Hematology 6/03/18

Hematopoiesis

 A physiologic process: production of blood cells from self-renewing and differentiating cells, aka
haematopoietic cells – stem cells. Without self-renewal-> cell pool is dying out-> aplastic anaemia.
Therefore. Self-renewal must always be dominant on diff.
 Yolk sack(Foetal life)->liver&skin->skeleton (long bone in foetal life)-> axial skeleton (adult)
 hematopoietic stem cells(CD34 allows recognition with flow cytometry/immunohistochemistry) ->
committed progenitors (still not identifiable)-> precursors(identifiable; after further diff)->mature cells
- in the bone marrow there are identifiable precursors already
- hematop s.c. are held in an undiff’ state, but it divides into: diff&same

FLT3 gene is important in leukocyte production, it is one of the genes mutated in acute myeloid
leukemia and FLT3-mutant acute myeloid leukemia is one of the most aggressive diseases that can be
cured only by allogeneic stem cell transplantation. Could it be the defected gene that can be cured with
retinoic acid?

 Midollo osseo struttura: progenitori emopoietici, sinusoidi, cellule reticolari, fibroblasti, adipociti,
matrice extracellulare. its histology; ratio b/n adipocytes and hematop cells can be obtained by a biopsy
whereas an aspirate gives the cytology.
 We examine bone marrow aspirates directly and send the bone marrow biopsies to the pathologist for
their examination.
 Hematopoietic stem cells are less than 0.5% of the stem cells. Identified by CD34. Can proliferate and
are self-renewal, and can differentiate to the progenitors. They flow from the BM to the peripheral
blood and are characterised by home: capability of spontaneously arriving to the BM.
 Blood cell production in man per day: RBC->2 x10^11, Neurtrophils-> 7X10^10, Platelets->2x10^11
 Other characteristics of hematop: recirculation b/n b.m -> peripheral blood , Homing- can reach b.m
s.c. niche spontaneously. Renewal(differentiation) capability increases and proliferating decreases
along hemaop.
 BM biopsy includes bone and hematop(aspirate only hematop, easier to asses each marrow cells.
Tissue from illium (posterior iliac spine): gives info on tissue architecture. Bone marrow sample varies in
its composition by age: elderly have 80% adipose cells and 20% Hematop and young people have 80%
heamatop and 20% adipose
 Common myeloid progenitor gives rise to: 1) erythroid precursor 2) megakaryocytes 3) myeloblast that
gives rise to granulocytes and monocytes(->macrophage)
 Common Lymphoid progenitor gives rise to: 1) B and T lymphocytes progenitors 2) NK
 One of the key players in self-renewal and development of haematopoietic cells is stem cell factor
(SCF). Absence of this factor is lethal. There are also other important glycoprotein growth factors,
which can regulate the proliferation and maturation, such as IL-2, IL-3, IL-6, IL-7. Other factors, termed
colony-stimulating factors (CSFs), specifically stimulate the production of committed cells. These
stimulate granulocyte formation and are active on either progenitor cells or end product cells. Three
CSFs are:
 - IL-3
- granulocyte-macrophage CSF (GM-CSF),
- granulocyte CSF (G-CSF): mobilizes the stem cells from the bone marrow to the peripheral blood to
differentiate to granulocytes.
- macrophage CSF (M-CSF). Same but to differentiate to macrophages.
-Erythropoietin is required for a myeloid progenitor cell to become an erythrocyte. On the other hand,
-thrombopoietin makes myeloid progenitor cells differentiate to megakaryocytes (thrombocyte-
forming cells).

 Erythropoiesis: EPO is produced in the kidney by the juxtaglomerular fibroblasts. 2 important

questions arise: 1) is the O2 saturation monitoring done in the arterial or venous blood? answer:
venous. Why?
2) why kidney? A. kidney assumes more blood than it needs. Tissue extraction (of O2 is very minimal
and stable. It’s probably an evolutionary adaptation. So venous blood not affected. Hence, it’s really
sensitive to changes in pO2.
 Erythrocytes: have biconcave shape, which acquires them good surface exchange. NO NUCLEUS. Life
span- 120 days (4 months). Macrophages mainly in spleen (Also in liver and BM) destroy them by
targeting the low membrane elasticity, rendered by lesions.
- Volume 90 Fl; Area 140 micro m^2 . diameter 8 mciron
 For reticulocytes you use different stains to illustrate cell counter. They make out only 0.5-2%. 20-100 x
10^9 per L. when they’re up-> haemolytic anaemia. Down-> might indicate BM problem (i.e.
hypoproliferative anaemia).
How are reticulocytes counted? With cyto fluorometry, by dying the ribosomal RNA with fluorescent
dye (orange).
-steps: erythroblasts (very basophilic because of the ribosomes that are required to synthesise Hb->
reticulocytes (only stays this way 1-2 days. They become acidophilic. -> nucleus is expelled, and we get
Erythrocytes

