Lecture 1 Introduction to blood diseases.

Cells of blood and hemapoietic organs.

Hematopoiesis and hematopoietic growth factors.
The methods of bone marrow laboratory examination

НижГМА, лечебный факультет,

кафедра госпитальной терапии,
цикл внутренние болезни,
клиническая гематология, 5 курс

К.м.н., доцент Волкова С.А., 2006 г.

 Hematology as the science

Historical aspects - The study of blood has a long history.

Humankind has probably always been interested in blood because it is likely
that even primitive people realized that loss of blood was associated with
death.
In bible references “to shed blood meant to kill”
When and in what manner blood was first examined is unknown.
Microscopic examination of the blood started in the seventeenth century.
The first description of the main part of hematology diseases appeared in the
nineteenth century.
The modern era of hematology started with the 1920s.
In the past 70 years hematology has become a science that uses all the
methods of such diverse scientific discipline as biochemistry, cell biology,
immunology, physical chemistry, molecular biology, genetic and nuclear
medicine.
 Hematology as the science

“…to shed blood meant to kill” Bible

General hematology diseases Oncohematology
 
diseases
Anemia Myeloid neoplasms
•Disorders of Iron Metabolism and Heme Synthesis Lymphoid neoplasms
•Megoloblastic Anemia (Vitamin B12-Deficiency, Folate Deficiency)  
•Hemolytic Anemia including Hereditary Disorders
•Other disorders associated with Anemia including Aplastic Anemia
and Anemia Associated with Renal disease
Disorders of hemostasis and coagulation
•Thrombocytopenia (immunologic and thrombotic Platelet
distraction)
•Inherited Coagulation Disorders
•Thrombosis
Nonmalignant disorders of leukocytes, the spleen and/or
immunoglobins
 Blood is biological fluid and includes:
The cells of blood and the blood plasma
Blood cells
Erythrocytes
or red blood cells (RBC)

• Anucleate, biconcave discoid cells filled

with hemoglobin, size - 7-7,5 micron
• life spans - 90-120 days
• The quantity –
Hb 135-175 g/l RBC 4,6-6,2x1012/l (Male)
Hb 120-160 g/l RCC 3,7-4,7x1012/l (Female)
↓ ↑
reactive
Anemia Erythrocytosis
tumor

The transport the respiratory gases: oxygen and carbon dioxide.

F.J. Schiffman Hematomal pathophysiology.

Blood cells
Leukocytes –White blood cells (WBC)
heterogeneous population of blood nuclear cells
Granulocyte: Neutrophyls (segmented, bands)
eosinphils
basophils
Agranulocytes:
Monocytes
Lymphocytes
• The quantity – 4,0-9,0*109/л
Different WBC count:
b. s. Eos. Bas. Lym. Mon.
1-6% 47-72% 0-4% 0-1% 19-37% 3-11%
60% 40%

Protection from foreign antigens.

 The biological functions of leukocytes
•Granulocyte and Monocytes play key roles in inflammation and
phagocytosis.
•B-lymphocytes confer immunity through production of specific, soluble
antibodies.
•T-lymphocytes direct a large variety of immunity functions, including killing
of cells that bear foreign molecules on their surface membranes.

WBC are the heterogeneity cell population and so in case of the

different quantity changes of WBC and/or WBC different count we
need to calculate the absolutely amount of each kind or more
important kind of leukocytes (neutrophils and lymphocytes)
 The most important quantity pathological
changes of WBC
Leucopenia: WBC < 4,0*10 9/l
Leucocytosis: WBC > 9,0*10 9/l
Neutropenia: neutrophils < 1,5*10 9/l
Agranulocytisis: neutrophils < 0,5*10 9/l
Lymphopenia: lymphocytes < 1,0*10 9/l
Absolutly lymphocytosis: lymphocytes > 5,0*10 9/l
 The most important quantity pathological
Examples
changes of WBC
WBC < 1,0*10 9/l Leucopenia, agranulocytosis,
s. - 5%, lym. – 95%. lymphopenia (aplastic
anemia)
WBC 30,0*10 9 /l,
s. - 15%, lym. – 85%. Leukocytosis due to absolutly
lymphocytosis (CLL)
WBC 100,0*10 9 /l,
blasts – 30% The error
s. - 35%,
lym. – 35%.
Blood cells
Platelets (Trombocytes)
•Very small anuclear cells (parts of
megacariocyte cytoplasm)
containing molecules required for
hemostasis
•The life spans – 8 days
the quantity -150,0 – 450,0*109 /l

