4.2 Special Collection Procedures
Key Takeaways
Blood cultures require strict aseptic technique — clean the site with chlorhexidine or povidone-iodine (NOT just alcohol), and clean the bottle tops with alcohol
Two sets of blood cultures from two different venipuncture sites are standard to distinguish true bacteremia from contamination
The glucose tolerance test (GTT) requires a fasting specimen, followed by a glucose drink, then timed blood draws at 30, 60, 120, and sometimes 180 minutes
Peak drug levels are drawn 30-60 minutes after drug administration (when the drug is at its highest concentration)
Trough drug levels are drawn immediately BEFORE the next scheduled dose (when the drug is at its lowest concentration)
Chain of custody specimens require strict documentation — the specimen must be in someone's possession at all times and tamper-evident seals must be applied
Arterial blood gas (ABG) specimens are typically collected by respiratory therapists or specially trained phlebotomists from the radial, brachial, or femoral artery
Cold agglutinin specimens must be kept at body temperature (37C) and transported in a pre-warmed container — cooling causes the antibodies to bind and invalidates the test
Beyond routine venipuncture and capillary puncture, phlebotomists must be familiar with specialized collection procedures that require additional skills, timing, or handling.
Blood Cultures
Blood cultures detect bacteremia (bacteria in the blood) and septicemia (systemic infection). They require the most stringent collection technique of any phlebotomy procedure.
Collection Protocol
| Step | Procedure |
|---|---|
| Site preparation | Clean with chlorhexidine (preferred) or povidone-iodine — NOT just alcohol |
| Drying time | Allow antiseptic to dry completely (30 seconds for chlorhexidine; 1.5-2 minutes for iodine) |
| Bottle top preparation | Wipe bottle tops with 70% isopropyl alcohol |
| Number of sets | At least two sets (one aerobic + one anaerobic bottle each) from separate venipuncture sites, usually drawn together or within about an hour; spaced draws only when ordered (for example, suspected endocarditis) |
| Volume | Fill each bottle to the recommended volume (about 10 mL per adult bottle) |
| Winged (butterfly) set | Fill the aerobic bottle first: air in the tubing goes into the first bottle |
| Needle and syringe | Follow the bottle manufacturer's instructions (bioMérieux BacT/ALERT says anaerobic bottle first; if less than the recommended volume is drawn, it says to fill the aerobic bottle first) |
Why Two Sets from Two Sites?
- Helps distinguish true bacteremia (both sets positive with the same organism) from contamination (only one set positive)
- A single positive culture may represent a skin contaminant (common organisms like S. epidermidis)
Timing
- Ideally collected before antibiotic therapy begins
- If the patient is already on antibiotics, collect just before the next dose (trough level)
- For fever spikes: some physicians order cultures to be drawn during temperature elevations
Glucose Tolerance Test (GTT)
The oral glucose tolerance test (OGTT) evaluates the body's ability to metabolize glucose over time. It is used to diagnose gestational diabetes and type 2 diabetes.
Standard GTT Protocol
| Step | Timing | Action |
|---|---|---|
| Fasting specimen | Time 0 | Draw fasting blood glucose (patient fasts 8-12 hours) |
| Glucose load | After fasting draw | Patient drinks 75g glucose solution (100g for gestational diabetes screening) within 5 minutes |
| 30-minute draw | 30 min post-glucose | May be optional depending on physician order |
| 1-hour draw | 60 min post-glucose | Blood draw |
| 2-hour draw | 120 min post-glucose | Blood draw |
| 3-hour draw | 180 min post-glucose | Only for gestational diabetes screening with 100g glucose |
Patient Instructions During GTT
- Patient must remain seated and not eat, smoke, or chew gum during the test
- Only water is permitted
- Patient should not exercise (physical activity lowers glucose)
- If the patient vomits the glucose solution, the test must be rescheduled
- All specimens are collected in gray-top tubes (sodium fluoride preserves glucose)
Peak and Trough Drug Levels
Monitoring drug levels helps ensure medications are at therapeutic (effective but not toxic) concentrations.
