Blood Sample Collection Protocol
Blood samples provide the highest DNA yield of all avian sample types accepted by SENO. This protocol covers three collection methods, safety procedures, and quality guidelines for submitting blood samples to our genetics laboratory.
Indications for Blood Collection
Blood samples are recommended when:
- Feather samples are unavailable (e.g., featherless chicks, molting birds)
- DNA fingerprinting or parentage verification is required (higher DNA yield improves resolution)
- Certain disease testing requires blood (e.g., blood-borne pathogen screening, complete blood count correlation)
- Sample age or storage history of alternative samples is uncertain
- Maximum DNA quantity is needed for archival biobanking
Important Considerations Before Collecting
| Factor | Consideration |
|---|
| Bird size | Minimum blood volume scales with body weight — do not exceed 1% of body weight in 24 hours |
| Stress level | Minimize handling time; stressed birds may experience complications |
| Anticoagulant | EDTA is preferred for DNA testing; heparin may inhibit PCR amplification |
| Time of day | Early morning collection reduces heat stress risk in warm climates |
| Recent feeding | Fasted birds tolerate venipuncture better; avoid post-prandial lipemic samples |
Collection Method Comparison
SENO accepts three blood collection methods. Choose the method that best suits your bird species, testing requirements, and shipping logistics.
| Method | Difficulty | DNA Yield | Shipping Ease | Best Use Case |
|---|
| Venipuncture (EDTA tube) | Advanced | Very high | Moderate (requires cold pack) | Large birds, biobanking |
| Blood spot (FTA card) | Moderate | High | Excellent (room temp) | International shipping, routine testing |
| Toenail clip (filter paper) | Easy | Medium | Excellent (room temp) | Small birds, neonates |
Method 1: Venipuncture (Recommended for Larger Birds)
Equipment Required
- Sterile syringe (1–3 mL) with 25–27 gauge needle
- EDTA blood collection tube (purple top, 0.5–2 mL)
- 70% ethanol or antiseptic wipe
- Cotton ball or gauze
- Disposable gloves
- Bird-specific restraint towel
- Permanent marker for labeling
Venipuncture Sites
| Site | Bird Size | Advantages | Disadvantages |
|---|
| Right jugular vein | All sizes | Easy access, large vessel | Requires trained restraint; left jugular is smaller |
| Brachial (wing) vein | Medium–large | Visible, easy access | Hematoma risk; vessel rolls |
| Medial metatarsal (leg) vein | Large birds | Safe for conscious birds | Smaller volume; slower collection |
Procedure
- Restrain the bird securely with a towel, exposing the chosen venipuncture site
- Locate the vein by visual inspection and gentle palpation
- Cleanse the site with 70% ethanol and allow to dry
- Insert the needle at a 15–30 degree angle, bevel up, with the needle pointing toward the heart
- Collect blood slowly — fill the syringe to the desired volume (0.1–0.5 mL for small birds; 0.5–1.0 mL for large birds)
- Remove the needle and apply firm pressure to the puncture site with sterile gauze for 30–60 seconds
- Transfer blood to the EDTA tube — gently invert 8–10 times to mix with anticoagulant
- Label the tube immediately with bird ID, collection date, and sample type
| Parameter | Specification |
|---|
| Collection Site | Jugular vein (right side), brachial vein, or medial metatarsal vein |
| Volume | 0.1–0.5 mL (small birds); 0.5–1.0 mL (large birds) |
| Anticoagulant | EDTA (purple top tube) preferred for DNA testing |
| Needle gauge | 25–27 gauge |
| Maximum draw | ≤ 1% of body weight per 24-hour period |
Method 2: Blood Spot on FTA Card (Preferred for International Shipping)
FTA cards contain proprietary chemicals that lyse cells, denature proteins, and stabilize nucleic acids at room temperature. This is the recommended method for clients shipping to SENO from abroad.
