Erythropoiesis and reticulatocyte count

1. PRESENT
BY
ANJU C ARJUNAN . B.Sc. MLT
LAB TECH II
DEPARTMENT OF LAB MEDICINE
DHAMAN HEALTH ASSURANCE HOSPITAL
KUWAIT
ERYTHROPOIESIS
&
RETICULOCYTE COUNT

2. CONTENTS
o INTRODUCTION
o SITES OF ERYTHROPOIESIS
o STAGES OF ERYTHROPOIESIS
o DURATION OF ERYTHROPOIESIS
o FACTORS NECESSARY FOR ERYTHROPOIESIS
o RETICULOCYTE COUNT

3. INTRODUCTION
RETICULOCYTES
 Reticulocytes are immature red blood cells.
 In process of erythropoiesis, reticulocytes develop and mature in the bone marrow
and circulate for about a day in the blood stream before developing into mature RBC.
HEMATOPOIESIS
 Hematopoiesis is the formation of blood cellular components.
 All cellular components are derived from hemopoietic stem cells.
 Bone marrow is the major site of hematopoiesis, it can occur in many other tissues
both during fetal development and after birth.

4. HEMATOPOIESIS

5. ERYTHROPOIESIS
Erythropoiesis is the process of the origin,
development and maturation of erythrocytes.
Sites of erythropoiesis:-
I - In fetal life:-
1. Mesoblastic stage :-
During the first two months of intrauterine life,
the RBCs are produced from mesenchymal cells of
yolk sac
2. Hepatic stage :-
From third month of intrauterine life, liver is
the main organ that produce RBC.
Spleen and lymphoid organs are also involved.
3. Myeloid stage :-
During the last three months of intrauterine
life, the RBCs are produced from red bone
marrow and liver.
II- In child hood:-
Occurs in all bones of the body
III- In adults :-
Occurs in the red bone marrow at the end
of the long bones & flat bones of the body.

6. STAGES AND DURATION OF ERYTHROPOIESIS
Multipotential hematopoietic stem cell (hemocytoblast)
Proerythroblast (pronormoblast)
Basophilic erythroblast(early normoblast)
Polychromatic erythroblast(intermediate normoblast)
Orthrochromatic erythroblast(late normoblast)
Polychromatic erythrocyte(Reticulocyte)
Erythrocyte

7. Stages of Erythropoiesis
1 2 3 4
5
6

8. Stage Size Nucleus Cytoplasm Hb Mitosis Diagram
Hemocytoblast 19-23
µm
Occupies whole of the cell.
-5-7 nucleoli present
- Chromatin remains closed.
-Occupies rim of the
cell and basophilic
in nature.
Absent Present
Proerythroblast/
Pronormoblast
15-20µm -2-3 nucleoli present
-Chromatin remains closed.
-Increases in size
-Basophilic in nature
Absent Present
Early Normoblast
/ Basophilic
Erythroblast
14-16µm
-Nucleoli absent
-Nucleus deceases in size
-Chromatin begins to open
-Increases in
volume.
-Less basophilic
Absent Present
Intermediate
Normoblast
10-14µm -Decreases in size and shows
cart wheel appearance
-Increases in size
-polychromatic in
nature
Starts to
appear Present
Late Normoblast
/ Orthochromatic
Erythroblast
8-10µm
7-8µm
-Decreases in size
-Pyknotic nucleus present
near the cytoplasmic
membrane.
-Occupies whole of
the cell.
- acidophilic in
nature.
Increase in
synthesis.
Stops
Reticulocyte 7-8µm
-Nucleus disappears
-Remaining of RNA can be
seen
- more acidophilic
in nature.
Decreases in
synthesis Absent
Mature RBC
7.2-
7.4µm -Absent
- deep pink/red in
color
limited to
38% of the
cell
Absent

9. Factors Necessary For Erythropoiesis
- Various substances are necessary for the development and maturation of
erythropoiesis.
- These factors are classified in to three categories namely,
* General factors
* Maturation factors
* Factors necessary for Hb synthesis.
1- General factors :-
general factors include,
• Erythropoietin
• Thyroxine
• Hemopoietic growth factors
• Vitamins

10. 1 .EYTHROPOIETIN :-
- Also known as hemopoietin or erythrocyte stimulating factor.
- It is a glycoprotein
- Secreted by peritubular capillaries of kidneys.
- Stimulus for secretion - hypoxia
Hypoxia
Affects kidney
Release of erythropoietic factor
Erythropoietinogen Erythropoietin

11. Actions :-
- Helps in production of proerythroblasts from stem cells and its maturation to RBC
- Release of matured RBC in to blood through the capillary membrane.(Even some
reticulocytes are release along with RBC)
2- Thyroxine :-
- Accelerates the process of erythropoiesis.
- In hyperthyroidism, polycythemia is common.
3- Hemopoietic growth factors :-
- Interleukins and stem cell factors induces the proliferation of pluripotent stem cells.
-IL3, IL6 and I L11 are involved.