-Normally in normoxia (normal oxygen content), most of HIF 1, which is a transcription factor, is
physiologically degraded. If oxygen tension decreases (in a condition known as hypoxia), less
transcription factor is degraded, therefore more HIF1 is available to bind erythropoietin gene and
stimulating transcription. This lead to an overproduction of erythropoietin, which circulates in the
peripheral blood and reaches the erythroid marrow
 -What happens in the bone marrow when EPO reaches it? The cell which is most influenced by it, which
means that it has a higher number of EPO receptors, is CFU-E (Colony Forming Unit Erythroid). The
number of these cells is redundant, we have many more than what we need, still due to the selection
of the species. Most of CFU-Es never bind a single molecule of EPO and die of apoptosis. most of CFU-
Es undergo apoptosis and the small number of them that bind EPO instead, starts differentiation and
maturation toward circulating RBC. Therefore, the concept about the expansion of erythropoiesis is
that EPO does not stimulate the erythroblasts but just prevents their apoptosis.
- Renal failure is the typical anemia in which the anemia-causing mechanism is related to reduction of
EPO.
-human recombinant EPO was developed in the late/middle 80s, and since then patients with severe
renal anemia (less than about 10 g/dl) are regularly treated with it. We now have Erythropoietins that
are administered every 2 weeks, therefore it is very easy.
- the abuse of EPO: Mainly by athletes, cyclists, long distance runners. It is really a major problem
because one risk of excessive expansion of erythropoiesis is high chance of thrombosis because high
levels of hematocrits can be dangerous.

 Hb structure: tetrameric protein with 4 globin chains (alpha, beta, gamma, delta, epsilon) and a
prosthetic group protoporphyrin ring with an Fe.
composition Hb of an adult:
-HbA: alpha2Beta2> 97%
-HbA2: alpha2gamma2<3% (2 may be increased in beta thalassemia or in people who are heterozygous
for the beta thalassemia gene.)
-HbF: alpha2gamma2<1% (foetus)
 Granulocitopoiesis: it’s important to identify the cells in the various stages of differentiation because
in some disease there’s an arrest in cancer. Example: acute promyelocytic leukaemia: was found to be
treated with retinoid acid. And saved 95% of pts.
Myeloblasts, promyelocytes-> myelocyte-> metamyelocyte-> band-cell-> segmented granulocyte
- Granulocyte colony-stimulating factor (G-CSF) and granulocyte macrophage colony-stimulating factor
(GM-CSF) are critical to neutrophil differentiation. Identifying with CDs.

-Neutrophils: poli-segmented nucleus (3-5 lobi). Specific granules (80-90%), primary granules (10-20%).
They only exist 6-12 hours in the peripheral blood.
#Granulocyte functions:
-neutrophils: protect the organism from foreign agents (CHEMOTAXIS, PHAGOCYTOSIS, ANTI-
MICROBIAL ACTIVITY).
-Eosinophis-> anti parasites& hypersensitivity
-basophils-> hypersensitivity (histamina)
 Megakaryocytpoiesis:
Promegakaryoblast-> megakaryoblast-> immature megakaryocyte-> mature megakaryocyte-> Platelet
-mature megakaryocyte: euploid 8-64 cr pair N, Diameter 20-50 mm, acidophilic cytoplasm,
multilobulated nucleus.
-platelet: production is 100x10^9 per day. Average life 8-10 days. 1/3 of them are in the spleen.
Destroyed in spleen and liver. Disc shape. Cytoskeleton (?). they have alpha- granules(protein and
coagulation factors) and dense corpus (nucleotides, amine genes)

 complete blood cell count (CBC): esame emocromo citometrico

- Erythrocyte count: males: 4.3 to 5.9 ,females: 3.8-5.2 per 10^12/L. Low RBC count (anemia):
trauma, RBCs destruction (haemolytic anaemia, hemoglobinopathies as sickle cell anaemia,
thalassemia, hereditary spherocytosis, enzyme defects as G6PD deficiency), acute or chronic
 bleeding from GI, nutritional deficiencies (iron, Vitamin B12, folate deficiencies), bone marrow
damage or disorders (leukaemia, MM, myelodysplasia, lymphoma), chronic inflammatory diseases,
kidney failure (decrease in erythropoietin production). High RBC count (polycythaemia):
polycythaemia vera, dehydration, lung disease, congenital heart disease, kidney tumour, smoking,
genetic causes.

