This document discusses potential alternatives to blood transfusions known as blood substitutes. It begins with some background on the need for blood substitutes due to limited blood supply and risks of transfusion. Two main types of blood substitutes are then described - volume expanders like crystalloids and colloids, and oxygen-carrying products like hemoglobin-based oxygen carriers and perfluorocarbon emulsions. The document focuses on hemoglobin-based oxygen carriers, outlining different acellular and cellular formulations and some examples that have undergone clinical trials. Overall it provides an overview of the history and development of blood substitutes.

2. N . V E E R A R A G A V A N
I I Y E A R P G
BLOOD SUBSTITUES

3. INTRODUCTION
 The transfusion of blood and blood products has become commonplace
since the first successful transfusion in 1818.
 Although the incidence of severe transfusion reactions and infections is
now very low, in recent years it has become apparent that there is an
immunological price to be paid from the transfusion of heterologous
blood, leading to increased morbidity and decreased survival in certain
population groups (trauma, malignancy).
 Supplies are also limited, and therefore the use of blood and blood
products must always be judicious and justifiable for clinical need

4. HISTORY

5.  1961- plaletet concentrates are reconized to reduce
mortality from hemorrhaging in cancer patients
 1972- process of apheresis.
 1983- stanford blood centre done screening centre
for AIDS
 2002- west nile virus identified as transfusion –
transmissible

6. INDICATIONS
 acute blood loss, to replace circulating volume and
maintain oxygen delivery
 perioperative anaemia, to ensure adequate oxygen
delivery during the perioperative phase
 symptomatic chronic anaemia, without haemorrhage
or impending surgery.

8. METHODS
 BLOOD PRODUCTS
 ARTIFICIAL BLOOD OR BLOOD SURRGOATE

9. BLOOD PRODUCTS
• Whole blood
• blood components :red cell concentrates, platelet
concentrates, fresh frozen plasma and cryoprecipitate
• plasma derivatives :albumin, coagulation factors and
immunoglobulins.

10. Key Elements
 Donors are chosen to exclude anyone whose blood
may harm the recipient
 Each donation is tested to establish the ABO and
RhD group of the donor’s red cells.

11. Screening tests
 Hepatitis B
 Hepatitis C
 HIV-1
 HIV-2
 HTLV
 Syphilis.
 Donations are leukodepleted as a precaution against
Creutzfeldt–Jakob disease
 (this may also reduce the immunogenicity of the
transfusion)

12. Whole blood
 consists of red blood cells, white blood cells and platelets
 Whole blood has a shelf life of 35 days.
 Citrate phosphate dextrose adenine (CPDA-1) is an
anticoagulant preservative in which blood is stored at 1°C to
6°C.
 The storage at 1°C to 6°C assists preservation by slowing
the rate of glycolysis approximately 40 times the rate at
body temperature.

13.  Citrate is an anticoagulant (prevents clotting by
binding calcium).
 Phosphate serves as a buffer.
 Dextrose is a red cell energy source
 Adenine allows RBCs to resynthesize adenosine
triphosphate (ATP).

14.  Advantages
Rich in coagulation factor
Metabolically active
 Disadvantage
Limited resource
Poor source of platelets

15. Packed RBC
 Packed red cells are produced by removing between 150-
200ml of citrated plasma from a unit of whole blood.
 Haematocrit = 60-70%.
 Storing red cells just above freezing allows survival for up
to 42 days.
 – but unfortunately decreases 2,3-DPG
 – ruins the platelets and neutrophils.

16.  The administration of packed RBCs is facilitated by
reconstituting them with a crystalloid or colloid; however,
not all crystalloids are suitable.(5% dextrose in water )
 If the solution contains calcium, clotting occurs.
 Solutions recommended for reconstituted packed
erythrocytes are 5% dextrose in 0.4% saline, 5% dextrose in
0.9% saline, 0.9% saline, and Normosol-R with a pH of 7.4.

17.  A unit of whole packed red cells will raise the hematocrit by
3% and the hemoglobin by 1-1.5 gm/dL
 OTHER PRBC PRODUCTS
 Irradiated packed red cells
 Washed packed red cells
 Cryopreserved packed red cells

18. Platelet Concentrates
 Component: platelets, 50 ml plasma
 cellular components that help in the clotting process.
 Platelets are stored for up to five days at room
temperature.
 Indication
 – used if there is a platelet
disorder
 – when massive blood loss
has occurred

19.  Platelets last for 3-5 days if stored on an agitator at 22°C and
at a pH of between 6.2 and 7.8.
 Each bag has a volume of 250-350ml.(apheresis-PC)
 Platelets should be inspected prior to infusion and packs
should be rejected, or referred for further opinion, if there is
any unexpected appearance such as discolouration.
 Platelets are not usually cross-matched with the recipient, but
where possible ABO specific platelets should be used.

