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Comparison of CD34 (+) Stem Cell Enumeration by Flowcytometry in Cord Blood, Bone Marrow & Peripheral Blood.

NAME: LAXMI RATHOR

ROLL NO: 10112017

FROM: FORTIS HOSPITAL NOIDA

GUIDE: Dr. NEENA VERMA

M.Sc. BIOCHEMISTRY


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CONTENT

INTRODUCTION.

AIMS &OBJECTIVES

RESULT AND DISSCUSSION


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INTRODUCTION

  • Stem cells are the cells with the ability to self replicate throughout the life of an organism and can differentiate or give rise to many cell types upon stimulation and given the right condition.

  • Stem cells have the potential to develop into functional mature cells having characteristics of different tissues or organs.


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TYPES OF STEM CELLS

Stem cells are a class of undifferentiated cells that are able to differentiate into specialized cell types .Commonly stem cells come from two main sources:

1. Embryos formed during the blastocyst phase of embryological development (embryonic stem cells)

2.Adult tissue (adult stem cells)Both types are generally characterized by their potency or to differentiate into different cell types (such as skin , muscle , bone (etc.)


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  • Use of stem cell Hematopoietic stem and progenitor cells (HSCs) is widely used for evaluation of graft adequacy of peripheral blood and bone marrow stem cell grafts.

  • In the present study, precursors in investigations of the signals that turn genes on and off. Condition such as cancer and birth defects are due to abnormal cell division and differentiation.

  • If the mechanisms are understood, new and better stratrgies for therapy can be developed. For use in testing new drugs. For example, new medication could be tested for safety on a wide variety of immature and differentiated cells.


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Aims & ObjectivesImportance of CD34+ Count

  • The CD34 antigen is present on immature hematopoietic precursor cells and hematopoietic colony-forming cells in bone marrow and blood, including unipotent progenitor cells.

  • An accurate measure of the CD34 cell count is a necessary for dose requirement protocols in stem cell transplantation. An incorrectly high result could lead to an infusate with less than the recommended threshold dose of CD34+ cells.


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What makes stem cells so important?

Stem cells make the 3 main types of blood cells: -

  • Red blood cells (Erythrocytes)

  • White blood cells(Leukocytes)

  • Platelets(Thrombocytes)

    Red blood cells:-Red blood cells (RBCs) carry oxygen from the lungs to all of the body,and then bring carbon dioxide back from the lungs to be exhaled,A blood test called the hematocrit show how much of your blood is made up of RBCs.


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  • white Blood Cells:-White blood cells (WBCs)fight infection caused by bacteria, and fungi.

  • There are different types of WBCs.

  • The most Important in Fighiting Infection are known as Neutrophills.

  • Another type of white blood cell the stem cell make are called lymphocyte.

  • These are immune cells that can make antibodies and help fight infection,andinclude the highly specialized :-

  • T-lymphocyte (T cells),

  • B-lymphocyte (B cells),

  • and natural killer (NK) cells.


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Platelets (thrombocyte):-Platelets are pieces of cell that seal damaged blood vessels and help blood to clot ,both of which are important in stopping bleeding.

  • A person whose platelets count drops ,below 150,000/mm is said to have Thrombocytopenia and may bruise more easily, bleed longer, and have noseblees,or bleeding gums.

  • Spontaneous bleeding (bleeding with no can be dangerous if bleeding occurs in the brain),or if blood begins to leak from intestine or stomach.


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Material &Method

The equipment used for CD34 enumeration was :-

  • BD FacsCalibur Flowcytometer.

  • For sample preparation a centrifuge REMI-8C was use.

  • Vortex mixer

  • Timer

    Fig.BD Facscalibur Flowcytometry


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Specimen Requirement

  • D-A/CPD/EDTA/HeparinA minimum of 100uL of specimen is required per test.

  • The specified anticoagulants required are as follows:

  • Normal and mobilized peripheral blood anticogulated with EDTA .

  • Fresh and thawed aphaeresis specimen’s anticoagulated with ACD-A or ACD-A/heparin/ EDTA.

  • Fresh and thawed bone marrow specimen’s anticoagulated with heparin, heparin/ACD-A, or EDTA .

  • Fresh and thawed cord blood anticoagulated with AC.


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Result & Disscussion

  • I performed CD 34 enumeration using Flowcytometry in 62 cases .These consisted of 14 samples of PB stem cell harvest ,2 pre -processed cord blood,20 post processed cord blood,8 post processed bone marrow,18 post processed bone marrow . These are represented below:

    Types of samples:

  • PB stem cell harvest sample: - 14

  • Preprocessed cord blood sample: - 2

  • Post processed cord blood sample: - 20

  • Preprocessed bone marrow sample:- 8

  • Post processed bone marrow sample:-18


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Age Distribution of cases:-The age ranged from 0 years to 65 years.

TheSex Distribution of cases:-

  • No. of males: - 41

  • No. of females: - 21

  • The cord blood is most frequent sample.


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Result of Cord Blood Samples:-

  • Abs stem cell counting for pre processed cord blood:-44.21

  • Stem cells % of W.BC for pre processed cord blood:-0.57

  • Abs stem cell counting for post processed cord blood:-97.22

  • Stem cells % of W.BC for post processed cord blood:-0.31

    Result of PBSCH Samples:-

  • Abs stem cell counting for PBSCH: - 124.11

  • Stem cells % of W.BC for PBSCH: - 0.411

    Result of Bone Marrow Samples:-

  • Bone marrow was the most precious sample.

  • Abs stem cell counting for pre processed bone marrow:-73.73

  • Stem cells % of W.BC for pre processed bone marrow:-0.58

  • Abs stem cell counting for post processed bone marrow:-235.05

  • Stem cells % of W.BC for post processed bone marrow:-0.81


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The analysis of Abs stem cells and % WBC of my showed the following samples:

  • Abs stem cell counting for PBSCH: - 124.11

  • Stem cells % of W.BC for PBSCH: - 0.411

  • Abs stem cell counting for pre processed cord blood:-44.21

  • Stem cells % of W.BC for pre processed cord blood:-0.57

  • Abs stem cell counting for post processed cord blood:-97.22

  • Stem cells % of W.BC for post processed cord blood:-0.31

  • Abs stem cell counting for pre processed bone marrow:-73.73

  • Stem cells % of W.BC for pre processed bone marrow:-0.58

  • Abs stem cell counting for post processed bone marrow:-235.05

  • Stem cells % of W.BC for post processed bone marrow:-0.81


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Figure 2.Show the absolute stem cell counts

  • Here, the horizontal lines show the name of blood samples.

  • The vertical line shows the absolute stem cell counts.


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Figure 3.Show the % of WBC

  • Here, the horizontal lines show the name of blood sample

  • The vertical lines show the stem cell as % of WBC.


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CONCLUSION

  • In this study we compared the yield of stem cells in cord blood, bone marrow, PBSCH samples,

  • The yield was highest in Bone marrow post processed sample.

  • CD34 enumeration done by Flowcytometric method is very fast and accurate method for counting of stem cell.


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THANK YOU


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