Specimen Preservation: Fixation, Stabilisation, Storage, Temperature and Quality Control
Specimen preservation is the deliberate use of time, temperature, chemical fixatives, anticoagulants, transport media and other controls to keep a patient specimen as close as possible to its biological state until the requested examination is completed. Preservation is part of the pre-analytical phase; a perfectly performed assay cannot repair a specimen that was allowed to clot, haemolyse, dry, warm, freeze, autolyse, grow contaminating organisms or lose its cellular and molecular targets.
Clinical safety principle: preservation is always test-specific. The correct condition for a culture may be harmful for a chemistry, cytology, coagulation, molecular or histology test. When a local laboratory SOP, referral laboratory or kit insert gives a different validated condition, follow that instruction, document it and do not improvise.
Learning outcomes
By the end of this lesson, a student should be able to:
- Define preservation and distinguish it from collection, transport, storage, fixation, stabilisation and processing.
- Choose an appropriate container, additive, preservative, temperature and maximum delay for common clinical specimens.
- Explain why blood, urine, stool, respiratory specimens, CSF, body fluids, tissue and cytology require different handling.
- Recognise when refrigeration, freezing, light protection, anticoagulation or immediate processing is required—and when it is contraindicated.
- Identify compromised specimens, record the effect on results, communicate with the clinician and apply laboratory acceptance/rejection policy.
1. Where preservation fits in the laboratory pathway
| Stage | What must be controlled | Typical failure | Clinical consequence |
|---|---|---|---|
| Clinical request | Correct test, indication, specimen, timing and urgency | “Blood test” without a defined test or timing | Wrong container or un-interpretable result |
| Collection | Patient identity, asepsis, volume, tube order, site and timing | Underfilled citrate tube; contaminated culture; wrong tube | Clotting error, false-positive culture or rejection |
| Initial preservation | Correct additive/fixative, prompt mixing, protection from light or evaporation | EDTA tube not mixed; tissue left dry; bilirubin exposed to light | Clots, autolysis, analyte loss or poor morphology |
| Transport | Sealed secondary packaging, temperature, time, orientation and biosafety | Leaking package, prolonged heat or inappropriate ice | Danger to staff and unreliable results |
| Receipt and processing | Identification, integrity, volume, time, condition and suitability for each test | Unlabelled or haemolysed specimen accepted without comment | Result assigned to wrong patient or analytically biased |
| Short/long-term storage | Validated retention temperature, aliquots, monitoring and traceability | Repeated freeze–thaw or unrecorded freezer failure | Loss of diagnostic material and inability to verify a result |
The World Health Organization quality approach requires each laboratory to define written, test-specific instructions for collection, preservation, transport, acceptance, storage and disposal. A temperature label such as “keep cold” is not enough: the SOP should state the permitted range, time window, container, additive and action after a deviation.
2. Key preservation concepts
| Term | Meaning | Examples | Important caution |
|---|---|---|---|
| Fixation | Irreversible chemical or physical preservation of cells/tissue, stopping autolysis and maintaining morphology. | 10% neutral buffered formalin for routine histology; alcohol fixation for some cytology; glutaraldehyde for electron microscopy. | Fixation prevents many downstream tests. Never place tissue intended for culture, flow cytometry, cytogenetics or many molecular assays in formalin unless the assay is validated for fixed material. |
| Stabilisation | Slowing or preventing change in an analyte, nucleic acid, cell or organism without necessarily fixing it. | Fluoride-containing tube for glycolysis inhibition; RNA stabilisation reagent; validated urine preservative. | Stabilisers are not interchangeable and can interfere with other assays. |
| Anticoagulation | Preventing clot formation so plasma, whole blood or cells remain available. | EDTA for CBC; sodium citrate for coagulation; heparin for many plasma chemistry tests. | The anticoagulant-to-blood ratio and correct mixing are essential; a clotted sample is not repaired by remixture. |
| Transport medium | Maintaining viability or nucleic-acid integrity during transit while limiting overgrowth or desiccation. | Cary–Blair for enteric bacteria; viral/UTM medium for respiratory swabs; Amies medium for selected bacterial swabs. | Transport medium is selected for the organism and test. It is not a general-purpose preservative. |
| Cryopreservation | Freezing at a validated temperature to slow degradation for later testing. | Aliquoted serum/plasma, selected molecular specimens, viable cells with a cryoprotectant. | Freeze–thaw cycles, frost-free freezers and poorly controlled thawing can destroy analytes or cells. |
| Storage/retention | Keeping a processed or unprocessed specimen for a defined period under monitored conditions. | Retained serum, paraffin blocks, slides, residual urine or microbiology isolates. | Retention duration is a local regulatory/SOP decision; it depends on specimen type, test, risk and clinical need. |
3. Universal rules before preserving any specimen
- Confirm the test menu and urgency. Ask which discipline will examine it—chemistry, haematology, coagulation, microbiology, histopathology, cytology, immunology or molecular diagnostics. The same specimen may need separate portions.
