HEMATOPOIESIS AND Hb estimation FINAL.ppt

Rbc

1. HEMATOPOIESIS
PRESENTED BY: DR PRANJALI DWIVEDI
FACULTY MODERATOR: PROF(DR) SHACHI AHALAWAT

2.  The process of formation of blood cells { RBC’S , WBC’S and platelets} , sites where
hematopoiesis occurs are known as hematopoietic tissues or organs [ bone marrow,
liver,spleen].
 Cells responsible for the function of hematopoiesis are first seen in yolk sac of embryo in
third week of embryonic life known as hematopoietic stem cells.
 WHAT ARE STEM CELLS ?
Cells capable of asymmetrically dividing , located in bone marrow
Has self renewing properties
HSC has ability to differentiate into any blood cell lines [ pluripotency]
The bone marrow and spleen forms a supporting system called “ Hematopoietic
microenvironment”.

3. Hematopoietic stem cells also take origin in mesoderm of aorta,
mesoderm of gonads and mesoderm of meso – nephrons.
HSC present in yolk sac migrate to other parts like liver (known as
dominant migration) , spleen and bone marrow.
Some hematopoietic stem cells also migrate to lymph nodes , spleen
known as minor migration.
Liver, lymph nodes and spleen continue as hematopoietic organs until
birth.

4. After birth hematopoietic activity takes place in bone
marrow.
Active bone marrow is called RED BONE MARROW
{VASCULAR} and inactive bone marrow is called as YELLOW
BONE MARROW {accumulated with fat cells}.
In newborn , if hematopoiesis is happening outside of bone
marrow it is called as “ EXTRA MEDULLARY
HEMATOPOIESIS”.
At puberty , hematopoeisis is limited to axial skeleton.

5. PROCESS OF HEMATOPOESIS:

7. Pleuripotent stem cells divides into two different types of multipotent stem cells
Multipotent stem cells are cells that have a capacity to self renew by dividing and to
develop into multiple specialized cell types present in a specific tissue or organ.
STEM CELLS TYPES:
PLURIPOTENT STEM CELLS :
• 18-23 micron
Giving rise to both: myeloid and lymphoid series of cells
 Self renewal.
MYELOID STEM CELLS:
Erythrocytes
Granulocytes : PMNs , Eosinophils and Basophils.
Thrombocytes.
LYMPHOID STEM CELLS:
Giving rise only to : Lymphocytes : T type mainly.

8. HAEMATOPOIETIC GROWTH FACTOR
Regulate proliferation, differentiation and maturation of hematopoietic proginator cells.
Influence the commitment of progenitors to specific lineages
Affect the function and survival of mature blood cells.on target cells.
HGFs may bind to specific cell receptors on the surface of the cells to directly induce their proliferation
and differentiation or may stimulate the production of other cytokines that then act on the target cells.

9. RED BLOOD CELLS
STAGES OF ERYTHROPOIESIS
PROERYTHROBLAST: largest (12-20 microns) with Basophilic cytoplasm, HAS A NUCLEUS WITH
STIPPLED CHROMATIN AND 1-2 NUCLEOLI.
EARLY ERYTHROBLAST:chromatin condenses, nucleoli lost, light basophilic cytoplasm 12-16
microns.
INTERMEDIATE ERYTHROBLAST: reddish tinge in cytoplasm as Hb production starts.size is 10-
12 microns and nucleus is condensed.

10. 
LATE ERYTHROBLAST: nucleus is densley pyknotic with dense Hb containing cytoplasm.

RETICULOCYTE: as reticulocytes mature into red blood cells , their RNA keeps on decreasing.
Peripheral blood fim shows polychromatic red cells. HIGHEST RETICULOCYTE COUNT IN HEMOLYTIC
ANEMIAS AND FOLLOWING THERAPY IN IRON FOLIC ACID/B12 DEFICIENCIES. REDUCED IN APLASTIC ANEMIA.

11. MATURE RBC: life span of 100-120 days, anucleate,inner portion less thick
than outer, central pallor ,major role to carry O2.
RED CELL MEMBRANE:The membrane provides mechanical strength and flexibility
to the red cell to withstand the shearing forces in circulation.
Red cell destruction: The life span of normal erythrocytes is about 120 days.
The senile red cells are

12. WHITE BLOOD CELLS
Neutrophils
Stages of Granulopoiesis :
Myeloblast: earliest recognizable cell in the granulocytic
maturation process. It is about 15 to 20 m in diameter, with a
large round to oval nucleus With basophilic cytoplasm.nucleus
has 2-5 nucleoli and nuclear chromatin is fine and reticular.

