RBCs RBCs - - Ageing in Health, Storage, Ageing in Health, Storage, Hypoxia Hypoxia - - Anemias Anemias (Thalassemia, (Thalassemia, Fanconi Fanconi ’ ’ s s , Renal), , Renal), High Altitude, High Altitude, Astronauts Astronauts - - neocytolysis neocytolysis , CD34 , CD34 + + Stem Cell Gene Therapy Research Initiatives* Stem Cell Gene Therapy Research Initiatives* Impington, Cambridge CB24 9LZ, UK Dr J N Mehrishi, PhD (Cambridge), FRCPath. Director, Initiator, Co-ordinator [email protected]. Tel +44-1223-57 36 28. skype <jaycantab> Data discussed: International Teams work Data discussed: International Teams work - - 1948 onwards 1948 onwards *(Independent of and separate from the University of Cambridge)
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RBCsRBCs--Ageing in Health, Storage, Ageing in Health, Storage, HypoxiaHypoxia--
Data discussed: International Teams workData discussed: International Teams work-- 1948 onwards1948 onwards
*(Independent of and separate from the University of Cambridge)
Hippocratic oath: Hippocratic oath: PremumPremum non non nocerenocere: do no harm : do no harm
Motto Motto -- University of Cambridge University of Cambridge Hinc lucem et pocula sacraTranslation: Here [we receive] light and sacred draughts;
"here" refers to - are metaphors for knowledge.
PhD degree awarded with the Trinitarian formula-...in nomine Patris et Filii et Spiritus Sancti-
in the name of the Father and of the Son and of the Holy Spirit.
Royal College of PathologistsRoyal College of Pathologists-- Sedes invenire et causas morborum –‘to seek the sites and causes of disease’ and help correct
them.I take these seriously!
Critical life and death decisions to assess
ANAEMIA -
shortage of circulating red blood cells (RBCs)
Containing Oxygen for delivery bound to
Haemoglobin (Hb) <11g/dL> INDIERCT.
RBCs at various stage of ageing, deterioration – in health and disease
(thalassaemia, sickle cell disease) - very heterogeneous
- Noninvasive and continuous Hb measurement with aspectrophotometric sensor (Rainbow DCI) with multiple wavelengths of light utilisation:
Masimo Pulse CO-Oximetry method shows the distinctive light-absorption characteristics of different haemoglobinspecies - proprietary algorithms to determine Hb levels spectrophotometry of
Would you decide the beauty of the wife from the photographs of some of these husbands?
Well- clinics use INDIERCT basis of levels of hemoglobin, Hb, (supplier of oxygen) : <11g/dLfor assessing anaemia (shortage of good red blood cells containing Hb for critical decisions (?life and death?)
Trying to decide the beauty of these women from the photographs of the husbands-Hb level Indirect – conclusion RBCs in anaemia imprecise!
contamination: serious membrane perturbationVideo clip : https://www.youtube.com/watch?v=_gbBX_cZrFcMehrishi JN. 2013. TRANSFUSION.;53:2667-2674. Current and historical perspectives on methodological flaws in processing umbilical cord blood. (Italian and French versions available)
For genetic engineering CD34+ cells obtained by a seriously flawed method: Miltenyi particles-black internalised and trapped:Isosurface imaging, TEM Confocal laserMicroscopy
Aanaemias:HbAanaemias:Hb (<11g/(<11g/dLdL--) old established INDIRECT) old established INDIRECT--complacent? complacent?
relying on heterogeneous RBCs: Suggest present day new knowledgrelying on heterogeneous RBCs: Suggest present day new knowledge e
directly using numbers/proportions of directly using numbers/proportions of ‘‘goodgood--functional RBCs.functional RBCs.
ANAEMIAS:ANAEMIAS:
Shortage of RBCs Shortage of RBCs
((HbHb) oxygen, blood ) oxygen, blood
lossloss--altitudealtitude--Trigger Trigger
EPO EPO ProducitonProduciton to to
BM. BM. Aanaemias:HbAanaemias:Hb
(<11g/(<11g/dLdL--) )
INDIRECT, INDIRECT,
Double check direct Double check direct
by by ‘‘good RBCsgood RBCs’’ ??
Left:Prchal JT. Clinical manifestations and classification of erythrocyte
1144 disorders. McGraw-Hill -Williams Hematology, 8e 2010, 1145
Eds Marshall A. Lichtman et al. With permission.