- Identify the patient at the bedside. Use the required identifiers, label immediately and record collection date/time, collector, source/site, relevant treatment and time-sensitive clinical information.
- Use the validated container. Check tube colour, additive, expiry, sterility, volume, lot where required and compatibility with the analyzer or referral laboratory.
- Preserve promptly. Mix additive tubes by gentle inversion as specified; do not shake. Start fixation, cooling, light protection or transport-media placement at once.
- Protect specimen quality. Prevent haemolysis, clotting, evaporation, desiccation, contamination, dilution, exposure to oxygen or light, and contact with unsuitable surfaces.
- Separate when necessary. Centrifuge and aliquot serum/plasma only according to the validated SOP. Use clean, leak-proof, labelled aliquots to avoid repeated opening and freeze–thaw.
- Document deviations. If time, temperature, volume, container or integrity is outside limits, do not silently proceed. Consult the laboratory, record the limitation, and recollect when a valid result is not possible.
- Preserve safety as well as accuracy. Treat all unfixed specimens as potentially infectious. Use leak-proof primary and secondary packaging, absorbent material and appropriate PPE.
Do not overgeneralise “refrigerate.” Refrigeration slows many chemical reactions but can damage cells, precipitate proteins, alter platelet function, reduce recovery of some organisms or change pH. A specimen for culture, CSF cell count, blood gas, cryoprotein, cold agglutinin or a specialised molecular assay may need a different condition.
4. Blood specimens
4.1 Whole blood and anticoagulant selection
| Specimen/handling | Common use | Preservation logic | Errors to avoid |
|---|---|---|---|
| EDTA whole blood (lavender/purple) | Full blood count, blood film, reticulocytes, HbA1c and selected molecular/flow tests | EDTA chelates calcium and preserves cellular morphology for a limited, test-specific period. Mix gently immediately after collection. | Clots from inadequate mixing; EDTA contamination of chemistry; excessive delay causing cell swelling, platelet clumping and morphology changes. |
| Sodium citrate whole blood (light blue) | PT/INR, aPTT, fibrinogen, D-dimer and specialised coagulation | Citrate reversibly binds calcium. Fill to the indicated mark to maintain the required blood:citrate ratio; gently invert. | Underfilling, high haematocrit without adjustment, clotted sample, traumatic draw or heparin contamination. Do not transfer blood between tubes. |
| Heparinised blood/plasma (green) | Many plasma chemistry tests, blood gases in validated systems, selected cytogenetic/flow applications | Heparin inhibits thrombin; the exact salt and tube type must match the assay. | Using lithium heparin for an assay affected by lithium; clotting from poor mixing; gel or separator incompatibility. |
| Fluoride/oxalate or other glycolysis-inhibiting tube | Glucose or lactate in settings where validated | Inhibits glycolysis after collection; it does not replace prompt separation when the method requires it. | Assuming every fluoride tube immediately stops glycolysis; using it for tests for which the additive interferes. |
| Serum | Biochemistry, serology, endocrine and immunology tests | Allow the required clotting time, then separate promptly according to the laboratory SOP. Keep capped and avoid prolonged contact with cells or fibrin. | Centrifuging before clot formation; haemolysis; fibrin strands; storing whole blood when serum was required. |
4.2 Serum and plasma preservation workflow
- Collect into the correct tube and gently invert additive tubes.
