Cell Separations

Cell Separation techniques aren’t something you’ll do every single day, but when you have a patient with a mix of their own red blood cells and transfused donor red blood cells circulating, these techniques become incredibly important, especially during transfusion reaction investigations!

Think of it like trying to sort laundry after someone mixed whites and colors – we need a way to separate the different populations of cells so we can examine them individually

The Core Purpose: Isolating Specific RBC Populations

The main reason we perform cell separations in the blood bank is to isolate the patient’s autologous (own) red blood cells from transfused allogeneic (donor) red blood cells when both are present in a sample

Why is this necessary?

• Transfusion Reaction Workups: This is the #1 reason! If a patient might be having a delayed hemolytic transfusion reaction, their antibody might be coating the donor cells, but not their own. We need to separate the populations to:
  • Perform a Direct Antiglobulin Test (DAT) specifically on the donor cell population (to detect antibody coating them)
  • Perform a DAT specifically on the patient’s cell population (usually expected to be negative in an alloimmune reaction, but could be positive if there’s also an autoantibody or drug issue)
  • Accurately phenotype the patient’s cells, especially if their pre-transfusion type is unknown or needs confirmation
  • Potentially phenotype the donor cells if needed (less common, usually rely on unit tag)
• Monitoring Bone Marrow/Stem Cell Transplant Patients: These patients can have mixed chimerism (both recipient and donor cells present) during engraftment. Cell separation might be needed for accurate phenotyping or other specialized testing

3. Investigating Mixed Field Agglutination If routine typing shows mixed field reactions that aren’t easily explained, separating cell populations might (rarely) help clarify the situation

The Main Method: Differential Centrifugation (Based on Cell Age/Density)

The most common method relies on the fact that red blood cells have a limited lifespan (about 120 days), and their density changes as they age

• Younger RBCs (Neocytes/Reticulocytes): Less dense. This includes newly produced patient reticulocytes and often the majority of cells in a relatively fresh unit of transfused blood
• Older RBCs (Gerocytes): More dense. These are the patient’s mature cells nearing the end of their lifespan

The Principle By spinning a sample very hard (microhematocrit centrifugation), we can separate the cells based on density

The Procedure (Simplified)

1. Wash Thoroughly wash the patient’s post-transfusion EDTA sample to remove all plasma
2. Centrifuge Place the washed red cells in microhematocrit tubes and centrifuge at high speed for a set time (e.g., 5-10 minutes)
3. Separate Layers Carefully score and break the microhematocrit tube to separate the layers:
   • Top Layer: Contains the least dense cells (enriched in reticulocytes and younger transfused cells - often considered the “donor enriched” fraction post-transfusion)
   • Bottom Layer: Contains the most dense cells (enriched in older patient cells - often considered the “patient enriched” fraction)
   • Middle Layer: Contains a mix of cells of intermediate age/density (often discarded or tested cautiously)
4. Harvest & Wash Suspend the cells from the top and bottom fractions separately in saline and wash them again
5. Testing Perform desired tests (DAT, phenotyping) on both the top and bottom fractions

Validation (Is the Separation Good Enough?)

• Often, we assess the separation by phenotyping the top and bottom fractions for antigens known to differ between the patient and the donor (if a pre-transfusion type is known or donor unit info is available). For example, if the patient is K-negative and received K-positive blood, the top fraction should be enriched for K-positive cells, while the bottom fraction should be predominantly K-negative
• It’s often not a perfect separation!: We usually achieve enrichment rather than pure populations. Interpretation requires acknowledging this limitation

Alternative / Specialized Methods

• Hypotonic Wash / Differential Lysis (for Sickle Cell Patients): Normal donor red cells (HbA) are more resistant to lysis in low-salt solutions than sickle cells (HbS). This property can sometimes be exploited to preferentially lyse sickle cells, leaving donor cells relatively intact for testing. Used specifically when dealing with sickle cell disease patients
• Immunomagnetic Beads / Affinity Columns: More sophisticated methods (usually research or highly specialized labs) using antibodies attached to beads or columns to bind and separate cells based on specific surface antigens. Not routine for post-transfusion workups

Post-Separation Testing

Once you have your enriched fractions:

• DAT: Perform on both fractions using polyspecific and monospecific AHG. A positive DAT primarily on the top (donor-enriched) fraction strongly suggests an alloantibody coating donor cells (transfusion reaction)
• Phenotyping: Type both fractions to confirm patient type (bottom fraction) and potentially donor contribution (top fraction)

Challenges and Considerations

• Timing: Separation is most effective when there’s a significant age difference between patient and donor cells (e.g., patient hasn’t been transfused very recently with old blood, or patient has high reticulocyte count)
• Patient Status: Conditions like hemolytic anemia (high reticulocytes) can affect the density profile of the patient’s own cells
• Degree of Separation: It’s rarely perfect. Results reflect enrichment, not purity
• Labor Intensive: Requires careful technique and time

Cell separation is a valuable tool, primarily for dissecting complex post-transfusion scenarios. Mastering the differential centrifugation technique and understanding how to interpret results from enriched fractions is key when investigating potential hemolytic transfusion reactions!

Key Terms

• Cell Separation: Techniques used to isolate specific populations of red blood cells from a mixed sample
• Autologous Cells: The patient’s own red blood cells
• Allogeneic Cells: Red blood cells from a donor
• Differential Centrifugation: Separation method based on differences in red blood cell density (related to cell age) using high-speed centrifugation (microhematocrit)
• Neocytes: Younger, less dense red blood cells
• Gerocytes: Older, more dense red blood cells
• Reticulocytes: Immature red blood cells, less dense than mature RBCs
• Top Fraction: Layer of cells collected after differential centrifugation containing the least dense cells (often enriched for reticulocytes/younger donor cells)
• Bottom Fraction: Layer of cells collected after differential centrifugation containing the most dense cells (often enriched for older patient cells)
• Enrichment: Achieving a higher concentration of a specific cell population in a fraction, but not necessarily a completely pure separation
• Mixed Field Agglutination: Agglutination pattern where clumps of agglutinated cells are mixed with unagglutinated cells, often indicating the presence of two cell populations reacting differently
• Chimerism: The presence in an individual of two or more genetically distinct cell lines (e.g., post-transplant)