In the past two decades, flow cytometry has been the
best diagnostic tool for several diseases in the fields of oncology,
hematology, immunology, and blood banking. FCM is now available in more and
more clinical institutes once the instruments are less expensive and more
The principle of FCM is the immunophenotyping of
specimens such as urine, CSF, blood, bone marrow and serous fluids. Optical and
fluorescence measurement of cells is realized, including DNA and RNA as well.
The size of the cell is measured via the forward light
scatter, and the complexity and fluorecence via the side light scatter. Main
compartments of the flow cytometer include the lasers and the detectors
(different detector for the fluorescence), the interrogation point where single
cells pass through the light beam, and the computer system where the analysis
is performed in a histogram Brown, M. and Wittwer, C., 2000 .
Fig. 8 Compartments of a flow cytometer (Source: Sanguine Bio
Fig.9 Example of histogram representation in FCM analysis. There
are small-sized cells with small
internal complexity on the left(lymphocytes), medium-sized cells with slightly
more internal complexity (monocytes), and large-sized cells with more internal
complexity on the right(other granulocytes) (Source: ResearchGate).
of FCM in PNH and related disorders. Today FCM is considered to be the method of choice in
detection of GPI anchor-deficient cells. Flow cytometers can now analyze
minimum six fluorescence parameters, leading to the detection of small
populations of deficient cells. Patients recommended for PNH testing with FCM
are those with clinical evidence of hemolysis, aplastic anemia, refractory
anemia-MDS without hemolysis, and unexplained hemoglobinuria and/or thrombosis
Parker et al., 2005, Borowitz et al., 2010.
In FCM CD59 is most commonly evaluated, as its
expression occurs in all hematopoietic cells. However, quantitation of CD55
also takes place. In order to exclude single isolated deficiency of GPI-AP, at
least two GPI-APs need to be assessed, with two different monoclonal antibodies
in two different cell lineages Brodsky, 2009.
10 Granulocytes are represented, 26% of which do not express CD55 and CD59,
located at the lower left quadrant. At the higher right quadrant, 74% of granulocytes are normal, expressing both
GPI-APs Source: Nissen-Meyer et al.,
Screening of only RBCs has led to false negative
results, especially when there was a recent hemolytic episode or blood
transfusion(the latter shows an increase in normal expression of CD55 and CD59
on the histogram). Granulocytes and monocytes are not influenced by blood
transfusion, that is why they present a more accurate picture of the PNH clone
size when measured Parker et al., 2005.