1. Hemopoiesis

2. Haematology: is the study of the blood and its diseases
Blood : is a suspension of non-dividing end-stage cells of three types namely, red cells (erythrocytes), White cells (leucocytes) and platelets (thrombocytes). The suspending fluid is the Plasma.

4. Function of Blood
Delivery of substances needed for cellular metabolism.
Removal of waste.
Defense against microorganisms & injury.
Maintenance of acid base balance.

5. Hemopoietic System
Hemopoiesis is the process of production of all blood cells.
Hemopoietic System is comprised of two major cell lines :
Myeloid
Lymphoid

6. Hemopoietic System
The earliest evidence of hemopoiesis ( mesoblastic phase) occur in the yolk sac of the embryo.
In this stage the hemopoietic system consists primarily of mesenchymal derived primitive erythroblast.
The hepatic phase begins in the second month of fetal life.
( megakaryocytes and granulocytes appears in the sinusoids of liver)
At wks gestation , the liver is the principle site of hemopoiesis till approximately 24 wks.
Bone marrow function starts from the 4th – 5th months of gestation and is the major site of Hemopoiesis till birth.

8. Hemopoietic System
hemopoietic system in normal individual in the first yr. of postnatal life is in both axial and radial skeleton.
Thereafter, there is a gradual regression of the hemopoiesis in the long bones until about the age of 15 , when the flat bones of the central skeleton ((pelvis, vertebral column, cranium, ribs ,sternum), and epiphysis of long proximal bones are the exclusive sites of hemopoiesis.
In normal adult the bone marrow is about 50% hemopoietic & 50% fatty which is capable of reversion to hemopoiesis if needed .

9. The Concept of STEM CELLS
Stem cells are cells from which all Hemopoietic elements originate. They are characterized by their ability of Self-renewal & Differentiation.
Stem cells require for their proliferation and differentiation :
- Certain regulatory factors (Hemopoietic Growth Factors).
- Suitable microenvironment, provided by the marrow space.
Stem cells Precursors End-stage cells
The End stage cells have a restricted life span, ranging from 4 months for RBC, days for platelets, hours for granulocytes. These cells have to be continuously replaced by bone marrow, and BM should produce daily some 2.5 billion red cells/kg, 2.5 billion red cells/kg platelets, and 1 billion granulocytes/kg. Actually any normal individual will produce a no. of hemopoietic cells equivalent to his body weight /year, to maintain his peripheral blood counts.

10. Bone Marrow Stroma
The bone marrow forms a suitable environment for the stem cell survival, self renewal and formation of differentiated progenitor cells.
It is composed of stromal cells, and microvascular network.
The stromal cells includes adipocytes, fibroblasts, osteoblasts, endothelial cells and macrophages which secrete extracellular molecules such as collagen, glycoprotein ( fibronectin and thrombospondin) and glycosaminoglycans to form the extracellular matrix.
In addition, stromal cells forms niches and provides several growth factors , cytokines , and adhesion molecules which support the stem cells survival.

13. Microscopical Features:
Erythroblasts or normoblasts
Microscopical Features:
Reduction in the cell size.
Loss of nucleoli.
Clumping of nuclear chromatin.
Hemoglobinization of the cytoplasm.
Loss of nuclei.
Proerythoblast
Reticulocyte
Early
intermediate
late
Mature RBC
Proerythoblast
The earliest Erythroid recognizable cell is the Proerythroblast, which is large cell with high N/C ratio, basophilic cytoplasm, and open nuclear chromatin with prominent nucleolus. The next stage is early normoblast, in which the cell loses its nucleolus, but cytoplasm remains blue. The next stage is the intermediate normoblast in which the N/C ratio gets smaller, while the cytoplasm becomes less basophilic, as Hb increases.while the nuclear chromatin becomes more condensed and clumped The next stage is the late normoblast in which the cytoplasm become more pink due to Hbinization, and nuclear chromatin more condensed. The nucleus is then extruded to form a reticulocyte, which remains normally about two days in the marrow and is then released to circulation and matures finally into a mature anucleated erythrocyte.
Normoblasts Intermediate Late
Red Cell precursors (in marrow)

15. Microscopical Features:
Reduction in the cell size.
Loss of nucleoli.
Granulation of cytoplasm ( primary and secondary).
Nuclear segmentation.
Stages of Maturation of the Granulocytic Series
In the bone marrow
Blood
Bone marrow

16. Blood leucocyte morphology

18. Megakaryocyte : the precursor of Platelets in the marrow
Microscopical Features:
Increased cell size.
Lobulation of the nuclei.
Cytoplasmic granulation.