| Level | When to Draw | Purpose |
|---|---|---|
| Trough | Immediately BEFORE the next scheduled dose | Measures the lowest drug concentration — ensures it stays above the minimum effective level |
| Peak | 30-60 minutes after drug administration (IV); 1-2 hours after oral dose | Measures the highest drug concentration — ensures it does not reach toxic levels |
Common Drugs Requiring Levels
- Vancomycin — antibiotic (trough and peak monitored)
- Gentamicin / Tobramycin — aminoglycoside antibiotics (nephrotoxic and ototoxic at high levels)
- Digoxin — cardiac medication (narrow therapeutic window)
- Lithium — psychiatric medication (narrow therapeutic window)
- Phenytoin (Dilantin) — anti-seizure medication
- Theophylline — respiratory medication
Critical: Timing is everything for peak and trough draws. Document the exact time of the draw and the time of the last dose on the requisition.
Arterial Blood Gas (ABG) Collection
ABG analysis measures blood pH, partial pressures of oxygen (PaO2) and carbon dioxide (PaCO2), and bicarbonate (HCO3-) to assess respiratory and metabolic function.
| Detail | Information |
|---|---|
| Who collects | Respiratory therapists, specially trained phlebotomists, nurses, or physicians |
| Common sites | Radial artery (preferred), brachial artery, femoral artery |
| Allen test | Performed before radial artery puncture to confirm adequate collateral circulation from the ulnar artery |
| Syringe type | Pre-heparinized ABG syringe |
| Handling | Remove all air bubbles immediately; cap syringe; place on ice; transport to lab within 30 minutes |
| Post-collection | Apply firm pressure for at least 5 minutes (10 minutes for patients on anticoagulants) |
The Allen Test (Modified Allen Test)
- Have the patient make a tight fist
- Occlude both the radial and ulnar arteries with your fingers
- Have the patient open their hand — the palm should be pale/blanched
- Release pressure on the ulnar artery only
- Positive (normal): Color returns to the hand within 5-10 seconds — safe to puncture the radial artery
- Negative (abnormal): Color does NOT return within 10 seconds — do NOT puncture the radial artery (inadequate collateral circulation)
Chain of Custody Specimens
Chain of custody (COC) applies to forensic specimens such as drug testing for legal purposes (employment, court-ordered, probation).
Key Requirements
| Requirement | Details |
|---|---|
| Documentation | Every person who handles the specimen must be documented (name, date, time, signature) |
| Continuous possession | The specimen must be in someone's custody at all times — no unattended storage |
| Tamper-evident seals | Applied in the patient's presence; patient verifies and initials the seal |
| Witnessed collection | Some protocols require direct observation of specimen production (urine) |
| Photo ID verification | Required for the donor/patient |
| Split specimen | A portion may be preserved in a separate container for independent confirmation testing |
Other Special Specimens
| Specimen Type | Special Handling |
|---|---|
| Cold agglutinins | Keep at body temperature (37C) — transport in pre-warmed container; cooling invalidates the test |
| Cryoglobulins | Keep at body temperature (37C) — similar to cold agglutinins |
| Ammonia | Transport on ice immediately; specimen degrades rapidly at room temperature |
| Lactic acid | Collect without tourniquet if possible; transport on ice; patient should not clench fist |
| Light-sensitive specimens (bilirubin, vitamin B12, folate) | Wrap in aluminum foil or amber-colored tubes to protect from light degradation |
| Coagulation specimens (PT/INR, PTT) | Must be tested within 4 hours of collection; keep at room temperature |
Bleeding Time Test
The bleeding time test (Ivy method) assesses platelet function and vascular integrity. While less commonly performed today (replaced by PFA-100 in many labs), it may still appear on the CPT exam:
Procedure
- Apply a blood pressure cuff to the upper arm and inflate to 40 mmHg
- Clean the inner forearm with alcohol and allow to dry
- Make a standardized incision using a bleeding time device (spring-loaded blade)
- Start the timer immediately
- Blot the blood every 30 seconds with filter paper — do NOT touch the wound directly
- Record the time when bleeding stops
- Normal bleeding time: 2-9 minutes (varies by method)
Clinical Significance
- Prolonged bleeding time may indicate: platelet disorders (thrombocytopenia), von Willebrand disease, aspirin or NSAID use, or vascular disorders
- Patients should be instructed to avoid aspirin and NSAIDs for 7-10 days before the test
2-Hour Postprandial Glucose
This test measures glucose levels 2 hours after a meal to assess glucose tolerance:
- Patient eats a standard meal (or glucose load)
- Blood is drawn exactly 2 hours after the meal begins
- Collected in a gray-top tube (sodium fluoride)
- Normal: Less than 140 mg/dL (2 hours postprandial)
- Impaired glucose tolerance: 140-199 mg/dL
- Diabetes: 200 mg/dL or higher
Cortisol and Diurnal Variation
Some hormones follow a diurnal (daily) pattern and must be collected at specific times:
| Hormone | Peak Time | Trough Time | Collection Notes |
|---|---|---|---|
| Cortisol | Early morning (6-8 AM) | Evening (4 PM - midnight) | AM and PM specimens may be ordered |
| Iron | Morning (highest) | Evening (lowest) | Best collected in the morning fasting |
| TSH | Early morning (highest) | Afternoon (lower) | AM collection preferred |
| Growth hormone | During sleep | During the day | Special stimulation tests may be ordered |
What antiseptic is recommended for skin preparation before blood culture collection?
