Blood: Ullmann's Encyclopedia of Industrial Chemistry

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Rainer Seitz1, Herbert König1, Johannes Dodt1 Print this page
1Paul-Ehrlich-Institute, Dept. of Haematology and Transfusion
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Medicine, Langen, Germany
Copyright © 2006 by Wiley-VCH Verlag GmbH & Co. KGaA. All rights
reserved. Advanced Product Search
DOI: 10.1002/14356007.a04_201.pub2 Search All Content
Article Online Posting Date: December 15, 2006 Acronym Finder
Abstract | Full Text: HTML
Abstract
The article contains sections titled:
1. Introduction
2. Composition, Physiology and Chemical Properties of Blood
2.1. Blood Cells
2.1.1. Hematopoiesis, Stem Cells
2.1.2. Red Cells, Hemoglobin
2.1.3. Leukocytes, Cytokins
2.1.4. Thrombocytes
2.2. Plasma Proteins
2.2.1. Analysis of Plasma
2.2.2. Coagulation Factors
2.2.3. Fibrinolysis System
2.2.4. Plasma Proteinase Inhibitors
2.2.5. Immunoglobulins
2.2.6. Albumin
3. Production
3.1. Blood and Plasma Donation
3.2. Substances Derived from Blood Cells
3.3. Fractionation of Plasma
3.3.1. Quality Requirements
3.3.2. Preparation of Intermediate Products
3.3.3. Purification of Plasma Proteins
3.4. Recombinant Proteins
3.5. Adverse Drug Reactions of Blood Products
3.5.1. Immunogenicity
3.5.2. Thrombogenicity
3.5.3. Transmittable Pathogens
4. Therapeutic Uses
4.1. Blood Components
4.1.1. Blood and Stem Cell Preparations
4.1.2. Fresh Frozen Plasma
4.2. Hemoglobin-Derived Oxygen Carriers
4.3. Cytokins
4.4. Coagulation Factors
4.5. Plasminogen Activators
4.6. Inhibitors
4.7. Immunoglobulins
4.8. Albumin
5. Economic Aspects
page 1 of 26
Blood is a multifunctional transport medium of vital importance for the body of higher organisms. Its composition is very
complex and includes various cell systems with peculiar functions, proteins, electrolytes, metabolites, and highly
sophisticated mediator systems. Both cells and plasma, which can be separated by centrifugation, can be used as and
further processed into medicines. Serum is the fluid remaining after coagulation of the blood. In this review, blood cell
systems and plasma proteins are referred to and medicinal aspects including blood and plasma collection and processing are
described. The medicines derived from blood and plasma, as well as basic research and clinical trials should keep an
important place because of the medicine as a whole benefits extremely from transfusions and, in particular, hemophiliacs
gain an enormous improvement of life quality and life expectancy.
[Top of Page]
1. Introduction
Blood is a multifunctional transport medium of vital importance for the body of higher organisms. The composition of blood is
very complex and includes various cell systems with peculiar functions, proteins, electrolytes, metabolites, and highly
sophisticated mediator systems. Its undisturbed circulation in an ubiquitous system of blood vessels with fine ramifications
throughout the whole organism, connections to the lymph vessels and interfaces with the extravascular compartments is
essential for the performance of all organs and enables numerous exchange processes. A sudden stop of blood circulation
will cause the death of the organism within a short time. The functions of blood can be allocated to the following categories:
1. Respiratory function: the blood provides the capacity to transport oxygen needed for the energy metabolism from the
lung to the organs, and of carbon dioxide back to the lung.
2. Regulatory function: besides by the nervous system, numerous specific body functions are controlled or influenced by
messenger substances such as hormones or cytokins that are transported by the blood; the production of blood cells
itself is a good example (see Section Hematopoiesis, Stem Cells). The blood has to provide also a stable internal
milieu; the pH is maintained within a narrow range around 7.4 by buffer substances and by transport of acid or basic
substances to metabolic or excretory organs. The blood is also involved in the regulation of further important
parameters, e.g., water household or temperature.
3. Nutritive and metabolic function: nutrients absorbed from the gastrointestinal tract are collected into the splanchnic
vessels, transported via the portal vein to the liver, the body's main synthetic and storage organ, and further
transferred to the tissues in order to maintain energy supply and their specific metabolic functions. Waste and toxic
products, as well as superfluous water and electrolytes are transported from the tissues to the sites of their elimination.
4. Protective function: the human body is permanently subject to external and internal attacks of agents disturbing its
homeostasis or even threatening the individual's life. The blood contains highly specialized cellular (various types of
phagocyte cells and lymphocytes) and humoral (e.g., immunoglobulins, complement, proteases, and inhibitors)
protective systems providing effective surveillance, defense, and repair.
The blood volume in humans is equivalent to about 6 to 8 % of the total body mass, which again underlines the significance
of this “fluid organ”.
[Top of Page]
2. Composition, Physiology and Chemical Properties of Blood

Blood can be obtained by donation into special containers that contain substances preventing coagulation and can be easily
separated by centrifugation into the cellular fraction and the liquid supernatant (the plasma); serum is the fluid remaining after
blood has undergone coagulation. Both cells and plasma can be used and further processed into medicines. Blood cell
counts and other relevant values are shown in Table 1.
Table 1. Typical values of selected cellular parameters measured in normal adult blood*; (Units are given as usually
used in clinical laboratories and text books; in some countries and publications SI-units are preferred for certain
parameters. The actual reference ranges may vary depending on the assay methods)
Parameter Value Remarks
Red cells
Erythrocyte (red cell) count m: 4.4 – 6.0 × 106/µL
f: 4.2 – 5.5 × 106/µL
Hemoglobin m: 14.0 – 18.0 g/dL f:
12.0 – 16.0 g/dL
Hematocrit (ratio cell m: 0.40 – 0.54 f:
volume:/ total blood volume) 0.37 – 0.47
MCH (mean corpuscular 27 – 32 pg mean hemoglobin mass per single red cell
hemoglobin)
MCHC (mean corpuscular 32 – 36 g/dL mean hemoglobin concentration in a single
hemoglobin concentration) red cell
MCV (mean corpuscular 80 – 94 fL
volume)
page 2 of 26
Reticulocytes 7 – 15 per 103 red
cells
White cells
Total white cell count 4.3 – 10.0 × 103/µL
Differential white cell count values obtained with microscopy of blood
(relative to total number): smears; values with automatic counters may
be different
Neutrophils, band form 1–5%
Neutrophils, segmented 45 – 70 %
Eosinophils 1–4%
Basophils 0–1%
Monocytes 2–8%
Lymphocytes 20 – 40 %
Platelets
Thrombocyte count 150 – 350 × 103/µL
* m = male, f = female proband.
2.1. Blood Cells

Several highly differentiated cell systems with specialized characteristics and functions are circulating in the blood. The cells
may be either nucleated such as leukocytes, in this case they are reactive to various stimuli, and actively mobile between
blood stream and tissues. Otherwise they are nonnucleated, have a restricted metabolism, and stay within the blood their
whole life-span. An example of the latter category are erythrocytes.
2.1.1. Hematopoiesis, Stem Cells

After the fetal period, the production of blood cells (hematopoiesis) takes place essentially in the red bone marrow within
spongious bones (e.g., vertebrae, sternum, pelvis), and in case of lymphatic cells also in lymph nodes and other sites (e.g.,
spleen, tonsils, appendix, Peyer's plaques, or during childhood in the thymus). In adults, the hematopoietic marrow has a
total mass of about 1000 to 1200 g and thus belongs to the largest organs. It possesses a special microenvironment, a
“matrix” of cells, connective tissue and adhesion proteins, which supports homing, proliferation, and maturation of
hematopoietic cells. There is a permanent turnover of mature blood cells, and the half lives of several types are short.
Therefore, great quantities of new cells have to be produced: for a healthy adult, the estimate is about 1011 granulocytes,
2 × 1011 erythrocytes, and 2 × 1011 thrombocytes every day.
The microscopic examination of bone marrow is a very important feature in the diagnosis of many hematological disorders.
Specimens can be obtained by needle biopsy from the pelvis (spina iliaca posterior) for histology, or by puncture and
aspiration from the sternum, and subsequent preparation of smears for cytological examination. The specimen can be
stained using conventional panoptic dyes or specific antibodies in order to visualize certain markers or structures. The bone
marrow gives at first glance the impression of a colorful, somewhat “chaotic” conglomerate of various cells of different size
and appearance (Fig. 1). However, the hematopoiesis is a complex and very thoroughly regulated process, which is
effectively controlled and can be adapted within a wide range to the actual needs of the organism in case of stressing
situations such as infections or blood loss.
Figure 1. Bone marrow (overview)
Panoptic Pappenheim stain (same stain used also in Figs. 3-5) of a smear of a punctate obtained by sternal puncture and
aspiration.
For the photographs in Figures 1 and 3-5, the authors are indebted to Mrs. Elke Eckstein, Philipps University Hospitals,
Marburg, Germany.
The proliferating and differentiating bone marrow cells can be divided into several lineages, according to the resulting mature
blood cells: (1) the “myeloid” lineages including the erythrocyte, thrombocyte, granulocyte of different types (neutrophil,
eosinophil, basophil), and monocyte/macrophage precursors, and (2) the lymphatic cell lines. All of these cell lineages are
ultimately derived from a common origin, the so-called “pluripotent stem cells” (Fig. 2). The latter are permanently
proliferating in order to maintain a constant pool of pluripotent stem cells and to produce a sufficient number of progenitor
cells for blood cell supply. Some stem and progenitor cells are permanently recirculated via the blood stream, which provides
the opportunity to harvest them for therapeutic purposes by apheresis. The first step of hematopoiesis is the evolution into
the myeloid or the lymphoid stem cells, and further into committed stem cells, which can be viewed as the start of the several
lineages. By proliferation and progressive differentiation numerous cells at different stages are generated, until the cells
reach the mature functional stage of blood cells and enter the blood via the ample bone marrow vessels (sinusoids). While
pluripotent and committed stems cells can not be discriminated by morphology, but only by specific surface antigens (used
as diagnostic markers), the more differentiated bone marrow cells can be identified by their typical microscopic appearance.
There are, e.g., progenitors of erythrocytes (erythroblasts, Fig. 3), granulocytes (myeloblasts, Fig. 4), or thrombocytes
(megakaryocytes, Fig. 5).
page 3 of 26
Figure 2. Scheme of cell line differentiation in hematopoiesis
BFU = burst-forming unit; E = erythrocyte; Epo = Erythropoetin; Eo = Eosinophil; GM = granulocyte-macrophage;

MEG = Megakaryocyte; Tpo = Thrombopoetin.
Figure 3. Erythropoiesis:
Large cell in the center is a proerythroblast; during further divisions and differentiation the cells become smaller, the nuclei
increasingly dense, the cytoplasm contains more and more hemoglobin. Finally, the nucleus is extruded. At the left
neutrophil granulocyte.
Figure 4. Myelopoiesis:
In the center myeloblast with sparse, basophilic cytoplasm, surrounded by more differentiated cells up to mature segmented
granulocytes.
Figure 5. Megakaryocytes:
Largest cells in bone marrow (compare surrounding cells)
A) Mature megakaryocyte with giant nucleus and “granulation”; B) megakaryocyte after shedding of platelets, leaving
nucleus behind.
The proliferation and differentiation of the lineages is controlled specifically by hematopoietic growth factors, which belong to
an important group of substances referred to as cytokins or “biologic response modifiers”. Their nomenclature is somewhat
confusing, due to historical reasons. A long-known substance is erythropoietin, a polypeptide produced by the kidneys in
response to reduced oxygen concentration, which stimulates the production of oxygen carriers, i.e., erythrocytes. A rather
“new” growth factor is thrombopoietin, which stimulates megakaryocytes [1], and shows about 50 % homology to
erythropoietin. Other factors were discovered by their ability to promote in vitro the growth of progenitor cell colonies, which
led to a nomenclature of the responsible cells as colony forming units (CFU) and of the growth factors as colony stimulating
factors (CSF), for example the granulocyte-colony stimulating factor, G-CSF. It was also noted that several of a group of
substances discovered as leukocyte-derived modifiers of inflammation, the interleukins, are also hematopoietic growth
factors. But also “classical” hormones, such as thyroid or adrenal hormones, have significant influence on blood cell
production. More detailed information about stem cell biology and their regulation can be obtained in [2], [3]. Much can be
learned also from the pathophysiology of bone marrow failure in acquired aplastic anemia [4]. However, in face of the rapidly
increasing knowledge with ongoing discovery of novel factors and interactions, it is impossible at present to provide a definite
and comprehensive description of the regulation of hematopoiesis. A provisional, simplified overview is given in Figure 2, a
selection of cytokins is listed in Table 2. A panel of mature blood cells is displayed in Figure 6 A – F.
Table 2. List of selected cytokins
Factor Major source(s) Main effect(s)
Granulocyte-colony monocytes, stimulation of generation and differentiation

stimulating factor (G-CSF) fibroblasts of neutrophils
Granulocyte- macrophage- T-cells, fibroblasts, stimulation and differentiation of progenitor
colony stimulating factor (GM- endothelium cells for granulocytes, monocytes, platelets,
CSF) erythrocytes
Erythropoietin kidney stimulation of erythropoesis, some effect
also on platelet production
Thrombopoietin liver stimulation of platelet production
Interleukin-1 (IL-1) monocytes enhancement of other growth factors, and of
immune reactions
IL-2 T-cells (helper cells) activation and proliferation of T-cells
IL-3 (multi-CSF) T-cells proliferation (self-renewal) and
page 4 of 26
differentiation of early progenitor cells
IL-4 T-cells, macrophages stimulation of B-cells
IL-6 macrophages, differentiation of B- and T-cells, acute-phase
fibroblasts, T-cells reaction (enhanced synthesis of fibrinogen
and other proteins)
IL-16 T-cells chemotaxis of CD4-cells, inhibits
proliferation of HIV in infected cells
Stem cell factor (SCF) fibroblasts and other stimulates CD34-positive stem cells
cells
Tumor necrosis factor (TNF macrophages enhancement of inflammatory reactions,
pro-coagulatory stimulation of endothelium,
antitumor effects
Interferons ( , , ) leukocytes, various antiviral, antiproliferative (some
fibroblasts, T-cells types of malignancy) and immune-
modulating effects
Platelet-derived growth factor megakaryocytes and growth stimulation of vessel wall and
(PDGF) platelets inflammatory cells
Figure 6. Mature leucocytes in the peripheral blood smear:
A) Band form; B) Segmented neutrophil; C) Eosinophil; D) Basophil; E) Monocyte; F) Lymphocyte
Compare size with surrounding erythrocytes and platelets.
2.1.2. Red Cells, Hemoglobin