Equipment Required
- FTA card (Whatman FTA or equivalent)
- Sterile lancet or needle
- 70% ethanol wipe
- Disposable gloves
- Desiccant pack
- Resealable plastic bag
- Permanent marker
Procedure
- Select a collection site — brachial vein or toenail clip (see Method 3)
- Collect fresh blood directly from the venipuncture site or free-flowing droplet
- Apply 1–2 drops to the FTA card's printed sample area (ensure the blood saturates the card, not pools on top)
- Air dry for 2–4 hours at room temperature (15–25°C) in a dust-free environment
- Do not stack wet cards — keep them separated until fully dry
- Place the dried card in a resealable bag with a desiccant pack
- Seal the bag and label with bird ID, date, collector name, and sample type
- No refrigeration required for shipping — the card stabilizes DNA at ambient temperature
| Step | Time Requirement | Critical Checkpoint |
|---|
| Blood application | Immediately after collection | Even saturation of the printed circle |
| Air dry | 2–4 hours | Card must be dry to the touch; no visible moisture |
| Packaging | After drying | Desiccant pack must be included |
| Shipping | Any time after drying | FTA cards stable for years when kept dry |
Troubleshooting FTA Cards
| Problem | Likely Cause | Solution |
|---|
| Blood pools on surface | Card surface is contaminated or waxed | Use a fresh card; touch drop gently to the paper |
| Insufficient blood volume | Droplet too small | Collect a larger drop; hold card directly at the site |
| Card smells moldy | Dried before packaging | Ensure 2–4 hour dry time; use fresh desiccant |
| DNA yield is low | Card got wet in transit | Double-bag with extra desiccant for humid climates |
Method 3: Toenail Clip Blood Collection (Minimally Invasive)
This method is suitable for very small birds (budgies, finches, canaries, chicks) where venipuncture is technically difficult or high-risk.
Procedure
- Restrain the bird gently but securely
- Select a toenail — avoid the dewclaw if present
- Clean the nail with 70% ethanol
- Clip the tip of the nail (approximately 1–2 mm) using clean nail clippers
- Collect the blood droplet on a FTA card or sterile filter paper
- Apply gentle pressure with a cotton ball or styptic powder to stop bleeding
- Repeat on another nail if more volume is needed (maximum 2–3 nails)
- Monitor the bird for 5 minutes after the procedure to confirm hemostasis
Limitations
- Yields only 1–3 small drops (10–50 μL total)
- Not suitable for EDTA tube collection (insufficient volume)
- Higher risk of sample contamination from the foot surface
Safety Precautions
| Precaution | Explanation |
|---|
| Use sterile, single-use materials | Prevents cross-contamination and infection at the puncture site |
| Apply pressure after collection | Ensures hemostasis; reduces hematoma risk |
| Dispose of sharps immediately | Use a puncture-resistant sharps container |
| Label tubes and cards immediately | Prevents sample misidentification |
| Change gloves between birds | Eliminates cross-contamination risk between different birds |
| Never reuse needles or lancets | Single-use only — reused instruments cause infection and sample contamination |
Post-Collection Handling
| Method | Storage Before Shipment | Maximum Hold Time | Shipping Condition |
|---|
| EDTA whole blood | 2–8°C (refrigerate) | 7 days | Cold pack (2–8°C) |
| FTA card (dried) | Room temperature (15–25°C) | Years | Room temperature |
| Filter paper spot | Room temperature (15–25°C) | 30 days | Room temperature |
Packing for Shipment
- EDTA tubes: Wrap in bubble wrap, place in a sealed plastic bag with absorbent material, ship with a cold pack in an insulated container
- FTA cards: Place in a resealable bag with desiccant, insert into a padded envelope
- Filter paper spots: Same as FTA cards; protect from moisture with desiccant
When Not to Use Blood Samples
Blood collection may be contraindicated in the following situations:
- Severely dehydrated or anemic birds (increased complication risk)
- Birds with clotting disorders or on anticoagulant therapy
- Extremely small neonates (< 10 g body weight) — use swab or feather instead
- Birds that cannot be safely restrained
- STAT testing where minimal handling time is critical — consider swab collection
Cross-References