Sample Collection_ADT

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.