Biochemical changes in stored blood

1. BIOCHEMICAL CHANGES
IN STORED BLOOD
BY
PROF./ MOUSTAFA RIZK
BLOOD TRANSFUSION : FROM BASICS TO ADVANCED
12-13/3/2008

2. A better understanding of the nature
and timing of molecular and
functional changes in stored RBCs
may provide strategies to improve
the balance of benefit and risk of
RBC transfusion .

3. The storage lesion
RBCs may be stored for up to 42 days
under controlled conditions before
transfusion.
However, numerous changes occur
in RBCs during storage (collectively
referred to as the "storage lesion")
that may alter their biological and
biochemical functions .

4. Retrospective cohort
studies
- A correlation between RBC storage
duration and morbidity and mortality rates
after transfusion suggesting progressive
storage lesions may be responsible for
adverse outcomes.
- RBC(STORED)-transfused patients had
worse outcomes than nontransfused patients
matched for clinical variables in several
studies .
Influence of erythrocyte concentrate storage time on postsurgical
morbidity in cardiac surgery patients.Leal-Noval SR, Division of Critical
Care, Department of Thoracic Surgery, Spain. Anesthesiology. 2004
Jan;100(1):193-4; author reply 194

5. Basran, S. et al. Anesth Analg 2006;103:15-20
The rate of in-hospital mortality presented by quartiles of maximum duration of storage of
transfused red blood cells (RBCs)

6. Retrospective cohort
studies
Patients who received older blood had
a significantly longer ICU stay,
possibly reflecting a higher level of
organ dysfunction.

7. Basran, S. et al. Anesth Analg 2006;103:15-20
The rate of acute renal dysfunction (ARD) presented by quartiles of maximum duration of
storage of transfused red blood cells (RBCs)

8. Basran, S. et al. Anesth Analg 2006;103:15-20
Intensive care unit (ICU) and hospital length of stay (LOS) presented by quartiles of
maximum duration of storage of transfused red blood cells (RBCs)

9. Retrospective cohort
studies
Moreover, stored RBC transfusion sometimes failed to
benefit:
1-Pediatric
2-Multiple trauma victims
3-Critical illness
4-Patients undergoing cardiac surgical procedures.
This raise the broader concern that RBC storage is
problematic and could be improved .

10. CASE STUDY
A 35-year-old man weighing 63 kg was presented for
elective repair of an atherosclerotic thoraco-abdominal
aortic aneurysm. He was a known hypertensive
pateint.
Routine biochemistry :
----------------------------
Na+ 135 mmol/l,
K+ 3.6 mmol/l,
Urea 5.1 mmol/l,
Creatinine 56 µmol/l
Haematological investigations were within normal
limits.

11. CASE STUDY
After anaesthetising the patient, the aneurysm was exposed. The
patient developed severe hypotension , which was treated with
rapid transfusion of :
- 1 litre of crystalloids,
- 1 litre of colloids,
- 4 units of warmed CPD-A stored whole blood .
Despite this, the hypotension persisted. This was immediately
followed by bradycardia, and ventricular fibrillation (VF). Internal
cardiac massage was started; the heart was found to be very
flabby.
In view of the VF and a `flabby' heart, hyperkalaemia was
suspected.
Blood transfusion was discontinued, even though the haemoglobin
was 7 g/dl and the hypovolaemia was treated by infusing crystalloid and
colloid solutions only.

12. CASE STUDY
The patient's blood was analysed for serum
potassium, which was 7.0 mmol/l.
The blood used for transfusion was noted to be
16 days old .
The biochemical analysis of the unwarmed bag
blood showed :
PH 6.8
Standard bicarbonate content 7.8 mmol/l,
Potassium 16.6 mmol/l.

13. Potassium levels in transfusion
recipients are determined by:
1-Amount of extracellular potassium
in the blood infused .
2-Pre-existing level of potassium in the
recipient .
3-Rate of transfusion of blood .
4-Renal excretion of potassium .
5-Acidaemia/acidosis .

14. What are the causes of
hyperkalaemia during blood
transfusion?

15. WHY ↑ K
AND ↓ PH
During storage there is a slow, but constant leakage of potassium from
the cells into surrounding plasma as a result of sodium/potassium
ATPase pump failure.
The plasma level of potassium may increase by 0.5-1 mmol/l per day
of refrigerator storage.Accordingly,
the total amount of extracellular potassium in a unit of whole blood
stored for 35 days is about 8.2 mmol.
On collection into a blood bag containing CPD or CPDA-1 solution (pH
5.5), the pH of blood decreases to approximately 7.0.
The pH continues to decline further and may be as low as 6.6 after
21-35 days of storage. This is not only due to citrate in the
anticoagulant, but also due to the metabolic processes in the red
blood cells, resulting in accumulation of fixed acids and CO2.

16. Potassium toxicity need to be
considered during resuscitation of
an acutely injured patient in the
accident & emergency department
or a patient bleeding in the operating
theatre after aortic surgery.
Fresher blood may be beneficial in
preventing these complications.

17. OTHER BIOCHEMICAL
CHANGES
Blood bank storage resulted in :
- ↓ Na+, Cl−, 2,3-DPG and ATP.
- ↑ lactate, pyruvate, ammonia, plasma
HB , intracellular Na , phosphate .