The red cells, the erythrocytes, are produced by proliferation and differentiation of precursor cells (erythroblasts) in the bone
marrow. The mature erythrocytes do not contain a nucleus and are no more capable of cell division. In the young
erythrocytes, during the first days after they left the bone marrow, some residues of RNA and organelles can be found by a
special stain. These cells are called reticulocytes. The normal life span of erythrocytes is about 110 d, thereafter they are
phagocytized and degraded in macrophages of the spleen and the so called reticulo-endothelial system (RES). The iron and
the protein of the erythrocytes is recycled for biosynthesis, the heme is degraded and metabolized into bilirubin, which is
transported by the blood stream into the liver, where metabolites are excreted in the bile.
Mature erythrocytes have a characteristic shape of a biconcave disk. Normal red cells have a diameter of 7.5 to 8.7 µm;
variations may be a diagnostic feature of disease states, e.g., smaller erythrocytes occur in iron deficiency, or larger cells in
vitamin B12 deficiency. Erythrocytes have an average volume of 90 fL, and their shape provides a particularly large surface
area of about 136 µm2, which is important for their function in gas exchange. Red cells show a pronounced deformability
which is needed for their passage through narrow capillaries. The erythrocytes are susceptible to alterations of the osmotic
pressure and will shrink in hypertonic and swell in hypotonic solution until they are lysed at a critically low osmolality.
Although the red cells do not contain a nucleus, they are quite differentiated cells which possess also an energy metabolism.
Glucose is utilized as a source of energy via the glycolytic pathway and the hexose monophosphate shunt; red cells lack,
however, the citric acid cycle. The energy is needed (1) to maintain the iron of hemoglobin in the divalent form; (2) to
maintain the electrolyte gradient with high potassium level inside the erythrocytes, (3) to protect hemoglobin and enzymes
from oxidation, and (4) to retain the biconcave shape of the cell.
The characteristic function of red blood cells, the transport of oxygen, is carried out by hemoglobin. Hemoglobin is a complex
of a tetrameric protein (globin) component and the heme (which is made up by a characteristic porphyrin ring system similar
to that found in chlorophyll, and divalent iron). The globin component consists of two alpha chains and two other chains. In
adults, most of the hemoglobin contains beta chains (giving a formula 2 2). This hemoglobin is named hemoglobin A. This
nomenclature must not be mixed up with the AB0 blood group system. In the fetal period, and in some hereditary disorders of
hemoglobin metabolism, hemoglobin contains other molecules instead of the beta chains, e.g., gamma chains in the fetal
hemoglobin ( 2 2, hemoglobin F). The binding of oxygen to hemoglobin is influenced by the temperature, pH value, and
the red cell content of 2,3-diphosphoglycerate. The oxygen dissociation curve shows a typical S-shape.
2.1.3. Leukocytes, Cytokins

All cells circulating in the blood, which are neither erythrocytes nor thrombocytes, are summarized by the expression
“leukocytes” or “white blood cells”. The leukocytes in the blood can be divided into granulocytes, monocytes, and
lymphocytes.
Granulocytes. The name “granulocytes” refers to the characteristic granules making up the specific appearance of these
blood cells under the light microscope. The granulocytes can be differentiated by the standard panoptic staining of blood
smears (e.g., Pappenheim or Wright) as neutrophil, eosinophil, or basophil granulocytes.
Neutrophil granulocytes normally comprise more than 50 % of the total leukocyte count. They are derived from precursor
page 5 of 26
cells in the bone marrow (myeloblasts and myelocytes). During the proliferation and increasing differentiation of these
precursor cells, the granules undergo a maturation process, and the nucleus changes from the big and round shape in
myeloblasts to the beanlike appearance of the so called “band forms” of the young granulocytes. During their further live
span, the nucleus of the neutrophil granulocytes undergoes progressive segmentation. While an increase of band forms in
the blood smear is a sign of an acute challenge, in most cases bacterial infection, the appearance of granulocytes with an
abnormally high number of segments may point to certain hematological diseases with impaired blood cell production.
Normally, the neutrophil granulocytes circulate only several hours in the blood circulation, before they extravasate and enter
the tissues. They are very reactive, nonspecific phagocytes, which can be viewed as the first line of combat against any
invasion of pathogenic or toxic agents. The neutrophil granulocytes play a significant role in the surveillance against bacteria
and fungi at outer surfaces of the body, e.g., the oral cavity. Therefore, a lack of functional granulocytes (e.g., in an acute
leukemia or bone marrow failure) may lead to characteristic, severe local infection in the mouth (gingiva, tonsils) or in the
perianal area. In individuals with normal bone marrow function, the production of neutrophil granulocytes can be increased
over a wide range in case of an acute challenge such as an acute bacterial infection, intoxication or major trauma, with an
increase of the total white cell count and the proportion of band forms in the blood. This increase is initiated by cytokins,
particularly IL-3, GM-CSF and G-CSF; IL-8 is a strong activator of neutrophil granulocytes. The neutrophil granulocytes are
activated by a variety of nonsoluble (e.g., pathogenic microorganisms) or soluble (endotoxins, leukotrienes, complement
factors, fibrinopeptides) stimuli. They are capable of active locomotion, and they are attracted to the site of the lesion by
chemotaxis. The phagocytosis is strongly enhanced by the so-called opsonization of particles, which means their coating by
molecules such as complement factors or immunoglobulins. The neutrophil granulocytes possess a very efficient apparatus
for intracellular killing of ingested microorganisms and degradation of foreign protein. They have the capacity to produce
highly reactive oxygen radicals by means of specific enzymes, such as myeloperoxidase, in their azurophilic granules. The
specific granules contain several enzymes such as acid hydrolases, alkaline phosphatases, and proteolytic enzymes,
particularly elastase, with a high capacity to degrade foreign materials. During activation and phagocytosis, oxygen radicals
and enzymes may leak out into the surrounding, where they may damage structures and proteins. Proteases from neutrophil
granulocytes, particularly elastase, are thought to contribute to extravascular fibrinolysis [5].
Eosinophil Granulocytes. The eosinophil granulocytes contain specific granules which are larger than those of neutrophils
and are stained red by eosin. Normally, they comprise 2 – 4 % of the total leukocyte count. The nucleus usually has two
segments. Besides enzymes like in neutrophil granulocytes, the specific granules contain a so-called “major basic protein”,
which makes up about 50 % of the granule content and plays a role in the killing of parasites and mammalian tissue cells.
Besides their ability to nonspecific phagocytosis, eosinophil granulocytes exhibit some characteristic features: The cytotoxic
destruction of parasites in cooperation with macrophages, cytotoxic damage to tissue cells (e.g., cells of the respiratory tract
in patients with asthma bronchiale), and neutralization of mediators from mast cells such as histamine, serotonin, and
bradykinin. During anaphylactic reactions, eosinophils contribute to the attenuation of the inflammatory process. Production
and function of eosinophils is controlled by cytokins (e.g., IL-5), and is also influenced by steroid hormones (decreased by
corticosteroids, increased by estrogens and androgens).
Basophil granulocytes have a very characteristic appearance due to their large and dark blue granules, which often hide the
nucleus. The granules contain high amounts of heparin and biogenic amines (histamine, serotonin) and bradykinin. The
effect of these mediators is generally an enhancement of inflammatory reactions by vasodilatation and prevention of
coagulation in order to maintain free access for inflammatory cells and mediators to the area of the lesion. Basophils possess
receptors for immunoglobulin E (IgE) on their surface and are specifically involved in type I allergic (anaphylactic) reactions,
where they cause severe hypotension or even lethal shock by massive release of histamine and bradykinin.
Monocytes belong to the group of large, phagocytizing cells which are called macrophages. They share a common
progenitor cell with the granulocytes in the bone marrow. The macrophages circulating in the blood stream are called
monocytes due to their round, oval or kidney-shaped nucleus, which is not segmented in contrast to granulocytes. After a
circulation time of a few days in the blood, the monocytes move to the tissues, where they may stay alive for months. The
macrophages in the tissues are called histiocytes, or in the liver also Kupffer cells. Macrophages may be found in all tissues
of the organism, but particularly rich of these cells are the liver, the spleen, lymphnodes, and bone marrow, as well as areas
of chronic inflammation, particularly granulomas. Macrophages are able to kill bacteria and to phagocytize necrotic material,
e.g., in tissue repair after a local infection has been overcome. Besides that macrophages have a very important function in
the specific immune defense due to their cooperation with lymphocytes. After phagocytosis and proteolytic digestion,
fragments of foreign material are transferred to the macrophage surface, where they are “presented” to lymphocytes.
Lymphocytes are produced in the bone marrow and in lymphatic organs such as the spleen, lymphnodes, tonsils, appendix,
Peyer's plaques, and — during childhood — the thymus. Lymphocytes in the peripheral blood can be identified as small to
mid-sized cells with a round nucleus and few cytoplasm. In contrast to other blood cells, the maturity stage and the specific
function of the lymphocytes cannot be differentiated by morphologic examination. In order to identify and quantitate the
different functional subsets of lymphocytes, the staining of characteristic antigens on the cell surface by specific antibodies
has been introduced. The antigens have been described in the “cluster of differentiation” (CD-) nomenclature, leading to a
large and still expanding list of antigens on blood and bone marrow cells (Table 3). The determination of the CD-antigens in
cell samples is nowadays a very important tool for the differential diagnoses of disorders of the hematopoietic and immune
system. A well known example is the disturbed balance between CD-4 and CD-8 lymphocytes in acquired immune deficiency
syndrome (AIDS). The CD-antigens resemble in many cases important functional receptors on the cell surface.
Table 3. Cluster of differentiation (CD)-nomenclature; examples of CD entities and their expression by blood cells
CD designation Remark Main cell reactivity
CD3 T-cell receptor complex T-cells

CD4 MHC class II and HIV receptor T-cells
CD8 MHC class I receptor T-cells
page 6 of 26
CD14 lipopolysaccharide (LPS) receptor macrophages, granulocytes, dendritic cells
CD19 surface Ig family B-cells
CD34 used for stem cell selection hematopoietic stem cells
CD41 GP IIb-IIIa (fibrinogen receptor) platelets
CD62P P-selectin activated platelets and endothelial cells
Lymphocytes can be divided into two main groups, the T-lymphocytes (which are primed in the thymus), and the B-
lymphocytes (which are primed in the bone marrow, the equivalent to a specific organ of birds, called Bursa Fabricii).
T-Lymphocytes. The group of T-lymphocytes includes cells with important regulatory function (so called helper or
suppressor cells), as well as cytotoxic cells. T-helper or inducer cells (TH) can be divided into subpopulations: Th1cells
support the proliferation and the function of T-lymphocytes and thus the cell mediated immunity, Th2cells support the
differentiation of B-lymphocytes to the antibody producing plasma cells. There is evidence that T-suppressor cells are
probably not a defined T-cell subpopulation of its own, “suppression” of the immune answer appears to be rather a special
functional state of cytotoxic or helper cells. For instance, Th1cells promote their own development, but inhibit at the same
time the development of Th2cells and vice versa.
A specific immune reaction is initiated by close contact and cooperation of an antigen-presenting cell (macrophages or
dendritic cells) and T-cells. The interaction proceeds on several levels. A nonspecific cell adhesion is mediated by adhesion
molecules, a specific interaction is brought about by molecules of the so-called major histocompatibility complex (MHC)
which can be divided into class I and class II molecules. The pattern of MHC-antigens of an individual is the basis for the
human leukocyte antigen (HLA) typing, which is useful for the assessment of compatibility in organ transplantation, as well as
in platelet transfusion. The MHC-proteins are important for the antigen presentation. MHC-molecules bind short peptides
derived from degraded antigens, class I peptides of viral proteins or endogenous (tissue derived) antigens, and class II those
of exogenous, e.g., bacterial proteins. The MHC-molecules bearing the antigen peptides react with a T-cell receptor which
recognizes specifically the presented antigen. By this way, T-cells with antigen specific T-cell receptors are activated and
produce and release cytokins. Particularly by IL-1 and IL-2, the immune reaction is initiated and enhanced, and the antigen
specific T-cell clones can proliferate and expand.
A subpopulation of T-cells possesses direct cytotoxic activity. These cells are able to attack specifically target cells
presenting the antigen by direct cell contact and a complex reaction involving the release of proteins which induce a
perforation of the membrane (perforine) and enzymes (granzyme A – H).
There are also effector cells which cannot be allocated to the T- or B-cell groups and are called natural killer cells.
B-Lymphocytes. The humoral immunity is a function of B-lymphocytes. They are stimulated by cytokins released from T
h2cells in cooperation with the antigen-presenting cells. On the surface of B-cells, their specific immunoglobulin is exposed in
a way similar to a receptor. If the antigen, e.g., a virus protein on cell surface, is presented in a coordinated and repetitive
arrangement, the B-cells may be activated directly without T-cell help [6].
A common principle of T- and B-cells is that a huge repertoire of preformed cells exists, each of which reacts specifically via
its T-cell receptor or surface immunoglobulin to a certain antigen. If the body is confronted with such an antigen, there will be
a clonal expansion of the lymphocytes which are able to react specifically with this antigen, and this expansion leads to a
strong cytotoxic or antibody-mediated immune reaction.
2.1.4. Thrombocytes
Thrombocytes, also called blood platelets, are the smallest blood cells. They contain no nucleus, but several specialized
organelles. Thrombocytes are derived from megakaryocytes in the bone marrow. In contrast to the other blood precursor
cells megakaryocytes do not undergo cell divisions, but endomitosis leading to a polyploid huge cell nucleus, surrounded by
a growing cytoplasm, which is roughly “granulated” at the end of maturation. The big “granules” are the preformed platelets,
which develop by progressive spreading of a membrane system throughout the megakaryocyte cytoplasm and thus
demarcation of the future platelets, and simultaneously development of the organelles. The maturation takes a few days, and
one megakaryocyte can produce up to about 2000 platelets. At the end of maturation, the platelets fall apart from their
mother cell and enter the blood stream, leaving the nude nucleus.
Thrombocytes have a life span of about one week in the circulation, where they are “a patrol”, the task of which is to detect
any disturbances of the integrity of blood vessels and to seal them rapidly in order to prevent excessive blood losses. Due to
this function the thrombocytes are the first line of hemostasis. Therefore, the time between a standardized cut into the skin
and the termination of bleeding (“bleeding time”) is mainly a function of the thrombocytes. As long as the thrombocytes
circulate through vessels with an intact endothelium, they are in a resting state; it has been calculated, that a platelet may
pass more than 8000 times through the whole circulation during his life span without being activated. Aged platelets are
cleared by the reticulo-endothelial system, mainly in the spleen. The spleen is also a kind of a storage pool which may host
about a quarter to a third of the total platelet mass and may release them into the circulation by contraction in case of a
challenge like a major injury. Spleenectomized individuals tend to have higher platelet counts in their blood.
Platelets have a discoid shape in the resting state and do not contain a nucleus. They possess characteristic and specific
organelles: The -granules contain proteins, mainly platelet factor 4 and -thromboglobulin — which are anti-heparin
factors — fibrinogen and coagulation factors, as well as thrombospondin, plasminogen activator inhibitor (PAI), and the
platelet derived growth factor (PDGF). A very characteristic type of granules are the dense bodies, which contain serotonin
page 7 of 26
and adenosine diphosphate (ADP). The so-called dense tubular system is involved in the production of prostanoids. The
open canalicular system is a system of channels spreading throughout the cell, enabling a rapid transport of the constituents
to the outside of the cell, which is important for the release reaction upon activation of the thrombocytes. The outer plasma
membrane of thrombocytes contains in the resting state only few negatively charged phospholipids, but a high concentration
of the negatively charged phosphatidyl serine is located at the inner layer of the cell membrane. Of functional importance are
also the cytoskeleton and in particular contractile proteins (actinin and myosin) which are important for the shape change
(see below), and the clot retraction.
The thrombocytes are very reactive cells and are rapidly activated, whenever they come into contact with other surfaces than
the intact endothelial cells. The are activated, in particular, by subendothelial structures such as collagen fibers or foreign
surfaces such as glass. In contrast to granulocytes, thrombocytes do not have the capacity of locomotion. They are passively
driven by the blood stream to the site of lesion. It has been estimated, that during the normal bleeding time of about five
minutes about 1012 platelets may pass the defect of the vessel wall, but only about 105 are found in the clot. The platelets
are particularly reactive in areas of fast and turbulent blood flow, where a considerable shear stress is imposed to them. This
is one of the reasons, why thrombocytes play a much more prominent role in arterial than in venous vessels. The activation
of thrombocytes leads to a rapid, microscopically visible reaction, the so-called shape change: The cell body changes from a
discoid to a spherical shape, and a number of pseudopods is extruded. The electrical charge of the outer membrane changes
by a fast transfer of phosphatidyl serine from the inner to the outer surface, and the platelets become “sticky”. The electrical
charge is also of great significance for the procoagulant function of platelets, since the activated platelet membrane is a
template for complexes of activated coagulation factors (see below). Besides the shape change, the thrombocytes undergo
also the so-called release reaction, which means the rapid extrusion of mediators, which are important for the activation of
surrounding thrombocytes and for other processes, such as the initiation of blood coagulation, and promotion of cell growth in
the injured vessel wall by PDGF. In the activation of platelets, presently three pathways can be differentiated. The first
pathway is mainly triggered by platelet adhesion to, e.g., collagen fibers, and involves the formation of prostanoids. By the
action of a phospholipase, arachidonic acid is liberated from the platelet membrane and subsequently metabolized to
prostanoids. An important step is the generation of cyclic peroxides by the enzyme cyclooxygenase, which is a target for the
antiplatelet effect of aspirin. The cyclic peroxides are further metabolized to thromboxane A2, which is one of the most potent
mediators of platelet activation and vasoconstriction (see also Cardiovascular Drugs; Prostaglandins). In endothelial
cells, there is also a cyclooxygenase pathway, which generates a counterpart to thromboxane A2,, prostacyclin, which is a
potent inhibitor of platelet activation and a vasodilator ( Prostaglandins). The second pathway of thrombocyte activation is
mediated by ADP, which is stored in high amounts in the dense body granules of the platelet. ADP acts via a specific P2T
purine receptor. The ADP-pathway is also stimulated by thromboxane A2, serotonin, and adrenaline (epinephrine). ADP may
be liberated from platelets also in a turbulent blood stream with high shear stress leading to cell damage. The third pathway
of platelet activation is mediated by thrombin, the key enzyme of blood coagulation (see below). Besides the three pathways
outlined above, there are additional agonists and antagonists, such as platelet activating factor or nitric oxide. Generally, after
activation by a primary agonist, the other pathways lead to a strong enhancement of the activation in a sense of a positive
feed back reaction. The intracellular events associated with activation by the above agonists, include an increase of the
intracellular calcium concentration, the formation of diacylglycerol and inositol triphosphate, leading to an activation of
proteinkinase C, and an increase of cyclic AMP. An overshooting reaction should be prevented by natural antagonists, such
as prostacyclin or nitric oxide.
The thrombocyte surface possesses a number of functionally important receptors, which have been mentioned above: A
purine receptor, a thromboxane A2 receptor, a thrombin receptor, an 2-adrenergic receptor, and a platelet activating factor
receptor. Besides that, there are a number of functionally important glycoproteins (GP), which act as receptors for specific
structures. Important examples are (1) the GP IIb-IIIa, a receptor for fibrinogen, von Willebrand factor (vWF, a multimeric
protein composed of up to 40 subunits each having an apparent molecular mass of 500 000), fibronectin, and vitronectin,
which is important for platelet aggregation; (2) GP Ib-IX, a receptor for von Willebrand factor, which is important for platelet
adhesion; and (3) GP Ia-IIa, a receptor for collagen. Platelet adhesion means the adherence of thrombocytes to
subendothelium or foreign surfaces, which involves adhesive proteins and particularly von Willebrand factor. Adhesion has to
be differentiated from aggregation, which means sticking of platelets to each other in the blood stream. Aggregation is
effectively inhibited by aspirin, blocking the thromboxane A2 pathway, while aspirin does not substantially influence adhesion.
During aggregation, fibrinogen forms “bridges” between GP IIb-IIIa of aggregating platelets.
2.2. Plasma Proteins

