Immunophenotyping and CD34 Expression

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Questions and Answers

What is the significance of enumerating CD34 stem cells after myeloablation?

It correlates with the engraftment success rate (correct)

It monitors the presence of lymphocytes in circulation

It determines the risk of graft-versus-host disease

It assesses the concentration of T-cells

What is the lower limit of CD34 cells required for adequate reconstitution of hematopoiesis per kg of body weight?

3x10^6 CD34 cells/kg

5x10^6 CD34 cells/kg

1x10^6 CD34 cells/kg (correct)

2x10^6 CD34 cells/kg

How are PBSCs typically collected for therapy following mobilization?

By apheresis after mobilization with G-CSF and plerixafor (correct)

Using surgical extraction methods

Through biopsy of bone marrow

From umbilical cord blood post-delivery

Which of the following is NOT a criterion for a routine clinical assay for hematopoietic progenitor stem cells?

Cost-effectiveness Signup and view all the answers

What does the ISHAGE protocol utilize for the analysis of hematopoietic stem cells?

Two-colour immunofluorescence and light scatter Signup and view all the answers

Which class of epitopes is characterized as being more widely distributed on endothelial cells and hematopoietic progenitor cells?

Class III epitope Signup and view all the answers

Which type of epitope is considered sensitive to enzymatic cleavage by glycoproteases?

Class I epitope Signup and view all the answers

What is a key characteristic of the CD34 molecule?

It is heavily glycosylated Signup and view all the answers

Which of the following statements about CD34 epitopes is accurate?

Class II epitopes are sensitive to glycoprotease cleavage Signup and view all the answers

What is one of the primary uses of the pan CD45 and pan CD34 monoclonal antibody combination?

To identify hematopoietic stem cells Signup and view all the answers

What is the purpose of using 7-AAD dye in the enumeration of CD34+ cells?

To assess the viability of the cells Signup and view all the answers

Which components are utilized for the absolute CD34 count in the single platform method?

Fluorescent beads and labeled antibodies Signup and view all the answers

What characteristic of 7-AAD makes it useful for identifying viable cells?

It strongly binds to GC-rich DNA regions Signup and view all the answers

What is the recommended minimum number of CD34+ events to be acquired for accurate enumeration?

100 CD34+ events Signup and view all the answers

Why is a known concentration of beads added to the sample?

To serve as a reference for absolute cell counting Signup and view all the answers

What is the primary sample type utilized in the G-CSF mobilization process?

Peripheral blood Signup and view all the answers

Study Notes

CD34 Overview

CD34 is a transmembrane glycoprotein present on hematopoietic stem cells, progenitor cells, and endothelial cells.
Almost all pluripotent and committed stem cells in colony-forming units (CFUs) express CD34.
Uncommitted progenitor cells are CD38- while committed progenitor cells are CD38+.
In normal conditions, CD34 positive cells constitute 1-2% of total bone marrow cells.

Importance of CD34 Enumeration

An adequate count of peripheral blood stem cells is essential for the successful recovery of hematopoiesis post-myeloablation.
The absolute count of CD34 cells correlates with engraftment success.
Approximately 2x10^6 CD34 cells/kg body weight is required for adequate hematopoiesis reconstitution, with a minimum of 1x10^6 CD34 cells/kg.
Peripheral blood stem cells (PBSCs) are collected through apheresis after stimulating with growth factors like G-CSF and plerixafor.

Immunophenotyping Principles

Routine assays for hematopoietic progenitor stem cells must be simple, sensitive, accurate, reproducible, and quick.
The ISHAGE protocol is a straightforward gating strategy using light scatter and two-color immunofluorescence analysis with pan CD45 and pan CD34 monoclonal antibodies.

CD34 Epitopes

CD34 epitopes vary and are classified based on their sensitivity to glycoproteases.
Class III epitopes are broadly distributed on endothelial cells and many hematopoietic progenitor cells, while Class I and II epitopes exhibit varying sensitivities to enzymatic cleavage.

Single Platform CD34 Enumeration

In 1998, a single-platform method for CD34 enumeration was established, using three-color flow cytometry with pan CD45, pan CD34 antibodies, and viability dye 7-AAD.
The sequential Boolean gating strategy helps to accurately identify true CD34+ cells.

7-Amino Actinomycin D (7-AAD)

7-AAD is a DNA-binding fluorescent compound that identifies viable cells, as it cannot penetrate intact cell membranes.
Strongly binds to GC-rich regions of DNA, making it useful for assessing cell viability.

Sample Types for Assay

G-CSF mobilized peripheral blood, fresh/frozen cord blood, leukapheresis products, and bone marrow samples can be used for CD34 enumeration.
Samples with leukocyte counts over 30x10^3/µL are diluted with PBS for accurate counting.

CD34 Enumeration Procedure

Mix 100µL of the sample with a premixed antibody cocktail and 7-AAD and incubate.
Use ammonium chloride lysing solution to lyse red blood cells before flow cytometry acquisition.
Viable cells are determined by the absence of 7-AAD staining.

Calculation for Viable CD34 Cells

The formula for viable CD34 cells per µL combines the number of viable CD34+ cells, the number of beads, dilution factor, and total beads counted.

Conclusion

Accurate enumeration of CD34+ cells is crucial for successful hematopoietic stem cell transplantation.
The ISHAGE protocol is a simple and efficient method, leading to reduced inter-laboratory variability.
Efficient assessment of cell viability is achieved using 7-AAD in flow cytometry.

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Description

This quiz focuses on the principles of immunophenotyping, particularly the role and expression of the CD34 transmembrane glycoprotein in various hematopoietic stem and progenitor cells. Participants will explore the significance of CD34 in distinguishing between uncommitted and committed progenitor cells in the bone marrow. Test your knowledge on this critical aspect of hematology and stem cell biology.