Hello friends welcome back to pathogenesis.
Sorry for delay in posting videos as we were
busy saving lives and taking care of COVID
patients.
This video is second part of basics of flow
cytometry.
After understanding the principles of a flow
cytometry machine, we next move to the uses
of flow cytometry in various haematological
disorders.
But before moving towards diagnosis of different
leukemias we will first understand normal
haematopoiesis in the bone marrow.
If we know how normal hematopoietic cells
behave in flow cytometry plots, then only
we can identify abnormal patterns in different
types of leukemias.
We will also understand about CD markers which
form the basis of flowcytometry are used to
label and identify different types of cells.
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Each type of cell in blood has its own unique
signature, whether it is a blast, lymphocyte,
monocyte, neutrophil or RBC. This signature
is defined by CD markers. No matter how many
times we read about CD markers and haematopoiesis,
we tend to forget it. So, in this video we
will learn to apply CD markers in a practical
way so that we can remember them.
Learning about CD markers is the key to understanding
and interpretation of flow cytometry.
What are CD markers?
The immune system works through leukocytes
interacting with each other, with other cells,
with infectious agents, and with other antigens.
These interactions are mediated by cell-surface
glycoproteins and glycolipids. It is these
protein and lipids that define the signature
of the cells and are known as CD markers.
Antibodies against these proteins and lipids
have provided powerful tools for analysis
of their structure, function, and distribution.
It provides an essential classification for
diagnostic and therapeutic purposes and have
been used widely in haematology, immunology,
diagnosis, and therapy.
Flow cytometry takes advantage of these CD
markers to identify a cell as lymphocyte or
promyelocyte or a blast. Flow cytometry analyse
each cell based on the CD markers present
and the number of those cells present in a
sample to label it as normal or leukemic.
USES of CD markers and Flow cytometry
CD markers are critical for the diagnosis
of lymphomas and leukaemia.CD Markers are
especially useful for identification of various
leukocyte populations using flow cytometry.CD
marker-specific antibodies have been widely
used for cell sorting and their identification
in flow cytometry-based machines
In addition, CD markers have become significantly
important for cancer treatment. Some therapeutic
antibody drugs have been designed to target
cells that have a CD marker for example Rituximab
to CD20 for lymphomas and leukemia treatment;
Alemtuzumab to CD52 for chronic lymphocytic
leukemia and T-cell lymphoma treatment.
Lets’ begin with learning about haematopoiesis
by studying CD markers on different types
of cells in the bone marrow.
Stem cells are positive for CD34, 117 and
negative for CD 38. CD 45 is also known as
common leukocyte antigen and is present on
all leucocytes. Granulocytes express CD13,
33, 11b and 15. Monocytes are positive for
CD64, 4, 14, 11b and 16. T lymphocytes express
CD3, 7, 5, 2, 4, and 8. T helper cells are
Cd4 positive. T regulatory cells are CD4 and
CD25 positive. Cytotoxic T cells are positive
for Cd8. B lymphocytes are positive for CD19,
20, 79a and 22. Thrombocytes or platelets
are positive for CD41 and 61. Natural Killer
cells are positive for CD16 and 56.
So, after getting an overview of CD markers
on different types of hematopoietic cells
lets understand normal haematopoiesis and
maturation.
A hematopoietic cell during its maturation
pathway from a blast to a mature cell undergoes
various immunophenotypic changes. Some markers
which already present on a blast or stem cell
are lost while others are gained sequentially
during maturation of a cell. Understanding
haematopoiesis, helps in understanding the
maturation pattern of a cell. Only when we
understand the normal haematopoiesis, we can
make use of flow cytometry to pick out what
is abnormal.
All hematopoietic cells are derived from multipotent
stem cells. This diagram depicts the overline
of bone marrow haematopoiesis which begins
with multipotent hematopoietic stem cell.