20. ASA GUIDELINESS
 Patients with severe thrombocytopenia (<20,000
cells/mm3) and clinical signs of bleeding usually require
platelet transfusion.
 However, patients may have very low platelet counts (much
less than 20,000 cells/mm3) and not have any clinical
bleeding.
 Patients such as these probably do not need platelet
transfusions.

21.  Individuals who have undergone trauma or require surgery
need higher platelet counts, probably 100,000 cells/mm3,
to maintain adequate hemostasis.
 Laboratory determinations and clinical evaluations must be
taken into account before a decision to transfuse platelets is
made.

22. Fresh Frozen Plasma
 FFP is collected as the supernatant after centrifuging a
donation of whole blood.
 It contains all the plasma proteins, Particularly factors V
and VIII, which gradually decline during the storage of
blood.
 shelf life – 2 years
 It is the first-line therapy in the treatment of
coagulopathic haemorrhage and used in reversal of
warfain therapy

23. Collection and storage of FFP
 It is frozen within 8 hours at -40 °C t0 -50 °C
 Under these conditions, the loss of Factors V and VIII is kept
to a minimum.
 Frozen packs are brittle and should be handled with care.
 The frozen plasma can be thawed using a dry oven (10 mins),
microwave (2-3 mins) or a water bath (20mins).
 Thawed FFP is best used immediately but may be stored at
4°C and infused within 24 hours.

24. Cryoprecipitate
 Cryoprecipitate is a supernatant precipitate of FFP
 rich in factor VIII and fibrinogen.
 It is stored at −30°C with a two year shelf life.
 It is given in low fibrinogen states or factor VIII
deficiency.
 Uses
 Factor VIII deficiency or hemophilia A
 Treatment of fibrinogen deficiencies

25. Transfusion reactions
 Hemolytic Reactions
 Allergic Reactions
 Febrile Reactions
 Bacterial Contamination
 Circulatory Overload
 Hypothermia
 Graft Versus Host Disease (GVHD)
 Transfusion related acute lung injury (TRALI)

26. ARTIFICAL BLOOD

27. Artificial Blood
 Artificial blood or blood surrogate is a substance
used to mimic and fulfil some functions of biological
blood,usually in the oxygen carrying sense.
 Main aim is to provide an alternative to blood
transfusion,which is transferring blood based
products from one person to another.
 It does not contain plasma,RBCs or WBCs.

28.  VOLUME EXPANDERS
1. Crystalloids
2. Colloids
 OXYGEN CARRYING BLOOD BASED PRODUCTS
1. Haemoglobin based oxygen carriers
2. Perflurocarbon oxygen carriers

29. Colloids
 Solutions that contain large molecules that don't pass the
cell membranes.
 5% albumin
 25% albumin
 10% dextran
 6% dextran

30. Crystalloids
 Solutions that contain small molecules that flow easily
across the cell membranes, allowing for transfer from the
bloodstream into the cells and body tissues
 It is subdivided into:
 Isotonic
 Hypotonic
 Hypertonic

31. Isotonic solutions
 Types of isotonic solutions include:
 0.9% sodium chloride (0.9% NaCl)
 lactated Ringer's solution
 5% dextrose in water
 Ringer's solution

32. Hypotonic solutions
• 0.45% sodium chloride (0.45% NaCl), 0.33% sodium chloride,
0.2% sodium chloride, and 2.5% dextrose in water
 Hypotonic fluids are used to treat patients with
conditions causing intracellular dehydration, when fluid
needs to be shifted into the cell , such as:
 1. Hypernatremia
 2. Diabetic ketoacidosis
 3. Hyperosmolar hyperglycemic state.

33. HYPERTONIC SOLUTIONS
 3% sodium chloride (3% NaCl):
 May be prescribed for patients in critical situations of
severe hyponatremia.
 Patients with cerebral edema may benefit from an infusion
of hypertonic sodium chloride
 5% Dextrose with normal saline (D5NS):
 Which replaces sodium, chloride and some calories

35. Why artificial blood?
 Increasing demand
 Decreasing supply
 Safety
 Infectious disease transmission
 Transfusion reactions
 Immunosuppression
 Cost

36. History
 In 1616, when William Harvey first described the circulation
of blood
 In 1665, the first recorded successful blood transfusion on dog
by Richard Lower
 Many materials used for transfusion that include milk, plant
resins, and sheep blood.
 In 1854, patients were injected with milk to treat Asiatic
cholera.

