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.

BACKGROUND AND RATIONALE:

Hemolysis is recognized as the leading preanalytical source of error in clinical laboratories, with emergency department settings reporting particularly high rates (range: 12-26%) due to specimen collection under time pressure, use of small-bore intravenous catheters, and challenging venous access. Hemolyzed specimens affect measurements of potassium, lactate dehydrogenase, bilirubin, and other analytes, resulting in clinical misinterpretation, repeated laboratory requests, and procedural delays in a time-sensitive environment.

Venous stasis created by tourniquet application is a physiological perturbation that promotes erythrocyte deformation and lysis through hemoconcentration and shear stress. Clinical guidelines (Clinical and Laboratory Standards Institute, CLSI H03-A6; Turkish Biochemistry Society Phlebotomy Guidelines 2015) recommend that the tourniquet should be released as soon as blood flow is established, ideally within 60 seconds, and not later than the completion of the first collection tube. However, in routine emergency nursing practice, the tourniquet is frequently maintained throughout the entire multi-tube collection sequence to maintain venous distension and reduce the likelihood of failed collection attempts.

This pragmatic trial directly tests whether adherence to guideline-recommended early tourniquet release translates into a clinically meaningful reduction in hemolysis rates compared to the commonly observed late release practice, in a controlled setting using standardized sphygmomanometer-applied venous stasis at 60 mmHg.

INTERVENTION DELIVERY:

Both interventions use a standard aneroid sphygmomanometer (not a conventional tourniquet rubber strap) inflated to 60 mmHg to standardize venous stasis pressure across all participants. This represents a methodological innovation that eliminates inter-practitioner variability in tourniquet application pressure.

Tube collection order (identical for both groups, per CLSI H03-A6):

1. Sodium citrate (blue cap, 2.7 mL) - coagulation studies
2. SST/gel separator (yellow cap, 5 mL) - serum biochemistry [PRIMARY OUTCOME TUBE]
3. K2-EDTA (purple cap, 3 mL) - complete blood count

Group A - Early Release Protocol: After confirming blood flow and completing the first tube (sodium citrate), the sphygmomanometer is deflated to 0 mmHg and removed. Subsequent tubes are collected without venous stasis.

Group B - Late Release Protocol: The sphygmomanometer remains inflated at 60 mmHg throughout all three tubes. It is deflated and removed only after the third tube (K2-EDTA) completes filling.

DIFFICULTY ASSESSMENT - A-DIVA Scale: All participants are assessed for venous access difficulty using the A-DIVA (Amsterdam Difficult Intravenous Access) Scale (van Loon et al., 2016) prior to blood collection. The A-DIVA scale assigns 0-1 points for each of five items: (1) history of difficult venous access, (2) no palpable vein, (3) no visible vein, (4) vein diameter <3 mm, (5) unplanned/emergency procedure. Total score ≥2 is classified as high-difficulty. The A-DIVA score is recorded as a covariate in statistical analysis.

HEMOLYSIS ASSESSMENT:

The Hemolysis Index (HI) is measured on all SST/yellow cap tube specimens by the clinical chemistry laboratory analyzer, reported on a six-category ordinal scale corresponding to free hemoglobin concentrations: (-) <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. Laboratory technicians performing HI analysis are blinded to group assignment throughout the study.

BLINDING:

Participants and healthcare providers administering the intervention cannot be blinded due to the procedural nature of the intervention. Outcome assessors (laboratory technicians performing hemolysis index measurement) are blinded to group assignment. Statistical analysis is performed on de-identified data.

PATIENT AND PUBLIC INVOLVEMENT (CONSORT 2025 Item 8):

Patients and the public were not formally involved in the design of this trial due to the pragmatic, low-risk, procedure-modification nature of the study. Findings will be disseminated through open-access publication and presented to nursing and laboratory staff at the participating institution.

DATA SHARING (CONSORT 2025 Item 4):

De-identified individual participant data and the data dictionary will be made available to qualified researchers upon reasonable written request to the principal investigator, following publication of the primary manuscript and approval by the institutional ethics committee, in accordance with applicable data protection regulations.

Inclusion Criteria:

• 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
• Accessible antecubital or forearm vein suitable for standard venipuncture (not requiring intravenous catheter placement for blood collection)

Exclusion Criteria:

• 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
• Declined informed consent
• Previously enrolled in this study (re-enrollment not permitted)
• Pregnancy (due to potential vascular changes affecting hemolysis rate)
• Known hemolytic anemia or other hematological condition associated with baseline elevated hemolysis

De-identified individual participant data, including the data dictionary, will be made available upon reasonable written request to the principal investigator following peer-reviewed publication, subject to institutional ethics committee approval and applicable data protection regulations.

Following a peer-reviewed publication

Reasonable written request to the principal investigator (emirunal@gmail.com).

Outcome assessors (laboratory technicians performing Hemolysis Index analysis) are blinded to group assignment. Specimens are labeled with participant ID only; group allocation information is not accessible to laboratory personnel. Participants and care providers cannot be blinded because of the intervention's procedural nature.