18. 2,3-DPG and ATP
1. During the first 3 weeks of storage the content of
2,3-DPG and ATP decreases.
2. In the presence of adenosine the concentration
of 2,3-DPG is maintained for periods up to 6
weeks at 4°.
3. On reincubation at 37° after storage for 3 weeks
at 4°, red cells lose their ability to utilize glucose.
This can be restored by adenosine; the ability to
utilize glucose appears to be related to levels of
ATP within the cell. Restoration can occur after 6
weeks of storage in the presence of adenosine.
Chemical Changes in Stored Blood, with Observations on the Effects of Adenosine
BY T. A. J. PRANKERD Medical Unit, University College Hospital Medical School, London
(Received 11 January 1956)

19. Bennett-Guerrero, Elliott et al. (2007) Proc. Natl. Acad. Sci. USA 104, 17063-17068
RBC 2,3-DPG (A), potassium (B), pH (C), lactate (D), pO2 (E), Hb O2 saturation (SO2) (F), cell-free Hb in
storage medium (G), and RBC surface phosphatidyl serine (PS) expression (H) as a function of storage
time

20. Effect of storage on RBC function
- One of the RBC's principal functions is
O2 delivery.
- Increases in O2 affinity in stored RBCs,
reflecting progressive decreases in 2,3-
diphosphoglycerate (2,3-DPG) over the
weeks of storage *.
- O2 delivery by stored RBCs is
deficient even early after processing and
before significant decline in 2,3-DPG .
*Roche Diagnostics (Mannheim, Germany) monitoring at 340 nm on a Shimadzu UV spectr.

21. Stored blood may lack vital
component
Much of the stored blood may lack a
component vital for it to deliver oxygen to
the tissues.
Nitric oxide, which helps keep blood vessels
open, begins breaking down as soon as
blood goes into storage, two research
teams report in separate studies to the
week's online edition of Proceedings of
the National Academy of Sciences,10/08/2007
Dr. Jonathan Stamler of Duke University, leader of one of the research groups.
Dr. Timothy McMahon, also at Duke, leader of the second research group

22. .
• It seems that researchers have found
a new reaction carried out by the hemoglobin mole
. Specifically, the researchers have
discovered a method of converting nitrate
salt stored in the blood cell into nitric oxide
by oxidized hemoglobin.
This supports the idea that hemoglobin has
undiscovered functions in the human body.
complexes of nitrite (left) and nitric oxide (right)

23. A
new reaction carried out by the hemog
• Haemoglobin, through a catalytic reaction
that does not change its own chemical
properties, converts nitrite salt to the
vasodilator nitric oxide. The nitric oxide
activity carried by haemoglobin escapes the
red blood cell to regulate blood flow.
• The paradox of how haemoglobin mediates
the conversion of nitrite to nitric oxide in a
way that it is not immediately destroyed in
the red cell and so it can be effective
biologically, now is solved.
Jjournal of Nature Chemical Biology, senior authors Daniel Kim-Shapiro, professor of
physics at Wake Forest, and Mark Gladwin, chief of the Vascular Medicine Branch of the
National Heart, Lung and Blood Institute of the NIH 5/9/2007

24. In the most recent study, the researchers
conclude that the nitrite-haemoglobin
reaction generates dinitrogen trioxide
(N2O3), which takes one of several
pathways from the red blood cell and
later separates into nitric oxide (NO) and
nitrogen dioxide (NO2).
Jjournal of Nature Chemical Biology, senior authors Daniel Kim-Shapiro, professor of
physics at Wake Forest, and Mark Gladwin, chief of the Vascular Medicine Branch of the
National Heart, Lung and Blood Institute of the NIH 5/9/2007

25. The O2 sensor role of hemoglobin
(Hb) subserves this RBC activity by
dispensing vasodilator S-nitrosothiol
(SNO) equivalents in proportion to
the degree of hypoxia in the tissues
it perfuses .

26. Effect of storage on RBC function
- Impairment in RBC-dependent
hypoxic vasodilation might underlie the
functional RBC storage lesion.

27. Effect of storage on RBC function
- However, less was known of how
storage influences the role of the RBC
in the O2-dependent regulation of
blood flow ("hypoxic vasodilation").

28. - Total Hb-bound NO and SNO-Hb
decreased markedly from 0 h (fresh
RBCs) to 3 h.
- Total Hb-NO and SNO-Hb were
similarly depressed in processed
samples and remained markedly
depressed for the 6 weeks of the
study.

29. Bennett-Guerrero, Elliott et al. (2007) Proc. Natl. Acad. Sci. USA 104, 17063-17068
SNO-Hb, related NO adducts, and vasoactivity of stored RBCs

30. CONCLUSION
"Banked blood is truly a national
treasure that needs to be protected ,
It can be life-saving, and in some
cases, it may be making things
worse. In principle, a solution to the
nitric oxide problem - to put it back -
needs to be proven in a clinical trial."

31. Thank YouThank You
PROF. / MOUSTAFA RIZKPROF. / MOUSTAFA RIZK