2.2.1. Analysis of Plasma
Plasma, the cell-free part of blood, contains numerous different proteins, each having certain and essential function(s).
Depending on the desired aim plasma proteins may be separated by different means. Generally, a distinction is made
between analytical and preparative purposes. These purposes dictate which technique to apply. For analyzation of plasma
electrophoretic techniques are preferred because they are both easy to perform and of remarkable resolving power. In this
case plasma is subjected to an electric field whereby the proteins are separated into different groups. The resolution depends
upon the electrophoretic technique applied. Using very common but relatively poor resolving techniques (e.g., zone
electrophoresis) plasma is separated into six major fractions, constituting the most prominent proteins: Albumin, 1-, 2-, -,
-globulins, and fibrinogen (Fig. 7). With more elaborated separation (and detection) techniques (see below), however,
plasma proteins can be separated into much more distinct fractions, hence allowing also the detection of minor constituents
(examples are given in Table 4): From Table 4 it becomes obvious that plasma proteins may be classified according to
several criteria, among others normal concentration, size, electrophoretic mobility, function.
Figure 7. Electrophoretic analysis of normal plasma. Typical separation of plasma proteins by electrophoresis
page 8 of 26
The anode is to the left. After electrophoresis the protein bands are stained and subsequently scanned by densitometry.
Table 4. Plasma proteins
Protein Plasma concentration, Biological function Mr

mg/mL
Albumin 40 – 50 transport, colloidosmotic 67 000

IgG Globulin 8 – 18 antibody 155 000
Fibrinogen 2 – 4.5 coagulation 340 000
Transferrin 2–4 iron transport 76 000
1-Proteinase inhibitor 2 – 3.5 inhibitor of serin proteases 54 000
2-Macroglobulin 1.5 – 3.5 general inhibitor of proteases 725 000
1-Lipoprotein 1.7 – 3 lipid, hormone transport 28 000

IgA Globulin 0.9 – 4.5 antibody 160 000
IgM Globulin 0.6 – 2.5 antibody 975 000
Haptoglobin 1-1, 2-1, 2-2 0.9 – 3 hemoglobin binding, 86 000
-Lipoprotein 0.6 – 1.5 lipid, hormone transport 27 000
Hemopexin 0.5 – 1.1 hemin binding 57 000
C3-Component 0.5 – 1.2 complement factor 185 000
2-HS Glycoprotein 0.4 – 0.85 opsonin 49 000
Inter- -trypsin inhibitor 0.4 – 0.7 trypsin inhibitor 160 000
1-Antichymotrypsin 0.3 – 0.6 chymotrypsin inhibitor 68 000
Ceruloplasmin 0.15 – 0.6 oxidase 132 000
Gc-Globulin 0.2 – 0.55 vitamin D3 binding 51 000
C1-Inhibitor 0.15 – 0.35 protease inhibitor 104 000
0.15 – 0.3 heparin binding 40 000
2-Glycoprotein I
C4 Component 0.2 – 0.5 complement factor 206 000
Prealbumin 0.1 – 0.4 thyroxin, retinol binding 55 000
Antithrombin III 0.15 – 0.2 inhibitor of activated coagulation 60 000
factors
C3-Proactivator 0.1 – 0.4 proteinase 90 000
C1q-Component 0.1 – 0.25 complement factor 400 000
C1r-component 0.1 complement factor 83 000
C9-Component 0.08 complement factor 71 000
Plasminogen 0.06 – 0.25 zymogen of plasmin 91 000
0.05 – 0.15 unknown 35 000
2-Glycoprotein III
Prothrombin 0.05 – 0.12 zymogen of thrombin 72 000
9.5S 1-Glycoprotein 0.04 – 0.08 constituent of amyloid deposits 308 000
Retinol binding protein 0.03 – 0.06 transport of retinol (vitamin A) 21 000
8S 3-Glycoprotein 0.03 – 0.05 unknown 220 000
Transcortin 0.04 cortisol transport 56 000
C1s-Component 0.02 – 0.04 complement factor, esterase 85 000
Factor XIII 0.01 – 0.04 fibrin cross-linking, 340 000
transglutaminase
Properdin 0.01 – 0.03 complement regulator 184 000
page 9 of 26
Thyroxin binding 0.01 – 0.02 binding of thyroxin, 54 000
glycoprotein
Factor X 0.01 zymogen of FXa 62 000
Zn 2-Glycoprotein 0.02 – 0.15 unknown 41 000
Lysozyme 0.007 – 0.02 bacteriolysis 14 500
Serum cholinesterase 0.005 – 0.015 cleavage of cholin esters 348 000
Factor V 0.005 coagulation cofactor 330 000
Factor IX 0.004 zymogen of FIXa 62 000
Protein C 0.004 zymogen of inhibitor of 62 000
coagulation
IgD Globulin 0.003 – 0.1 antibody 175 000
IgE Globulin 0.001 – 0.0014 antibody 190 000
Factor VII 0.0005 zymogen of FVIIa 50 000
Factor VIII 0.00015 coagulation cofactor 280 000
The size of a given protein is determined by the number of amino acids it is composed of and by posttranslational
modifications. Most plasma proteins are modified by attachment of nonprotein side chains to the molecules (typical examples
are sugar residues and phospholipids).
The electrophoretic mobility is determined by the relative amounts of positively, negatively, or uncharged amino acids, as well
as by structure and size, depending upon which individual separation technique is applied (native gel electrophoresis,
denaturing gel electrophoresis under reducing and nonreducing conditions, respectively, and isoelectric focusing, the latter
being the system with the highest resolving power). Also, combinations are possible, e.g., isoelectric focusing followed by
denaturing SDS gel electrophoresis.
The numerous proteins of plasma execute manifold functions. Some prominent examples are immunoglobulins as antidotes
against foreign molecules, albumin for transport function, coagulation factors for sealing lesions, cytokins for the exchange of
information between cells, numerous inhibitors for, e.g., the control of enzymatic reactions against overshooting.
To characterize single protein components of plasma these must normally be isolated from the other plasma constituents.
For this purpose they are subjected to a series of separation techniques, each taking advantage of a different characteristic
of the protein in question. Typical examples for fractionation sequences are salt (or alcohol) precipitation, hydrophobic
interaction chromatography, ion exchange chromatography, affinity chromatography (if available), gel filtration, in certain
cases reversed-phase chromatography.
2.2.2. Coagulation Factors