RIGHT: Data kindly provided by Prof. Ferruh Artunc – Tübingen
Chronic kidney disease is a silent devastating Chronic kidney disease is a silent devastating
diseasedisease-- linked to many other disease states linked to many other disease states ––
usually discovered when patient tested for usually discovered when patient tested for
something else.something else.
AnaemiaAnaemia AFFECTS ~10% of the world AFFECTS ~10% of the world
population.population.
Renal Renal anaemiaanaemia-- treatment by EPO (expensive treatment by EPO (expensive
Basis of Direct numbers of Basis of Direct numbers of ‘‘goodgood’’
RBCs after rapid, inexpensive isolated RBCs after rapid, inexpensive isolated
populations is precise populations is precise -- preferable preferable
Genetic Disease,Blood Disorders, Immune Enzyme deficiencies, DNA damage- ‘homing’defect, Hypoxia - Regulation of Oxygen levels tightly controlled by Red Blood Cells (RBCs) via
hemoglobin, Hb - Fe++ // Fe+++ flip flop-pick up – transport and deliver to tissues
Hypoxia sensed in the kidneys –
Triggers erythropoietin Production for RBCs
RBC Life span 120d: eliminated, recycled
Defective - (i) Renal anaemia: ~80 days
RBC survival (ii) Thalassemia : ~55 days
Thalassemia, Genetic Disease,Blood Disorders, Immune Enzyme deficiencies, DNA damage- ‘homing’ defect, Hypoxia - Regulation of Oxygen levels tightly controlled by Red Blood Cells (RBCs) via
hemoglobin, Hb - Fe++ // Fe+++ flip flop-pick up – transport and deliver to tissues
Immune Deficiencies- gene mutation -X chromosome that encodes a component (or chain) shared by the T-cell growth factor receptor and other growth factor receptors.
This component is referred to as γcSCID-X1 and SCID related to adenosine deaminase defect. Enzyme deficiencies.
Fanconi’s anaemia- DNA damage- ‘homing’ defect.
Take patient’s cells or stem CD34+ cells- modify: with-without using electroporation to introduce large molecules of DNA-retroviruses or CRISPRCas9 or the newer CRISPRPf1:
is not a true estimate – Rethinking wiser for precision – Quantify Hb directly from
RBCs various ages isolated easily-30¢/sample
More reliable estimates of Hb needed
Correlated with the Y-RBC subset-obtained by PRERCOLL isolation
RBCs High negative charge keeps awayThe highly negatively charged macrophages
When RBCs–ve charge decreased ~25-30%
the resident macrophages
get closer to RBCs - position themselves
- to grab damaged/old RBCs
- for elimination by the RES.
RBC:120d life span:with ageing-biomaterials, water are lost
volume (v) decreases � �density (d) increases (m ÷ v = d) Sialic acid (‘homing’ mol) charge decrease; ageing, storage lesion, thalassemiaCells-PERCOLL fractionated –lightest density –young- Y-RBCs densest density –old- O-RBCs. Hb lost as vesicles- more from old, aged RBCs, 20% Hb lost, particularly in the last half of its life (in storage).
Passage of a red blood cell through a single-pore Photo-mounting of two independent scanning electron micrographs.
Capillary with Red Blood Cell (TEM x32,830). This image is copyright Dennis Kunkel at www.Dennis Kunkel.com, used with permission.http://www2.estrellamountain.edu/faculty/farabee/BIOBK/biobookcircsys.html
Blood elements are notoriously heterogeneous – as are the cord blood cells.The lightest: Young RBCsSeparated By PERCOLL discontinuous Density centrifugation of RBCs :Over various PERCOLL-buffer solutions To obtain The Densest- Old RBCs that bear decreased sialic acid related charge by 25-30%. ζ- potential: lower by: 7mV.
RBC monitoring- burden of stroke- substantially higher
younger age - higher fatality cf . others
Changing life style - from hunter gatherers- diet-
Shortage of – folic acid, vitamin B complex, iron-
Incidence of Anaemia is high, risks of diabtes II
Great need-increased focus on monitoring AnaemiaAnaemia
reducing
Aboriginal stroke incidence and improved outcomes
Strongly recommend – establish blood RBC subsets,
markers – sound rational bases for management.
AnaemiaAnaemia: : Aboriginal-Torres
Strait Islander populations
Proposals for blood cell studies consistent with current national policy and program initiatives including the National Strategic Framework for Aboriginal and Torres Strait Islander Health.
