Sample Collection, Preservation and Its Estimation

• Instructor: Dr. Shashank Kamble

Introduction

• Quality laboratory results begin with correct and complete sample collection.

• Examples of samples include:

  • Blood

  • Sputum

  • Urine

  • Feces

  • Saliva

  • Solid tissues

  • Other body fluids

Broad Objective

• Ensure correct collection of specimens.

• Properly place the specimen in the right container.

• Transport the specimen to the laboratory correctly and timely.

Aims of Sample Collection

• Accuracy: Ensure analytical results represent actual analyte concentration in patient to reflect physiological/pathological state.

• Safety: Minimize patient discomfort and complications.

• Avoid Recollection: Efficient sample collection to prevent the need for repeat sampling.

Example Biological Samples

• Blood (venous, arterial, capillary - whole blood, serum, plasma)

• Urine

• Feces

• Saliva

• Solid tissues

• Amniotic fluid

• Cerebrospinal fluid

• Synovial fluid

• Peritoneal fluid

• Pleural fluid

• Pericardial fluid

Blood Sample Collection

• To collect large amounts of blood, use an evacuated tube system with interchangeable glass tubes to avoid multiple venepunctures.

• Evacuated tubes are pre-prepared either with or without additives and possess sufficient vacuum to draw a predetermined blood volume.

Blood Collection Equipment

• Sterile needles

• Sterile syringes/Plain vacutainer

• Blood tubes

• Alcohol pads

• Tourniquet

Types of Blood Collection

Capillary Blood

• Most frequently obtained from a finger or thumb.

Venous Blood

• Typically collected from a vein in the front elbow or forearm.

Arterial Blood

• Commonly obtained from radial, brachial, or femoral artery.

• Less frequently examined; used for blood gas determinations.

• Note: Capillary blood is similar in composition to arterial blood.

Anatomy of Veins and Arteries

• Cephalic vein

• Brachial artery

• Basilic vein

• Median cubital vein

• Ulnar artery

• Radial artery

• Deep palmar arch

Blood Collection Methods

• Venipuncture: Procedure that involves inserting a needle into a vein, typically for laboratory testing.

  • Steps:

    • Clean area with antiseptic.

    • Apply pressure with an elastic band.

    • Insert needle, collect blood into a vial/syringe, remove needle, apply pressure.

• Capillary Puncture: Sharp tool (lancet) used to puncture skin and collect blood; useful for infants and young children.

• Arterial Sampling: Direct puncture of an artery for blood gas collection.

Fingerstick Sampling

• Minimally invasive and quick collection method from the fingertip.

• Reduces stress and anxiety, especially in children.

• Limited blood loss with the possibility of at-home testing and no need for a phlebotomist.

Anticoagulants

General Information

• Plasma/whole blood requires anticoagulant to prevent coagulation.

Types of Anticoagulants

1. Heparin

   • Inhibits thrombin formation.

   • Does not alter red cell volume or subsequent determinations.

   • Quantity: 2 mg/10 mL blood.

2. EDTA (Ethylenediaminetetraacetic acid)

   • Chelates calcium ions preventing coagulation.

   • Quantity: 20 mg/10 mL blood.

   • Suitable for DNA assays but problematic for cytogenetic analysis.

3. Oxalates (Potassium oxalate)

   • Precipitates calcium ions; most commonly used oxalate.

   • Quantity: 30 mg/10 mL blood.

4. Sodium Citrate

   • Converts calcium into non-ionized form.

   • Quantity: 30 mg/10 mL blood.

   • Note: Citrated plasma not suitable for calcium estimation.

5. Sodium Fluoride

   • Acts as an anticoagulant and preservative in blood glucose estimation.

   • Prevents red cell metabolism and has antibacterial properties.

   • Quantity: 10 mg/1 mL blood; used with oxalate for glucose estimation (3:1 mixture).

Blood Collection Tubes Color-Coding

• Red-Top Tubes: No additives; used for serum samples.

• Lavender-Top Tubes: Contain EDTA; used for complete blood cell counts.

• Green-Top Tubes: Contain heparin.

• Blue-Top Tubes: Contain sodium citrate.

• Black-Top Tubes: Contain sodium oxalate.

• Yellow-Top Tubes: Contain acid-citrate-dextrose (ACD) solution.

• Grey-Top Tubes: Contain a glycolytic inhibitor.

