Quiz 2

Basic Hematological Method

Hematology Laboratory Test
-Simple observation of the blood and its component
-highly automated methods which includes examination at the molecular level
Manual Cell Counting (wbc, rbc, platelets)

Hemocytometer or counting chamber (Heart of Diluting pipette

manual cell count) Diluent (commercially/ or laboratory prepared
NEUBAUER reagent)
*Principle of performance (for all the cells) is the same. They only differ in dilution, diluting fluid and area in the
counting chamber where cells are counted.

RBC Diluting Pipette WBC Diluting Pipette

Big Bulb (101) Small Bulb (11)

Dilution Factor- Amount of blood vs. Total amount of the solution/ volume
Depth Factor- Distance between the chamber and cover slip 1.0 (constant)
Area Factor

Hemoglobin Determination

Sahli Helige Method Cyanmethemoglobin Method

Uses weak acid Drabkins Reagent
Uses color comparator Spectrophotometer

Microhematocrit (packed cell volume)

Volume of packed RBCs that occupies the given volume of the whole blood.
**Capillet is filled with blood, centrifuge, and PCV is read using Microhematocrit reader.
Blood Indices or RBC Indices

MCV- Mean Cell Volume MCH- Mean Cell Hemoglobin

MCHC- Mean Cell Hemoglobin Concentration
**Use to differentiate types of Anemia
Reticulocyte- Last immature RBC stage/ Matures within 2-3 days in the bone marrow and 1 day in the peripheral
blood.
Reticulocyte Count (Supravital Stain- Methylene Blue & Brilliant Cresyl Blue)

Rate of settling of RBCs from plasma after the General screening test
bone marrow
Uses Wintrobe tube/ Westgren tube
Peripheral Blood Examination

Provides valuable information about the patients condition

WBC and platelet estimation
WBC differential count
Evaluation of the morphology of the cells RBC/WBC (anomalies and inclusion bodies)
WBC Differential Count

Percentage of the different WBCs are determined

100 WBCs are differentiated/ Classified and counted respectively
Special blood cell counter is used
Done under OIO microscope field
Staining Blood Smear
Blood Smear

Thick to thin transition, with feathery edge (tongue shape)

Romanowsky Stains (Wrights stain & Giemsa stain)
CBC Inclusions

WBC count
WBC differential count
Hemoglobin determination
Hematocrit determination
Platelet count estimation
RBC count estimation
Hematopoietic System and Hematopoiesis/ Hemopoiesis (blood formation)

Continuous regulated process of blood cell Cell proliferation

production Cell differentiation
Cell renewal Cell maturation
Hematopoiesis

The process leads to the formation, development and specialization of all the functional blood cells that are
released from the bone marrow to the blood circulation.
***Adults- restricted to the bone marrow
Erythropoiesis- RBC Granulopoiesis- GRANULOCYTES (Basophil,
Eosinophil, Neutrophil)
Lymphopoiesis- WBC
Megakaryopoiesis- MEGAKARYOCYTES
Monopoiesis- MONOCYTES
Thrombopoiesis- PLATELETS
Lymphoiesis- LYMPHOCYTES
Fetal Development- occurs in different areas of the developing fetus
Hematopoietic Tissue

Organs involved in blood production

Medullary Hematopoiesis

Involves the formation of normal blood cell in the marrow

Vertebrae, Ribs, Sternum, Pelvis, Skull, Scapulae, Proximal end of the long bones
***Occurs in the bone marrow bounded by sinusoids
Extramedullar Hematopoiesis

Involves the formation of blood cell outside the bone marrow

Spleen, Liver, Lymph nodes, areas of Lymphatic tissues
Bone Marrow

produces Platelets, RBC, Granulocytes

also serves as one of the formation sites of lymphocytes, monocytes, plasmacytes
***the size of the cell decreases as it matures
Yellow Bone Marrow

filled with fat

fat occupies the reserved space and mixes with the solution and disappears as the blood cell increasefor
regeneration to occur on demand without interfering with vital structure
PRE-NATAL HEMATOPOIESIS
Mesoblastic Phase/ Yolk Sac Phase