As mentioned above, coagulation factors have evolved as a response of organisms against injuries. It is now generally
accepted that the system of coagulation may be divided into two branches, the extrinsic system and the intrinsic one (see
Fig. 8). The extrinsic branch represents the immediate and direct response to lesions of the organism. This part of the
coagulation system is composed of factor VII/VIIa and tissue factor, two coagulation components which come into contact
with each other only in the case of disturbances of the normal tissue organization. The intrinsic branch (represented by
factors XII, XI, and IX) may be interpreted as an additional amplification mechanism which is turned on, once the extrinsic
system has become activated. As shown in Figure 8, both branches are interconnected at the level of coagulation factor X.
Downstream of factor X both systems ultimately lead to the formation of fibrin by thrombin. From Figure 8 it can also be
derived that normal coagulation is restricted to the vessel walls due to the dependence of the necessary events upon
phospholipids. As the complete system is composed of a cascade of protease zymogens (i.e., enzyme precursors), this
system is prone to overshooting and must be under tight and rapid control. This is fulfilled by specific and efficient inhibitors,
such as antithrombin (AT) and tissue factor pathway inhibitor (TFPI). In addition, thrombin is transformed from a procoagulant
protein into an anticoagulant protein by interaction with thrombomodulin.
Figure 8. Scheme of blood coagulation
The events of the clotting cascade are illustrated schematically here. Only the unactivated coagulation factors are presented
with the exception of factor IIa (thrombin) for simplicity. The organism's immediate response to a tissue lesion is shown on
the left (FVII/TF to thrombin). The contact activation pathway (via FXII to FIX) is also illustrated. The whole process is
dependent upon phospholipids which are contained in the cellular components of the system (vessel wall and platelets).
TF = Tissue factor; HMWK = High molecular weight kininogen: Pk = prekallikrein
Coagulation factors are listed according to their historical order of discovery by giving the term coagulation factor (often
shortened to factor) followed by roman numbers. Most of the coagulation factors must be altered before they can fulfill their
physiological function. These proteolytically modified coagulation factors are labeled by adding the letter “a” for activated.
Coagulation Factor I (Fibrinogen). Fibrinogen [9001-32-5], Mr 340 000, is the most prominent coagulation factor in plasma
(3 mg/mL). Fibrinogen is the precursor of fibrin [9001-31-4], which is generated as the final event of clot formation. During
normal coagulation fibrinogen is converted to fibrin by thrombin. Other enzymes of nonplasma origin, however, can induce
clotting as well, e.g., papain and several proteolytic components of certain snake venoms. Several microorganisms, such as
Staphylococcus also can cause coagulation by cleaving fibrinogen.
page 10 of 26
Fibrin, the cleavage product of fibrinogen is poorly soluble in aqueous salt solutions. This low solubility is, in the course of
coagulation, further reduced by activated factor XIII (see below) to allow clot stabilization.
Coagulation Factor II (Prothrombin). Prothrombin [9001-26-7], Mr 72 000, is contained in normal plasma at a concentration
of 100 µg/mL. It is a glycosylated protein containing about 5 % carbohydrate. Like several other coagulation factors it is
produced in the parenchymal cells of the liver. For normal prothrombin synthesis vitamin K is required, as is for the synthesis
of other zymogens of the coagulation pathways. During activation to the serine protease thrombin the protein's molecular
mass is nearly halved to 37 000 whereas the other coagulation factors (see below) are shortened by few amino acids only
when activated (the leaving peptide termed activation peptide). Although thrombin is the final protease of the coagulation
cascade it plays an important role in regulating its own activity and production. This is accomplished by both, feedforward
activity in activating cofactors V and VIII and negative feedback activity by activating inhibitors of coagulation including its
own (protein C and protein S). Thus, thrombin is not only a procoagulant but simultaneously an important regulator of normal
hemostasis. A rather common genetic variation in the prothrombin gene (G to A transition) at position 20210 leads to
elevated prothrombin levels in plasma and is considered to increase the risk for venous thrombosis in people affected.
Coagulation Factor III (Thromboplastin, Tissue factor). Thromboplastin [9035-58-9] was initially discovered as a poorly
defined substance that in coagulation experiments ultimately led to thrombin formation. It was then found to be composed of
one part protein noncovalently bound to one part of heterogeneous phosphatides. The protein component is tissue factor (Mr
47 000), a glycoprotein with Ca2+- dependent affinity to factors VII and VIIa. Unlike the other coagulation cofactors it does not
circulate in the blood but is integrated into cellular membranes not normally in contact with the blood stream. A tissue injury
results in exposure of tissue factor to blood, thus enabling its contact with FVII/ FVIIa. It is assumed to stabilize factor VIIa in
a proteolytically active conformation to catalyze the conversion of FIX and FX to FIXa and FXa, respectively. The designation
factor III meanwhile has been replaced by listing the respective components individually, namely tissue factor, phospholipids.
Coagulation Factor IV (Calcium). Calcium ions are required for most individual steps of coagulation as catalyst. The cation
acts as a bridge between phospholipids in cellular membranes and carboxy glutamyl residues in certain coagulation factors.
This results in a conformational change in the respective coagulation factor leading to formation of a complex which can be
activated by the protease produced in the previous step of the coagulation cascade. Similar to the situation with FIII, calcium
is the currently used denomination.
Coagulation Factor V (Plasma Accelerator Globulin). Factor V, Mr 350 000, is the inactive precursor to factor Va (see
factor VI, below). Like factor VIII (see below) it is not a zymogen. Its concentration in plasma is about 10 µg/mL. After
activation of factor V by thrombin, the resulting factor Va serves as a cofactor for the activation of prothrombin to thrombin by
activated factor X, thus facilitating feed forward amplification of the clotting process. A genetically determined defect termed
“factor Va Leiden” (Arg 506 is exchanged for Gln) results in activated protein C (APC) resistance, thus leading to an
increased risk for thromboembolic complications. This genetic defect is almost exclusively restricted to humans of the
western hemisphere. In this population, however, this defect is quite evident (one out of ten people is affected by this
mutation).
Coagulation Factor VI (Activated Factor V). Unlike all other terms for coagulation factors, factor VI is the designation for
(just) the activation product of another clotting factor (FV, see above), therefore it is somewhat confusing and its use is
discontinued.
Coagulation Factor VII (Proconvertin). Proconvertin [9001-25-6], Mr 50 000, is the least prominent vitamin K dependent
coagulation factor with a plasma concentration of 0.5 µg/mL. Together with tissue factor and phospholipids, activated factor
VII (FVIIa) forms the initiator of coagulation as a response to tissue damage by activating factor X (extrinsic coagulation
pathway) and by activating factor IX (intrinsic pathway). In contrast to the other coagulation factors some (about one in
thousand molecules) activated factor VII is reported to circulate in the blood.
Coagulation Factor VIII (Antihemophilic Globulin A). Factor VIII [9001-27-8], Mr 280 000, circulates in plasma in tight
association with von Willebrand factor. Due to its tight association with vWF the molecular mass of FVIII has long been
overestimated. The physiological function of this tight association is assumed to lie in transport and protection of FVIII. In
addition, vWF mediates the association of FVIII with platelets (see Section Thrombocytes). The normal plasma concentration
of FVIII is 150 ng/mL, classifying it as a trace protein. Hereditary defects in FVIII synthesis are the cause for hemophilia A,
leading to severe bleeding disorders. After activation by thrombin (or FXa), FVIIIa serves as a cofactor for the activation of
FX by FIXa at a cellular phospholipid surface.
Coagulation Factor IX (Christmas Factor, Antihemophilic Factor B). Like the other factors of the prothrombin complex
(FII, FVII, FX, and protein C and protein S) factor IX [9001-28-9], Mr 62 000, is a glycoprotein. It also contains several -
carboxy glutamic acid residues that are essential for its function. FIX has a plasma concentration of 4 µg/mL. Similar to the
situation with FVIII, genetically determined deficiency in this coagulation factor results in a bleeding disorder and a prolonged
partial thromboplastin time, called hemophilia B in this case. Factor IX is the final zymogen of the intrinsic coagulation
pathway. Once activated, the resulting enzyme together with activated FVIII, phospholipids, and calcium forms the tenase
complex, that activates factor X.
Coagulation Factor X (Stuart Prower Factor). Factor X [9001-29-0] is the link between the intrinsic coagulation pathway
and the extrinsic one, the former pathway being considered as an amplification mechanism of the latter. After activation by
either FIXa (intrinsic) or FVIIa (extrinsic) to FXa this enzyme, with FVa as cofactor, phosholipids, and calcium ions forms the
prothrombinase complex that converts prothrombin to thrombin. It can be interpreted as a feed forward amplification that
activated factor X together with tissue factor can activate FVII, thus enabling the activation of additional FIX and FX
molecules. Factor X has an apparent Mr of 67 000 and is contained in normal plasma at a concentration of 10 µg/mL. Unlike
the other procoagulants of the prothrombin complex nonactivated FX is composed of two peptide chains held together by a
page 11 of 26
disulfide bridge.
Coagulation factor XI [9013-55-2], Mr 160 000, is a zymogen involved in the intrinsic coagulation pathway resulting by
contact with foreign material (contact activation). It is composed of two identical peptide chains held together by a disulfide
bond. After activation by factor XIIa (see below) it will activate FIX. The plasma concentration of FXI is 5 µg/mL.
Coagulation factor XII (Hagemann Factor), [9001-30-3], Mr 90 000, is the first zymogen in the contact activation of the
coagulation system and the only one which is not dependent upon the presence of calcium ions for function. Its normal
plasma concentration is 40 µg/mL. No bleeding disorders are reported as a consequence of a deficiency in this factor, people
affected rather carry an increased risk for thromboembolic disorders. FXII plays an important (although not fully understood)
role not only in the coagulation system but also due to its link to the kallikrein and plasmin systems.
Coagulation Factor XIII (Fibrin Stabilizing Factor). Unlike the aforementioned zymogens factor XIII [9013-56-3] is not the
precursor of a protease, but of a transglutaminase. Once activated by thrombin, the enzyme stabilizes the fibrin clots by
introducing covalent links between lysine and glutamine residues in individual fibrin molecules thus rendering the clot
insoluble. It also introduces covalent links between other plasma proteins, e.g., fibrin, vitronectin, and 2-plasmin inhibitor.
Plasma FXIII is a heterodimer (FXIIIA2B2) composed of two subunits each, called FXIII-A (the proenzyme) and FXIII-B
(formerly called FXIII-S). Activation of FXIII to FXIIIa is accomplished by splitting of an activation peptide from the A-chains.
The plasma concentration of FXIII ( Mr 320 000) is 20 µg/mL.
Protein C, Mr 62 000, is the zymogen of an inhibitor of coagulation. Like FX it is a two-chain molecule. Its plasma
concentration is 5 µg/mL. After activation by thrombin bound to thrombomodulin (a “receptor”-like protein on the surface of
endothelial cells), protein C together with its cofactor (activated protein S, see below) will inactivate activated factors V and
VIII, respectively. Thus it acts as a modulator of coagulation by interference with FXa generation as well as thrombin
formation. Due to its charge characteristics protein C is found in the prothrombin complex (factors II, VII, IX, X) during plasma
fractionation. Genetically determined deficiency in protein C results in elevated risk for thromboembolic complications.
Protein S, Mr 69 000, has a plasma concentration of 10 µg/mL. Besides its cofactor function for activated protein C it has an
anticoagulant effect via direct interaction with FVIIIa. Similar to the situation with protein C, genetically determined protein S
deficiency results in an increased risk for thromboembolic complications.
2.2.3. Fibrinolysis System