Trombocytopenia trombocytosis
I – 150,0 – 100,0*10 9 /l
Tumour
II – 100,0-50,0*10 9/l Reactive
450,0-600,0*10 9/l > 600,0*10 9/l
III – 50,0-0*10 9/l
Platelets provide hemostasis through their abilities to adhere, aggegate
and provide a surface for coagulation reactions. Angiotrophical function.
 Hematopoiesis

•Despite these extreme structural and functional differences among the

cells of the blood, all of the blood cells are the progeny of the
hematopoietic stem cell.
•The joint progenitor of blood cells is the hematopoietic Pluripotential
stem cell.
•The processes involved in production of all the various cells of the blood
from the hematopoietic stem cells are collectively called hematopoiesis.
•Hematopoiesis – is the multistage process of the divisions and
differentiations of the hematopoietic pluripotential stem cell as the result
of which RBC, WBC and platelets come forward in the peripheral blood.

•Hematopoiesis begins early during embryogenesis. And so the

hemopoitic organs can be divisible into the embryonic and the adult.
 The hemapoieitic organs

The embryonic hemopoitic organs The adult hemopoitic organs

The yolk sac (6-10 weeks of The bone marrow - is the exclusive
gestation) site of postnatal hematopoiesis
(myelopoiesis and lymphopoiesis)
under normal circumstances.
The fetal liver and the spleen Lymphoid organs:
(10 weeks – second trimester) Central:
The bone marrow (from third The bone marrow and
trimester) The thymus
Peripheral:
The spleen
Lymph nodes
Peyer patchers in the gut
 The two types of bone marrows:
Yellow marrow (Inactive) composed primarily of fat.
red marrow (active in hematopoiesis)
•In early childhood, the medullary cavities of virtually all bones are active
sites of hematopoiesis. Total marrow space is about 1.6 liters. It is nearly
100% active red marrow.

• During adolescence and adulthood, the site of hematopoiesis gradually

shifts from the long bones of the skeleton to the more central flat bones
(i.e., the skull, vertebrae, ribs, sternum, and pelvis). Total marrow space in
the adult is about 4 liters. About half of this is active.
 The distribution of the active
red bone marrow

Location % of total active marrow

Pelvis 40
Vertebrae 28
Cranium-mandible 13
Ribs 8
Sternum 2
Ends of long bones 8
 Hematopoietic Stem Cells
Hematopoietic stem cells (HSC) make up a unique clone of cells that are capable of
differentiating into the multiple cell lines of hematopoietic system. A certain number
of the uncommitted pluripotential stem cells differentiate or become "committed" and
give rise to the various myeloid and lymphoid cell lines.
HSC are believed to be present in red bone marrow and in all major organs that make
up the reticuloendothelial system, as well as in the peripheral blood.
Stem cell proliferation and differentiation is believed to be under direct influence of:
hematopoietic growth factors that are present in the local milieu of the
reticuloendothelial system,
stromal cells and other cells that make up the unique microenvironment of the bone
marrow.
Most evidence for the existence of these pluripotential HSC comes from in vitro studies
and animal models. Such studies have shown regenerative capabilities of the
marrow and hematopoietic system after infusion of certain populations of
mononuclear cells following complete hematopoietic ablation.
The pluripotential HSC can be identified by the monoclonal antibodies CD34.
Morphologically, they are believed to be similar to large immature lymphocytes and to
be diffusely distributed throughout the marrow.
 The uncommitted
pluripotential HSC

The "committed" myeloid stem The "committed"

cell lymphoid stem cell.

Specific myeloid cell lines : Specific lymphoid cell lines:

Erythropoiesis B-lymphopoesis
Granulopoiesis T-lymphopoesis
Monopoiesis
Thrombopoiesis
 The essential differential stages of hemopoietic cells
into each line of hematopoiesis:

Blast – the morphologically identified precursor of the hematopoietic growth

(myeloblast, erythroblast, monoblast, vtgacarioblast, lymphoblast).
Pro- cytes – the maturated cell – the cell in the process of differentiation
(promyelocyte, promonocyte)
-cytes – the mature cell – the terminal stage of differentiation (the erythrocyte).
Granulocytopoiesis
 Erythropoiesis
Ebl

PrNbl

BasNbl

PolyNbl
During the erythropoiesis, approximately 10% to 15% of the
OxyNbl erythroid precursors never completely mature and are
destroyed in the bone marrow. If this % more then 15%, it is
Ret ineffective erythropoiesis.

RBC
 Thrombopoiesis
MK-blast

Megakaryocytes are specialized bone marrow cells that differentiate

from the myeloid stem cell and are responsible for the production of
platelets. Platelets are actually cytoplasmic fragments and as such
ProMK-cyte are not complete cellular elements. They are shed from the mature
megakaryocytes.