- Hb: to diagnose anaemia: lower than 13 in m and 12 in F; and erythocytosis: more than 17 in M and
less than 16 in F g/dl. Never diagnose anaemia based just on RBC count; consider Hb and RBC size.

- Haematocrit (Hct): 39% to 50% for men; 36% to 47% for women. Anaemia: abnormally low
haematocrit (if also low MCV and high RDW: chronic iron-deficient anaemia. Also leukaemia).
Polycythaemia: abnormally high haematocrit. Haematocrit is an important parameter only in
Polycythaemia Vera, in which there is an increase in RBCs and WBCs production in the bone
marrow. A type of therapy called Venesection of blood volume of 400 ml every week or month and
its target is to bring the haematocrit levels (RBC percentage) below 45

- MCV mean cell volume: 80-100 fL. The MCV value is typically increased in all megaloblastic
anaemias, anaemias (macrocytic anaemia) due to B12/folic acid deficiency, while it is typically
decreased in a patient with iron deficiency (microcytic anaemia)

- Mean corpuscular hemoglobin (MCH): 27 to 32 picograms. Calculated by total Hb/ RBC count

- Mean corpuscular hemoglobin concentration (MCHC): 32 to 36 g/dL. MCHC is diminished

("hypochromic") in microcytic anemias, and normal ("normochromic") in macrocytic anemias (due
to larger cell size, though the hemoglobin amount or MCH is high, the concentration remains
normal). MCHC is elevated ("hyperchromic") in hereditary spherocytosis, sickle cell disease and
homozygous hemoglobin C disease.
calculated by dividing Hb by Hct

- Red cell distribution width (RDW or RCDW): 11.5% to 14.5%. It measures variation in RBC size.
When anemia presents with normal RDW, think about Thalassemia. When anaemia presents with
high RDW: Iron Deficiency Anaemia (low MCV), folate and vitamin B12 deficiency anemia (high
MCV), mixed deficiency (Iron + B12 or folate) anemia, recent hemorrhage (normal MCV).

- Reticolocytes: 0.5-2% or 20-10X 10^9/L

- Platelet count: 100-400 10^9/L. Thrombocytopenia, low pc (< 150,000): cancer treatments such as
drugs or chemotherapy, as well as radiation, drugs, autoimmune disorders. Thrombocytosis, high
pc (> 400,000): anemia in which blood cells are destroyed earlier than normal, infections, surgery,
trauma, cancer, medicines, CML, polycythemia vera, primary thrombocythemia, recent spleen
removal

- Mean Platelet Volume (MPV): 7.5 to 11.5 femtoliters. High MPV: destruction of platelets
(inflammatory bowel disease, immune thrombocytopenic purpura (ITP), myeloproliferative
diseases and Bernard-Soulier syndrome. It may also be related to pre-eclampsia, and recovery
from, transient hypoplasia). Low MPV (thrombocytopenia when it is due to impaired production as
in aplastic anemia).
{blood volume in ml is 70x weight in KG}

- WBC (white blood cell) leukocyte count: 4-11 10^9/L: High WBC count: leukocytosis (infections,
inflammation, leukemia, tissue necrosis, allergic responses). Low WBC count: leukopenia (bone
marrow damage, bone marrow disorders such as myelodysplastic syndrome, vitamin B12 or folate
deficiency, lymphoma, autoimmune disorders, sepsis, diseases of the immune system.

- Neutrophils: 40% to 60% of the total. May indicate bacterial or acute vital infection

- Lymphocytes: 20% to 40%. Higher with some viral infections, or chronic lymphocytic leukaemia (CLL). Can
be decreased by HIV infection.

- Monocytes: 2% to 8%. May be raised in bacterial infection, tuberculosis, malaria, monocytic leukaemia.

- Eosinophils: 1% to 4%. Increased in parasitic infections, asthma, or allergic reaction.

- Basophils: 0.5% to 1%. May be increased in bone marrow related conditions (leukaemia or lymphoma)

-Cut off values for transfusions are normally 8 g of hemoglobin for RBC transfusion and 10 000 (?) for
platelets transfusion.

 After RBC destruction, globin is taken to the amino acid pool, iron will be removed and part of it will be
stored in ferritin within the macrophage and the other part will be released to Transferrin protein in
peripheral blood. Transferrin protein also carries iron from macrophages to blood. Iron is released from this
side of the macrophage back to transferrin through Ferroportin protein. Protoporphyrin 9, in the
macrophage is turned into Biliverdin and after than transformed into bilirubin. Bilirubin will be eventually
transported to the liver.