13. Promyelocyte: is slightly larger in size than
myeloblast. Primary or azurophil granules appear at
the promyelocyte stage. The nucleus contains
nucleoli as in myeloblast stage, but nuclear
chromatin shows slight condensation.

14. Myelocyte: characterized by the appearance of
secondary or specific granules(neutrophilic,
eosinophilic, or basophilic).”DAWN OF
NEUTROPHILIA”- initially, specific neutrophilic
granules appear only in golgi region near the
nucleus, more condensation of chromatin, nucleoli is
absent, cytoplasm present is greater in amount,last
cell capable of cell division.

15. Metamyelocyte: the nucleus becomes indented
and kidneyshaped, and the nuclear chromatin
becomes moderately coarse. Cytoplasm contains
both primary and secondary granules.

16. Band stage (stab form): This is characterised
by band-like shape of the nucleus with
constant diameter.

17. Segmented neutrophil(polymorphonuclear neutrophil):
With Leishman’s stain, nucleus appears deep purple
with 2 to 5 lobes which are joined by thin
filamentous strands. Nuclear chromatin pattern is
coarse.

18. Neutrophils have a life span of only 1 to 2
days in circulation.
FUNCTION OF NEUTROPHILS
In response to infection and inflammation, neutrophils come and lie closer to
endothelium to adhere to its surface (sticking)
Followed by escape of neutrophils from blood vessels to extravascular tissue
(emigration) guided by chemotactic factors.
Chemotactic factors: bacterial factors, C3a and C5a, breakdown components
of neutrophil, fibrin fragments and leukotriene B4.
PHAGOCYTOSIS : 3 steps: antigen recognition, engulfment and killing of
organism.

19. EOSINOPHIL:The nucleus is often bilobed and the cytoplasm
contains numerous, large, bright orange-red granules.
Maturation time for eosinophils in bone marrow is 2 to 6 days
and half-life in blood is less than 8 hrs. In tissues, they reside in
skin, lungs, and GIT.Play an important role in phagocytosis,
antihelmenthic and allergic response.

20. BASOPHIL: Basophils are small, round to oval
cells which contain very large, coarse, deep purple
granules. The nucleus has condensed chromatin and
is covered by granules. Basophils are also involved in
some cutaneous basophil hypersensitivity reactions
[histamine in basophil plays an important role in
hypersensitivity].

21. MONOCYTES:The monocyte is a large cell (15-20
m), with irregular shape, oval or clefted (often
kidney-shaped) nucleus and fine, delicate chromatin.
Cytoplasm is abundant, blue-grey with ground glass
appearanceand often contains fine azurophil
granules and vacuoles.Phagocytic cells, antigen
presenting cells, produce cytokines.

22. LYMPHOCYTES: The nucleus is round or slightly
clefted with coarse chromatin and occupies most of
the cell.The cytoplasm is basophilic, . On
immunophenotyping, there are two major types of
lymphocytes in peripheral blood: B lymphocytes (10-
20%) (humoral immunity) and T lymphocytes (60-
70%) ( cell mediated immunity). About 10-15% of
lymphocytes are of natural killer (NK) cell type ( kills
virus infected cells and some neoplastic cells).

23. B LYMHOCYTES: arise from lymphoid stem cells.
Primary development in bone marrow later migrate to
lymph nodes and spleen. B cells undergo differentiation
and proliferation to form plasma cells which secrete and
memory cells have lifespan of many years and upon
restimulation.
The fuction of B LYMPHOCYTES is production of antibodies
after differentiation to plasma cells.
T LYMPHOCYTES: originate from progenitor cells in
bone marrow and undergo maturation in thymus, circulate
in peripheral blood and get transported to secondary
lymphoid organs

24. FUNCTION OF T LYMPHOCYTES:
CD4 + T helper cells:
Recognition of antigen presented by antigen presenting cells
Assisting macrophages to kill intracellular pathogens.
Stimulation of B cells to produce antibodies.
CD8+ CYTOTOXIC CELLS: kill target cells via release of
granzymes and perforins.
Regulatory T cells: prevent reactions against self antigen.

25. Importance of Human leukocyte
antigens:
Organ transplantation
Recognition of foreign antigen and immunity.
Association between certain HLA antigen and diseases like
ankylosing spondylitis, type 1 DM, SlE, Myastenia gravis
and sjogren’s syndrome.
Paternity testing
Transfusion medicine: provision of HLA matched platelets
to patients who have become refractory to platelet
transfusions due to formation of HlA antibodies following
RDP transfusion.