It further gives rise to common myeloid progenitor
and common lymphoid progenitor cells. Common
myeloid progenitor cells are the origin of
mature RBCs, platelets, neutrophils, eosinophils,
basophils and monocytes. In today’s video
we will learn all about granulopoiesis or
development of granulocytes.
Haematopoiesis begins with Stem cells which
are positive for CD34 and CD117 and negative
for CD38.
Blasts have immunophenotypic profile similar
to stem cells or we can say that blasts may
have one or more markers expressed by the
stem cells.
Myeloblasts develop from common myeloid progenitor
cells. They mature through myeloblast, promyelocyte,
myelocyte, metamyelocyte to form neutrophils.
Myeloblasts or blasts of myeloid lineage express
CD34 or CD117 or HLA-DR.
And as granulocytes mature through the stages
of myeloblast, promyelocyte, myelocyte, metamyelocyte,
band from and neutrophils, there is total
loss of CD34 and CD117.
Promyelocytes are CD34 and HLA DR negative
but express CD117. Acute promyelocytic leukemia
or APML arises from promyelocytes and has
characteristic CD markers on flow cytometry
plots.
CD13 and CD 33 are expressed by all stages
of granulocytes but can be downregulated or
upregulated in some type of leukemia where
they act as leukemia associated aberrant immunophenotype
or LAIP on flow cytometry
CD15 is expressed from promyelocyte stage
to neutrophils with increasing intensity.
CD16 is a maturity marker and is expressed
in band forms and neutrophils.
CD 11b is a monocytic as well as maturity
marker of granulocyte series.
Moving on to haematopoiesis in Monocytic Lineage
Monoblasts also arise from common myeloid
progenitor cells. They progress to form promonocytes
and finally monocytes. Macrophages are tissue
monocytes.
Monoblasts are CD34, CD117 and HLA-DR+. While
CD34 and CD 117 expression is lost during
maturation to monocytes, expression of HLA-DR
is retained in monocytic lineage cells after
blast stage.
CD13 and CD33 are expressed through out maturation
cycle but CD33 is brighter in monocytes.
Monocytes during haematopoiesis gain CD11b,
CD14 and CD64 from the stage of promonocytes
with increasing expression to mature monocytic
stage. They are also known as the monocytic
markers.
Acute myelo-monocytic leukemia, acute monoblastic
leukemia, juvenile myelomonocytic leukemia
are few of the hematopoietic disorders arising
from disorders of monocytic series which we
will be learning in upcoming videos.
Now we will be moving next to erythropoiesis
or development of RBC’s and megakaryopoiesis
or development of PLATLETS.
Erythrocytes and megakaryocytes originate
from megakaryocyte erythrocyte progenitor
cell which develops from common myeloid progenitor
cell.
Megakaryoblast is the precursor of mature
megakaryocyte. It shows CD34 expression along
with weak expression of CD41 and CD61. CD38
and CD42 are absent. As they matures they
lose CD34 and gain CD38 along with very strong
expression of CD41, CD61 and CD42.
RBC cycle begins from proerythroblast leading
to basophilic erythroblast, polychromatophilic
erythroblast, ortho-chromatophillic erythroblast,
reticulocyte and finally RBCs.
A proerythroblast is characterized by positivity
for CD34 and CD117 which are immaturity markers,
along with CD36, CD71 and CD235a positivity.
CD34 and CD117 are lost from next stage onwards.
CD235a which is a pan-erythroid marker is
present across all stages of erythrocyte.
Whereas, CD 36 and CD71 are lost on mature
RBCs.
Flow cytometry utilises maturation and development
patterns during haematopoiesis as foundation
for diagnosis of various disorders.
After covering CD markers in myeloid, monocytic,
erythroid lineage in this video we can now
understand the origin of most myeloid disorders.
In our next video we will be covering of hematopoieis
of lymphoid cells including B-cell and T-cell
development along with their respective CD
markers and origin of various lymphoid hematopoietic
disorders.
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