- For serum, allow clot formation according to the tube and local SOP; do not force clotting by shaking or heating.
- Centrifuge using the validated time, relative centrifugal force and balancing procedure.
- Inspect for haemolysis, icterus, lipaemia, fibrin, insufficient volume and wrong tube.
- Aliquot separated serum/plasma into labelled, leak-proof tubes if testing is delayed or multiple departments will test it.
- Use the laboratory’s validated short-term refrigeration and long-term freezing conditions. Record freezer identification and temperature excursions.
Many routine chemistry samples tolerate a limited period at controlled room temperature or refrigeration, but stability differs by analyte. Potassium, phosphate, glucose, lactate, bilirubin, enzymes, hormones, drugs and antibodies do not all behave the same way. A result must never be labelled “stable” without reference to the method, specimen type and validated time.
4.3 Time- and condition-sensitive blood tests
| Test/specimen | Preservation priority | Why mistakes matter |
|---|---|---|
| Blood gas | Analyse immediately in a suitable heparinised syringe; eliminate air bubbles and avoid delays. Cooling, if used, must follow the analyzer/laboratory SOP. | Cells continue consuming oxygen and producing carbon dioxide; pH and gas tensions change. Air contamination drives values toward room-air equilibrium. |
| Blood culture | Use sterile culture bottles, correct blood volume and aseptic skin preparation. Inoculated bottles are generally transported promptly and are not routinely refrigerated. | Small volume lowers sensitivity; contamination causes false bacteraemia; inappropriate temperature can reduce recovery. |
| Ammonia, lactate and selected metabolic tests | Rapid transport and method-specific temperature/light requirements; contact the laboratory before collection where possible. | Cellular metabolism changes concentration quickly, leading to dangerous over- or under-estimation. |
| Bilirubin, porphyrins and light-sensitive analytes | Protect from light using an approved amber tube, foil or light-protective bag as specified. | Photodegradation can produce a falsely low result. |
| Cold agglutinins/cryoproteins | Collection and transport may require warm handling until serum separation, followed by a validated cold phase. | Cooling the whole blood too early can precipitate proteins or cause cell agglutination before the test is performed. |
| Flow cytometry and cellular assays | Use the requested anticoagulant, adequate cell count and rapid delivery; follow the receiving laboratory’s stability window. | Cell death, antigen loss and altered viability can make an immunophenotype uninterpretable. |
Blood case
A citrate tube for an aPTT is filled only halfway and arrives after a long delay. The patient is receiving heparin. The problem is not solved by adding saline or transferring blood: the citrate:blood ratio is wrong and the delay may alter the sample. Contact the laboratory, document the rejection/limitation and recollect in a correctly filled tube when clinically safe.
5. Urine preservation
Urine is biologically active. Cells lyse, casts disintegrate, bacteria multiply, glucose is consumed, ketones evaporate or disappear, bilirubin degrades and pH changes when a specimen stands at room temperature. The correct preservation depends on whether the request is urinalysis, microscopy, culture, timed chemistry, toxicology or molecular testing.