Renal anaemia: CKD: RBC life span ~80d
Patients: before, during and after~4 weeks
treatment with
rHuEPO v BAY 87-2243, FG4516 FG-
4592, AKB-6548, GSK1278863
Blood fractionated (count numbers and proportions of
Y-RBC::O-RBC)
Flow cytometry :cell subsets labelled with specific
Ligands for surface molecules- changes correlate with
Hb, MCV, MCHCSNA-FITC, CD47? CD55?
• new strategies to treat anemia are
still an evolving and fascinating area of experimental and clinical research. At present, the most promising class of agents seems to be HIF stabilizers, as evident in the number of molecules currently under development.
• This class of drug stimulates erythropoiesis by physiologic concentrations of endogenous EPO,whichmay translate into a clinical advantage because concerns for ESA safety are higher at the high doses.
• However, these theoretical advantages will need to be demonstrated clinically in large trials.
• Conversely, the class needs to be proved safe in light of the potential risks for increases in levels of VEGF and related factors and the possibility of widespread stimulation of complex pathways leading to unexpected side effects.
• The final judgment of benefit/risks of these agents will be possible only after the completion of large long term safety studies testing hard end points.
Discovery of sialic acid-Neural cell
sphingolipids-Klenk-Tay-Sachs, surface
Stroma,
Gunnar Blix 1894-1981discovery of sialic acids.
Ernst Klenk 1896-1971
Uhlenbruck, Faillard et coll.
UhlenbruckHouse- CologneDiscussion-Mehrishi- 1970Cat RBC structureUnique- deeper cleftsGlycolipid carboxylsExposed by pronase
Pilot Study Proposal by Mehrishi based on extensive
CKD Patients: males femalesBefore, during at end of 4-12 wksTreatment with (i)rHuEPO (ii)BAY, FG4592-Hb etc.
Microsamples of Blood- (CPDA-1)0.1-1.0 ml Monitoring test-
Quantitative, rapid, inexpensive ($1/sample)
CKD Patients: Renal Anaemia
males females
Before, during at end of 4-12 wks
Treatment with EPO and
the related agents
Versus
Pyrolyl Hydroxylase inhibitors
• new strategies to treat anemia are
still an evolving and fascinating area of experimental and clinical research. At present, the most promising class of agents seems to be HIF stabilizers, as evident in the number of molecules currently under development.
• HIF- PHD2 inhibitors- This class of drug stimulates erythropoiesis by physiologic concentrations of endogenous EPO,whichmay translate into a clinical advantage because concerns for ESA safety are higher at the high doses.
• However, these theoretical advantages will need to be demonstrated clinically in large trials.
• Conversely, the class needs to be proved safe in light of the potential risks for increases in levels of VEGF and related factors and the possibility of widespread stimulation of complex pathways leading to unexpected side effects.
• The final judgment of benefit/risks of these agents will be possible only after the completion of large long term safety studies testing hard end points.
Hemoglobin (Hb)Structure of human hemoglobin. The proteins 2 αααα and 2 β
subunits are in red and blue, and the iron-containing heme groups in green.
Hemoglobin (HbA) Sickle cell Hb-glu substituted by val HbS
heme group
Sickle cell anaemia
Sickled and other red blood cells
Hb- sickle cell disease- Pauling
Linus Pauling during a conversation with the
protein chemist William Castle during an
Over night train journey between Denver and
Chicago discussed sickled red cell shape-
structure changes.
Pauling guessed that this might reflect a
structural difference between normal
(HbA) and sickle-cell haemoglobin
(HbS) which could be detected by the
pH- electrophoretic mobility
relationships:
Found: isoelectric points – revealing!
suggesting diff
HbA 6.87 (-ve ion) compositions
HbS 7.09 (+ve ion)
Sickle Cell Disease (HbS) – Itano/Pauling: Electrical approach indicated protein AA Changes
Hemoglobin preparations from Patients with sickle cell disease compared
with normal and with a trait: pH- electrophoretic mobility plots Isoelectric points: HbA: 6.87 (-ve ion) HbS: 7.09 (+ve ion)from Harvey Itano's dissertation. 1949.
