September 16, 2024

Intro to human blood components, cell types, and cell markers

Blood components as separated by density gradient

1. Plasma: yellow layer on top, 55% of blood volume

2. Buffy coat (leukocytes and platelets): white layer in the middle, <1% of blood volume

  • White blood cells (leukocytes) = granulocytes (neutrophils, eosinophils, basophils) and PBMCs/agranulocytes (monocytes, dentritic cells, T cells, B cells, NK cells)
  • Platelets (thrombocytes) = the blood clotting cells, the smallest blood cell type
  • Related: buffy cone refers to the leukocyte-rich waste product from plateletpheresis procedures. It isn't the same as buffy coat , which is a layer from the density gradient, but both are sources of leukocytes.

3. Red blood cells (erythrocytes): red layer at the bottom, 44-45% of blood volume. 

  • Adult red blood cells are anucleated (don't have a nucleus). The nucleus is lost during cell maturation.
  • Fetal red blood cells do have a nucleus . This affects cell density makes it more difficult to use density gradient medium protocols to separate cord blood into the three layers, compared to adult peripheral blood.

Learn more about blood components and blood cell types:

Buffy coat components (leukocytes and platelets)

Leukocytes (white blood cells) = PMNs + PBMCs, part of the buffy coat

Learn more about white blood cells:
PMNs: polymorphonuclear cells ("granulocytes"), white blood cell subgroup
  • Neutrophils (50-70% of all leukocytes) - stain well with neutral stains
    • First cell to arrive when tissue is infected
    • Defends against invading organisms & produces puss in infected tissue
    • Nucleus has 2-5 lobes depending on the age of the cell
    • Short-lived, fragile cells with short life-span (not good for single cell experiments). When cleaning up the buffy coat to further isolate PBMCs, this is one of the major cell types you're trying to separate away from PBMCs.
    • Detect cytokines (e.g. released at site of infection) and travel there
    • They are phagocytes , meaning cells that can do phagocytosis to eat other cells including invading bacteria. "Phagocyte" is a cell function term, not a cell lineage term. A lot of immune cells can perform phagocytosis.
    • Contain granules called lysosomes , cell organelles that contains lysozymes, enzymes that breaks down other cell components such as bacterial cell walls
  • Eosinophils (2-4% of all leukocytes) - stain well with acidic stain like eosin
    • Nucleus has 2-3 lobes
    • Contain granules that deal with allergic reactions, parasitic worms, autoimmune diseases
    • Release cytokines and chemical signals for wound healing
  • Basophils (<1% of all leukocytes) - stain best with basic stains (show up dark blue)
    • Release cytokines, histamines, and other signaling molecules
    • Largest cell size among granulocytes
  • PMNs can kill large cells that are too big for phagocytosis
  • Promote allergies and asthma
PBMCs: peripheral blood mononuclear cells ("agranulocytes"), white blood cell subgroup
  • PBMCs have granules also but they're smaller and not as easy to see, historically called "agranular".
  • Lymphocytes (>80% of PBMCs)
    • T cells - part of adaptive immunity
      • CD4+ T cells (25-60% of PBMCs): regulatory T cells & helper T cells
      • CD8+ T cells (5-30% of PBMCs): killer T cells, kill self-cells with problems, e.g. cancerous cells, infected cells
    • B cells - produce antibodies, part of adaptive immunity
      • CD19+ B cells (5-10% of PBMCs) 
      • Activated by helper T cells
    • Natural killer (NK) cells - detect human cells with abnormal surface markers and kills them, e.g. infected cells, cancer cells
      • CD56+ CD3- NK cells (10-30% of PBMCs)
  • Monocytes (2-8% of all leukocytes, 2-10% of all PBMCs) - larger cells (25-30 um diameter) that take in other cells using phagocytosis. Important for innate immunity.
    • CD14+ monocytes (5-10% of PBMCs) - progenitor cells that can differentiate into dendritic cells, macrophages, and sometimes other cell types
    • Phagocytosis of pathogens and dying self-cells (e.g. apoptotic cells).
  • Dendritic cells (1-2% of PBMCs)
    • Antigen-presenting cells that link innate and adaptive immune systems
Other:
  • Macrophages - phagocytes that live in tissue, not blood. They won't be part of the PBMCs isolated from peripheral blood, but they can be derived from monocytes (which are in PBMCs from blood).
    • Scavenger cells involved in foreign cell detection, antigen presentation to T cells, and cytokine release to initiate inflammation. They also eat dead and dying self-cells to clean up the body. 
    • Macrophage functions vary by tissue type. In placenta, fetal-derived macrophages are called Hofbauer cells.
    • IL-6 promotes monocytes to differentiate into macrophages instead of dendritic cells. Citation: Chomarat et al. 2000, Nature Immunology
Learn more about PBMCs:

Platelets (Thrombocytes), also part of the buffy coat

  • Platelets are small and colorless, anucleated (no nucleus), and shaped like a plate.
  • Only 1-5 um diameter, which means they pass through most cell filters which creates problems for single cell and single nuclei sequencing. Comparatively, mammalian nuclei are 5-20 um diameter.
  • They are technically fragments of cells, not actual cells . 
  • They appear as tiny dots under the microscope that don't fluoresce with the acridine orange/propidium iodide (AOPI) fluorescence dye used for cell counting and viability staining. 
    • Live cells show green fluorescence with AOPI.
    • Nuclei, chromatin (DNA), and dead cells show red fluorescence with AOPI. 
    • Platelets don't fluoresce with AOPI.
  • Platelets are activated by buffers containing calcium and magnesium ions (undesirable)
  • Inhibit platelet activation by using buffers containing bovine serum albumin (BSA)
  • If platelet contamination is a big issue in your sample, you can reduce platelet counts with a slow spin step ( 120 rcf for 10 minutes, brakes off, room temperature) followed by discarding the platelet-rich supernatant. You just need to be careful not to lose your other cells since 120 rcf is a very low speed and might not be enough to create a good cell pellet. If working with limited sample, first try 200 rcf and see if that removes enough platelets with the supernatant.

Learn more about platelets:

Blood cell type markers (incomplete)

CD3 (CD3A, CD3D... gene) = T cell marker, not expressed by B cells or monocytes

CD4 = helper and regulatory T cell marker, not expressed by killer T cells

CD8 (CD8A gene) = killer T cell marker, not expressed by expressed by helper or regulatory T cells

CD11C (ITGAX gene) = conventional dendritic cell marker

CD14 = monocyte marker, only classical monocytes?

CD16 (FCGR3A gene) = natural killer (NK) cell marker, also expressed by non-classical monocytes but NOT classical monocytes (see Villani et al 2018).

CD34 = hematopoietic stem cell (HSC) marker, the cell type that most people are trying to pull out of cord blood

CD45 (PTPRC gene)  = pan-leukocyte marker (expressed by white blood cells), cell marker for nucleated hematopoietic cells

CD66b = eosinophil and neutrophil marker. It's sometimes called a granulocyte cell marker but beware that it's not expressed by all/any(?) basophils. [To do: update after learning more about CD66b and basophils]

FOSB = activated (but not naive) CD4+ T cell marker. It's a marker to separate CD4+ T cells.

GNLY = natural killer (NK) cell marker, also expressed by GZMH+ cytotoxic T cells (but not GZMK+ cytotoxic T cells)  (see Shi et al 2022)

GZMH = cytotoxic T cell marker & CD16+ natural killer cell marker (see Shi et al 2022)

HLA-C = monocyte marker

LYZ = monocyte marker

MS4A1 = B cell marker

MS4A7 = monocyte marker, expressed by CD16+ (but not CD14+) monocytes (see Shi et al 2022)

MZB1 = B cell and plasmacytoid dendritic cell marker


Cell markers list is a work in progress...

Miltenyi Biotec non-gradient methods to isolate blood cells



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