The organisms needs an efficient and fast system to produce a clot in any case of violation of blood vessels. The generation
of a fibrin matrix is also important for wound healing and inflammatory reactions. However, of equal importance is an efficient
system for fibrin degradation. This fibrinolytic system is needed for the prevention of overshooting clotting, or at least, for
recanalization and repair after thrombotic events. Intra- and extravascular fibrinolysis is also necessary for removal of the
fibrin matrix along with the progressive “organization” during the healing of wounds and inflamed areas.
The main fibrinolytic system in the blood is the plasmin pathway. It is activated by the proteolytic cleavage of the proenzyme
plasminogen [9001-91-6], Mr 93 000, a protein with a plasma concentration of about 2 µM (200 mg/L). In its N-terminal part
plasminogen contains five characteristic domains, the so-called kringle domains, and the rest of the plasminogen molecule
contains the active center of a serine protease. The kringle domains are important for the binding of plasminogen to fibrin and
thus the interaction with the substrate of the activated enzyme. The activation takes place by cleavage between Arg560-
Val561. The activation of plasminogen does not involve a complex cascade, such as the coagulation system. However,
several physiologic pathways of activation exist. The most important pathway involves the tissue-type plasminogen activator
(t-PA), which is synthesized predominantly in endothelial cells. The expression of t-PA can be enhanced by several
mediators, the most important being thrombin. t-PA contains also two kringle domains, binds to fibrin and activates
plasminogen. Further domains of t-PA, whose physiologic significance is not yet clear, are a so-called “finger” domain, which
is also found in fibronectin, and a domain which is similar to epidermal growth factor. Another important physiologic
plasminogen activator is the urokinase-type plasminogen activator (u-PA), which may be found as “pro-urokinase”, a single
chain molecule (scu-PA), and the cleaved form urokinase, devoid of the N-terminal epidermal growth factor-like and kringle
domain. The name pro-urokinase is somewhat misleading, since also this molecule has already some fibrinolytic activity. It
has been speculated that pro-urokinase circulates bound to some “inhibitor”, which gets lost by binding to fibrin, where pro-
urokinase can be readily further activated by proteolytic cleavage, e.g., by traces of plasmin. The fibrinolytic system is also
activated by the so-called “contact activation” involving kallikrein and FXIIa, which is at least in part mediated by conversion
of scu-PA to urokinase.
Plasmin is not only involved in fibrin cleavage, but has also a variety of other substrates [7]; of importance for hemostasis are
the proteolytic cleavage of t-PA and scu-PA enhancing its own activation, and on the other hand the inactivation of
coagulation factors V and VIII.
Plasmin can degrade both fibrinogen and fibrin at several cleavage sites. The result are several fibrin(ogen) degradation
products of different size (X, Y, D, E). These fibrin(ogen) degradation products, particularly the large X-fragments, lead to a
disturbance of fibrin polymerization and have thus an anticoagulatory effect. Fibrin degradation products (FDP) can be
detected by several laboratory tests; specific tests are available for fibrinogen or fibrin degradation products. The
demonstration of FDP in the plasma may be associated with an abnormal activation of fibrinolysis, e.g., during a
disseminated intravascular coagulation. An isolated overstimulation, called hyperfibrinolysis can rarely be found in patients
with certain tumors, e.g., prostate carcinoma. In diseases with a very strong inflammatory reaction, such as sepsis,
pancreatitis, or polytrauma, a massive release of proteolytic enzymes from neutrophil granulocytes, particularly elastase,
occurs which may contribute to intra- and extravascular fibrinolysis [5].
page 12 of 26
2.2.4. Plasma Proteinase Inhibitors
Most of the proteins involved in the coagulation and fibrinolytic system are proteolytic enzymes. These proteinases belong all
to the class of serine proteinases. Their activity must be regulated because otherwise they would be a threat to the host that
they are designed to protect. This problem has been solved, for instance, by the evolution of a series of proteins which
decrease the activity of the proteolytic enzymes and which are called proteinase inhibitors (Table 4). These proteinase
inhibitors are also class specific and therefore this chapter is dealing with proteinase inhibitors of serine proteinases. The
inhibitors rapidly form tight stoichiometric complexes with proteinases in a substratelike manner which leads to inactivation of
the proteolytic enzyme. Cleavage of the inhibitor may occur, if at all, at a much slower rate compared with a substrate. In the
case of serine proteinase inhibitors, inhibition results from an interaction of the proteinase substrate binding site and a region
of the inhibitor which is known as reactive site loop. In the case of canonical inhibitors like the kunins, the conformation of the
reactive site loop is highly complementary to the surface of the enzyme and resembles a substrate bound to the specificity
site [8], [9]. Unlike the Kunitz-type inhibitors, antithrombin has a reactive site loop which is not held rigidly by disulfide bonds
but is able to swing in a hingelike manner. In the resting state, the reactive site loop projects away above the bottom of the
serpin. When the reactive center docks with thrombin, the Arg393–Ser394 reactive site peptide bond is cleaved. At this
stage, the complex is covalently linked. Normally, in the next step a substrate would dissociate after cleavage regenerating
the active enzyme. In contrast, antithrombin remains linked to thrombin by one arm of the now cleaved reactive site loop.
This arm becomes inserted into the body of the inhibitor as an additional -pleated sheet. This leads to a stable complex in
which the enzyme is crushed against the body of the inhibitor, distorting it, which results in irreversible inhibition [10]. It
should be mentioned that 2-macroglobulin is unique with respect to its completely different mechanism of inactivating
proteinases and its ability to inhibit members of the four classes of proteinases [11].
Proteinases which initiate and amplify the coagulation and fibrinolytic systems are thought to have a restricted ability to
cleave peptide bonds. Their role is to activate the zymogen of other components of the system usually by a single peptide
bond cleavage. Proteinase inhibitors act to limit the activation by rapidly complexing key enzymes of the amplification
system. However, they do not completely shut down the pathways, but rather inhibit systemic activation and thereby localise
coagulation and fibrinolysis. The physiological importance of proteinase inhibitors may be seen from their relative plasma
concentration. If one excludes albumin and immunoglobulins, about 20% of the remaining proteins are proteinase inhibitors.
Although there are inhibitors of cysteine and metallo proteinases in plasma, most of the inhibitors regulate the serine
proteinases. At this time, inhibitor concentrates of antithrombin III (AT III), 1-proteinase inhibitor ( 1-PI), and C1-Inhibitor
(C1-Inh) are commercially available. Therefore, these three inhibitors are described in the following.
Antithrombin III, AT III; Mr 58 000, [12], [13] is a single chain glycoprotein and a member of the serpin family. It is synthesized
in the liver and circulates in plasma with a half-life of about 3 days. The primary structure exhibits 432 amino acid residues,
six of which form 3 intramolecular disulfide bonds. The carbohydrate moiety of the protein contributes to the molecular mass
by about 15%. The plasma concentration of AT III is about 2.6 mol/L and the reference activity is given with 70-120%. Two
isoforms exist in plasma, AT- and AT- . About 90% of the plasma AT III is present as the -isoform. The two isoforms
differ in their degree of glycosylation: the -form exhibits four glycosylation sites, whereas the -form lacks the carbohydrate
at Asp135. AT- has a greater affinity for heparin and for both the intact and injured vessel wall [14].
Antithrombin III is the major inhibitor of thrombin, factor Xa, and factor IXa but it also inactivtes factors XIa and XIIa as well as
serine proteinases which do not belong to the coagulation system like plasmin, kallikrein, and complement C1.
Inhibition of proteinases by AT III is slow if compared other proteinase–proteinase inhibitor complex formation. The inhibitory
action of AT III is greatly accelerated (about hundredfold) by heparin or related polysaccharides [15]. Among the most
pertinent are heparan sulfate proteoglycans which are found on or underlying the vessel wall where anticoagulant activity
occurs. While the order of magnitude of the rate enhancement for inhibition of thrombin, factor IXa, and factor Xa by
antithrombin in the presence of heparin there is a fundamental difference in the mechanism of enhancement of inhibition.
Thrombin inhibition is accelerated because the heparin chain provides a template to bring the reactive site of the inhibitor and
the active site of thrombin in close proximity. This is observed with heparin oligosaccharides of size greater than 18
monosaccharide units but not for those of smaller size [16], Even though they are still inducing a conformational change in
antithrombin which leads to the optimal inhibitory conformation of the reactive site loop [17]. The affinity of heparin to the
thrombin-antithrombin (TAT) complex is much lower than to the free enzyme and heparin therefore dissociates from TAT
complexes. The complexes are removed from circulation by receptors in the liver (half-life < 5 min). In contrast, accelerated
inhibition of factor Xa is mainly achieved by the induced conformational change of AT III which is observed even with a
pentasaccharide.
Antithrombin deficiency may be acquired or inherited. The pattern of inheritance is usually autosomal dominant (non-sex-
linked) with levels of biologically active inhibitor around 40-70% of normal. Deficiencies of AT III but also of protein C and S
are the most common known genetic causes for thrombosis. Venous thrombosis is a serious medical problem which affects
annually one in thousand individuals causing morbidity and death. Commonly, antithrombin deficiencies are divided in two
groups, type I and type II, based on the results from immunological and functional assays [18], [19]. Type I is characterized
by a 50 % reduction of antigen (and activity) caused by heterozygous mutation of the gene. Homozygous AT III type I
deficiency appears to be not compatible with life. Type II deficiencies are those cases where the antigen level is normal but
functional activity is reduced (usually by a heterozygous mutation). Type II deficiencies are subdivided in mutations affecting
the reactive site (RS), the heparin binding site (HBS). Acquired AT III deficiencies are due to either decreased liver synthesis,
increased loss, increased consumption, or drug-induced.
1-Proteinase inhibitor, Mr 52 000, 1-PI) is a single-chain glycoprotein and a member of the serpin family. It is synthesized
primarily in the liver but also in mononuclear phagocytes, intestinal epithelium and kidney parenchyma. 1-PI circulates in
the blood with a half-life of 3-5 days and is cleared by the reticulo endothelial system. The primary structure exhibits 394
amino acid residues, containing one free cysteine residue and three carbohydrate side chains [20].
page 13 of 26
1-PI inhibits a large number of serine proteinases including trypsin, chymotrypsin, cathepsin G, plasmin, thrombin, tissue
kallikrein, factor Xa, plasminogen, and neutrophil elstase. During acute phase response 1-PI is upregulated in order to
control neutrophil derived proteases at inflammatory sites. It is implicated that 1-PI plays also a role in immune response
which include inhibition of lymphocyte response to phytohemagglutinin, inhibition of chemotaxis, and enhancement of
pokeweed mitogen driven IgG synthesis [21].
Protease inhibitor and individual alleles are named according to their isoelectric focusing characteristics (PI M, PI S, PI Z). In
addition to the polymorphism at gene level, the protein exhibits microheterogeneity due to differences in glycosylation and N-
terminal processing. Inheritance of 1-PI alleles is autosomal codominant: Each allele contributes to the total serum
concentration and overall isoelectric focusing pattern. Phenotypes are PI MM (two normal alleles) PI ZZ (homozygous
inheritance of two defect Z alleles) and PI SS (homozygous inheritance of two defect S alleles) [22]. 1-PI-deficiency affects
about one in 6000 caucasian americans but only one in 40 000 to one in 100 000 black americans. 95 % of all 1-PI-deficient
patients belong to the PI ZZ which is characterized by a plasma level of 1.8 - 3.6 mg/mL (10 - 28 mg/mL for normal
phenotypes, PI MM; threshold level for protection against lung damage due to proteolytic activity 10 - 12 mg/mL). Clinical
manifestation of 1-PI-deficiency is emphysema in adults and liver disease in children.
C1-Inhibitor, Mr 104 000, is a single-chain glycoprotein which belongs to the serpin family. It is mainly synthesized in the liver.
Other sites of biosynthesis are skin fibroblast as well as monocytes. C1-Inhibitor circulates in the blood with a half-life of
about 67 to 72 hours. Normal plasma levels are in the range of 0.2 mg/mL. The native molecule consists of 478 amino acid
residues (including two disulfide bonds) and exhibits an unusually high carbohydrate content of about 50 % [23].
The inhibitor is involved in the regulation of several proteolytic systems including the complement (C1s, C1r), coagulation
factor XIa, fibrinolytic (plasmin), and contact (factor XIIa, kallikrein) systems. Hereditary or aquired deficiency of C1-Inhibitor
may result in accumulation of vasoactive substances. Sudden death due to acute larynx edema or nonitching, hard, painless
swelling of soft tissues, and coliclike pains might result. In type I hereditary angioedema (HAE), an insufficient synthesis and
elevated turnover of a normal C1-Inhibitor results levels of 5 - 30 % of normal. In type II, functionally inactive inhibitor is
detected and the product of the normal allele is found in low concentrations. In aquired angioedema (AAE) type I, C1-inhibitor
and C1 are diminished and in type II of AAE an anti-C1-Inhibitor autoantibody interferes with complex formation between
enzyme and inhibitor [23].
2.2.5. Immunoglobulins
Immunoglobulins, also called antibodies ( Immunotherapy and Vaccines – Antibodies) are produced by B-lymphocytes
(see Section Leukocytes, Cytokins) ( Immunotherapy and Vaccines – Immune Response). Their function is to “recognize”
and bind specifically certain molecules, their corresponding antigens. The human organism possesses about 106 – 108
different antibody specificities and is thus able to react to a great variety of antigens. The immunoglobulins make up a
specific humoral immunity and play an important role in the body's defense against pathogenic microorganisms and toxic
substances.
As already outlined above, each B-cell carries its own specific immunoglobulin on the outer cell surface, which functions as
an antigen receptor. If a certain antigen enters the organism, it binds selectively to corresponding antigen receptors (surface
immunoglobulins) on B-cells. By this mechanism, B-cells, which are able to produce suitable antibodies, are selected and
activated. Supported by cytokins produced by T-helper cells, a clonal proliferation and differentiation into plasma cells is
initiated. The plasma cells produce and secrete high amounts of the specific antibody which mediates the humoral immune
answer. The antibody production can last over different periods of time up to many years; in many cases, after the antibody
production is down regulated, so called memory cells remain which may reside permanently in lymphatic organs and also
may recirculate. By this mechanism, a fast and strong immune reaction can be initiated in case of repeated antigen
exposure.
The basic structure of immunoglobulins is that of a Y-shaped four-chain polypeptide (Fig. 9). The polypeptide is a symmetric
tetramer of two heavy (i.e., high molecular) chains and two light (i.e., low molecular) chains each. The C-terminal portion of
these chains contains the so-called constant regions, while the N-terminal parts (the end of the two branches of the “Y”) have
a very variable structure, which is responsible for the high diversity of binding properties corresponding to the numerous
antigens, which can be recognized. The variability is brought about by gene rearrangements during the maturation of the B-
cell precursors into mature B-cells (see [24]). While the two branches of the “Y”, also called Fab, contain the variable parts
responsible for antigen recognition, the C-terminal part, called Fc, is important for effector functions of the immunoglobulins,
e.g., complement binding and interactions with inflammatory cells. Immunoglobulins can be cleaved by enzymes like papain
or pepsin, which can be utilized to produce fragments with particular characteristics (Fig. 10). Such a cleavage may be of
interest in order to achieve a different distribution into body compartments of the Fab-fragments compared to intact
immunoglobulins, but may also be utilized if only antigen binding, but not the effector functions mediated by Fc are required.
Figure 9. Schematic structure of immunoglobulin: VL and VH are variable regions, CL and CH constant parts of light and
heavy chains
page 14 of 26
Figure 10. Products of enzymatic cleavage of IgG.
2.2.6. Albumin
Albumin is the most abundant plasma protein, with an average concentration of 36 – 50 g/L. It has a molecular mass of
66 000 and is the only plasma protein of this size without carbohydrate content. Albumin is synthesized by the liver. The half
life of albumin is about 19 d, it is catabolized in many tissues and some loss occurs through the intestinal tract.
The most important function of albumin is to maintain the oncotic pressure, which is necessary to retain fluid within the
vasculature against the hydrostatic blood pressure. While in the arterial part of capillaries the hydrostatic pressure is higher
leading to extravasation of fluid, the hydrostatic pressure decreases towards the venous part of the capillaries below the
oncotic pressure, which leads to an influx of fluid. This mechanism is of critical importance for the exchange of fluid and
metabolites between the extra- and intravascular space and thus for the homeostasis of the body as a whole. Besides this
function, albumin is a carrier protein for numerous substances, both physiological compounds as well as medications.
[Top of Page]
3. Production
As outlined in Chapter Composition, Physiology and Chemical Properties of Blood, there are numerous constituents of the
blood that can be used as medicines. The majority of products on the market are derived from human blood. The use of
animal material is for several reasons only of minor importance. Plasma proteins can be produced also by recombinant
techniques, which is so far mainly used for coagulation factors for hemophilia treatment and for thrombolytic drugs.
3.1. Blood and Plasma Donation

The first attempt to transfer blood from one individual to another was in 1492, when blood from three young men was given to
the Pope Innocent VII; unfortunately all four died [25]. Also trials with sheep blood were disappointing. A major step forward
was finding a way to prevent clotting of the blood; the first nontoxic anticoagulant was sodium phosphate, which was found
out by BRAXTON HICKS in 1869. Another breakthrough was the discovery of AB0 blood groups by KARL LANDSTEINER in 1901.
Now the stage was set for further refinements and improvements in the collection, storage, and application of blood
transfusions. This new medical discipline was strongly stimulated by World War II, when blood donation programs were
developed, preservatives such as acid citrate-dextrose (ACD) were invented, and techniques for blood fractionation were
developed. Of major importance for the development of a plasma industry was the work of COHN, who developed techniques
for plasma fractionation (see Section Fractionation of Plasma).
The fundamental prerequisite for the manufacture of blood components and plasma derivatives remains the willingness of
millions of people to donate their blood. One of the most important issues in the whole area is therefore the ongoing
information, motivation, and protection of blood donors. Experience has shown that severe diseases, particularly infections
with viruses such as human immune deficiency virus (HIV) can be transmitted by blood. In order to protect the recipients of
blood products from such risks, careful selection of donors and testing of their donations is necessary. In order to ensure this,
standards have been developed by organizations for blood collection (blood systems) and regulatory authorities, both on
national and international levels. Although these standards are more or less comparable in developed countries, the
organization of blood systems and the particulars of regulations and guidelines in this field may differ considerably not only
from continent to continent, but also from country to country. Essential points of donor selection are the physical examination
in order to verify a good health status, and an interview or questionnaire in order to find out whether there is any obstacle in
the medical history or the behavior of the prospective donor. The latter point may be rather delicate, since, e.g., individuals
with unsafe sexual behavior are not acceptable as blood donors.
If a donor is found suitable, the collection of his/her blood can start (in some countries the very first donation of an individual
may be discarded as a measure of precaution). For the proper collection of blood, the rules of good manufacturing practice
(GMP) have to be strictly followed. This includes suitable premises, well trained personal, and a quality assurance system.
Crucial is the maintenance of hygiene from disinfection of the venipuncture site to a clean environment for processing and
storage of blood components. The term blood components covers all blood-derived products for direct use as transfusion
(e.g., erythrocyte or platelet concentrates, fresh or fresh frozen plasma). The blood is primarily collected into plastic blood
bags. The collecting bags are usually centrifuged in order to produce different types of components. Numerous modifications
exist, it is for instance possible to produce platelet concentrates from platelet rich plasma (supernatant of a “soft” spin of the
blood) or from the so called buffy coat (white layer containing leukocytes and platelets on top of the red cells after a “hard”
spin of the blood). In order to avoid a bacterial contamination of the products, the industry developed closed systems, i.e., a
collection bag and additional bags in which the components can be transferred after separation, interconnected by
appropriate tubes. For some special components it is necessary to open the system (an example is the addition of
cryopreservatives to stem cells). For that purpose so called sterile connecting devices (SCD) have been developed which
automatically cut and reconnect tubes with high temperature in order to avoid contamination. Of crucial importance for
transfusion medicine is the careful documentation of the donation and all relevant data. The components must not be
released before the negative results of safety relevant tests (serological markers for HIV and hepatitis B and C infections,
additional parameters depending on the country) are available. Careful documentation and labeling is also necessary
page 15 of 26
because a confusion of components (“the wrong blood to the wrong patient”) is a prominent cause of severe and potentially
fatal complications.
Blood collection can also be carried out by apheresis techniques. This means, that a part of the blood is separated from the
donation and the rest is given back to the donor. In the beginning, this was done by so called “manual” apheresis. An
example was the manual plasmapheresis, where blood was taken from the donor and separated into plasma, which was
used and the red cells which were retransfused into the donor. In the meantime techniques for “automatic” apheresis have
been developed, which involve an extracorporal circulation of blood. In principle, blood flows from the donor via a tubing
system into a machine, where a continuous flow differential centrifugation takes place with outlet tubes leading the desired
component(s) into collection bag(s), and the rest of the blood back to the donor. By this way plasma can be obtained, as well
as cellular components such as platelets or stem cells or erythrocytes.
The above described collection techniques may be used to obtain both blood components for transfusion and source plasma
for the manufacture of plasma protein concentrates. The origin of the source plasma, i.e., donor population (geographical and
epidemiological background), technique of collection (recovered from whole blood or plasmapheresis) and handling (freezing,
storage, transport) may have significant impact on the quality of this important raw material. In order to enable the production
on an industrial scale, source plasma is usually pooled; the size of these plasma pools varies considerably according to the
facilities used and the product and can be up to several ten thousand donations. The fractionation of these plasma pools is
described below.
3.2. Substances Derived from Blood Cells

