Proper labeling
• Prompt transportation/incubation according to laboratory protocol

Key concept

Poor aseptic technique → contamination → false-positive culture → inappropriate antimicrobial treatment.

The exact antiseptic agent, collection volume, number of sets, and timing should follow the current institutional SOP and microbiology protocol.

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2.38 Specimen Collection and the Laboratory Quality System

Specimen collection is part of quality assurance.

A useful model is:

Patient → Identification → Collection → Labeling → Transportation → Processing → Analysis → Reporting

An error at the beginning of this chain can affect every subsequent step.

Three major quality stages

Phase	Examples
Pre-analytical	Collection, labeling, transportation
Analytical	Testing and measurement
Post-analytical	Validation, interpretation, reporting

For MLT students, mastering specimen collection is therefore not simply a phlebotomy skill—it is a fundamental laboratory quality and patient-safety competency.

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1.39 Practical Checklist for Blood Collection

Before collection:

☐ Verify patient identity
☐ Verify request form
☐ Confirm required tests
☐ Select appropriate specimen container
☐ Check required volume
☐ Perform hand hygiene
☐ Wear PPE
☐ Prepare collection equipment

During collection:

☐ Select appropriate site
☐ Apply appropriate antisepsis
☐ Perform venepuncture/skin puncture correctly
☐ Avoid excessive suction
☐ Collect sufficient volume
☐ Release tourniquet appropriately
☐ Apply pressure after needle removal

After collection:

☐ Mix anticoagulated specimen gently
☐ Label immediately
☐ Inspect specimen for clot/hemolysis/leakage
☐ Dispose of sharps safely
☐ Transport promptly
☐ Store under appropriate conditions

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1.40 Key Differences: Blood vs Bone Marrow Collection

Feature	Blood specimen	Bone marrow specimen
Collection	Skin puncture/venepuncture	Aspiration/trephine biopsy
Invasiveness	Relatively low	Invasive
Common specimen	Whole blood/plasma/serum	Marrow aspirate/core
Main laboratory use	Routine hematology and other tests	Investigation of marrow disorders
Processing	Tube-based or direct testing	Smears, anticoagulated material, tissue fixation
Major concern	Hemolysis, clotting, wrong tube, delay	Clotting, inadequate sample, poor smear, inappropriate fixation
Personnel	Trained phlebotomy/clinical staff	Authorized trained clinician
Laboratory role	Collection and analysis	Specimen preparation, processing and examination