15. In order to separate red and white blood cells, which of the following methods can be used?
    (1) Ion-exchange chromatography and FACS
    (2) Hydrophobic chromatography and density gradient centrifugation
    (3) Density gradient centrifugation and FACS
    (4) Hydrophobic chromatography and FACS

Introduction

Separating red blood cells (RBCs) and white blood cells (WBCs) is a crucial procedure in both clinical and research settings for diagnostic purposes and various therapies. Multiple separation techniques exist, but among the most efficient and widely used are density gradient centrifugation and fluorescence-activated cell sorting (FACS). This article explores these two methods, explains why they are suitable for separating RBCs and WBCs, and compares them to other techniques like ion-exchange chromatography and hydrophobic chromatography.

Density Gradient Centrifugation: Principles and Application

Density gradient centrifugation is a technique that separates cells based on their density differences by spinning blood samples in specialized media such as Ficoll or Percoll.

• How it works: When centrifuged, cells distribute in layers according to their density. RBCs are denser and settle at the bottom, whereas the less dense WBCs form a distinct layer called the “buffy coat” above the RBC layer.

• Advantages: It is relatively inexpensive, simple, and effective for initial fractionation of blood components.

• Limitations: It provides separation based on density but not specific cell types, so further sorting may be required for pure populations.

Fluorescence-Activated Cell Sorting (FACS): Precision Sorting

FACS is a specialized flow cytometry technique that allows for very precise separation of cells based on fluorescent labeling of specific surface markers.

• How it works: Cells are labeled with fluorescent antibodies specific to cell types (e.g., CD markers). The cells flow in single file through a laser beam, where fluorescence signals are detected, and cells are sorted accordingly into different containers.

• Advantages: High specificity and purity, ability to sort multiple cell types in one run.

• Limitations: Requires advanced equipment and fluorescent labeling reagents, making it more costly.

Why Density Gradient Centrifugation and FACS Together?

Combining the two methods is a common strategy:

• Use density gradient centrifugation first to enrich and separate RBCs and WBCs from whole blood.

• Follow with FACS for sorting specific subpopulations of WBCs with high purity based on their markers.

Comparison with Other Techniques

• Ion-exchange chromatography: Mostly used for protein separation, not typically for whole cells like RBCs and WBCs.

• Hydrophobic chromatography: Primarily separates proteins or molecules based on hydrophobic interactions, not cells.

Therefore, options involving ion-exchange or hydrophobic chromatography paired with FACS are not ideal for RBC/WBC separation.

Correct Answer

Among the options, density gradient centrifugation and FACS (Option 3) is the correct combination for separating red and white blood cells efficiently and specifically.