MK-cyte

Trombocytes
Lymphocytes are derived from
committed stem cells that originate
from the pluripotent hematopoietic
stem cell.

Once committed early lymphoid

cell further differentiates and gives
two major classes of lymphocytes:
B-lymphocytes and T-
lymphocytes.

Both classes of lymphocytes appear

morphologically similar when viewed
through the light microscope.
 The methods of blood and bone marrow laboratory examination:
•Cell Counts and morphological analysis of blood cells and bone
marrow cells
•Immunodiagnosis:
radioimmunoassay (RIA)
enzyme-linked immunoassay (ELISA)
fluorescent antibody techniques, including flow cytometry.
•Cytogenetics - microscopic analysis of chromosomes.
•Molecular Genetics and Hematology
Southern Blot analysis
Northern Blot analysis
In situ hybridization (FISH)
Polymerase Chain Reaction (PCR)
 Morphological methods: the blood analyses

Cell counts may be determined either manually or by

automated hematology analyzers
Manual counts are carried out after appropriate dilution
of the sample in the hemocytometer or the chamber.
The hemocytometer is a spatially contracted counting
chamber that contains a specific volume. Cells may
then be counted with microscope.
Automated methods for cell counts use two main types
of technology:
– Depend on changes in impedance in electric flow
– use differences in light scatter properties
Morphological methods: the blood analyses
 The components of the
needle for bone marrow
The needle aspiration

The needle core

 The instruments for bone marrow
aspiration

The hypo

The bone marrow

needle

When the needle in the same assy we can make

bony puncture as a rule the sternum puncture.
 In the same assy we can aspirate some of the
jelly-like marrow substance.

We get out the jelly-like

marrow substance on Petri dish
Manual counts are carried out after appropriate dilution of
the sample in the chamber – we get the date about aspirate
cellulary according the absolutely quantity myelocariocytes
and megacariocytes

The jelly-like marrow substance is smeared onto the glass

slides for myelocariocyte different count
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 Morphological methods: the bone marrow analyses
Index number of bone marrow aspirate

The normal absolute amount 50,0-150,0 x 10 9/l

nuclear contended cells of bone
Morrow (myelocariocytes)
Different myelocariocyte count according to cell lines
Myeloid cells (granulocytopoiesis) 60-70%
Erythroid cells (erythrocytopoiesis) 20%
Lymphocytes (lymphocytopoiesis) 10-15%
Plasma cells (lymphocytopoiesis) 2%
Megas, Monos, fibroblasts, etc 1%
Myeloid:Erythroid ratios 3:1
The universalize needle for bone
marrow aspiration and biopsy
 Morphological methods: the bone marrow analyses
Bone marrow biopsy
The substrate for analysis is the bone column which is processed by
histological processing. After that we can estimate the tour tussue
types in histological specimens of bone marrow. They are
• hematopoietic,
• connective,
• bony
• adipose tissues

The bone marrow biopsy is the more accurate for determining the
cellularity and sometimes for processes involving the bone.
 3

The biopsy evaluates cellular production and is a function of age. A

normocellular marrow for a child under 2 years is virtually 100% red
marrow. A normocellular marrow for a mature adult is 40-50% fat,
60-40% hematopoietic elements.
 Immunodiagnosis
All immunologic assays are based on either the primary binding
of antibody to antigen or the secondary phenomena resulting from such
interaction.
Some assays are designed to detect the presence of antibody
against known antigens, anti-immunoglobulin, anticomplement, antiviral
antibodies.
Methods than depend on the primary binding of antibody to
antigen include:

•radioimmunoassay (RIA)
•enzyme-linked immunoassay (ELISA)
•fluorescent antibody techniques, including flow cytometry.
 Immunodiagnosis
Fluorescent antibody techniques is the main for
immunophenotyping analysis cells of blood and bone marrow with
monoclonal antibody named as Cluster Differentiation (CD) Antigens.
Cluster Differentiation (CD) Antigens or surface marker
molecules are cell-membrane proteins (glycoproteins) or receptors
(antigens) that allow identification of multiple cell lines at different
stages of maturation.
Cluster differentiation molecules are present not only on
lymphocytes and other hematopoietic cells but on virtually all cells.
Their presence and identification on malignant cells frequently
help to provide the necessary information to confirm malignancy of the
clone and to determine from what tissue or cell line the malignant clone
is derived.
Cytogenetics – microscopic analysis of
chromosomes.
FISH-method
Thank you for attention