| Urine request | Preferred preservation approach | Key cautions |
|---|---|---|
| Routine urinalysis and microscopy | Examine promptly. If delayed, use the laboratory’s validated refrigeration and return the sample to an acceptable testing condition before analysis. | Refrigeration may cause crystals or precipitates; do not interpret a cold, visibly precipitated sample without considering this effect. |
| Urine culture | Send promptly in a sterile container. Where immediate processing is impossible, use a validated refrigerated interval or boric-acid preservative tube according to the microbiology SOP and manufacturer’s fill line. | Boric acid is not suitable for every assay and may inhibit or alter organisms if the tube is under- or over-filled. Do not use preservative tubes for routine chemistry unless validated. |
| Timed urine (for example, 24-hour collection) | Use the preservative specified for the analyte, a clean collection container, complete start/finish times and keep under the required temperature conditions. | Missed urine, wrong start time, evaporation, contamination or unrecorded preservative makes the total excretion calculation invalid. |
| Urine cytology | Send fresh promptly or place in the cytology preservative recommended by the receiving laboratory. | Cells degenerate rapidly; a preservative suitable for cytology may be unsuitable for culture or molecular testing. |
| Urine for molecular assays | Use the collection tube and stabiliser specified by the assay; avoid repeated freeze–thaw and send with complete test information. | Inhibitors, dilution and wrong preservative can cause invalid or false-negative amplification. |
Practical urine checklist
- Use a clean, dry, leak-proof, correctly labelled container.
- Record whether the specimen is midstream, catheter, suprapubic, first morning or timed.
- Do not add household chemicals or unvalidated preservatives.
- Separate aliquots for culture, chemistry, microscopy and cytology when requested.
- Report delay, refrigeration or preservative use to the laboratory.
6. Stool preservation
Stool contains enzymes and a large microbial population; protozoal trophozoites degenerate and bacterial composition changes quickly. The preservative is determined by the target: enteric culture, ova and parasites, toxin/antigen, molecular pathogen panel or inflammatory marker.
| Investigation | Preservation/transport | What must not be done |
|---|---|---|
| Routine bacterial culture | Send a fresh stool specimen rapidly. If delay is unavoidable, use an approved enteric transport medium such as Cary–Blair according to the receiving laboratory. | Do not put a culture specimen into formalin or PVA; preservatives can kill or inhibit organisms and invalidate culture. |
| Ova and parasite examination | Examine fresh within the validated window or place portions in complementary preservatives such as 10% formalin and a suitable permanent-stain preservative (for example PVA/SAF) according to the parasitology SOP. CDC guidance notes that preservation is needed when examination cannot occur promptly and that formalin and PVA have complementary advantages. | Prevent urine, water, soil and disinfectant contamination. Do not assume one preservative detects every parasite or replaces concentration and staining. |
| Viral, toxin, antigen or multiplex molecular testing | Use the kit-specific sterile container, transport temperature and permitted delay. Some assays require fresh/refrigerated stool; others use a dedicated collection medium. | Do not transfer stool into a preservative without checking the assay validation. |
| Faecal calprotectin and other chemistry | Use the manufacturer’s container and validated temperature/time limits. | Prolonged heat, dilution, wrong container or stool mixed with urine may alter the result. |
When parasitology is requested and delay is expected, divide the stool before preservation: one portion for methods requiring fresh material and one in the specified preservative. Label each portion with the preservative and test.
7. Respiratory specimens and swabs
| Specimen | Preservation strategy | High-yield pitfalls |
|---|---|---|
| Sputum for bacterial culture | Collect deep lower-respiratory sputum, not saliva, into a sterile wide-mouth container; transport promptly under the laboratory’s validated conditions. | Salivary contamination, leaking container and delayed processing cause misleading flora and poor recovery. |
| Specimen for tuberculosis testing | Use the programme/laboratory container and follow its instructions for smear, culture and NAAT. Protect from leakage and deliver promptly. | Do not add formalin to a specimen intended for culture or molecular TB testing. Follow local biosafety and referral requirements. |
| Nasopharyngeal, oropharyngeal or other viral swab | Use the synthetic swab and viral/UTM medium specified by the assay; maintain the validated temperature and time window. | Cotton or calcium-alginate swabs and dry transport may inhibit some molecular or viral tests. |
| Bacterial wound/throat swab | Use an appropriate bacterial transport system (for example Amies-based medium where specified) and record the anatomical site. | A swab without site information cannot be interpreted reliably; transport medium for viruses is not automatically suitable for bacteria. |
| Bronchoalveolar lavage or aspirate | Use sterile, leak-proof container(s); divide for bacterial, fungal, mycobacterial, cytology and molecular studies before adding any test-specific preservative. | One preservative or one container cannot serve all departments. Communicate with the laboratory before collection for complex cases. |
8. Cerebrospinal fluid (CSF)
CSF is a low-volume, high-priority specimen. Cells lyse rapidly and organisms may be few; therefore collection tubes, volume allocation, urgency and temperature must be coordinated with the laboratory and clinical team.