37.  Other materials that were tried during the 1800s include
hemoglobin and animal plasma.(Chang, 2004)
 In 1883,there was a creation of Ringer's solution.
 In research using part of a frog's heart, Sydney Ringer,
found that the heart could be kept beating by applying
the solution.
 (Hoffman et al., 1990)

38.  In 1966, experiments with mice suggested a new type of
blood substitute, perfluorocarbons (PFCs).
 In 1968, the rat's blood replaced with a PFC emulsion and
it lived for a few hours and recovered fully after blood was
replaced. (Sarkar, 2008)

39. Ideal Artificial Blood
 Increased availability that would rival that of donated
blood, even surpass it
 Oxygen carrying capacity, equaling or surpassing that of
biological blood
 Volume expansion
 Universal compatibility: elimination of cross matching
 Pathogen free: elimination of blood contained infections

40.  Minimal side effects
 Survivability over a wider range of storage
temperatures
 Long shelf life
 Cost efficient
 (Squires, 2002)

41. Types of Blood Substitutes
 1) Perfluorocarbons (PFCs), chemical compounds
which can carry and release oxygen
 2) Haemoglobin-based oxygen carriers (HBOCs)
derived from humans, animals, or artificially via
recombinant technology

42. Haemoglobin-based oxygen carriers
 Hboc are prepared from devasted RBC, bacteria,bovine .
 High-level production of recombinant Hb using
simple Escherichia coli expression system has been
reported by Hoffman et al in 1990.
 Human hemoglobin is obtained from donated blood that
has reached its expiration date
 One unit of hemoglobin solution can be produced for
every 2 units of discarded blood

43. Hemoglobin (Hb)
 The structure of Hb was determined in 1959 by Max
Perutz.
 Molecular weight 64.5 kDa
 Tetrameric protein comprised of two α and two ß globin
subunits that fold into compact quaternary structure (α 2
ß 2).
 Each α and ß subunit contain an iron-heme group that
binds to oxygen molecule allowing for transport.

44.  Research on hemoglobin-based fluids dates back to the 1920s
when the stroma of the cells was lysed to obtain hemoglobin
 Problems with free hemoglobin include
 osmotic diuretic effects
 renal toxicity
 coagulation abnormalities
 short half-life
 vasoactive effect

45.  Two types
 Acellular Hboc
 Cellular Hboc

46. Acellular Types

47.  Cross-linked HBOC - intramolecular covalent bonds were
formed between globin chains in order to prevent their
detachment.
 Polymerised HBOC- Hb molecules are cross-linked
intermolecularly to increase the molecular size.
 Conjugated HBOC- Inert polymers are attached to the
surface of Hb molecules.

48. Hemopure
 One of those products is HBOC-201 (Hemopure; Biopure Corporation),
made from bovine blood.
 It is universally compatible and is stable at room temperature for up to
3 years.
 Patients who received Hemopure had an increased number of serious
adverse events. The vasoconstrictive properties of Hemopure may have
caused myocardial infarction in susceptible patients
 Studies have been proposed to coinfuse a nitric oxide donor such as
nitroglycerin in a fixed ratio, in a single bag compound or as separate
infusions(OPK BIOTECH)

49. Oxyglobin
 Approved for veterinary use.
 It consists of chemically stabilized bovin haemoglobin in a
balanced salt solution and contains no red blood cells.
 The cross-linked haemoglobin, several tetramers bound together, works
by circulation in the plasma and supplying oxygen to tissues.
 Drawback:
 risk of transmission of diseases

50. Polyheme
 Outdated human donated blood (pyridoxylated human Hb)
 shelf life of about a year at room temperature
 They reported that patients can be resuscitated with
PolyHeme, without using stored blood, for up to 6 units in
12 hours after injury
 Drawback:
 increased number of myocardial infarctions

51. Conjugated HBOCs
 Inert polymers are attached to the surface of Hb molecules.
 Due to unique characteristics, low toxicity, and lack of
immunogenicity or antigenicity in body, PEG can be the
best polymer for conjugation.
 Hemospan is a PEG-conjugated Hb, which is under clinical
trial as an oxygen carrier. This modification has been
shown to increase the circulation half-life of the product.

