Early vs. Late Tourniquet Release and Phlebotomy-Induced Hemolysis in the Emergency Department: The TOURNI-ED Randomized Trial (TOURNI-ED)

Early Versus Late Release of Sphygmomanometer-Applied Venous Stasis and Phlebotomy-Induced Hemolysis in the Emergency Department: A Parallel-Group Randomized Controlled Trial

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.

Study Overview

• Status: Not yet recruiting
• Conditions: Hemolysis; Venous Stasis; Phlebotomy; Blood Specimen Collection
• Intervention / Treatment: Procedure: Sphygmomanometer-Applied Venous Stasis with Varied Release Timing During Phlebotomy

Detailed Description

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.

• Study Type: Interventional
• Enrollment (Estimated): 792
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Emir Ünal; Phone Number: +905327766010; Email: emirunal@gmail.com

Study Locations

• Turkey (Türkiye)
  • Istanbul
    • Pendik, Istanbul, Turkey (Türkiye), 34899
      • Marmara University Pendik Training and Research Hospital
      • Contact: Emir Ünal; Phone Number: 05327766010; Email: emirunal@gmail.com

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

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

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Prevention
• Allocation: Randomized
• Interventional Model: Parallel Assignment
• Masking: Single

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Early Release Group (Group A); Venous stasis is applied using a sphygmomanometer inflated to 60 mmHg. The sphygmomanometer is released (deflated to 0 mmHg) immediately after the first blood collection tube (sodium citrate, 2.7 mL, blue cap) completes filling. The second (SST/gel separator, 5 mL, yellow cap) and third (K2-EDTA, 3 mL, purple cap) tubes are collected without active venous stasis. The tube collection order follows the CLSI H03-A6 guidelines for all participants.	Procedure: Sphygmomanometer-Applied Venous Stasis with Varied Release Timing During Phlebotomy; A 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).; Other Names: Tourniquet Release Timing; Venous Stasis Duration; Phlebotomy Technique Modification
Active Comparator: Late Release Group (Group B); Venous stasis is applied using a sphygmomanometer inflated to 60 mmHg. The sphygmomanometer is maintained at 60 mmHg throughout the multi-tube collection sequence and released (deflated to 0 mmHg) only after the third and final collection tube (K2-EDTA, 3 mL, purple cap) has filled. This reflects current common nursing practice in the emergency department setting.	Procedure: Sphygmomanometer-Applied Venous Stasis with Varied Release Timing During Phlebotomy; A 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).; Other Names: Tourniquet Release Timing; Venous Stasis Duration; Phlebotomy Technique Modification

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
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+.	Measured at the time of laboratory analysis, within 2 hours of blood collection

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
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.	Measured at the time of laboratory analysis, within 2 hours of blood collection
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.	Within the same emergency department visit (up to 24 hours post-collection)
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.	Measured during the blood collection procedure
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.	During the blood collection procedure and up to 30 minutes post-procedure

Collaborators and Investigators

• Sponsor: Marmara University Pendik Training and Research Hospital

Publications and helpful links

General Publications

• Ayten S, Koşer M, Cumhur A, et al. Comparison of the Hemolysis Rate According to Biochemistry Test Results of Patients Admitted to The Emergency Department with the Results of the Hemcheck Device. Eurasian J Emerg Med. 2025;24(2):126-31. doi:10.4274/eajem.galenos.2024.80947
• Lv L, Li C, Wei W, Sun S, Ren X, Pan X, Li G. Optimization of sparse-view CT reconstruction based on convolutional neural network. Med Phys. 2025 Apr;52(4):2089-2105. doi: 10.1002/mp.17636. Epub 2025 Feb 2.
• Tomar SL, Carden DL, Dodd VJ, Catalanotto FA, Herndon JB. Trends in dental-related use of hospital emergency departments in Florida. J Public Health Dent. 2016 Jun;76(3):249-57. doi: 10.1111/jphd.12158. Epub 2016 Apr 22.
• Calleja R, Mielke N, Lee R, Johnson S, Bahl A. Hemolyzed Laboratory Specimens in the Emergency Department: An Underappreciated, but Frequent Problem. J Emerg Nurs. 2023 Sep;49(5):744-754. doi: 10.1016/j.jen.2023.06.001. Epub 2023 Jun 27.
• Ersoy S, Ilanbey B; Kirsehir, Turkey. A Single-Center Prospective Study of the Effects of Different Methods of Phlebotomy in the Emergency Department on Blood Sample Hemolysis Rates. J Emerg Nurs. 2023 Jan;49(1):134-139. doi: 10.1016/j.jen.2022.08.005. Epub 2022 Sep 20.
• Wollowitz A, Bijur PE, Esses D, John Gallagher E. Use of butterfly needles to draw blood is independently associated with marked reduction in hemolysis compared to intravenous catheter. Acad Emerg Med. 2013 Nov;20(11):1151-5. doi: 10.1111/acem.12245.

Study record dates

Study Major Dates

• Study Start (Estimated): June 1, 2026
• Primary Completion (Estimated): June 1, 2027
• Study Completion (Estimated): August 1, 2027

Study Registration Dates

• First Submitted: May 23, 2026
• First Submitted That Met QC Criteria: May 23, 2026
• First Posted (Actual): June 1, 2026

Study Record Updates

• Last Update Posted (Actual): June 1, 2026
• Last Update Submitted That Met QC Criteria: May 23, 2026
• Last Verified: May 1, 2026

More Information

Terms related to this study

• Keywords: randomized controlled trial; emergency department; hemolysis; blood collection; venous stasis; phlebotomy; tourniquet; hemolysis index
• Additional Relevant MeSH Terms: Vascular Diseases; Cardiovascular Diseases; Pathologic Processes; Disease Attributes; Skin Diseases; Skin Ulcer; Varicose Veins; Leg Ulcer; Pathological Conditions, Signs and Symptoms; Skin and Connective Tissue Diseases; Emergencies; Varicose Ulcer; Hemolysis
• Other Study ID Numbers: 09.2026.829

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: 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.
• IPD Sharing Time Frame: Following a peer-reviewed publication
• IPD Sharing Access Criteria: Reasonable written request to the principal investigator (emirunal@gmail.com).
• IPD Sharing Supporting Information Type: STUDY_PROTOCOL; SAP; ANALYTIC_CODE

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No