Core rules
- Label tubes in collection order and record the tube used for each test according to local policy. Commonly, separate portions are allocated for chemistry, microbiology, cell count and special studies, but local order may differ.
- Send CSF immediately by hand or approved urgent transport. Never leave it on a ward bench or in a pneumatic system if local policy prohibits this.
- For suspected bacterial meningitis, prioritise immediate microbiology processing. Do not routinely refrigerate a CSF intended for bacterial culture unless the receiving laboratory specifically directs it; some organisms are temperature-sensitive.
- Do not freeze CSF intended for routine cell count or morphology: freezing destroys cellular detail. Viral, molecular or specialised tests may require refrigeration or freezing only under the assay SOP.
- Record the opening pressure, appearance, collection time, antibiotics already given and traumatic-tap information where relevant.
| CSF purpose | Preservation priority | Reason |
|---|---|---|
| Cell count/differential | Immediate delivery and processing; avoid freezing. | Leukocytes and erythrocytes deteriorate, changing the apparent count and differential. |
| Gram stain/culture | Immediate microbiology handling; use sterile container and appropriate temperature per local SOP. | Low organism burden and fragile pathogens make delay dangerous. |
| Chemistry | Prompt separation/analysis as requested; avoid contamination from unsuitable tubes. | Glucose, protein and lactate can change with cells, organisms and delay. |
| Viral/molecular testing | Use assay-specific refrigerated/frozen aliquot if not tested promptly. | Nucleic acid stability and inhibitors differ by assay. |
9. Serous, synovial and other body fluids
Pleural, peritoneal, pericardial, synovial and other aspirated fluids may require several parallel portions. Before collection, clarify whether the requests include cell count, chemistry, Gram stain/culture, mycobacterial/fungal culture, cytology, crystals, pH or molecular tests.
| Requested examination | Preservation approach | Key point |
|---|---|---|
| Cell count/differential | Fresh fluid promptly, or the anticoagulant specified by the laboratory (often EDTA for selected cell-count applications). | Clotting traps cells and falsely lowers the count; prolonged delay causes degeneration. |
| Culture | Sterile container; inoculate blood-culture bottles when validated and appropriate; send promptly. | Do not place culture fluid in formalin or cytology fixative. |
| Fluid pH | Collect anaerobically in a heparinised blood-gas syringe, expel air and analyse rapidly according to the laboratory protocol. | Air, lidocaine, residual saline and delay can change pH; a routine tube is not equivalent to a blood-gas syringe. |
| Cytology/cell block | Send fresh promptly or use the cytology preservative specified by the department; provide volume and clinical site. | Cells can degenerate or adhere to the container. Do not fix the entire sample before checking whether microbiology or flow studies are needed. |
| Synovial crystals | Use a clean container, avoid EDTA or other additives unless validated, and examine promptly for intracellular crystals. | Delay, contamination and wrong anticoagulant can obscure crystals or mimic them. |
10. Tissue, biopsy and surgical specimens
10.1 Decide the destination before placing tissue in a container
A tissue specimen may be needed for histology, microbiology, immunofluorescence, flow cytometry, cytogenetics, electron microscopy and molecular testing. Once placed in formalin, it cannot be used as fresh sterile tissue for culture or many viable-cell studies. The surgeon, clinician and pathology laboratory must agree on specimen division before collection whenever possible.