70% isopropyl alcohol only
Chlorhexidine or povidone-iodine
Hydrogen peroxide
Benzalkonium chloride
A trough drug level should be drawn:
30 minutes after the drug is administered
2 hours after the drug is administered
Immediately before the next scheduled dose
At the same time every day regardless of dosing
During a glucose tolerance test (GTT), the patient vomits 30 minutes after drinking the glucose solution. What should the phlebotomist do?
Continue the test and draw specimens at scheduled times
Give the patient another glucose drink immediately
Cancel the test and reschedule for another day
Draw only the remaining scheduled specimens
The Allen test is performed before arterial puncture to assess:
The patient's pain tolerance
Whether the vein is suitable for puncture
Adequate collateral circulation to the hand
The patient's blood pressure
Which of the following specimens must be transported on ICE? (Select all that apply)
Select all that apply
Ammonia
Arterial blood gas (ABG)
Blood cultures
Lactic acid
Cold agglutinins
Bilirubin
Match each special specimen to its transport requirement:
Match each item on the left with the correct item on the right
Ammonia
Cold agglutinins
Bilirubin
Blood cultures
During chain of custody specimen collection, _____ seals must be applied in the patient's presence and the patient must initial them.
Type your answer below
Why are two sets of blood cultures collected from two different venipuncture sites?
To ensure enough blood volume for the laboratory
To distinguish true bacteremia from skin contamination
Because the first set is always unreliable
To test for different types of bacteria at each site
The Allen test is considered POSITIVE (normal) when:
The patient reports pain during the test
Color does NOT return to the hand after releasing the ulnar artery
Color returns to the hand within 5-10 seconds after releasing the ulnar artery
The radial pulse becomes stronger after the test
Arrange the steps of the glucose tolerance test (GTT) in the correct order:
Arrange the items in the correct order
Verify patient has fasted 8-12 hours
Draw blood at 1-hour mark
Draw blood at 2-hour mark
Patient drinks 75g glucose solution within 5 minutes
Draw fasting blood glucose specimen
What should the phlebotomist do if the patient exercises (walks around the floor) during a GTT?
Continue the test — exercise does not affect results
Note the exercise on the requisition — results may be affected as exercise lowers glucose
Cancel the test immediately
Give the patient a second glucose drink to compensate
Peak drug levels are drawn:
Immediately before the next scheduled dose
30-60 minutes after IV drug administration
24 hours after the last dose
At the same time every day regardless of dosing
Capillary blood glucose levels are typically _____ than venous blood glucose levels.
Lower
Higher
The same as
Unrelated to
During a blood culture collection using a winged (butterfly) collection set, which bottle should be filled FIRST?
The anaerobic bottle
The aerobic bottle
Either bottle — order does not matter
A discard tube must always be drawn first
Which of the following drugs has a narrow therapeutic window and commonly requires peak and trough monitoring?
Acetaminophen (Tylenol)
Vancomycin (antibiotic)
Lisinopril (blood pressure)
Metformin (diabetes)
During the bleeding time test, the blood pressure cuff should be inflated to:
20 mmHg
40 mmHg
80 mmHg
120 mmHg
Cortisol levels follow a diurnal pattern with the HIGHEST levels occurring at what time?