19. Life span of blood cells in peripheral circulation
- Red cells : 120 days.
- Granulocytes : ~ 1day.
- Platelets : 7-10 days.

20. Normal Blood Film

21. Bone marrow examination
In certain conditions a bone marrow examination is required for diagnosis or follow-up of patients. There are two types of marrow procedures :
1. Bone marrow aspirate : done from iliac crest or sternum, in which a specimen is aspirated using a wide bore needle from the active marrow, smeared, stained and then examined for any abnormalities.
2. Bone marrow biopsy : here a core of bone marrow tissue is taken, and processed and stained as in histopathological specimens (H&E stain)

22. Bone marrow sets
Aspirate Set
Biopsy Set

23. Common sites for Bone marrow procedures in adults
Best x x
Manubrium Sternii
Post. Superior Iliac spine

24. Bone marrow aspiration
Bone marrow aspirate smear
Fragment
Higher magnification of trail.
Trail

25. Bone marrow section stained with H&E stain
Bone Marrow Biopsy
Bone marrow biopsy slide-core of BM
Bone marrow section stained with H&E stain

26. Indications for BMA with or without BMB
Unexplained anemia (microcytic , macrocytic)
Unexplained thrombocytopenia
Pancytopenia
Leukoerythroblastic blood picture
Acute leukemia
CML
CLL
NHL
Multiple myeloma
Fever of unknown origin
Storage disease
Follow up after treatment for acute leukemia

27. Hematologic investigations

28. Hematologic investigations
CBC: provide information about quantities of each cellular component as well as data on red blood cell size & hemoglobin content.
CBP: CBC + Blood film
Blood should be anticoaggulated either with EDTA (CBC) or sodium citrate (coaggulation tests).

30. Normal Values For Peripheral Blood
Female Male
Erythrocytes (per µl) 4.8±0.6x ±0.8x106
Hemoglobin (g/dl) ±2 16 ±2
Hematocrit (%) ±5 47 ±5
Reticulocytes (%) __________________________________________
Mean corpuscular volume (MCV; fl) 80-95
Mean corpuscular hemoglobin (MCH; pg)
Mean corpuscular hemoglobin concentration (MCHC; %)

31. Blood Film

32. Haemoglobin Estimation
The first and foremost investigation in any suspected case of anaemia is to carry out haemoglobin estimation.
Several methods are available but most reliable and accurate is the cyanmethaemoglobin (HiCN) method employing Drabkin's solution and spectrophotometer
If the haemoglobin value is below the lower limit of the normal range for particular age and sex, the patient is said to be anaemic
In pregnancy, there is haemodilution and, therefore, the lower limit in normal pregnant women is less (10.5 g/ dl) than in the non-pregnant state.

33. Clinical Significance Of Hb Measurement
A decrease or increase in hemoglobin concentration must be reported ,as it is a sign of disease requiring investigations
A decrease in Hb concentration is a sign of anemia
While an increase can occur due to;
Reduced oxygen supply (congenital heart disease , emphysema)
Polycythemia

34. Haematocrit or Packed Cell Volume
It is the amount of packed red blood cell, following centrifugation, expressed as a total blood volume
Normal Value
Male: %
Female: 37-47%
Roughly, the haematocrit value is 3 times the Hb concentration

36. Clinical Significance
A decrease in the haematocrit value is a suitable measurement for detection of anaemia, also in case of hydremia (excessive fluid in blood as in pregnancy)
An increase is an indication decrease oxygen supply (as in congenital heart disease, emphysema) or as in polycythemia and dehydration
The value of haematocrit is used with haemoglobin and red cell count for the calculation of MCV, MCH and MCHC