| Purpose | Correct preservation | High-yield cautions |
|---|---|---|
| Routine histopathology | Place promptly in adequate 10% neutral buffered formalin (approximately 4% formaldehyde) with sufficient volume; tissue should be thin enough for penetration. | Use a wide-mouth leak-proof container. Do not compress, dry, freeze or leave tissue in saline for routine histology. Do not use formalin for culture or viable-cell studies. |
| Microbiology culture | Send a separate piece fresh in a sterile container, without formalin; use approved transport conditions. | A swab is often inferior to tissue or aspirate. Clearly identify site and suspected organism. |
| Immunofluorescence (for example renal or skin biopsy) | Use the receiving laboratory’s validated medium, such as Michel-type medium where specified, or fresh handling for immediate delivery. | Formalin can destroy or reduce immunoreactants needed for direct immunofluorescence. |
| Flow cytometry | Send fresh viable tissue/cell suspension in the specified medium and temperature; arrange rapid delivery. | Formalin fixation kills cells and prevents a viable immunophenotype. |
| Cytogenetics/FISH and some molecular studies | Use fresh tissue or the laboratory’s validated medium, with rapid transport and adequate tumour content. | Do not assume formalin-fixed paraffin-embedded tissue is interchangeable with fresh tissue; fixation duration affects nucleic-acid quality. |
| Electron microscopy | Use a very small piece in glutaraldehyde as directed; reserve separate tissue for light microscopy and other studies. | Large pieces fix poorly. Glutaraldehyde is not a substitute for routine formalin and can interfere with other testing. |
| Frozen section or selected enzyme/lipid studies | Send fresh, unfixed tissue immediately on the validated medium; do not immerse in formalin. | Freezing must be controlled; an unplanned freezer or dry ice may cause artefact or biosafety problems. |
10.2 Formalin fixation essentials
- Use 10% neutral buffered formalin for routine histology when requested; the “10%” label refers to a formalin solution approximately equivalent to 4% formaldehyde.
- Use a generous fixative:tissue volume (commonly around 10:1) so the specimen is fully immersed; follow local SOP for large specimens.
- Open or slice large specimens as directed by pathology so fixative can penetrate; do not slice a specimen when orientation or margins must first be documented.
- Do not allow tissue to dry before fixation. Do not top up a leaking container with water or saline.
- Record cold-ischaemia time, fixation start time and relevant orientation/margins where required.
- Formalin is toxic and potentially carcinogenic: use labelled containers, ventilation, PPE, spill procedures and approved disposal.
10.3 Bone marrow, skin and small biopsies
- Bone marrow aspirate: prepare smears immediately; place additional material in the anticoagulant and transport medium requested for flow, cytogenetics, molecular tests or culture.
- Bone marrow trephine: place promptly in the fixative specified by histopathology; avoid drying and do not put all marrow material into formalin if fresh studies are required.
- Skin biopsy: clarify whether the indication is routine histology, direct immunofluorescence, culture or molecular testing before choosing formalin, Michel-type medium or fresh sterile handling.
- Small endoscopic biopsies: prevent desiccation, use separate labelled containers for different sites and provide the site and clinical question.
