White Blood Cell Staining Methods

What are we distinguishing by staining?

Residual white blood cells in donated blood products may pose potential risks during transfusion. These leukocytes can trigger immune-mediated reactions, including fever and chills, and may negatively affect the quality of blood bags. For more information, read the previous article.

For this reason, it is important to verify the presence and level of residual white blood cells in stored blood bags. Accurate leukocyte detection helps support blood product quality control and reduces the risk of transfusion-related adverse reactions.

Romanowsky stain

In blood sample analysis, red blood cells (RBCs) and white blood cells (WBCs) can be distinguished by using differential staining methods.

A commonly used approach is Romanowsky staining, including Wright’s stain and Giemsa stain, which helps visualize the morphological differences between blood cell types.

Because RBCs lack nuclei, they typically appear pink to red after staining. In contrast, WBCs contain nuclei and other intracellular components, which usually stain blue to purple. These staining patterns make it easier to identify and differentiate RBCs from WBCs in whole blood samples.

Fluorescent stain

In whole blood cell analysis, red blood cells (RBCs) and white blood cells (WBCs) can also be differentiated using fluorescent nucleic acid stains, such as acridine orange (AO) and propidium iodide (PI).

AO and PI bind to nucleic acids, including DNA and RNA. Since mature mammalian RBCs lack a nucleus, they typically show little to no fluorescence signal with nucleic acid-based staining. WBCs, however, contain nuclei and intracellular nucleic acids, allowing them to be selectively stained and detected. AO-stained WBCs are commonly observed as green fluorescent cells, while PI-stained cells can appear red depending on membrane permeability and staining conditions.

This fluorescence-based staining method is useful for distinguishing nucleated cells from non-nucleated RBCs in blood samples. As a result, it provides a rapid and reliable approach for WBC detection, RBC and WBC differentiation, and whole blood sample quality assessment.

If you would like to know more about the fluorescence staining, check the following article:

Ch.4-2 Fluorescence dye solution (PI / AO / DAPI)

What staining techniques should you choose?

Different clinical findings can be obtained depending on the staining method used.

Common clinical findings using Romanowsky staining and fluorescent staining for white blood cells include:

* Identifying and counting different types of white blood cells

* Assessing the morphology of white blood cells

* Detecting abnormalities in white blood cells, such as those associated with leukemia, anemia, and infections

* Supporting blood and bone marrow pathology analysis

* Detecting malaria and other blood parasites

* Monitoring the response to treatment for blood disorders

Both Romanowsky staining and fluorescent staining are valuable tools for diagnosing and monitoring blood disorders.

However, there are differences between the two methods. In this comparison table, PI is used to represent fluorescent stains.

Feature	Romanowsky stain	Propidium iodide stain
Type	Basic	Acidic
Dyes used	Azure B and methylene blue	Propidium iodide
Sensitivity	More sensitive	Less sensitive
Speed	More time-consuming ( > 15 mins)	Faster ( < 5 mins)
Ease of preparation	More difficult	Easier
Preferred use	Diagnosing blood disorders	Rapid screening

Distinguishing WBCs from RBCs is important for assessing potential transfusion-related risks and ensuring the quality of donated blood products. Romanowsky staining helps differentiate blood cells based on color and morphology, while fluorescent staining detects nucleic acids present in WBCs.

These staining methods can be used to identify different WBC types, assess WBC morphology, detect abnormal cells, and monitor treatment responses in blood disorders. Both Romanowsky staining and fluorescent staining are valuable tools for blood cell analysis and the diagnosis and monitoring of blood-related diseases.

Reference

1. Horobin RW. How Romanowsky stains work and why they remain valuable - including a proposed universal Romanowsky staining mechanism and a rational troubleshooting scheme. Biotech Histochem. 2011 Feb;86(1):36-51. doi: 10.3109/10520295.2010.515491. PMID: 21235292.

2. NanoEntek, Ch.4-2 Fluorescence dye solution (PI/AO/DAPI), Accessed Jun. 2023.

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[ADAM rWBC2]

Residual Leukocyte Counter

Adam rWBC 2

[ADAM rWBC HT]

Residual White Blood Cell Counter

Adam rWBC HT