Role of the electric charge
• Importance- keep separate
• When decreased- eliminated
• Isolation of cells
• Cord blood stem cells
86
Huang, Wu & Mehrishi+ et al. Human red blood cell aging: correlative changes in surface charge and cell properties. J. Cell Mol. Med. 15 (2011) 2634–2642
RBCs 120d life span heterogeneity RBCs 120d life span heterogeneity
2x 102x 106 6 /sec renewal/sec renewal--
revealed by revealed by electropherogramelectropherogram
Marina Marina KamenevaKameneva et al. et al. Cambridge: SeamanCambridge: Seaman-- 1963 1963
PittsburghPittsburgh MehrishiMehrishi--1966 1966
Huang, Wu (Guangzhou), Huang, Wu (Guangzhou), MehrishiMehrishi (Cambridge) et al. 2011(Cambridge) et al. 2011
RBC age distribution in male and female bloodRBC age distribution in male and female blood
0
10
20
30
40
50
60
Per
cen
t
Young RBCs Middle age RBCs Old RBCs
MalesMales FemalesFemales
The picture is based on data published by The picture is based on data published by MicheliMicheli et al. , 1984et al. , 1984
RBCRBC
DeformabilityDeformability
RBCRBCSedimentationSedimentation
RateRate
Low Low
ShearShear
ViscosityViscosity
RBCRBC
MechanicalMechanical
FragilityFragility
p<0.05
p<0.001
p<0.05
p<0.001
Dif
fere
nce
(%
)
Mechanical properties of old and young red blood cellsMechanical properties of old and young red blood cells
0
20
40
60
80
100
120
140
160
180
200
"Young" RBCs "Old" RBCs
0
5
10
15
20
25
30
35
40
45
50
Hem
ato
crit
, %
Difference in male (red) and female (green) hemorheological paraDifference in male (red) and female (green) hemorheological parametersmeters
p<0.001
50
60
70
80
90
100
110
RB
C d
efo
rma
bil
ity
, %
p<0.001
0
10
20
30
40
50
60
70
80
90
100
RB
C a
gg
reg
ati
on
, d
iffe
ren
ce (
%)
p<0.005
0
2
4
6
8
10
12
14
ES
R (
mm
/hr
)
p<0.01
Oxygen Delivery Index (hematocrit/blood viscosity )Oxygen Delivery Index (hematocrit/blood viscosity )
5
6
7
8
9
10
Male blood
Female blood
p<0.001
Oxy
gen
Del
iver
y I
nd
ex
0
5
10
15
20
25
30
35
40
45
50
Hem
ato
crit
, %
Difference in male (red) and female (green) hemorheological paraDifference in male (red) and female (green) hemorheological parametersmeters
p<0.001
50
60
70
80
90
100
110
RB
C d
efo
rma
bil
ity
, %
p<0.001
0
10
20
30
40
50
60
70
80
90
100
RB
C a
gg
reg
ati
on
, d
iffe
ren
ce (
%)
p<0.005
0
2
4
6
8
10
12
14
ES
R (
mm
/hr
)
p<0.01
Oxygen Delivery Index (hematocrit/blood viscosity )Oxygen Delivery Index (hematocrit/blood viscosity )
5
6
7
8
9
10
Male blood
Female blood
p<0.001
Oxy
gen
Del
iver
y I
nd
ex
·
The mechanical properties of blood of premenopausal women are superior to men, and place them at lesser risk for cardiovascular diseases than men in any age group.
·Male blood possesses an increased viscosity, RBC aggregability and RBC rigidity. ·RBCs of males were found to have mechanical fragility higher than for womens RBCs.
• Old RBCs (reproductive age)
< 50% Old RBCs males
• women of reproductive age have almost half as many old RBCs than in men -and almost x2 as many young RBCs than in men,
• young RBCs females (reproductive age)
x2 young RBCs males
• Old RBCs-
• increased mechanical fragility and aggregability, decreased deformability as compared to young RBCs.
• Decreased deformability and increased aggregabiJity of RBCs cause an increase in blood viscosity
• known as risk factors of cardiovascular diseases
•
• men possess a higher number of old RBCs with suboptimum mechanical properties than premenopausal women,
• who due to monthly blood loss
• (sort of a blood transfusion?)
• have a higher number of young cells and a lower number of old RBCs than their male counterparts
• -results of Kameneva et coll. suggest that an elevated hemorheological risk for males is associated with the age distribution of RBCs.
• This, in addition to significantly higher hematocrit, may be the reason for the increased risk of morbidity and mortality from cardiovascular diseases of men as compared to women of reproductive age.