52.  MP4 is another PEG–Hb conjugate designed as an oxygen
carrier. This product did not cause vasoconstriction in
animal models, and its efficacy to deliver oxygen to hypoxic
tissues was demonstrated; MP4 is now under human
clinical trials.
 In addition to PEG, other polymers have been used to
conjugate Hb, including benzene tetracarboxylate
dextran,hydroxyethyl starch (called HRC 101), and albumin

53. CELLULAR HBOC’s
 Third-generation hemoglobin substitutes have begun to address
the deficiencies of earlier formulations.
 Hb is encapsulated in a cell-like structure
 The mixing of phospholipids and cholesterol in the presence of
free hemoglobin forms a sphere with hemoglobin in the center.
 Encapsulation of Hb by a phospholipid layer (liposome-
encapsulated Hb [LEH]) prolonged its half-life and shelf-life
comparing to acellular products

54.  These liposomes have oxygen dissociation curves similar to red
cells, and administration can transiently achieve high circulating
levels of hemoglobin and oxygen-carrying capacity.
 Research is still in the preclinical testing stage; progress in
prolonging the half-life and elucidating the effects on the immune
system, particularly reticuloendothelial sequestration, is crucial
before clinical testing can begin.
 Hb vesicle is a PEGylated product with increased serum half-life
and decreased recognition by the immune system

55. Clinical applications
 One of the main problems limiting the application of these
products is their inability to convert Fe3+ to Fe2+, which is
an important function of RBCs
 Met-Hb with low oxygen-carrying capacity was produced,
showing that such complications can be avoided by
attaching reducing agents to Hb surface in this product
series

56. Current status of Hb0c

57. Perfluorocarbons
 Perfluorochemicals (PFCs) are colorless, inert, and
apparently nontoxic liquids with low boiling point
temperatures and are insoluble in water and alcohol
 First demonstration on O2 capacity by Clark in 1966

58.  PFCs have two challenges to overcome for use as blood substitutes.
 The first is that the liquid form is immiscible in water; thus, PFCs must
be suspended as microdroplets with the use of emulsifying agents.
 The second is that unlike hemoglobin, the oxygen that is dissolved in
PFCs has a linear relationship to the partial pressure of oxygen,
whereas hemoglobin has a sigmoidal disassociation curve favoring full
loading at normal atmospheric oxygen levels. Thus, the FIO2 that has
to be applied is too high..

59. Fluosol-DA
 Fluosol-DA was the first accepted PFC-based RBC
substitute, which is an emulsion of perfluorodecaline and
perfluorotripropylamine.
 Oxygen-carrying capacity of Fluosol-DA is only 7.2% at
37°C, which is lower than RBCs
 The use of this product entails complications such as
pulmonary reactions supposedly due to complement
activation by the emulsifying agent in Fluosol-DA and can
be prevented by steroid injection

60. Second generation PFC’s
 Formulated to allow more oxygen-carrying capacities, with alterations
in the emulsion properties.
 Such new compounds can also be stored at 4° C, whereas previous
solutions had to be frozen.
 OxyFlour™ and Oxygent™ are among the second-generation PFC-
based blood substitutes which are rejected by clinical trials due to some
side effects such as complications in determining the effective dose for
OxyFlour™ administration and also increased risk of stroke following
administration of Oxygent™.

61.  However, except some changes in clotting factors, no
specific interaction between blood components and
administered PFCs has been reported.
 Administration of PFC-based products can result in mild
thrombocytopenia (10%–15% reduction in platelet count)
as well as flu-like syndrome

62. Third generation PFCEs
 Perftoran and PHER-O2
 Pulmonary complications has been reported with the use of
Perftoran and, PHER-O2 is in reasearch
 (Modery et al., 2013)

63. Current status PFC

64. Other Promising Technique
 There is a possibility of using stem cells as a means of producing an
alternate source of transfusable blood.
 A study performed by Giarratana et al. (2013) describes a large scale
ex-vivo production of mature human blood cells using hematopoietic
stem cells.
 A team of IIT-Madras scientists from the department of engineering
design has been successful in creating enough red blood cells from stem
cells. (Narayan, 2013)
 To date, the use of red blood cells (RBCs) produced from stem cells in
vitro has not proved for routine transfusion.(Kim, 2014)

65. CONCLUSION
 Blood supply demand are increasing as compared to blood
donations in the world.
 Artificial blood is especially useful in circumstances when donor
RBC units are unavailable or when transfusion of real blood is
not an acceptable option.
 Two distinctly different classes of oxygen carriers are being
developed, each capable of transporting and delivering oxygen to
peripheral tissues.

66.  Most of the initial attempts at synthesizing blood substitutes were not
favorable because of significant adverse effects.
 However, Considering the need, there are several companies still
working on the production of a safe and effective artificial blood
substitute.
 Though, there are many challenges in this aspect, advancing science
and technology may result in development of better blood substitutes in
future for overcoming the need for biological blood transfusions in the
operative and trauma settings.

67. references
 Sabiston textbook of surgery
 Bailey & love short practice of surgery
 Journals