37. Red Cell Indices Mean Cell Volume(MCV))
It is calculated from PCV and red cell count as follows:
MCV = PCV/RBC ( fl)
Normal value: fl
It decrease in iron deficiency anaemia and haemoglopinopathies
It is increase in megaloblastic anaemia and chronic haemolytic anaemia

38. Mean Cell Haemoglobin Concentration (MCHC)
It is calculated from the haemoglobin and PCV as follows:
MCHC = Hb/PCV g/dl
Normal value: g/dl
It is usually decreased in iron deficiency anaemia (microcytic hypochromic anaemia)

39. Mean Cell Haemoglobin (MCH)
It is calculated from the haemoglobin and erythrocyte count as follows:
MCH = Hb/RBC pg
Normal value: pg
It is decrease in iron deficiency anaemia and thalassaemia (microcytic hypochromic anaemia)
It is recognized by the pale colour of the red cell in the peripheral blood film
It is increase in macrocytic anaemia (vitamin B 12 and folic acid)

40. Peripheral Blood Film Examination
Normal RBC :
The normal human erythrocytes are biconcave disc, 7.2 um in diameter, and the thickness of 2.4 um at the periphery and 1 um in the center
The biconcave shape render the red cell quite flexible so that they can pass through capillaries whose minimum diameter is 3.5 um
More than 90% of the weight of the red cell consist of haemoglobin.
Normal red cells (normochromic): have uniformly coloured haemoglobin in side the cell with a small clear paler region in the center

41. Size variation:
Normal: normal size ( ), and normal diameter (6-8u) called Normocytes
Macrocytes: increase size of cells having diameter > 8 u and MCV > 95fl
Microcytes : decrease size of cells having diameter < 6 u and MCV < 80fl

42. Size variation Anisocytosis: variation in the size of RBC.
Poikilocytosis: variation in the shape of RBC.
Anisopoikilocytosis: variation in both size & shape of RBC.

44. WBC and Platelets WBC : 4-10 x109/l Differential Count :
Neutrophils, Lymphocytes, Eosinophils, Basophils, and Monocytes.
Platelets : X 109/l

45. Blood leucocyte morphology

46. Leucocytes in Blood
Leucocyte counts range in a normal adult between 4-10 x 109/L.
Normally majority of the cells seen are neutrophils (2-7x 109/l), followed by lymphocytes (1-3 x109/l), monocytes (0.2-1x109/l) , eosinophils ( x 109/l) and basophils ( x 10 /l).
Determination of the proportion of various leucocyte types in the blood is called Differential leucocyte count.

47. Terms used to denote changes in leucocytes numbers:
Leucocytosis : increased no. of leucocytes above 10.0 x 109/L.
Leucopenia : Reduced total leucocyte count below 4.0 x 109/L.

48. Neutrophilia Bacterial infection. Inflammation & tissue necrosis.
Neoplasms.
Acute hemorrhage or hemolysis.
Drugs.

49. Lymphocytosis Viral infections.
Chronic infections: Tuberculosis, brucellosis.
Chronic lymphoid leukemia.

50. Eosinophilia
Allergic diseases.
Parasitic diseases.
Drug sensitivity.

51. Platelets in Blood Platelets count normally ranges between
150 to 450 x 109/L.
Increased Platelets above 450 is called thrombocytosis.
Reduced Platelets below 150 is called thrombocytopenia.

52. Reticulocytes Count
Reticulocyte count are juvenile red cells containing RNA reminent .
Reticulocyte stained with special stain called methylen blue.
(normal %) is done in each case of anaemia to assess the marrow erythropoietic activity.

54. Red Cell Distribution Width RDW
Range of variation in red blood cells volume
Normal Range 11.5 – 14.5%)
Its helpful in differentiating types of anemia.

55. Erythrocyte sedimentation rate ESR
The rate with which the RBC settle over a specified time period.
Male: up to 15mm|hr
Female: up to 20 mm|hr
It is non specific, not diagnostic
It is an indicator of the disease course

56. Erythrocyte sedimentation rate ESR
Increased in : Malignant diseases, Inflammatory diseases, Bacterial infections.
Decreased in : Polycythemia.