MACS Handbook

Blood

1 Introduction

Peripheral blood or whole blood carries the various blood cells — erythrocytes or red blood cells, leukocytes or white blood cells, and thrombocytes or platelets — suspended in blood plasma. Plasma is mostly water, but contains also proteins, glucose, ions, hormones, and clotting factors. Removing clotting factors from plasma generates serum. 
The most abundant cells in blood are red blood cells. These contain hemoglobin, an iron-containing protein that reversibly binds oxygen and thus facilitates its transport throughout the body. Thrombocytes stop bleeding by clumping and clotting blood vessel injuries. Mammalian thrombocytes have no cell nucleus; they are fragments of cytoplasm derived from megakaryocytes in bone marrow that enter blood circulation. Approximately 25–40% of platelets are stored in the spleen and released as needed, and the remainder circulate freely in the blood with a turnover of about 10 days. Finally, the various subtypes of leukocytes play essential roles in the immune response.
All cellular components of blood arise from hematopoietic stem cells (HSCs). Located in bone marrow, these pluripotent cells divide to produce specialized progenitor cells of more limited potential: common lymphoid progenitor cells and common myeloid progenitor cells. Common lymphoid progenitor cells give rise to T and B lymphocytes and natural killer (NK) cells, whereas common myeloid progenitor cells give rise to monocytes, dendritic cells, and granulocytes (including neutrophils, basophils, and eosinophils). Finally, erythrocytes, and thrombocyte-producing megakaryocytes- arise from their respective common progenitor.

2 Leukocytes

Given their role in the immune response, leukocytes are of particular interest to understand mechanisms of disease and health and thus, are commonly isolated from blood and blood products. Classification of leukocytes can be based on their origin — myeloid or lymphoid — or on the morphology of their nucleus — mononuclear or polynuclear — and the presence or absence of granules in their cytoplasm, which results in two groupings: the peripheral blood mononuclear cells (PBMCs) or agranulocytes and the polymorhponuclear leukocytes (PMNs) or granulocytes. 

2.1 Peripheral blood mononuclear cells (PBMCs)

PBMCs include lymphocytes (T, B, and NK cells), monocytes, and dendritic cells. Among other characteristics, T and B lymphocytes are distinguished by their site of maturation and antigen receptors. T cells mature in the thymus and express the T cell receptor TCR, whereas B cells mature in bone marrow and carry the B cell receptor BCR. 
T cells are CD3+ and further classified into helper T cells (TH) that express CD4 and cytotoxic T cells (TC) that express CD8. B cells are characterized by CD19 expression and respond to pathogens by producing large quantities of antigen-specific antibodies to neutralize foreign objects like bacteria and viruses, and opsonize (mark) them for easy recognition by other immune cells. NK cells are CD56+CD3 and derive from the same progenitor cells as T and B cells, but lack antigen receptors. Finally, monocytes are the largest type of PBMCs and express CD14. They circulate in the bloodstream for 1–3 days and then typically move into tissues throughout the body where they can differentiate into macrophages and myeloid-lineage dendritic cells.

At a glance: PBMC cell types 

Cell typeFrequency % 
(range in healthy individuals)
MarkersFunction
CD4+ T cells25–60%CD3+CD4+Coordinate adaptive immunity through activation and regulation of other immune cells
CD8+ T cells5–30%CD3+CD8+Destroy cancer cells or cells that are infected or damaged
B cells5–10%CD19+Secrete antibodies as part of humoral immune response
NK cells10–30%CD56+ CD3-Trigger lysis or apoptosis of infected cells
Monocytes5–10%CD14+Take up foreign objects via phagocytosis, perform antigen presentation, and produce cytokines
Dendritic cells1–2%Process and present antigen material to T cells

2.2 Granulocytes

Granulocytes are characterized by the presence of granules in their cytoplasm and a variable nucleus shape, hence the name polymorphonuclear leukocytes(PMNs). Neutrophils, eosinophils, and basophils are all granulocytes that release the contents of their prominent granules upon activation. Neutrophils are the most abundant leukocyte in blood and the first responders to a site of inflammation, where they exert their phagocytic function. Eosinophils (also known as acidophils) are thought to be involved in attacking multicellular parasites, such as such as worms. Basophils appear in many specific inflammatory reactions, particularly those that cause allergic symptoms. Basophils contain the anticoagulant heparin, which prevents blood from clotting too quickly, and the vasodilator histamine, which promotes blood flow to tissues.
MACS Handbook:

Granulocytes (human)

3 Blood-based sources of leukocytes

The most commonly used source for leukocytes is peripheral whole blood. However, blood products such as buffy coat, Leukocyte Reduction System Chambers (LRSC), and leukapheresis material such as Leukopak are increasingly used as sources. For certain applications, especially stem cell research, cord blood and bone marrow are more suited as starting material. Cell number and composition differs among blood and the various blood products, making different sources more suitable as starting materials for any given application or research question.

At a glance: Cell types, absolute numbers and frequencies in whole blood and blood products
Whole bloodBuffy coatLRSCLeukopak
ratio to PBMCsratio to PBMCsratio to PBMCsratio to PBMCs
Erythrocytes 5x109  cells/mL4x1011 cells/buffy coat5x1010 cells/LRSC8x109 cells/half Leukopak
1000 400501.2
PBMCs2x106 cells/mL1x109 cells/buffy coat1x109 cells/LRSC 7x109 cells/half Leukopak
1111
Leukocytes5x106 cells/mL 2x109 cells/buffy coat1x109 cells/LRSC7x109 cells/half Leukopak
2,5211
Average percentage of total leukocytes
Whole blood
(standard deviation)
Buffy coat
(standard deviation)
LRSC
(standard deviation)
Leukopak
(standard deviation)
Pan T cells22.5%
(3.84)
53.8%
(6.12)
53.8%
(6.12)
54.46%
(12.44)
CD4+ T cells22.5%
(3.84)
24.4%
(6.57)
32.39%
(6.6)
31.92%
(6.4)
CD8+ T cells

6.8%

(1.32)
10.8%
(4.60)
15.04%
(4.5)
17.13%
(8.1)
B cells5.2%
(2.32)
7.2%
(2.42)
11.33%
(3.3)
12.52%
(4.6)
Monocytes8.4%
(1.32)
10.7%
(2.83)
20.28%
(5.7)
19.08%
(7.1)
NK cells

4.4%

(2.42)
4.1%
(2.37)
10.21%
(4.0)

7.65%
(3.0)
CD3+/CD56cells0.8%
(0.67)
1.4%
(2.05)
1.75%
(1.3)
4.33%
(7.1)
Eosinophils3.2%
(1.59)
1.3%
(0.80)
1.88%
(2.3)
1.07%
(0.9)
Neutrophils53.8%
(6.12)
34.4%
(11.44)
2.89%
(1.7)
3.91%
(3.4)
Frequencies of cells were determined with the 7-Color Immunophenotyping Kit, human; number of leukocytes and erythrocytes was determined by Sysmex.

3.1 Whole blood

All major immune cells can be found in the most natural condition in peripheral blood freshly drawn from healthy donors, patients, or participants in clinical trials. However, a typical whole blood sample is rarely larger than 10 mL, and can often be smaller if, for example, when the donor is a child. Larger volumes can be acquired from healthy donors but usually no more than 500 mL are drawn per donation. This greatly limits the number of rare cells that can be acquired from whole blood. The advantage of whole blood as a source is the freshness of the material, which is essential for sensitive cells like neutrophils. 
Anticoagulants are commonly used in samples to prevent blood clotting. Heparin, ethylenediaminetetraacetic acid (EDTA), and citrate are among the most commonly used additives. Heparin binds to the enzyme inhibitor antithrombin III (AT), causing a conformational change that results in its activation, which subsequently inactivates thrombin, factor Xa and other proteases involved in blood clotting. EDTA strongly and irreversibly chelates divalent cations such as calcium ions, thereby preventing blood from clotting. Finally, citrate is also used to bind calcium.

3.2 Buffy coat

The buffy coat is the fraction of a blood sample that contains white blood cells after removing erythrocytes and plasma. After blood donation, a 450-mL blood bag is centrifuged, separating leukocytes from plasma (top fraction) and erythrocytes (bottom fraction). Generally, blood banks are only interested in plasma and erythrocytes, and those parts are extracted from a blood bag. The remaining material (buffy coat) is concentrated leukocytes with a reduced number of erythrocytes and granulocytes and varies in volume from 30 to 80 mL. Readily available and relatively inexpensive, buffy coats are a popular source of PBMCs.