Begins around 14th to 19th day of embryonic development after fertilization

High demand of fetus of Oxygen in mesenchymal cell (yolk sac)
Characterized by the development of primitive erythroblasts that produce measurable embryonic
hemoglobin and nucleated
No Leukocytes and Thrombocytes at this phase
Does not contribute significantly to definitive hematopoiesis
Occurs intravascularly (within a developing vessel)
***blood cell in the yolk sac originate in the angiogenic cell cluster (blood island)
Hepatic Phase

4th to 5th week gestational

Characterized by recognizable cluster of developing erythroblast, granulocytes, and monocytes
Production of Megakaryocytes
Liver: chief site of hematopoiesis (peak 3rd month) and retaining activity until 1-2 weeks after birth
Spleen, Kidney, Thymus, Lymph Nodes, contribute to the process
-Spleen/Kidney (B-Cell)
-Thymus (T-Cell)
-Lymph Nodes (Produces RBC, Granulocytes, Megakaryocytes)
Lymphoid cells begins to appear
Occurs extravascularly
Detectable levels at the hemoglobin (Hb) F, HbA and HbA2

Medullary Phase/ Myeloid Phase

Starts in the 5th month of
gestation
Occurs in the developing
medulla bone marrow cavity
Mesenchymal cells
(embryonic cells) core of
the bone and differentiate
into skeletal cells and
hematopoietic blood cells
Myeloid activity starts in this
stage
Myeloid-Erythroid Ratio
approaches the adult level of
3:1 by 21 weeks of gestation
On the 6th month: Bone
Marrow is the primary site of hematopoiesis
Erythropoietin (EPO) Granulocyte Colony- Stimulating Factor (GCSF), Granulocyte-Monocyte Colony
Stimulating Factor (GM-CSF), Fetal Hemoglobin (HbF) HbA2 and adult hemoglobin can be detected
(Erythropoietin is a hormone)
POST-NATAL HEMATOPOIESIS
Newborn: 80-90% of bone marrow is active in producing nucleated RBCs, Lymphocytes and Hemoglobin
Young Adult (Around 20 years old) 60% of bone marrow is active
Older Adult (Around 55 years old) 60% yellow marrow and 40% red marrow

Red Bone Marrow

Composed of extravascular cord that contains all the developing blood cell lineages stem and progenitor
cell, adventitial cells and macrophages.
Main Function: production and proliferation of blood cells that are divided into three compartments/ cell
types (Stem cells, Progenitor cells and Precursor cells)

Cell Compartments/ Types

Stem Cells (Pluripotent or Multipotent cells)
differentiate into specific cell lines: Stem cells differentiates Colony Forming Units (CFU): differentiate
into either of pathways
Giving rise to the primitive B or T Lymphocytes
Giving rise to erythrocytes, granulocytes and megakaryocytes (thrombocytes)
Progenitor Cells (Unipotent stem cells)
Differentiate only into a single cell line. These cells develops into BFU-E, CFU-E, CFU-MEG, CFU-GM
(CFU-cell performing unit, BFU-blast performing unit, E-erythrocytes, MEG-megakaryocytes
Precursor Cells
These cells mature into blast fomrs (myeloblast, megakaryoblasts, pronomoblast and lymphoblast)

Bone Marrow Cell Production

Bone marrow evaluation is based on Myeloid Erythrocyte Ratio (M:E Ratio)
Normal M:E Ratio 3-4:1
Most cell production in the bone marrow is myelocytic and is mostly granulocytic
***Myelocytic pool of cells takes 3 days to develop into mature cells peripheral blood
Cell Proliferation and Differentiation
All blood cells originated from Pluripotent Stem Cell (PPSC)
Differentiation is determined by Protein Factors and influence by Growth Factors
Growth factors are regulatory glycoprotein hormones (example: Erythropoietin, Interleukin and Colony
Stimulating Factors)
Hematopoietic stem cells develop into Colony Forming Units (CFU) Granulocytes, Erythrocytes,
Monocytes and Megakaryocytes (CFU-GEMM)
The CFU-GEMM differentiates into the following Peripheral Blood.
BFU-FCFU-EErythrocytes
CFU-MEGThrombocytes
CFU-GMCFU-MMonocytes
CFU-GMCFU-GSegmented Neutrophils
CFU-EOEosinophils

(NEXT TOPIC ERYTHROPOIESIS)