BACKGROUND:
Hemolysis is the most common preanalytical error in emergency department (ED) laboratories, affecting 17-26% of blood samples collected in the ED and leading to test cancellations, repeat venipuncture, delayed diagnoses, and increased healthcare costs. Venous stasis created by tourniquet application during phlebotomy is a recognized contributing factor to hemolysis. While clinical guidelines recommend releasing the tourniquet once blood flow is established, the optimal timing of tourniquet release in relation to tube filling sequence has not been systematically evaluated.
OBJECTIVE:
The primary objective of this trial is to determine whether early release of sphygmomanometer-applied venous stasis (released after the first tube fills) reduces hemolysis rates compared to late release (released after the last tube fills) during routine phlebotomy in ED patients triaged as green or yellow category.
DESIGN:
Single-center, parallel-group, superiority randomized controlled trial with 1:1 allocation ratio. The trial was prospectively registered prior to the enrollment of the first participant.
PARTICIPANTS:
Adult patients (≥18 years) presenting to the emergency department with triage category green (semi-urgent) or yellow (urgent), for whom blood collection is indicated as part of routine clinical care. Patients requiring blood collection from an intravenous catheter, those with known coagulation disorders, and those who decline to participate are excluded.
INTERVENTIONS:
Group A (Early Release): Sphygmomanometer inflated to 60 mmHg for venous stasis; tourniquet released immediately after the first tube (sodium citrate, blue cap) completes filling. Remaining tubes (SST/gel, yellow cap; K2-EDTA, purple cap) are collected after release.
Group B (Late Release): Sphygmomanometer inflated to 60 mmHg; tourniquet maintained throughout all tube filling and released only after the last tube (K2-EDTA, purple cap) completes filling. Tube collection order follows the CLSI H03-A6 standard for both groups.
PRIMARY OUTCOME:
Hemolysis rate, defined as the proportion of serum separator tube (SST/yellow cap) samples with a Hemolysis Index (HI) ≥ 1+ (corresponding to free hemoglobin ≥50 mg/dL), is assessed by the clinical chemistry laboratory analyzer. The outcome assessor (laboratory technician) is blinded to group assignment.
SECONDARY OUTCOMES:
(1) Distribution of ordinal hemolysis index categories (-, 1+, 2+, 3+, 4+, 5+) in SST samples; (2) Proportion of hemolyzed samples requiring repeat blood collection; (3) Total blood collection duration (seconds) from sphygmomanometer inflation to last tube filling completion; (4) Complication rate (hematoma, nerve injury, vasovagal reaction, arterial puncture, multiple puncture attempts).
SAMPLE SIZE:
A total of 792 participants (396 per group) are required based on an assumed hemolysis rate of 12% in the late release group and 6% in the early release group (50% relative risk reduction), α=0.05 (two-tailed), 80% power (Fleiss with pooled variance), plus 10% dropout buffer.
RANDOMIZATION:
Simple randomization using a computer-generated random number list (randomizer.org). The allocation sequence is maintained by a designated person not involved in enrollment or data collection. Allocation is revealed sequentially at the point of care.
STATISTICAL ANALYSIS:
Primary analysis: Chi-square test comparing hemolysis rates between groups (intention-to-treat population). Secondary analyses: Mann-Whitney U test for ordinal HI distribution; logistic regression for adjusted odds ratio. Bonferroni correction applied to multiple secondary comparisons (adjusted α = 0.017). Per-protocol analysis performed as a sensitivity analysis. Missing data handled using complete case analysis with sensitivity analysis.
Age 18 years or older
Presenting to the emergency department and triaged as green (semi-urgent) or yellow (urgent) category according to the Emergency Severity Index (ESI) or equivalent institutional triage system
Blood collection (venipuncture) indicated as part of routine clinical care by the attending emergency physician
Ability to provide written informed consent
Blood collection performed via an existing intravenous catheter or central venous access device
Known or suspected coagulation disorder (e.g., hemophilia, thrombocytopenia with platelet count <50,000/uL, current anticoagulant therapy with active bleeding)
Active upper extremity injury, infection, lymphedema, or arteriovenous fistula at the potential collection site
Triage category red (resuscitation) at the time of blood collection
Parallel
Sphygmomanometer-Applied Venous Stasis with Varied Release Timing During Phlebotomy
Procedure/SurgeryA standard aneroid sphygmomanometer is used in place of a conventional tourniquet rubber strap to apply venous stasis at a standardized pressure of 60 mmHg prior to venipuncture. Blood is collected in three tubes in the following order: (1) sodium citrate tube (blue cap, 2.7 mL), (2) serum separator tube/SST (yellow cap, 5 mL), (3) K2-EDTA tube (purple cap, 3 mL). The intervention variable is the timing of sphygmomanometer release: immediately after tube 1 (Group A, early release) versus after tube 3 (Group B, late release).
Hemolysis Rate (Proportion of Hemolyzed Serum Separator Tube Specimens)
The proportion of serum separator tube (SST/yellow cap) specimens with a Hemolysis Index (HI) of ≥1+ (corresponding to free hemoglobin concentration ≥50 mg/dL), as measured by the clinical chemistry laboratory analyzer. The HI is reported on a six-category ordinal scale: (-) \<50 mg/dL; (1+) 50-99 mg/dL; (2+) 100-199 mg/dL; (3+) 200-299 mg/dL; (4+) 300-500 mg/dL; (5+) \>500 mg/dL. A specimen is classified as hemolyzed if HI ≥1+.
Ordinal Distribution of Hemolysis Index Categories
Distribution of specimens across six Hemolysis Index categories (-, 1+, 2+, 3+, 4+, 5+) in SST/yellow cap tubes, analyzed as an ordinal variable to characterize severity of hemolysis beyond the binary primary outcome.
Proportion of Specimens Requiring Repeat Blood Collection Due to Hemolysis
The proportion of participants whose SST/yellow cap specimen was reported as unsuitable for analysis due to hemolysis and for whom a repeat blood collection was clinically requested by the attending physician, resulting in a second venipuncture within the same emergency department visit.
Total Blood Collection Duration
Time in seconds from sphygmomanometer inflation (venous stasis initiation) to completion of filling of the last collection tube (K2-EDTA), measured using a stopwatch by the healthcare provider performing the collection.
Procedural Complication Rate
Harms were systematically assessed. The proportion of participants experiencing any of the following procedure-related complications: (1) hematoma at puncture site, (2) nerve injury (persistent paresthesia \>30 minutes), (3) vasovagal reaction (syncope or pre-syncope), (4) accidental arterial puncture, (5) requirement for more than two venipuncture attempts. Complications are reported by the performing healthcare provider at the time of the procedure.
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