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Blood Bank

Frozen/Deglycerolized RBCs

This section delves into Frozen and Deglycerolized Red Blood Cells, a specialized component involving cryopreservation for extended storage. While not used routinely for most transfusions, it’s a vital tool for managing inventories of rare blood types, autologous units, and strategic reserves

What Are Frozen/Deglycerolized RBCs?

  • Definition: These are Red Blood Cells that have been treated with a cryoprotective agent (glycerol), frozen solid at ultra-low temperatures for long-term storage, and then thawed and washed (deglycerolized) to remove the glycerol before transfusion
  • Concept: Freezing allows RBCs to be stored for years instead of weeks, but requires special processing both before freezing and after thawing

Purpose / Indications (Why Freeze RBCs?)

The primary reason is long-term storage (up to 10 years or potentially longer). This is particularly useful for:

  • Rare Blood Units: Maintaining an inventory of RBC units with rare antigen phenotypes (e.g., negative for high-frequency antigens, specific combinations needed for patients with multiple antibodies)
  • Autologous Units: Storing a patient’s own blood for extended periods before planned surgeries (less common now due to shorter maximum liquid storage times and improved surgical techniques, but still an option)
  • Military / Strategic Reserves: Stockpiling blood for deployment or emergency preparedness in remote locations
  • Patients with Multiple Alloantibodies: Ensuring availability of compatible units when finding liquid-stored compatible blood is difficult

The Process: Freezing and Thawing/Deglycerolizing

This is a multi-step process requiring specialized equipment and reagents:

  1. Selection of RBC Unit Usually, relatively fresh RBCs (often < 6 days old) are selected for freezing to ensure optimal post-thaw quality
  2. Glycerolization (Adding Cryoprotectant)
    • Why Glycerol? Glycerol acts as a cryoprotective agent. It penetrates the RBC membrane and reduces the amount of intracellular water, preventing the formation of large, damaging ice crystals during freezing. Without it, freezing would destroy the cells
    • Methods: A concentrated glycerol solution is added slowly to the packed RBCs with mixing until an intracellular equilibrium is reached. Two main methods exist based on the final glycerol concentration:
      • High Glycerol (40% weight/volume): Most common method. Allows for slower, uncontrolled freezing rates and storage in mechanical freezers at -65°C to -80°C
      • Low Glycerol (20% weight/volume): Requires rapid, controlled-rate freezing and much colder storage temperatures (-120°C or colder, often using liquid nitrogen vapor phase at -196°C). Less common due to complex storage needs
  3. Freezing and Storage (Frozen State)
    • The glycerolized RBC unit is placed in a protective overwrap and frozen
    • Storage Temperature: ≤ -65°C (for high glycerol method)
    • Expiration (Frozen): 10 years (FDA standard; may be longer under specific protocols)
  4. Thawing
    • When needed, the frozen unit is removed from the freezer
    • Thawed rapidly in a monitored water bath or approved thawing device at 30-37°C until all ice is melted
  5. Deglycerolization (Removing Cryoprotectant - CRITICAL STEP)
    • Why Deglycerolize? Glycerol is hypertonic. If glycerolized RBCs were infused directly, the high intracellular glycerol concentration compared to the patient’s plasma would cause water to rush into the cells, leading to massive osmotic lysis (hemolysis)
    • How: This is essentially a washing process performed using automated cell washers. The thawed unit undergoes sequential washes with solutions of decreasing tonicity (decreasing salt concentration) to gradually remove the glycerol from the RBCs without lysing them. Typical wash solutions might include 12% NaCl, 1.6% NaCl, and finally isotonic 0.9% NaCl (often with added dextrose)
    • The final product is RBCs suspended in an isotonic saline/dextrose solution

Properties of Deglycerolized RBCs

The final product after thawing and washing is different from standard liquid-stored RBCs:

  • Composition: Primarily RBCs suspended in saline/dextrose solution
  • Reduced Plasma: Virtually all plasma proteins are removed during the washing process
  • Reduced Leukocytes: The washing process significantly reduces the number of WBCs (similar effect to leukoreduction by filtration)
  • Reduced Platelets: Platelets are removed during washing
  • Reduced Potassium & Additives: Accumulated potassium and byproducts from the initial liquid storage period (before freezing) and any additive solutions are washed away
  • Cell Recovery: Some RBC loss occurs during the freeze-thaw-wash cycle. Acceptable recovery is typically ≥ 80% of the original RBCs

Storage and Expiration (Post-Deglycerolization)

This is a key limitation:

  • Storage Temperature: 1°C to 6°C (like standard RBCs)
  • Expiration: Highly dependent on whether the deglycerolization process was performed in an open or closed system:
    • Open System: If the process potentially exposed the unit to ambient air (traditional methods), the expiration is 24 hours due to the risk of bacterial contamination
    • Closed System: If performed using FDA-approved sterile connecting devices and closed washing systems, the expiration can be extended, often up to 14 days, depending on the specific system’s validation and labeling. This significantly improves logistics

Quality Control

QC measures may include:

  • Adequate RBC recovery
  • Acceptable levels of residual glycerol (measured indirectly by osmolality or directly)
  • Acceptable levels of free hemoglobin (hemolysis)
  • Sterility testing (especially for longer post-thaw storage)

Administration

  • Compatibility: Standard ABO/Rh typing and crossmatch procedures are required
  • Filtration: Administer through a standard blood filter (170-260 microns)
  • Infusion Time: Must be completed within 4 hours of spiking the bag or removal from controlled 1-6°C storage

Advantages

  • Extended Storage (10+ years): Primary benefit, crucial for rare units and strategic reserves
  • Reduced Plasma Proteins: Beneficial for patients with severe allergic reactions (similar to washed RBCs)
  • Leukocyte Reduced: Reduces risk of FNHTRs, HLA alloimmunization, and CMV transmission
  • Lower Potassium Load: Removes accumulated potassium from the supernatant

Disadvantages

  • Complex and Time-Consuming Process: Requires specialized equipment, trained personnel, and significant processing time (cannot be provided “on demand” quickly)
  • Costly: Higher cost due to equipment, reagents, and labor involved
  • RBC Loss: Inevitable loss of some RBCs during processing
  • Short Post-Thaw Shelf Life: Especially with open systems (24 hours), limiting flexibility
  • Logistics: Requires careful inventory management and planning for thawing/deglycerolization

Key Terms

  • Cryoprotective Agent: A substance (like glycerol) that protects cells from damage during freezing
  • Glycerolization: The process of adding glycerol to RBCs before freezing
  • Deglycerolization: The washing process used after thawing to remove glycerol from RBCs
  • Osmotic Lysis: Rupture of cells due to rapid water influx caused by osmotic imbalances
  • Open System: A processing method where the sterility of the component may be compromised (limits post-process expiration)
  • Closed System: A processing method maintaining sterility throughout (allows longer post-process expiration)
  • High/Low Glycerol Method: Refers to the final concentration of glycerol used, impacting freezing rate and storage temperature requirements