Midnight
Early morning (6-8 AM)
Afternoon (2-4 PM)
Evening (8-10 PM)
A patient is scheduled for a 3-hour glucose tolerance test (100g glucose) for gestational diabetes screening. How many total blood specimens will be collected?
2 specimens (fasting and 2-hour)
3 specimens (fasting, 1-hour, 2-hour)
4 specimens (fasting, 1-hour, 2-hour, 3-hour)
5 specimens (fasting, 30-min, 1-hour, 2-hour, 3-hour)
Which of the following are requirements for collecting chain of custody (forensic) specimens? (Select all that apply)
Select all that apply
Photo ID verification of the donor
Tamper-evident seals applied in the donor's presence
Continuous documented possession of the specimen
Collection by any available staff member without documentation
Donor initials on the specimen seal
Specimen can be left unattended in the laboratory
Match each timed collection to its correct timing:
Match each item on the left with the correct item on the right
Trough drug level
Peak drug level (IV)
Fasting glucose
2-hour postprandial glucose
Newborn Screening & Pediatric Considerations
Newborn Screening (NBS) / Heel Stick
Newborn screening is one of the most important applications of capillary puncture. Every state requires newborn screening for certain genetic and metabolic disorders, though the specific panel varies by state.
Collection Requirements
| Parameter | Requirement |
|---|---|
| Timing | 24-48 hours after birth (after the infant has fed) |
| Site | Medial or lateral plantar heel surface |
| Depth | No deeper than 2.0 mm (US procedures based on CLSI; WHO's outer limit is 2.4 mm) to avoid the calcaneus |
| Specimen | Filled circles on Guthrie filter paper card |
| Drying | Air dry horizontally for 3-4 hours — NEVER stack, heat-dry, or touch the circles |
| Filling | Apply blood to ONE side only; blood must soak through to fill the entire circle |
Common Conditions Screened
- Phenylketonuria (PKU) — inability to metabolize phenylalanine
- Congenital hypothyroidism — thyroid hormone deficiency
- Galactosemia — inability to metabolize galactose
- Sickle cell disease — abnormal hemoglobin
- Cystic fibrosis — mucus-producing gene defect
- Biotinidase deficiency — inability to recycle biotin
Key Exam Fact: If the newborn is discharged before 24 hours of age, the specimen should still be collected before discharge AND a second specimen collected within 1-2 weeks. Early collection may miss conditions like PKU that require protein intake to become detectable.
Blood Cultures from Pediatric Patients
When blood cultures are ordered for pediatric patients:
- Use age-appropriate blood volume (typically 1-3 mL per bottle for infants)
- Strict aseptic technique is critical — contamination rates are higher in pediatrics
- A single blood culture set may be acceptable in neonates due to limited blood volume
- Use butterfly (winged infusion) sets for better control in small veins
- Always calculate maximum safe blood draw volume (no more than 2.5-5% of total blood volume)
Maximum Blood Volume Calculation for Pediatric Patients
Total blood volume in children is approximately 80 mL/kg. The maximum safe draw should not exceed 2.5% of total blood volume in a single draw or 5% in a 24-hour period.
Example: A 4 kg neonate has approximately 320 mL total blood volume. Maximum single draw = 8 mL. Maximum in 24 hours = 16 mL.
US procedures based on CLSI say an infant heel puncture should go no deeper than ____ mm to avoid the calcaneus (heel bone).
Type your answer below
Which of the following are correct procedures when collecting a newborn screening specimen on filter paper? (Select all that apply)
Select all that apply
Apply blood to one side of the filter paper only
Blood must completely fill each printed circle
Stack the cards to dry more quickly
Allow the card to air dry in a horizontal position for 3-4 hours
Use the first drop of blood for the filter paper circles
Blood should soak through and be visible on the reverse side of the card
A phlebotomist must calculate the maximum safe blood draw for a 3 kg neonate in a 24-hour period. If the neonate's total blood volume is approximately 80 mL/kg, what is the maximum volume that can be drawn?
6 mL
12 mL
24 mL
48 mL
Sections you finish are checked off in the contents.