11. Cytology specimens
| Cytology type | Preservation | Common failure |
|---|---|---|
| Fine-needle aspirate | Prepare air-dried and/or alcohol-fixed smears as requested; rinse residual material into the cytology preservative or medium if cell block/molecular testing is planned. | Allowing all material to dry or placing all material in formalin before smears are made. |
| Body-fluid cytology | Send fresh rapidly or place in the laboratory’s validated cytology preservative; keep enough material for cell block and ancillary tests. | Delay causes degeneration; excess blood, low volume or wrong preservative reduces yield. |
| Cervical cytology | Use the approved liquid-based vial or make an immediate properly fixed smear according to the screening programme. | Air-drying, lubricant contamination, wrong vial and inadequate transformation-zone sampling. |
| Urine cytology | Prefer a fresh, properly collected specimen or validated cytology preservative; send promptly. | First-morning concentrated urine may contain degenerated cells; a delay without preservation lowers sensitivity. |
12. Temperature, light and physical protection
| Condition | Potential benefit | Risks and examples |
|---|---|---|
| Controlled room temperature | Maintains some cells, organisms and reactions when validated; useful for urgent hand-delivery of selected specimens. | Heat accelerates metabolism and degradation. “Room temperature” should be defined by the SOP, not a personal guess. |
| Refrigeration (commonly 2–8°C when specified) | Slows cellular metabolism and bacterial overgrowth for many chemistry, urine and selected molecular specimens. | May damage cells, alter gas values, precipitate proteins/crystals or reduce recovery of temperature-sensitive organisms. Never apply indiscriminately. |
| Freezing | Extends stability for validated aliquots, selected serology/chemistry and molecular materials. | Freeze–thaw damages cells, clotting factors and some proteins; use aliquots and a monitored freezer. Some specimens must never be frozen. |
| Deep freezing (for example −70/−80°C) | Longer-term preservation of selected nucleic-acid, viral, research or biobank material. | Requires validated assay, backup power/monitoring and controlled packaging. It is not a default for routine clinical samples. |
| Light protection | Prevents photodegradation of bilirubin, porphyrins and selected drugs/analytes. | Foil or amber protection must not obscure the label or compromise biosafety. |
| Orientation and evaporation control | Maintains anticoagulant contact, prevents leakage and limits concentration changes. | Loose caps, excessive headspace, prolonged uncapped storage or repeated opening cause contamination and evaporation. |
13. Preservation decision algorithm
- What is the exact test? If uncertain, call the receiving laboratory before collection.
- Is the target alive, cellular, chemical, antigenic or nucleic-acid based? This determines whether fixation, cooling, transport medium or rapid processing is appropriate.
- Does the specimen require a separate portion? Divide before adding a preservative that could compromise another test.
- What is the validated time window? Record collection and preservation times; arrange urgent hand transport for critical specimens.
- What temperature and light conditions are validated? Use a monitored device when required and document excursions.
- Can the specimen be aliquoted? Aliquot once, label every aliquot and avoid repeated freeze–thaw.
- Is the container intact and traceable? Reject or qualify specimens that are unlabelled, leaking, grossly contaminated, wrong-container, insufficient or outside stability limits.
14. Quality control: acceptance, rejection and corrective action
| Finding | Why it matters | Action |
|---|---|---|
| Unlabelled or mismatched specimen | Risk of wrong-patient result | Follow positive-identification policy; usually reject and recollect. Never relabel from memory. |
| Leaking, broken or contaminated exterior | Biohazard and possible loss/dilution | Protect staff, document incident and request recollection when possible. |
| Wrong container or preservative | Additive may interfere or destroy the target | Consult the test SOP; do not transfer blindly to a new container. |
| Clotted anticoagulated blood | Cell counts and coagulation results are invalid | Reject and recollect; a clot cannot be mixed away. |
| Haemolysis, lipaemia or icterus | Interference or in-vivo disease may affect measurement | Assess severity and analyte-specific impact; report qualification or recollect. |
| Insufficient volume | Cannot perform required tests or repeats | Prioritise urgent tests only after discussion; recollect if safe. |
| Delay or temperature excursion | Stability may be exceeded | Document times/temperatures, check validation and contact the laboratory before release. |
| Specimen placed in formalin when culture/flow was needed | Viability lost; culture or cellular studies may be impossible | Notify clinician promptly and recollect if possible; record a pre-analytical nonconformity. |
Rejection is a patient-safety action, not punishment. If a compromised specimen is processed because recollection is impossible, the report should state the limitation and the clinician should interpret it with that information.
15. Practical cases
Case 1: “Cold everything”
A nurse refrigerates a CSF sample for suspected bacterial meningitis because the laboratory is far away. This can delay urgent processing and may harm recovery of temperature-sensitive organisms. Call the laboratory/referral centre and use the validated urgent pathway; do not apply the urine-storage rule to CSF.
Case 2: One biopsy, four tests
A renal biopsy is placed entirely in formalin, but direct immunofluorescence and culture were also requested. The specimen cannot simply be “taken back out.” Before collection, divide tissue into correctly labelled portions for histology, immunofluorescence, microbiology and any fresh-cell study.