3.3 Leukocyte reduction system chamber (LRSC)

LRSC, also known as buffy cone, is the waste product of plateletpheresis, a process by which thrombocytes are separated and collected from drawn blood, and the remaining blood portions are returned to the donor. Leukocytes are collected as an unwanted component with the platelets and must be removed to prevent rejection problems during platelet transfusion. Leukocytes are filtered out by leukoreduction and collected into an LRSC. Thus, the LRSC contains a high density of leukocytes with a low concentration of neutrophils.

3.4 Leukopaks

A good source for large quantities of leukocytes are commercially available Leukopaks. This is an enriched leukapheresis product collected from peripheral blood consisting of a variety of blood cells including monocytes, lymphocyte, and erythrocytes. The number of erythrocytes is low compared to leukocytes (on average 1:1), as is the number of granulocytes. Commercial Leukopaks are available in a few standard sizes, ranging from an entire Leukopak to ½ and ¼ sizes. Although the number of PBMCs per Leukopak is variable, it generally remains within a certain range. 
In some cases, donors are injected with G-CSF (granulocyte colony stimulating factor) to induce leukocyte production and trigger migration of stem cells from bone marrow into blood. Therefore, mobilized Leukopaks have higher numbers of leukocytes compared to immobilized Leukopaks, and frequencies of the various PBMCs differs as well. Most Leukopaks are from healthy donors, but it is possible to request Leukopaks for a disease state, such as allergies and diabetes.

4 Cord blood as a source of leukocytes

Cord blood remains in the placenta and the umbilical cord after birth and is collected to harvest stem cells that can be used to treat hematopoietic and genetic disorders. Cord blood contains all elements found in whole blood, but is also rich in hematopoietic stem cells (CD34+). The number of CD34+ cells varies greatly among individual donors, but typically 1–4 million CD34+ cells can be collected per cord. Cord blood is used increasingly on an experimental basis as an alternative source of stem cells typically obtained from bone marrow. Most cord blood transplants have been performed in patients with blood and immune system disorders. 

5 Bone marrow as a source of leukocytes

Bone marrow is a semi-solid tissue found within the spongy or cancellous portions of bones. In birds and mammals, bone marrow is the primary site of new blood cell production or hematopoiesis. The tissue consists of marrow adipose tissue, supportive stromal cells and two types of stem cells: mesenchymal (MSC) and hematopoietic (HSC). HSCs which are destined to mature into the various blood cells, including leukocytes, whereas MSCs generate the different specialized cells found in skeletal tissues, including cartilage cells (chondrocytes), bone cells (osteoblasts), and fat cells (adipocytes). Stem cells are typically harvested directly from the red marrow in the iliac crest of the pelvic bone.

6 Sample preparation of blood

Whole blood can be used directly for the isolation of target cells. In some cases, however, isolation is more effective from a population of PBMCs that can be generated by density gradient centrifugation or erythrocyte lysis, though the latter is considered only a “quick and dirty” method. The starting material for the generation of PBMCs may also include buffy coats, LRSCs, and Leukopaks.
The most common density gradient centrifugation method uses Ficoll Paque®, a solution of high molecular weight sucrose polymers. A blood sample is centrifuged in a tube containing a density gradient medium to separate its components according to their densities. Erythrocytes are denser than plasma, and so become packed into the bottom of the tube to make up 45% of total volume. This volume is known as the haematocrit. Leukocytes and platelets form a narrow cream-colored coat (buffy coat) immediately above the erythrocytes. Finally, plasma makes up the remaining contents of the tube and is a pale yellow fluid accounting for just under 55% of the total volume. The buffy coat contains lymphocytes, monocytes, and platelets and is harvested for subsequent two centrifugations in a buffered salt solution to wash the PBMCs and remove the platelets. The resulting PBMCs can be used for further cell subtype isolation or other experimental procedures. A protocol for the isolation of PBMCs from blood by density gradient centrifugation can be downloaded from the Related Resources panel to the right.