Components from Blood Collection

• From 10 mL of blood:

  • Plasma or serum: 6-7 mL

  • Lymphocytes and mononuclear cells: 10-20 x 10^6 cells/mL

  • Erythrocytes: 5 x 10^6 cells/mL; 10-15 mg HB

Changes in Blood upon Storage

• Loss of CO2

• Conversion of glucose to lactate

• Increase in plasma inorganic phosphate

• Formation of ammonia from nitrogenous substances

• Conversion of pyruvate to lactate

• Material passage through red cell envelope

Types of Blood for Testing

• Whole Blood: Used for ammonia, hemoglobin, lactate, and pH measurement.

• Plasma: Extracted after mixing blood with anticoagulants; used for ascorbic acid, bicarbonate, glucose, chloride, and fibrinogen estimation.

• Serum: Liquid left after coagulation; used for protein, albumin, bilirubin, and cholesterol estimation.

Blood Culture Requirements

• Venous blood volume: 0.5 – 2 mL for infants, 2-5 mL for children, 5-10 mL for adults.

• Timing: As early as possible, before starting antibiotics.

• Transport: In blood culture bottles (with glucose broth or bile salt broth) at ambient temperature.

DNA Extraction Sources

• Possible sources from blood include RBC, leukocytes, serum, plasma, and blood clots.

Serum Fatty Acid Processing

• Measure within 2 weeks at 4°C, a few months at –20°C, and within a year at –80°C.

Sample Storage Considerations

• Critical to maintain records of identity, storage history, temperature fluctuations, and effects of storage duration.

Urine Collection Overview

• Non-invasive and easily obtainable; evaluates body metabolic processes, exposure to agents.

• Collection type depends on tests.

Types of Urine for Collection

1. First Morning Urine: Collected after sleeping; must be preserved if not delivered within 2 hours.

2. Random Urine: Any time collection, suitable for routine screening.

3. Fractional Collection: Discard first morning urine, then collect second for concentration comparison.

4. Timed Collection: Over 12-24 hours for day-to-day comparison.

Urine Collection Practices

• Use clean containers (50-3000 mL); preservatives may be needed; must record total volume, mix well, and create aliquots.

Tissue Collections

• Confirm clinical diagnosis through histological analysis; examine tumor characteristics.

• Collect more material than for pathological evaluation; include both tumor and normal tissues.

Tissue Storage Methods

• Formalin-fixed paraffin-embedded specimens and frozen tissues at –70°C.

Adipose Tissue Analysis

• Feasible and low-risk collection, provides long-term dietary intake reflections.

Exhaled Air Collection

• Evaluate exposure to solvents; source for susceptibility markers (e.g., caffeine breath test).

Hair Analysis

• Easy to collect; reflects disease conditions and provides a permanent record of trace elements.

Nail Clippings

• Simple to collect; minimal processing required; reflects trace elements and dietary intake.

Buccal Cell Collection

• Non-invasive method for PCR analysis, measures mutations.

Saliva Collection and Analysis

• Efficient source for biological material; useful for endogenous and xenobiotic compounds.

• Contains organic (mucin, amylase) and inorganic constituents.

Saliva Collection Technique

• Mouth wash and gargle to get rid of food particles prior to collection.

Tests for Saliva Analysis

• Various tests conducted to identify amylase, mucin, calcium, and inorganic phosphate.

Sample Measurements

• Evaluate levels of corticosteroids, antibodies (e.g., HIV-1), and cotinine.

Feces Analysis

• Examine infectious markers and oncogenes.

Semen Collection Considerations

• Evaluate endocrine/reproductive factors; sexual abstinence required.

• Should reach the lab within one hour.

Temperature Requirements for Specimen Storage

• Maintain optimal temperatures for specimens:

  • -20°C: Urine

  • -70°C: DNA, Serum, Hormones

  • -120°C: Hormones, carotenoids

Storage Protocols

• Freezers require monitoring systems for alerts and backup equipment in case of failures.

Sample Shipping Requirements

• Depends on time, distance, climate, transport method, and type of specimen.

• Use polyurethane boxes with dry ice or liquid nitrogen for low-temp requirements.

Swab Collection Techniques

1. Throat Swab: Techniques and locations for collection.

2. Nasopharyngeal Swab: Proper technique for sample retrieval.

3. Rectal Swab: Convenient for certain patients; limitations noted.

References

• Laboratory practical for practical biochemistry – 2nd edition – Shivaraja Shankara YM.

• Practical Haematology by Dacie, Lewis.

• Textbook of Microbiology – Ananthanarayan and Paniker’s (8th edition).

• Textbook of Medical Biochemistry – 8th edition - Chatterjee.

• Internet.

Acknowledgments

• Thank You.