Case 3: Delayed urine culture
A urine specimen sits overnight at room temperature. Bacterial multiplication and pH change may produce a misleading colony count. Recollect if possible or follow the laboratory’s policy; never add boric acid after collection as an improvised rescue.
Case 4: Haemolysed potassium
A visibly haemolysed serum tube gives a high potassium. Haemolysis may be in-vitro or clinically significant. Check the specimen and clinical context, document the interference and recollect urgently when a safe decision depends on potassium.
16. Examination-ready summary
| Remember | Exam answer |
|---|---|
| Best preservation | Correct test-specific container + additive/fixative + time + temperature + transport + documentation. |
| Formalin | Routine tissue morphology; not for culture, viable flow studies or many fresh molecular/cytogenetic tests. |
| EDTA | Cell counts and selected whole-blood assays; inadequate mixing causes clots and delay changes morphology. |
| Citrate | Coagulation; fill to the mark because the blood:citrate ratio is critical. |
| CSF | Urgent hand delivery; do not freeze routine cell-count specimens; follow local microbiology temperature instructions. |
| Stool | Fresh/transport medium for culture; formalin/PVA or SAF for selected parasitology; never use one preservative for every request. |
| Urine | Prompt testing; validated refrigeration or preservative for delay; boric acid is test-specific. |
| Quality system | Written acceptance/rejection criteria and monitored storage/transport protect both patients and staff. |
17. Quick self-test
- Why is preservation considered part of the pre-analytical phase?
- Distinguish fixation from stabilisation.
- Why must a citrate coagulation tube be filled to its indicated volume?
- Why is a clotted EDTA sample unsuitable for a full blood count?
- Why should blood-culture bottles usually not be refrigerated after collection?
- When can urine boric acid be useful, and why is it not a universal preservative?
- Which stool preservatives are used for complementary parasitology methods, and why must culture stool remain separate?
- Why must CSF for routine cytology/cell count not be frozen?
- What happens if a biopsy intended for culture is placed in formalin?
- Why must a body-fluid pH sample be protected from air?
- List three analytes or specimen types that may require light protection or special temperature handling.
- What information should be recorded after a temperature excursion?
- How should a leaking or unlabelled specimen be managed?
- Why are separate aliquots useful?
- What is the safest action when the correct preservation condition is uncertain?
Answer guide
1. Because errors before analysis can permanently change the specimen and cannot be corrected by the analyzer. 2. Fixation preserves structure, usually irreversibly; stabilisation slows change while retaining the target for a particular test. 3. The citrate:blood ratio controls calcium binding and coagulation results. 4. The clot traps cells and produces unreliable counts. 5. Cooling may reduce recovery of some organisms and delays urgent processing. 6. It can maintain urine culture stability when validated; it is not suitable for every test and must be filled correctly. 7. Formalin and PVA/SAF support different parasite detection methods; formalin can inhibit culture. 8. Freezing destroys cellular detail. 9. Formalin kills organisms and prevents many viable-cell studies. 10. Air changes gas tension and pH. 11. Examples include bilirubin/porphyrins, blood gas, ammonia/lactate, cryoproteins, CSF and viable cells. 12. Collection/preservation time, temperature, duration, condition, test, action and responsible staff. 13. Protect staff, document and follow rejection/recollection policy. 14. They reduce repeated opening and freeze–thaw while allowing different tests. 15. Contact the receiving laboratory and follow its written SOP.
Authoritative resources and further reading
- WHO Laboratory Quality Stepwise Implementation tool: sample collection, preservation and transport
- WHO LQMS: sample storage, retention and disposal
- WHO Laboratory Quality Management System handbook
- WHO Laboratory Biosafety Manual, fourth edition
- CDC DPDx: stool specimen collection and preservation
- CDC: collection, storage and preservation of specimens before shipping
Local practice note: Exact holding times, temperatures, preservatives and retention periods are method- and laboratory-specific. Students should use this chapter to understand the reasoning, then follow the current SOP of the Ugandan laboratory or referral service performing the test.