Some attempts were made in the past to produce crude blood cell extracts or homogenates for medicinal purposes. These
preparations are poorly characterized and have been part of alternative medicine in some countries.
However, there are also some examples for specific medicines derived from blood cells. From blood leukocytes -interferon
can be obtained ( Interferons – Natural Leukocyte IFN). A preparation of hemin (derivative of the heme obtained from the
hemoglobin of outdated erythrocytes), is used in severe hepatic porphyrias. An old desire in medicine, which had a
renaissance in context with the experience of HIV transmission by blood products, is to find blood substitutes, which means
nonerythrocyte oxygen carriers. Besides several synthetic substances such as perfluorocarbons, a current approach is to use
modified or cross-linked hemoglobins. A limitation of this approach is that higher concentrations of free hemoglobin may be
toxic, e.g., for the kidneys. Besides their capacity to transport oxygen, the hemoglobin-based blood substitutes appear to
have also some impact on vascular tonus and blood pressure and other indirect effects; such effects may be of therapeutic
use.
3.3. Fractionation of Plasma

3.3.1. Quality Requirements
In any kind of medical therapy, assessment of the risk-benefit as well as the development of therapeutic standards is an
important consideration for the use of a medicinal product. Historically, in the case of hemophilia patients substitution therapy
with plasma or plasma-derived products was of such a life-saving importance that other aspects were not considered
adequately. However, with the HIV catastrophe this attitude has changed and virus safety of plasma and plasma derived
products are issues of highest priority in health policy, the medical community, and the public interest. Although virus safety
is obviously of major interest, besides the transmission of viruses and other hitherto unknown pathogens, possible other
safety aspects must be considered. Examples are the development of antibodies (inhibitors) to plasma components and the
risk of thrombogenicity due to the administration of activated coagulation factors.
In general, the whole manufacturing process contributes to the quality of a product. In the case of blood preparations, this
process already starts with the collection of the source material, plasma for fractionation. Measures taken are the selection of
donors, testing of single donations and subsequently pooling of single plasma donations to plasma pools, testing of the
plasma pools, and the selection of suitable methods for the purification including virus inactivation/elimination steps. All
manufacturing steps should be performed under validated conditions and according to cGMP yielding a product of consistent
quality, safety, and efficacy. Regulatory approaches in different continents are still not entirely harmonized but their essential
elements are similar. For the EU, the blood and plasma collection for both the manufacture of plasma for transfusion and
plasma for fractionation is regulated by Directives [26], whereas the quality standard for plasma for fractionation, e.g., quality
attributes, viral marker testing of plasma pools as well as freezing, storage, and transport conditions are provided by the
monograph “Plasma for fractionation” of the European Pharmacopoeia (Ph. Eur.) [27]. In addition, there are monographs for
plasma derived products available, e.g., human Factor VIII, Factor IX, Prothrombin Complex Concentrates, Fibrinogen,
Albumin, Immunoglobulins, etc. setting quality standards for these products. Monographs are legally binding documents.
Guidelines of the Committee for Medicinal Products for Human Use (CHMP), the scientific working arm of the European
Medicines Agency (EMEA) in London [29] and its working groups, e.g., the Biological Working Party (BWP), provide further
guidance on the manufacture of plasma derived medicinal products [28]. These guidance documents consider all steps in the
manufacture of a plasma-derived product with a focus on infectious pathogens.
3.3.2. Preparation of Intermediate Products

Typical examples of plasma fractionation leading to low or intermediate purity are the Cohn fractionation procedure or
derivatives thereof [30]. This technique is up to now in use for the production of major protein constituents of plasma (e.g.,
albumin and immunoglobulins). The Cohn fractionation takes advantage of the different physicochemical properties of
proteins resulting in their different behavior at a given temperature in aqueous solutions of differing pH, salt, and alcohol
concentration (see Fig. 11). For the purification of less abundant components it is now a widely common technique to
separate plasma into the cryoprecipitate (obtained by thawing frozen plasma at or around 4 °C) and into so-called cryo-poor
plasma (proteins which stay in solution after this procedure). Redissolved cryoprecipitate has long been in use for the
treatment of hemophilia A, because coagulation factor VIII is enriched in this plasma fraction. Cryoprecipitate is also used as
a source for fibrinogen which may then be marketed as tissue glue (in combination with thrombin). The cryo-poor plasma
fraction is up to now in use for the preparation of intermediate-purity products, such as immunoglobulins, albumin, and
page 16 of 26
prothrombin complex concentrates (PCC). A derivative of the latter is activated prothrombin complex (aPCC) marketed as
factor eight inhibitor bypassing activity (FEIBA) or Autoplex. PCC has long been used for the treatment of, e.g., hemophilia B
while it is now occasionally in use for the treatment of acquired coagulation disturbances (e.g., reversal of anticoagulation by
treatment with Marcumar during surgery). Activated PCC is given in certain cases of hemophilia A to patients with inhibiting
antibodies against FVIII. The PCC is obtained from cryo-poor plasma by batch adsorption to anion-exchange media at
relatively low pH, followed by washing and step elution of the coagulation factors. In some protocols a barium citrate
precipitation step (or similar) is included in the process. Essentially the same procedure is performed for the preparation of
Autoplex (FEIBA), followed by an activation step in order to obtain (among others) activated FVII.
Figure 11. Cohn fractionation of plasma.
Plama fractionation according to Cohn and co-workers Plasma is fractionated by stepwise adding ethanol, adjusting pH, and/
or dilution. Although not illustrated here, the temperature is also carefully controlled. Each step (the corresponding
designations in the figure are as in the original article) is followed by removing the resulting precipitate. The individual
precipitates contain mainly fibrinogen (no. I), -, - globulin, and prothrombin (no. II/III), -globulin (no. IV-1), -, -
globulin (no. IV-4), and albumin (no. V).
3.3.3. Purification of Plasma Proteins

The Cohn fractionation is up to now in use for the preparation of albumin and immunoglobulins, representing the major
constituents of plasma proteins. Since the late 1980s, however, have not only analytical techniques been very much
improved, but the same is true for preparative applications. Typical examples are FIX and FVIII high-purity concentrates
which are now available. Both coagulation factors are characterized by their relative scarcity as compared to albumin and
immunoglobulins. These novel developments became available through the now widespread use of affinity chromatography
(see Section FVIII High Purity Concentrates.) as well as novel chromatography media and column designs (e.g., radial flow
technology) allowing higher flow rates, thus shortening the purification process.
FIX High-Purity Concentrates. In contrast to PCC — the therapeutic for the treatment of hemophilia B in former years —
FIX is purified by gradient elution of proteins from anion- exchange columns, followed by adsorption to at least group specific
affinity columns, e.g., heparin or dextran sulfate modified media. Also in use are chromatography techniques employing
immobilized monoclonal antibodies. These techniques lead to purities such that the target protein is at least the major
component in these products.
FVIII High Purity Concentrates. Compared to high purity FIX concentrates the purity of so-called high purity FVIII
concentrates is much less striking. This, however, is not surprising, considering that on a molar basis FIX is hundred-fold
more concentrated in plasma than FVIII (see below). One major contaminant in several commercially available concentrates
is serum albumin which is added after purification in order to stabilize FVIII. Beginning with cryoprecipitates, FVIII is purified
using anion-exchange chromatography, in certain cases followed by affinity chromatography on monoclonal antibody media.
Alternative purification strategies take advantage of the association of FVIII with von Willebrand factor. Being an unusually
large protein complex, this association was successfully subjected to size exclusion chromatography, a technique which is
normally characterized by poor resolution.
Although not a purification step, it should be mentioned here that for safety reasons all medium- or high-purity products on
the market are subjected to two complemantary virus inactivation steps (e.g., heat treatment or solvent detergent treatment)
prior to or after filling of the final containers. Before this procedure is performed the proteins are transferred into a buffer
system capable of stabilizing the fragile coagulation factors. These stabilizing additives (mainly sugars and/ or amino acids,
albumin) represent a contamination. Similarly, antithrombin III and/ or heparin are added to certain coagulation factor
concentrates. This step is performed to prevent activation of coagulation factors or to inactivate those which were converted
to proteases during production. These components may be considered a contamination, at least from a biochemical view.
Generally, the purification of proteins for therapeutic use is considered to be beneficial by removing possibly hazardous
contaminants. However, the procedures performed during purification may also be detrimental for the proteins to be purified.
This may hold true especially for the purification of coagulation factors as many of these are progenitors of proteases and
must not be activated, otherwise the therapeutics may lead to thromboembolic complications. Also, they are extremely
sensitive against denaturation (due to their multi-subunit structure as in the case of, e.g., FVIII). The denaturation may lead to
the formation of neoantigens which in turn will cause the development of inhibiting antibodies.
3.4. Recombinant Proteins

In order to circumvent the risk of contamination of coagulation factor preparations by human pathogens (Transmittable
Pathogens) recombinant gene technology has been introduced for the production of several therapeutics instead of purifying
these from human plasma. Recently, certain member states of the European Union (Ireland, UK, Sweden) have decided to
use recombinant coagulation factors exclusively where available. Among others, FVIIa, FVIII, and FIX have been cloned and
expressed in eucaryotic cells of nonhuman origin from which they can subsequently be purified. The purification strategies
follow the same procedures as outlined above for the purification from human plasma to yield products free of human
viruses. However, recently the problem of other pathogens has become apparent. At least during the initial phase of
eucaryotic cell cultivation serum of animal origin is needed for feeding, thus raising the risk of contamination by nonhuman
pathogens which eventually will adapt to the new host. Moreover, there is a theoretical risk that potentially novel pathogens
may contaminate recombinant protein preparations. Here, prion proteins, the putative causative agent of bovine spongiforme
encephalitis (BSE) or Creutzfeld Jacob disease in man (CJD) should be mentioned [31]. In addition, due to the expression in
cells of nonhuman origin, posttranslational modifications may differ in proteins from recombinant sources which eventually
lead to the recognition of these products as foreign and the development of antibodies against these agents. Apart from
producing recombinant proteins in vitro in eucaryotic cells, expression systems have been developed which allow the
production in living animals where the proteins of interest are delivered into the milk and can be purified from this source.
page 17 of 26
3.5. Adverse Drug Reactions of Blood Products
3.5.1. Immunogenicity
Proteins are increasingly used as therapeutic agents (see Enzymes – Nonindustrial Enzyme Usage and Enzymes –
Therapeutic Enzymes). It has been recognized that these medicinal products frequently induce an immune reaction leading
to antibody formation. The consequences of an immune reaction to a therapeutic protein range from transient appearance of
antibodies without any clinical consequences to severe life threatening conditions. Potential clinical consequences are severe
allergic reactions, reduction of efficacy, and induction of autoimmunity, including antibodies to the endogenous protein. Of
further concern is the interference of antibody formation with clinical parameters, e.g., recovery, residence time, and the
laboratory assays to measure the therapeutic protein [32], [33], [34].
Many factors may influence the immunogenicity of therapeutic proteins. They can be considered to be patient-, disease-, or
product-related. Patient-related factors might predispose patients to an immune response, e.g., genetic background, ethnic
origin, immune and infection status, and treatment with immune-suppressive medicines. Disease-related factors include
various immune disorders. Product/treatment-related factors might also influence the likelihood of an immune response, e.g.,
intensity of treatment (dose frequency, duration of treatment), route of administration, source of protein (human or animal or
recombinant origin), manufacturing process (contaminants, impurity profile), formulation and stability characteristics
(degradation products, aggregates) of a given protein.
Development of antibodies against blood components is of special importance for the blood coagulation factors VIII and IX
which are used for the treatment of hemophilia A or B patients [35]. These events are the most serious complications during
the treatment of hemophiliacs besides the possible transmission of blood-borne pathogens. The antibodies, also called
“inhibitors”, can interfere with the biological function of the coagulation factor [36] or can enhance for example the clearance
of the coagulation factor [37]. Both events can lead to life threatening complications for the patients. Patients who develop
inhibitors are classified as low or high responder according to the level of antibodies. Low responders are those who produce
a low level of inhibitor (< 5 B.E.) and who do not react with an increase of inhibitor in case of new coagulation factor
injections. High responders are those patients with a high titer of inhibitor (> 5 B.E.) and who react with an increase of
inhibitor in case of new coagulation factor injections. Low responders can be treated with higher doses of the coagulation
factor concentrate which overcome the inhibitor, whereas for the treatment of high responders other therapies like treatment
with factor VIIa or activated prothrombin complex concentrates have to be considered.
Antibodies directed towards functional epitopes of the factor VIII molecule are currently evaluated in vitro by their capacity to
inhibit the procoagulant activity of plasma factor VIII. Quantitation is obtained by mixing/incubating dilutions of the inhibitor
containing plasma with a fixed amount of factor VIII. The active amount of inhibitor, expressed in units per milliliter plasma, is
the reciprocal of the plasma dilution that neutralizes factor VIII activity. For routine analysis, the New Oxford assay (source of
factor VIII is a concentrate) and the Bethesda method (source of factor VIII is a pooled normal plasma) have been used [38],
[39]. It is now recommended to use a modification of the Bethesda method by which the progressive decrease of pH during
incubation is prevented, which can lead to false positive results [40]. Inhibitors can also be detected independent of their
functional activity using enzyme-linked immmunosorbent assay (ELISA) techniques.
Inhibitor development against the substitutive product is considered to be due to either the type of substitute or the severity of
hemophilia. Patient-related factors for the development of antibodies are poorly understood and a relationship between gene
defect and inhibitors is not manifest. Results for hemophilia A patients, however, point out that patients with severe gene
defects are more prone to inhibitor development, perhaps due to the absence of circulating factor VIII and the lack of immune
tolerance induction [41], [42]. Product-related factors could also be responsible for inhibitor development which was observed
in hemophilia A patients treated for several years with factor VIII products without developing inhibitors. When these patients
had been exposed to a certain factor VIII product, they developed inhibitors after a variable length of exposure days [43].
3.5.2. Thrombogenicity
Thromboembolic complications are the major adverse events during the treatment of patients with prothrombin complex
concentrates (PCCs) [44-47]. Prothrombin complex concentrates contain the coagulation factors II, VII, IX and X, protein Z,
as well as the coagulation inhibitors protein C and S. These products are indicated for the treatment of patients with
decreased levels of factors of the prothrombin complex due to vitamin K deficiencies, overdosing of oral anticoagulants, e.g.,
vitamin K, or decreased synthesis caused by liver dysfunctions. The three component PCCs (factor II, IX and X) have
previously been used for the treatment of hemophilia B but have been replaced by high-purity factor IX concentrates or
recombinant factor IX. Different from PCCs are those products (FEIBA, Autoplex) which contain activated factors of the
prothrombin complex and which have another indication.
Prothrombin complex concentrates are standardized with respect to their factor IX content. According to the Ph. Eur.
monograph [27], PCCs should exhibit an equal composition of the prothrombin complex factors and their content of protein C
and S should be indicated. In addition, most of the PCCs have heparin and antithrombin III added. PCCs have to comply with
requirements of the nonactivated partial thromboplastin time and the thrombin fibrinogen clotting time. As these parameters,
however, have been found to be of low predictive value with respect to in vivo correlation, additional measures should be
followed by the treating physician in order to minimize the risk of thrombotic complications [46], [47].
During a 4-years survey from 1987 to 1990, 72 case reports on complications with PCCs were published [48]. Major risk
factors of thrombogenicity have been reported to be (1) predisposing factors, i.e., the patient's underlying disease; (2)
therapy factors, i.e., dosage (zymogen overload leading to a prothrombotic state) and concomitant therapy; and (3) quality of
the PCCs. With respect to the quality aspect of PCCs, activated coagulation factors II, VII and IX as well as the absence or
low levels of antithrombin III and protein C and S have been discussed to cause the clinical signs of thrombogenicity which
manifest as phlebitis or venous thrombosis, pulmonary embolism, cerebral or myocardial infarction, disseminated
intravascular coagulation with bleeding, and organ failure due to consumption of coagulation factors and fibrin formation.
Coagulant active phospholipids, which have been detected in PCCs, were also discussed as potent thrombogenic triggers
potentiating the effect of activated coagulation factors [49]. During the last few years, scientific data were accumulating which
showed without a doubt that prothrombin overload is causing thromboembolic complications while treating patients with
page 18 of 26
PCCs [50-52].
3.5.3. Transmittable Pathogens