26. THROMBOPOIESIS:Process of formation of platelets.

27. HEMOGLOBIN ESTIMATION

28. Hemoglobin is the major constituent of the red cell cytoplasm, accounting for approximately
90% of the dry weight of the mature cell.
It is comprised of heme and globin.
Hemoglobin is tetramer consisting of two pairs of similar polypeptide chains.
Each of the four chains is attached to heme which is a complex of iron in ferrous form and
protoporphyrin.
Major type of hb in adults is HbA and it has 2 alpha globin chains and 2 beta globin chains.
The gene that codes for the formation of alpha globinchains is located on chromosome 16.
The gene that codes for the formation of beta globin chains is on chromosome 11.
INTRODUCTION:

29. HEMOGLOBIN VARIANTS:

30. Heme has the ability to bind oxygen reversibily and carry it to tissues.
It also facilitate the exchange of carbon dioxide between the lungs and tissues.
FUNCTION OF HEMOGLOBIN:

31. Elderly renal insufficiency, inflammation,
testosterone deficiency, diminished
erythropoiesis, myelodysplasia reduce hb
concentration.
Exercise - increase hb concentration
Posture - lying to sitting increases hb
concentration.
Altitude - increases hb concentration
Smoking - increases hb concentration.
PHYSIOLOGICAL VARIANTS IN HB
CONCENTRATIONS:

32. To Determine presence and severity of anemia.
Screening of polycythemia.
Response to specific therapy in anemia.
Estimation of red cell indices
Selection of blood donors.
INDICATIONS FOR HB ESTIMATION:

33. COLOUR COMPARISION BETWEEN STANDARD AND TEST SAMPLE BY:
Visual methods:
1.Sahil’s acid hematin
2.Tallqvist hemoglobin chart
3.WHO hemoglobin colour scale
4.Oxyhemoglobin method
Specific gravity method
Photoelectric method:
1.Cyanhemoglobin method
2.Oxyhemoglobin method
3.Alkaline hematin method
COLORIMETRIC METHODS:

34. GASOMETRIC METHOD:
OXYGEN CARRYING CAPACITY IS MEASURED .
It does not measure carboxyhemoglobin , sulfhemoglobin, methemoglobin.

35. Iron content of hemoglobin is first estimated.
Indirectly hemoglobin is derived – (100 gms of Hb contains 374 gms of iron).
Time consuming method.
THIS METHOD IS USED TO CALIBRATE ALL OTHER METHODS OF HB ESTIMATION.
CHEMICAL METHOD:

36. Used in mass screening like selection of donors.
Rapid and simple.
A drop of blood is allowed to fall in copper sulphate solution
of specific gravity of 1.053 from height of 1 cm becomes
encased in a sac of copper proteinate which prevents
dispersion of fluid for 15 seconds.
Specific gravity 1.053 is equivalent to 12.5 gms|dl hb level.
If drop sinks, specific gravity is higher than copper sulphate.
Specific gravity method:

42. PRINCIPLE:
Blood is mixed with Drabkins solution.
DRABKINS SOLUTION: Ph 7.0 – 7.4
Potassium ferricyanide
Potassium cyanide
Potassium dihydrogen phosphate
Non- ionic detergent
Distilled water.
Erythrocytes are lysed producing an evenly distributed Hb solution.
Potassium ferricyanide converts Hb to methemoglobin.
Methemoglobin combines with potassium cyanide to form cyanmethemoglobin.
Absorbance is measured in spectrophotometer at 540 nm.
To obtain amount of unknown Hb sample, its absorbance is compared with standard cyanmethemoglobin.
Take 5ml of Drabkins solution and to it add 20 microlitres of EDTA blood. Mix well and wait for 5 mins.
CYANMETHEMOGLOBIN METHOD:

49. 7 PARTS DIFFERENTIAL ANALYSER

52. PRINCIPLE:
Based on the ability of oxyhaemoglobin to dissociate oxygen at
decreasing oxygen tensions by spectrophotometric measurement.
Hb is converted to oxyhaemoglobin by reaction with ammonia and the
colour of solution is measured in a photocolorimeter.
The advantages of this method are : simple , quick, its reliability is
not affected by increased bilirubin level.
Disadvantage: the solution of oxyhaemoglobin fades very quickly.
Oxyhemoglobin method:

53. REFERENCES FROM:
WINTROBE’S CLINICAL HEMATOLOGY
DACIE AND LEWIS PRACTICAL
HAERMATOLOGY

54. THANK YOU !THANK YOU !