Summary (Summary (KamenevaKameneva))•• Male blood has higher viscosity due to higher Male blood has higher viscosity due to higher
hematocrit and RBC aggregation and lower RBC hematocrit and RBC aggregation and lower RBC deformability deformability
•• Men possess a higher number of old RBCs and a Men possess a higher number of old RBCs and a
fewer number of young RBCs than premenopausal fewer number of young RBCs than premenopausal womenwomen
•• Old RBCs demonstrate an increased ability to Old RBCs demonstrate an increased ability to
aggregate and decreased deformability as aggregate and decreased deformability as compared to young RBCscompared to young RBCs
•• Oxygen delivery index is significantly lower Oxygen delivery index is significantly lower (p<0.001) for male blood than for blood of (p<0.001) for male blood than for blood of
•• prepre--menopausal femalesmenopausal females
•• Increased blood viscosity and Increased blood viscosity and aggregabilityaggregability of of RBCs and decreased deformability of RBCs are RBCs and decreased deformability of RBCs are known risk factors of cardiovascular diseases.known risk factors of cardiovascular diseases.
•• The difference in the mechanical properties of The difference in the mechanical properties of male and female blood places men at higher risk male and female blood places men at higher risk of cardiovascular diseases than preof cardiovascular diseases than pre--menopausal menopausal womenwomen
•• Blood donation or regular, small phlebotomy Blood donation or regular, small phlebotomy might help to improve rheological properties of might help to improve rheological properties of blood and reduce the risk of cardiovascular blood and reduce the risk of cardiovascular diseases in men and postdiseases in men and post--menopausal menopausal women!!!?women!!!?
Summary (cont.)Summary (cont.)
Thalassemia
• Thalassemia (is a form of inherited autosomal recessive blood disordercharacterized by abnormal formation of hemoglobin. The abnormal hemoglobin formed results in improper oxygen transportand destruction of red blood cells. Thalassemia is caused by variant or missing genes that affect how the body makes hemoglobin, the protein in red blood cells that carries oxygen
Thalassemia- Hb- genetic disorder-
Liverpool- Army- Singapore- Gurkha girl- anaemia suspected not responding. With foresight and enterprising, Weatherall using car batteries hooked up a high electrophoresis set up to diagnose thalassemia- a genetic disorder- not enough Hb made or defective-apparently had not asked for permisison and almost got court martialled. Affects large populations- a health and a financial burden. RBC lives for 120 days but 55d in thalassemia- cell surface charge is decreased- quantified by the machine- such as the CAM-APPARATUS or similar Zeiss.
Thalassemia:Monitoring blood test (0.1 to 1ml)
• Decreased expression of CD47 and CD55
• surface molecules on density-based subsets of red cells of β-thalassaemiaintermedia patients compared to red cells of healthy blood donors
• Angela Risso, Elena Mansutti & Mehrishi
• (June 2015- JBCP)
β-thalassaemia intermediapatients density based- red
cells- high countρn = 1.078 g/cm3 versus ρ > 1.1092 g/cm3)
and very dense RBCs (ρ ≥ 1.12 g/cm3).
Cf sickle cell disease: ρ > 1.12 g/cm3
Thalassemia 1:Lightest. 2: Middle 3: Densest
β-thalassaemia intermedia patients
% RBCn vs RBC Thal
Density Healthy S1-A S1-B S2-A S2-B
Low 6.85±2.17 0.05 1.3 1.4 2.5
Middle 73.58±6.06 0.5 40 37.7 54.2
High 19.57±7.84 0.45 57.9 47.5 43
Very high 0.005±0.001 99 1.78 13.9 3.75
• RBCs (Y-RBC numbers::O-RBCs) of patients with renal anaemia treated with rHuRPO versus one of the 3 new proteins-pyrolyl hydroxylase inhibitors.
• blood fractionated (numbers and proportions of Y-RBC::O-RBC) and flow cytometry of cell subsets labelled with SNA-FITC, CD47? CD55?
• This will then be correlated with the Hb, MCV, MCHC and the clinical state in renal anaemia.
• FG-4592 stabilizes the activities of HIF, a cytosolic transcription factor, leading to activation of the genes associated with erythropoiesis, including erythropoietin and enzymes involved in iron metabolism
• New strategies to treat anemia are
still an evolving and fascinating area of experimental and clinical research. At present, the most promising class of agents seems to be HIF stabilizers, as evident in the number of molecules currently under development.
• HIF- PHD2 inhibitors- This class of drug stimulates erythropoiesis by physiologic concentrations of endogenous EPO,whichmay translate into a clinical advantage because concerns for ESA safety are higher at the high doses.
• However, these theoretical advantages will need to be demonstrated clinically in large trials.
• Conversely, it needs to be proved safe in light of the potential risks for increases in levels of VEGF and related factors and the possibility of widespread stimulation of complex pathways leading to unexpected side effects.
• The final judgment of benefit/risks of these agents will be possible only after the completion of large long term safety studies testing hard end points.