12. 4.Vitamins :-
-vitamins such as Vit B ,Vit C, and Vit D are necessary for erythropoiesis.
-Vitamin c helps in utilization of iron, thus maintain the differentiation of hemopoietic progenitors .
(Vitamin c act as an enzyme co-factor and enhances mobilization of ferrous form of iron to
transferrin, thus increasing bioavailability.)
Maturation factors :-
* Vitamin B12
* Intrinsic factor
* Folic acid
Vitamin B12 (Cyanocobalamin):-
- Also known as extrinsic factor
-Essential for synthesis of DNA .So its deficiency leads to failure in maturation and division of cell
thus leads to pernicious anemia (larger and fragile cells ).

13. 2- Intrinsic factor :-
- Essential for the absorption of vitamin B12
3- Folic acid :-
- Essential for the synthesis of DNA
- Deficiency causes failure in maturation, leads to anemia in which the cells are larger and appear in
megaloblastic (Proerythroblast ) stage.
Factors necessary for Hb formation:-
Various materials are essential for the formation of hemoglobin in the red blood cells. The deficiency
of these substances decreases the production of Hb leads to anemia. These factors are as follows
* Proteins and amino acids
* Iron
* Copper – necessary for the absorption of iron from GI tract.
* Cobalt and nickel – essential for the utilization of iron during Hb formation.
* Vitamins – vitamin C, Riboflavin, Nicotinic acid and Pyridoxine are also essential for the formation
of Hemoglobin.

14. RETICULOCYTE COUNT
Aim :-
Reticulocyte count is done to determine if red blood cells are being created in the bone
marrow at an appropriate rate.
Automated retic count :-
- Most fully automated blood counters now incorporate with reticulocyte counting capacity.
- By using various dyes and fluorochromes, which combines with RNA of reticulocytes.
- Following binding of the dye, fluorescent cells can be enumerated using a flow cytometer.

15. Manual method:
Principle:-
Reticulocyte counts are juvenile red cells ,which contains remnants of ribosomal
ribonucleic acid (rRNA). Ribosomes have the property of reacting with certain basic dyes
such as azure b, Brilliant cresyl blue or new methylene blue and precipitated in
cytoplasm which can be seen as a blue or purple granules and as a network or reticulum.
Procedure :-
- Take 2-3 drops of anticoagulated blood in a small test tube and add equal volume of
supravital stain to it.
- Mix well and incubate the mixture at 37° C for 15-20 minutes.
- Mix again and prepare a wet preparation or dry preparation.

16. Wet preparation:-
- A drop of mixture is placed on a slide and place a cover slip on it.
- The drop should be small, so that the layer of fluid under cover slip will be thin .
- examine under oil immersion and number of reticulocytes present /1000 RBC should be counted
and expressed as percentage.
Dry preparation:-
- Prepare a thin smear with the mixture.
- The smear can either be examined directly or after it has dried.
- 1000 RBC s are counted and number of reticulocytes seen during the count reported as a
percentage of the total number of RBC.
- Smear should not be counter stained
Observation:-
Reticulo filamentous material – deep blue
Non reticulated cells - diffused shades of pale greenish blue.

17. STAINED SMEAR OF RETICULOCYTE

18. Calculation:-
Reticulocyte % = Number of reticulocytes counted X 100
Number of RBC counted
Reticulocyte index = Observed reticulocyte % X Measured Hb or PCV
Normal Hb or PCV
Absolute reticulocyte count = Reticulocyte % X RBC count / cu.mm
100
Normal values :-
in infants - 2 – 6 %
in adults - 0.5 – 2.5 %

19. Significant :-
Increases in :-
- Hemolytic conditions ( marrow has to produce more cells to try compensate
for their loss )
- loss of blood
- Increased bone marrow activity.
- Hemolytic anemia
- Erythroblastosis fetalis
- Kidney cyst or tumors.
Decreased in :-
- Bone marrow suppression ( due to certain drug, radiation therapy )
- Anemia caused by low iron levels, or low vitamin b12 or folate.

20. REFERENCE
 Dacie & Lewis. Practical Hematology. Text book , 12 edition.2017
 K. Sembulingam & Prema Sembulingam. Essential of medical physiology. Text book. 3 rd.
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Clin Lab Anal. 2001;15(5):267-94.
 Piva E, Brugnara C, Spolaore F, Plebani M. Clinical utility of reticulocyte parameters. Clin
Lab Med. 2015 Mar;35(1):133-63.
 Moras M, Lefevre SD, Ostuni MA. From Erythroblasts to Mature Red Blood Cells: Organelle
Clearance in Mammals. Front Physiol. 2017;8:1076.
 Noulin F, Borlon C, van den Eede P, Boel L, Verfaillie CM, D'Alessandro U, Erhart A.
Cryopreserved reticulocytes derived from hematopoietic stem cells can be invaded by
cryopreserved Plasmodium vivax isolates. PLoS One
 Mast AE, Blinder MA, Dietzen DJ. Reticulocyte hemoglobin content. Am J Hematol. 2008