Hemolysis - the rupture of RBCs - releases hemoglobin into the sample, which can contaminate DNA preparations, inhibit downstream enzymatic reactions, and reduce overall DNA quality. It occurs due to mechanical shear stress, osmotic imbalances, or temperature fluctuations that compromise RBC membranes. In automated systems, factors such as pipetting speed, needle gauge, and centrifuge acceleration can exacerbate this. For labs running high-throughput automated systems, minimizing hemolysis is critical to ensure consistent, high-purity DNA yields essential for PCR, NGS, and other molecular assays.

1. Sample Collection:

Effective sample collection is the first step in minimizing hemolysis. The choice of anticoagulant is critical. EDTA, one of the most commonly used anticoagulants, prevents blood clotting by binding calcium, a vital factor in the clotting process. But, calcium is also crucial for maintaining the structural integrity of red blood cell (RBC) membranes. When EDTA chelates calcium, it destabilizes RBC membranes, making them more susceptible to rupture. Heparin is a gentler alternative that prevents clotting without removing calcium. However, it can inhibit PCR enzymes and is generally avoided in molecular workflows. Citrate works by binding calcium in a way that is reversible. This helps maintain a stable environment for RBCs without causing too much damage. But because it can dilute the blood sample, you might get less DNA in the end.

2. Temperature Control

Temperature management plays a pivotal role in preventing RBC lysis:

• Sample Transport: Blood must be transported at 4°C and processed within 24 hours when possible. Delays or temperature fluctuations increase hemolysis, thereby reducing DNA quality.

• Cold Storage: Maintaining samples at 4°C slows metabolic processes and preserves membrane integrity. For long-term storage, -80°C freezing is standard, but freeze-thaw cycles should be minimized.

• Equilibration Prior to Extraction: Before automated processing, samples should be warmed to room temperature (~20–22°C) to prevent thermal shock. Sudden temperature changes cause RBC membranes to rupture easily, especially when combined with mechanical stress.

3. Centrifugation

Centrifugation is necessary to separate plasma or buffy coat from whole blood, but incorrect settings can increase hemolysis. For initial plasma separation, a speed of 1,500 × g for 10 minutes is recommended, which effectively separates plasma without damaging RBCs. For cfDNA extraction, a higher speed of 5,000 × g may be used without significant RBC lysis. The type of centrifuge rotor also matters—fixed-angle rotors reduce shear stress compared to swing-bucket designs. Finally, gradual acceleration and deceleration minimize shear forces that can rupture RBCs.

4. Buffer Chemistry

Chemical composition of buffers is crucial in maintaining RBC integrity while achieving efficient cell lysis. The ideal lysis buffer must efficiently lyse nucleated cells while preserving RBC integrity. Non-ionic detergents, such as Triton X-100 at concentrations between 0.1–0.5%, provide a gentle approach, ensuring that nucleated cells are lysed without damaging RBCs. Maintaining buffer pH between 7.4 and 8.0 is essential for membrane stability. Additionally, a low salt concentration (<150 mM NaCl) reduces osmotic stress on RBCs, preventing them from swelling and bursting.

5. Automated Pipetting Precision

Automated systems rely on precise liquid handling, which directly affects hemolysis rates. Pipetting speed is a critical factor—high speeds can create mechanical shear forces that rupture RBCs. Optimal settings (~200 µL/s) ensure gentle handling. Accurate tip positioning prevents bubbles, which can increase mechanical stress. Finally, gentle mixing strategies—such as slow pipetting over vortexing—maintain sample homogeneity without excessive mechanical force.

How Manta Tackles Hemolysis: Proprietary Chemistry Meets Engineering Excellence

Manta is an automated DNA/RNA extraction system designed for precision, reliability, and high-quality yield. Built with advanced engineering and proprietary chemistry, Manta is specifically optimized to handle diagnostic samples, including whole blood, without compromising DNA integrity. Whether you are processing a few samples or running high-throughput workflows, Manta ensures consistent performance and minimal sample loss through an integrated approach combining:

• Specialized Lysis Chemistry: The proprietary buffer formulation balances efficient WBC lysis with RBC preservation. This selective lysis reduces hemoglobin contamination and preserves DNA integrity, critical for downstream NGS and PCR.

• Temperature-Controlled Workflow: Manta’s sample racks and reagent reservoirs maintain optimal temperatures during all process steps, minimizing thermal stress.

• Fine-Tuned Liquid Handling: High-precision pipetting hardware controls flow rates and tip immersion depth with sub-millimeter accuracy, drastically reducing mechanical shear.
  
  

This combination allows Manta to consistently produce DNA of superior purity (A260/A280 > 1.8; A260/A230 > 2.0), with low hemolysis rates even in demanding high-throughput environments.

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