One immanent risk of blood products is the transmission of pathogens. This issue is therefore of particular concern and
appropriate measures have to be taken by the manufacturer to prevent the transmission of infections by the use of plasma-
derived medicinal products [53]. Those pathogens include HBV, HCV, HIV-1 and HIV-2, HAV, and Virus B19. During the
recent years, emerging pathogens, e.g., variant CJD (vCJD) and West Nile virus (WNV) had to be added to this list of
pathogens with importance for the safety of blood products. Therefore, the collection of the source material, plasma for
fractionation, is of great importance in the assurance of the quality and safety of medicinal products derived from blood.
Factors which should be considered for the collection of plasma (blood) include various factors like suitability of donors,
screening of donations and epidemiology of the population from which plasma is collected. The risk assessment for medicinal
products derived from plasma (blood) should consider the amount of virus which may contaminate the manufacturing plasma
pool versus the capacity of the manufacturing process to remove or inactivate the pathogens. By considering the amount of
starting material needed to manufacture a single dose of product, the probability of potential virus contamination can be
estimated [54].
A recently emerged infection in North America is the WNV which belongs to the Flaviviridae family. Clinical symptoms in
humans occur 2-14 days after infection and viraemia within 1-3 days after infection and lasts 1-11 days. Thus, an infected
person could be viraemic before symptoms are seen. As mentioned above, the viral safety of plasma-derived medicinal
products involves several approaches and re-evaluation those for the purpose of WNV lead to the assurance that the
measures in place today are adequate to assure viral safety of plasma-derived products with respect to WNV [55].
Tracing recipients of blood transfusions from donors who subsequently developed vCJD has revealed possible cases of
secondary transmission. This finding is consistent with the observation for sheep transfusion studies that infectivity in blood
can be transmitted during preclinical phase of infection. To date many studies have been performed, which investigate the
contribution of various manufacturing steps to reduction of infectivity. However, caution is needed in interpretation of the data
since the effectiveness of a step is dependent on a number of variables, e.g., process conditions and state of the agent in the
sample. Consequently, effectiveness of removal may vary from process to process. As vCJD is an emerging disease,
uncertainties still exist about the number of cases of vCJD that will occur and the transmission of the infective agent by blood
donations. Today, no screening test is available and therefore approaches are considered to identify donors who may
present a higher risk. Therefore it is recommended to exclude donors who have spent a cumulative period of one year in the
United Kingdom from donating blood/plasma for fractionation. In view of the lack of adequate information on vCJD, it is
prudent to recall batches of plasma-derived medicinal products where a donor to a pool subsequently develops vCJD. Also
medicinal products containing plasma-derived products as excipient should be recalled [56]. This issue is kept under review
of the CHMP and its working groups.
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4. Therapeutic Uses
Medicines derived from blood and plasma play an important role in the medical therapy in general. In most cases, they are
used to replace the respective components or proteins in case of their loss or insufficient production. Particularly, the
development of contemporary surgery, which is often associated with blood loss, would not have been possible without the
possibility of transfusion. Also in hematology and oncology, and of course in the treatment of bleeding disorders, blood
products are indispensable.
4.1. Blood Components

4.1.1. Blood and Stem Cell Preparations
Erythrocytes. The most important components in terms of the amounts used and the potentially immediate life-saving effect
are those containing erythrocytes. Whereas initially whole blood was the mainstay of hemotherapy, nowadays erythrocyte
concentrates are prevailing. These concentrates are conventionally produced from whole blood donations by centrifugation,
and can be obtained also by modern apheresis techniques. With contemporary additives, erythrocyte concentrates may have
a shelf life of up to 7 weeks. It is a well-documented experience [57] that immunization of blood recipients against donor
antigens is mainly mediated by contaminating leukocytes. Immunization can cause severe allergic reactions or impair the
therapeutic effect to subsequent transfusions. Also other unwanted effects of transfusions have been attributed to their
leukocyte content. Thus, “leukodepletion”, the removal of leukocytes by filtration or modification of apheresis, is increasingly
recommended, despite the increase of expenses for the concentrates.
There are several guidelines [59], but no rigid rule regarding the indication for erythrocyte transfusion. In every individual
patient, the indication should be considered carefully. For instance, young, otherwise healthy patients tolerate lower Hb-
levels than elderly patients, particularly those with cardiovascular disorders. Chronic anemia is far better compensated than
acute blood loss. Therefore, the conventional recommendation of 10 g/dL Hb and below as a trigger for transfusion is at best
a rough “rule of thumb”.
Platelets. A lack or functional disturbances of thrombocytes lead to a severe bleeding tendency. The typical clinical pattern is
petechiae and bleeding from mucous membranes (e.g., nose, gingiva) but also life-threatening CNS bleedings. A bleeding
tendency may be present at thrombocyte counts around 30 000/µL; a clear increase of the bleeding risk necessitating platelet
transfusions occurs below 10 000/µL. Functional disturbances may be inherited such as defects of GP IIb-IIIa
(thrombasthenia Glanzmann), or of GP Ib-IX (Bernhard Soulier Syndrome). More frequent are acquired functional defects in
liver disease or renal insufficiency, or by medications such as analgesics or antibiotics. On the other hand, platelet inhibition
may be a target of therapy in thromboembolic diseases, particularly in arterial events such as myocardial infarction or stroke.
The classical substance for platelet inhibition is aspirin, but a number of new substances against specific receptor pathways
are currently developed and evaluated.
page 19 of 26
Leukocytes. The use of leukocyte (e.g., granulocyte) concentrates is still restricted to very special circumstances, since
considerable problems occur with unwanted effects, e.g., acute lung injury.
Stem Cells. An increasingly important field is the production and use of hematopoietic stem cells [61]. Stem cells can be
obtained from aspirated bone marrow, from peripheral blood by apheresis, and from placenta via the umbilical cord; also fetal
liver contains stem cells. Currently, the most important use is for “rescue” of hematopoiesis after otherwise lethal
chemotherapy and/or radiation for treatment of several malignancies, or after damage or aplasia of the bone marrow (bone
marrow or stem cell transplantation). Also “autologous” stem cells taken from the patient and reinfused after intensified
antineoplastic treatment are in use; strategies are developed to purify the CD-34 positive stem cells, or to remove malignant
cells (“purging”) from the preparation. Still in the experimental stage are approaches to augment the stem cells in culture (“ex
vivo expansion”), or to manipulate them by incubation with antigens and cytokins in order to obtain a specific immunological
therapy. Of so far unforeseeable importance are stem cells as a vehicle for gene therapy.
4.1.2. Fresh Frozen Plasma

Most of the whole blood donations are separated into cells and plasma; plasma can also be obtained by plasmapheresis (see
Section Blood and Plasma Donation). Plasma can be used as source for industrial production of protein concentrates, or as a
component for direct transfusion. For the latter purpose, plasma is usually rapidly frozen as soon as possible (fresh frozen
plasma) and stored in a freezer in order to preserve the sensitive proteins such as coagulation factors.
Plasma as a therapeutic component has the advantage that it is the physiologic fluid part of blood with a balanced content of
proteins, particularly albumin, coagulation factors, inhibitors, and immunoglobulins, as well as electrolytes and other natural
constituents. Therefore, plasma is in principle an ideal substitute in case of blood loss, in combination with red cells as
clinically appropriate. However, it is no more recommended to use plasma just for volume replacement, since it is available in
limited amounts only, and since alternatives, so-called “plasma expanders”, e.g., dextrans or hydroxyethyl starch, are
available. Moreover, plasma still carries a low residual infectious risk; there are strategies to keep plasma in quarantine until
the donor is tested again after at least 6 months, to exclude the possibility of donations during the diagnostic window, i.e., the
time between infection and the detectability of virus markers. There are also attempts to apply virus inactivation steps to
single donations, or to pooled plasma, which is subsequently divided again into units of the usual size.
The main indication of plasma transfusion is the prevention and treatment of profound and complex disorders of blood
coagulation, such as consumptive thrombohemorrhagic disorders, in surgical and intensive care patients. In the treatment of
those disorders, e.g., disseminated intravascular coagulation, a multitude of factors and inhibitors are consumed, and plasma
may be a suitable way to replace all of them in a balanced way. Large volumes of plasma may have to be transfused in order
to obtain relevant replacement in full-blown consumption, potentially leading to fluid overload; some clinicians use therefore
concentrates alone or in combination with plasma. The therapy of such complex disturbances, however, is far from being
standardized, and should be directed by experienced specialists taking into account the individual clinical picture. In the
treatment of hemophilia or other single-protein deficiencies, suitable concentrates should be used. In general, the indications
for plasma transfusion are hard to define, and should be further evaluated in clinical studies.
4.2. Hemoglobin-Derived Oxygen Carriers

The driving forces behind the development of “artificial blood” are the avoidance of the risk of infection by allogeneic blood
transfusion, and to overcome the problems posed by blood grouping and the limited shelf life of erythrocyte concentrates. In
fact, hemoglobin isolated from blood, which may be modified e.g., by cross-linking, is capable of effective oxygen transport.
These preparations may elicit also some increase of blood pressure, which might be interesting in particular circumstances.
The original hope to have a new, virtually unlimited source of oxygen carriers which would supersede conventional blood
components has been darkened so far by the finding of substantial toxicity of larger doses of these carriers. Free hemoglobin
outside the “parent” erythrocytes is bound to a specific transport protein, named haptoglobin, and has to be cleared from the
circulation. If the capacity of haptoglobin and the clearance system is exhausted, free hemoglobin is excreted via the kidney,
and may lead to kidney failure similarly as in the case of massive myoglobin release in severe trauma (“crush kidney”). Also
the new hemoglobin-derived substances are still limited in their dose. It might turn out that they have a potential in the
emergency ambulance, e.g., for immediate on-site resuscitation of accident victims. Hemoglobin-derived substances are also
evaluated for other indications, e.g., oxygen delivery and pressor effect in septic shock, or sensitization to radiotherapy in
cancer patients. However, the intriguing approaches appear to need substantiation by further research.
4.3. Cytokins
A growing number of cytokins have effects of potential therapeutic interest (see Section Leukocytes, Cytokins and Table 2).
Here, only some examples already in clinical use are mentioned.
One of the first therapeutic cytokins was interferon ( Interferons), due to its antiviral and potential antineoplastic effects.
Interferon is used in chronic viral hepatitis and may lead to clinical remission in part of the patients [63]. It is not a universal
antitumor drug, but in several malignancies, e.g., in chronic myeloid leukemia, interferon is a valuable part of the therapeutic
repertoire.
Increasingly used in hematology and oncology are also the hematopoietic growth factors such as G-CSF or GM-CSF. The
may be useful to stimulate granulopoiesis in patients during the aplastic phase after chemotherapy, and also for
“mobilization” of stem cells from the bone marrow in order to harvest them by apheresis. A new, promising, substance is
thrombopoietin, which stimulates platelet production. Another substance which is conventionally classified as a hormone
should also be mentioned here: erythropoietin, which is physiologically produced by the kidneys. Recombinant erythropoietin
has proved to be of great value for patients with chronic renal failure, which used to be dependent on transfusions before.
Erythropoietin is also used in certain hematological disorders.
4.4. Coagulation Factors
page 20 of 26
Congenital coagulation factor deficiencies mostly affect the level of a single factor, while acquired deficiency (e.g., due to
impaired synthesis in liver disease, loss or dilution of blood, or consumption in sepsis) includes usually many or all factors. All
coagulation factors are contained in plasma, which may be used for the treatment of complex factor deficiencies (see
Section Fresh Frozen Plasma), concentrates are recommended in cases where specific factors are to be administered. This
concept of a targeted therapy helps to avoid fluid overload, and an unwanted and possibly overshooting increase of factors
which are normal or even elevated at baseline. This chapter covers treatment of congenital, one-factor deficiencies. There is
a trend to high purity concentrates, since the lacking factor can be substituted in a more controlled fashion, and impurities are
kept low. Such impurities could be associated with unwanted effects, such as immunosuppression, or thrombogenicity [65],
[44].
The classical indication for the use of concentrates are the hemophilias A (lack of factor VIII), and B (lack of factor IX). Both
are congenital disorders mediated by X-chromosomal inheritance, and thus affect almost exclusively males. The disease can
be expressed with different severity depending on the underlying gene defect; the severe hemophilias (levels of < 1 % of the
normal plasma level) are usually manifested already in early childhood, when the babies start crawling and moving around
and thereby may hit themselves mildly, which would be easily compensated by a healthy coagulation, but may lead to severe
bleeding in hemophiliacs. The typical symptoms are large hematoma of the skin, and bleeding into muscles and joints;
bleeding at other sites including the particularly life-threatening central nervous system bleeds also occur. The frequency of
hemophilias is about 1 in 10 000 males. Being a congenital defect, hemophilia necessitates life-long treatment. The therapy
may be given in case of symptoms (“on demand treatment”), many patients with severe hemophilia are substituted with a
fixed continuous schedule of usually two to three injections per week (“prophylactic treatment”); a typical prophylactic single
dose would be 30 iU per kilogram of body weight.
While during the early 1980s a disastrous epidemic of HIV infections, transmitted by coagulation factor preparations, hit the
hemophiliac population, the safety of the concentrates has been increased fundamentally in the meantime by careful
selection and testing of blood donors, and virus inactivation procedures. Currently, the most important problem of treatment
is the occurrence of so-called “inhibitors”, which means neutralizing antibodies, during therapy. These inhibitors usually occur
during the initial phase of treatment around the 10th to the 20th exposure to the factor, which is “strange” to the recipient's
immune system. The inhibitors only rarely cause allergic symptoms (predominantly those against factor IX). Therefore they
may develop unnoticed and may cause severe problems in case of bleeding episodes, since the effect of the transfused
factor is impaired or even blocked. It is recommended to check the patients regularly for inhibitors, particularly during initial
treatment. Inhibitors occur much more often in hemophilia A, which is also the far more frequent form of the hemophilias. The
treatment of such inhibitor patients has two aims: (1) to stop and control acute bleeding and (2) to eliminate the inhibitor. For
stopping and controlling of bleeding, high doses of the involved factor, PTC, “activated” PTC, or (in hemophilia A patients
only) porcine factor VIII or, as a newer approach, recombinant factor VIIa are in use. For the elimination of inhibitors, also
referred to as “immunetolerance treatment”, prolonged treatment with frequent and high doses of factor VIII (“Bonn protocol”),
as well as a combination with extracorporal removal techniques and immunosuppressive treatment (“Malmö protocol”) have
been developed [66]. The elimination of factor IX inhibitors is particularly problematic, since allergic and anaphylactic
reactions and, in case of high doses of factor IX, immune complex disorders such as nephritis [67] may develop. The
occurrence of inhibitors is a frequent problem (in hemophilia A in up to 50 % of patients). In two clusters, inhibitors with
unusual characteristics were associated with certain products [68], [43], which had to be taken off the market.
Another congenital bleeding disorder, which is even more frequent than hemophilia, is von Willebrand disease. However,
since there are a lot of different mutations and thus functional and clinical categories, the classification of severity and the
therapeutic consequences are much less clear-cut than in the hemophilias. A lack or dysfunction of von Willebrand factor
may lead to a reduction of factor VIII levels and/or disturbances of platelet adhesion; the clinical symptoms resemble in many
cases more a platelet defect with prolonged bleeding time, petechiae, and mucous membrane bruising. Since the inheritance
is autosomal (chromosome 12), both sexes are equally affected. It is strange that the clinical severity may differ from one
affected family member to the other, and even during time course in an individual. By far the most patients have mild
symptoms, and it is not unusual to detect the disease in an adult. Mild forms may be treated with a vasopressine analogue
(DDAVP), which mobilizes residual von Willebrand factor from its production site, the endothelial cells; this therapy is
effective only a limited time (several days). In any case, an accurate diagnosis should be established first, and the effect
should be evaluated in a test treatment, before, e.g., surgery is to be covered by DDAVP; in some forms of dysfunctional von
Willebrand factor DDAVP may even be contraindicated. In some countries, factor VIII concentrates containing von Willebrand
factor are registered for substitution in severe forms of von Willebrand disease.
Other congenital coagulation factor deficiencies are less frequent or even rare; an example is factor XIII deficiency with an
estimated prevalence of 1 in 1 × 106 people. Factor XIII is a fibrin-stabilizing factor, which is important to make clots stable
and resistant against premature fibrinolysis. Characteristic symptoms are umbilical cord bleeding of newborns, repeated re-
bleeding after initial hemostasis, wound healing disturbances and spontaneous abortions. Unfortunately, there is a quite high
frequency of central nervous system bleeds. In the past, a concentrate had been manufactured from placenta; nowadays, for
treatment, a plasma-derived concentrate is registered in some countries. The significance of an acquired factor XIII
deficiency needs further elucidation; there are intriguing data pointing to a role of factor XIII in wound healing [69].
4.5. Plasminogen Activators

Plasminogen activators are used in the thrombolytic therapy of severe thromboembolic disorders such as myocardial
infarction and lung embolism. The physiologic task of the fibrinolytic system is the prevention of an overshooting clotting.
However, if clinically significant thrombosis occurs, the natural fibrinolysis may take considerable time (several months) in
order to remove the fibrin in the thrombus. In many cases, the thrombus is perpetuated by “organization”, i.e., the conversion
into a connective tissue “scar”. Particularly in emergencies like myocardial infarction, a very fast recanalization is needed.
The first, and still widely used substance was streptokinase, a plasminogen activator derived from streptococci.
Streptokinase forms a complex with a plasminogen, and this complex activates free plasminogen to plasmin. The
disadvantage of streptokinase is mainly its antigenicity. Also a number of physiologic plasminogen activators exist, and
numerous attempts have been made to modify them by recombinant techniques. The most widely used are urokinase and t-
page 21 of 26
PA. While urokinase, similarly as streptokinase, produces free plasmin leading to a massive degradation of fibrinogen and
coagulation factors and thus to a pronounced disturbance of coagulation, t-PA acts predominantly at the fibrin clot and has
therefore also been named a “fibrin specific” thrombolytic agent. The initial hope that this would reduce bleeding
complications was not fulfilled at the high t-PA doses needed for rapid thrombolysis. However, t-PA appears to act somewhat
faster than the other thrombolytic agents which might be an advantage in certain emergencies. However, urokinase and
particularly t-PA are very costly.
Compared to a thrombophilic state due to a congenital deficiency of coagulation inhibitors (see Section Inhibitors), inherited
disorders of the fibrinolytic system are much less frequent and the clinical significance is less clear. Though there are some
reports about an enhanced thrombotic risk for individuals suffering from disturbances of t-PA expression or release, and from
a plasminogen deficiency [70], there was recently a report of a total plasminogen deficiency in several patients who did not
experience serious thromboembolism [71].
4.6. Inhibitors
The congenital inhibitor deficiencies usually pertain to a single inhibitor — although evidence points to a higher prevalence of
combined defects than in coagulation factors (Section Coagulation Factors — whereas an acquired reduction usually
includes several inhibitors. The organism contains several proteolytic systems and respective protease inhibitors; this chapter
is focused on the inhibitors relevant in the hemostatic system. Congenital deficiencies of inhibitors of the fibrinolysis system
may cause severe, hemophilia-like bleeding, but are very rare; there are no specific concentrates available. Congenital
deficiencies of coagulation inhibitors have attracted increasing interest in the past decade, and “thrombophilia” has been
coined as a new term.
A total deficiency of antithrombin III is lethal, and total deficiency of protein C is associated with the life-threatening purpura
fulminans of newborns. In contrast to, e.g., hemophilia (see Section Coagulation Factors), most symptomatic patients with
deficiencies of the coagulation inhibitors antithrombin III, protein C, and protein S are heterozygotes with plasma levels of
around 50 % of normal, which makes the diagnostic discretion from borderline normals sometimes difficult. The affected
family members show an increased tendency to mostly venous thromboembolism at a relatively young age; in antithrombin III
deficiency, up to 80 % of the affected individuals may experience a thrombotic event before the age of 40 years. In contrast
to hemophilia, there is no indication for prolonged substitution, with the exception of particular risk situations like major
surgery and in the peripartal period in pregnancy and after delivery. Usually, it is recommended that the patients are treated
by prophylactic oral anticoagulation (cumarin treatment) after the first thrombotic manifestation. A problem in patients with
protein C or protein S deficiency is that these proteins are also vitamin K-dependent and their level is further lowered during
cumarin treatment. Particularly in the initial phase of anticoagulation, a critical dysbalance between coagulation factors and
protein C may occur; a typical clinical complication is the so-called cumarin necrosis.
In some countries, antithrombin III concentrates are licensed, which are used predominantly in acquired deficiency, as in
disseminated intravascular coagulation. However, a clinical study failed to show a reduction of mortality in severe sepsis [72].
Recently, recombinant activated protein C was licensed for treatment of severe sepsis [73]. Well-controlled clinical studies
are extremely difficult to perform in those severe and complex disorders.
4.7. Immunoglobulins
The most common application of immunoglobulins is the replacement of endogenous immunoglobulins in patients with
immune deficiency syndromes compromising the specific immune defense. In addition, high doses of immunoglobulins may
be used to overcome certain autoimmune disorders, such as idiopathic thrombocytopenic purpura (ITP) [74]. A further
therapeutic segment is the application of specific immunoglobulins, which can be gained from immunized donors. A typical
example is immunoglobulin against the rhesus antigen D, which is used for prophylaxis of immunization of rhesus negative
mothers by rhesus positive babies, which could lead to a severe hemolytic disorder in further rhesus positive offspring.
Immunoglobulins may also be produced by recombinant techniques in order to obtain antibodies with specific
pharmacological effects. Examples are antibodies against the platelet GP IIb-IIIa (see Section Thrombocytes) as a potent
antiplatelet drug in high risk situations such as thrombolysis for myocardial infarction, or antibodies with specific antitumor
action, which may be conjugated with antineoplastic substances.
4.8. Albumin
The use of albumin has been scrutinized following a meta-analysis of Cochrane and co-workers, who suggested an
increased mortality. Though this conclusion was not substantiated, it is recommended that the indication for human albumin
should focus on the use of albumin for restoration and maintenance of circulating blood volume where volume deficiency has
been demonstrated and use of a colloid is appropriate [75]. Haemodynamic parameters should be monitored in patients
receiving albumin due to the risk of hypervolaemia and cardiovascular overload.
[Top of Page]
5. Economic Aspects
Blood is a very precious resource from the ethical standpoint, since it is a generous gift of voluntary donors. Blood and
plasma and the derivatives thereof are also of high economical value, since there are considerable expenses for the
infrastructure of donation centers, donor selection and testing, storage of labile components, handling and transportation of
source plasma, and industrial production of concentrates, which includes plasma fractionation, purification of proteins and
virus inactivation steps. Furthermore, the quality and safety of the products should be continuously refined by ongoing
research, for instance on new pathogens or their potential to cause immunological or thrombotic side effects. The treatment
of patients with blood and plasma products is therefore expensive, particularly for patients who need lifelong treatment for
congenital diseases such as hemophilia or immune deficiencies. This is one of the reasons, why a functional blood system
and particularly supply of plasma products is so far guaranteed mainly in the developed countries; it is estimated that
currently about 80 % of all hemophiliacs worldwide do not receive medically adequate treatment.
page 22 of 26
Since the financial budgets of the health care systems are limited also in economically strong nations, there is an increasing
pressure to use these resources as economically as possible. The decisions in every single country are strongly influenced
by medical traditions and ethical views. However, a scientific basis for estimation of the cost vs. benefit ratio of certain
expensive therapeutic interventions is increasingly advocated. A typical example is the competition between plasma-derived
and recombinant coagulation factors. This necessitates well-designed basic research and clinical trials, as well as solid
comprehensive concepts requiring the input of scientists, clinicians, health care suppliers, regulators, politicians and, of
course, the patients and the citizens, who pay for the social systems.
Despite discussions about health care budgets, it should not be forgotten, how immense the benefit of the development of
transfusion is for medicine as a whole, and how dramatically the life expectancy and the quality of life of hemophiliacs
improved by the introduction of replacement therapy. Thus, also in the modern medicine of increasingly sophisticated
invasive procedures and the upcoming paramount of gene therapy, the medicines derived from blood and plasma should
keep an important place.
[Top of Page]
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[Top of Page]
Abbreviations used in this article
AAE: acquired angioedema
ACD: acid citrate-dextrose
a-PTC: activated prothrombin complex
BFU-E: burst-forming unit-erythrocytes
BSE: bovine spongiforme encephalitis
CD: cluster of differentiation
CJD: Creutzfeld Jacob disease
CFU: colony forming unit
CPMP: Committee of Proprietary Medicinal Products
CSF: colony stimulating factors
Eo: eosinophil
page 25 of 26
EMEA: European Medicines Evaluation Agency
FDP: fibrin degradation products
FEIBA: factor eight inhibitor bypassing activity
GM-CSF: granulocyte-macrophage colony stimulating factor
GMP: good manufacturing practice
GP: glycoprotein
HAE: hereditary angioedema
HIV: human immune deficiency virus
HLA: human leukocyte antigen
HBS: heparin binding site
HMWK: high molecular weight kininogen
Ig: immunoglobulin
IL: interleukin
ITP: idiopathic thrombocytopenic purpura
LPS: lipopolysaccharide
MCH: mean corpuscular hemoglobin
MCHC: mean corpuscular hemoglobin concentration
MCV: mean corpuscular volume
MEG: megakarocyte
MHC: major histocompatibility complex
PA: plasminogen activator
PAI: plasminogen activator inhibitor
PDGF: platelet-derived growth factor
PTC: prothrombin complex
1-PI: 1 -proteinase inhibitor
PK: prekallikrein
RES: reticulo-endothelial system
RS: reactive site
SCD: sterile connecting device
SCF: stem cell factor
TAT: thrombin-antithrombin
Tpo: thromopoetin
TNF: tumor necrosis factor
vWF: von Willebrand factor
[Top of Page]
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Abstract | Full Text: HTML

2. Composition, Physiology and Chemical Properties of Blood

Parameter Value Remarks

* m = male, f = female proband.

2.1. Blood Cells

2.1.1. Hematopoiesis, Stem Cells

Figure 1. Bone marrow (overview)

Figure 2. Scheme of cell line differentiation in hematopoiesis

BFU = burst-forming unit; E = erythrocyte; Epo = Erythropoetin; Eo = Eosinophil; GM = granulocyte-macrophage;

Largest cells in bone marrow (compare surrounding cells)

Table 2. List of selected cytokins

Factor Major source(s) Main effect(s)

Granulocyte-colony monocytes, stimulation of generation and differentiation

Figure 6. Mature leucocytes in the peripheral blood smear:

A) Band form; B) Segmented neutrophil; C) Eosinophil; D) Basophil; E) Monocyte; F) Lymphocyte

Compare size with surrounding erythrocytes and platelets.

2.1.2. Red Cells, Hemoglobin

2.1.3. Leukocytes, Cytokins

CD designation Remark Main cell reactivity

CD3 T-cell receptor complex T-cells

2.2. Plasma Proteins

Table 4. Plasma proteins

Protein Plasma concentration, Biological function Mr

Albumin 40 – 50 transport, colloidosmotic 67 000

2-Macroglobulin 1.5 – 3.5 general inhibitor of proteases 725 000

1-Lipoprotein 1.7 – 3 lipid, hormone transport 28 000

2.2.2. Coagulation Factors

Figure 8. Scheme of blood coagulation

TF = Tissue factor; HMWK = High molecular weight kininogen: Pk = prekallikrein

2.2.3. Fibrinolysis System

Figure 10. Products of enzymatic cleavage of IgG.

3.1. Blood and Plasma Donation

3.2. Substances Derived from Blood Cells

3.3. Fractionation of Plasma

3.3.2. Preparation of Intermediate Products

Figure 11. Cohn fractionation of plasma.

3.3.3. Purification of Plasma Proteins

3.4. Recombinant Proteins

3.5.3. Transmittable Pathogens

4.1. Blood Components

4.1.2. Fresh Frozen Plasma

4.2. Hemoglobin-Derived Oxygen Carriers

4.4. Coagulation Factors

4.5. Plasminogen Activators

Abbreviations used in this article

AAE: acquired angioedema

ACD: acid citrate-dextrose

a-PTC: activated prothrombin complex

BFU-E: burst-forming unit-erythrocytes

BSE: bovine spongiforme encephalitis

CD: cluster of differentiation

CJD: Creutzfeld Jacob disease

CFU: colony forming unit

CPMP: Committee of Proprietary Medicinal Products

CSF: colony stimulating factors

FDP: fibrin degradation products

FEIBA: factor eight inhibitor bypassing activity

GM-CSF: granulocyte-macrophage colony stimulating factor

GMP: good manufacturing practice

HAE: hereditary angioedema

HIV: human immune deficiency virus