From: D. Robert Sutherland (rob.sutherland@utoronto.ca)
Date: Fri Dec 20 1996 - 10:05:13 EST
While the 'Milan' protocol for CD34 enumeration in cytokine-mobilised PB suggested by Ulrik Sprogoe-Jakobsen was an improvement over some earlier techniques, this simple protocol cannot accurately enumerate CD34+ cells in more heterogeneous samples such as bone marrow (or CD34+ cells selected therefrom e.g., from Cellpro columns) (see Experimental Hematology 22: 1003-1010, 1994). Similarly, it is not too good on cord blood and can be badly 'fooled' if samples are not in highly viable condition, i.e., it cannot discriminate between staining of bona fide CD34+ cells and non-specific staining of non-CD34+ cells exhibiting low side scatter. This can be a real problem if the sample is contaminated with significant numbers of dead/dying/apoptopic lymphoid cells. It also relies upon an isotype-matched control to set the positive cell analysis region for CD34+ cells. An increasing number of investigators have concurred that this is a dangerous practice for rare event analysis such as CD34+ cell detection in PB etc., where CD34+ cell numbers can be in the 0.02%-o.04% range. Some isotype matched controls stain more events non-specifically than are specifically stained by the CD34 antibody used. Other isotypes may stain virtually nothing in a particular sample, leading to the detection of many more CD34+ events than in fact are contained in a particular sample. We have developed a two-colour (CD34/CD45), four parameter CD34+ cell detection protocol for ISHAGE (Journal of Hematotherapy 5:213-226, 1996) that addresses a number of these issues and is more broadly useful than the single fluorescence parameter analysis at the heart of 'Milan'-type protocols. While the recommended gating strategy may require a little more attention, accurate rare event analysis warrants this extra effort and in our experience, the ISHAGE protocol is far more accurate, sensitive and reliable compared to relatively unsophisticated FL1- (or FL2-) versus side scatter analysis. In the ISHAGE protocol, the isotype control is simply used to enumerate non-specifically stained events that otherwise exhibit the staining and light scatter characteristics of true CD34+ cells. We have also recently demonstrated that dead cells are 'gated out' by the sequential, cumulative gating strategy at the heart of this protocol. While some (vested interest) groups have suggested that this gating strategy using CD45 as a counter stain results in the ISHAGE protocol 'missing' CD34+/CD45- cells, we have yet to detect such events in the hundreds of samples of normal marrow, PB, cord blood or cytokine-mobilised blood. Like others, we too have noted occasional pre-B type leukemias that express very low levels of CD45. However, non-malignant CD34+ cells all appear to express CD45, albeit at levels lower than expressed by lymphocytes. The ISHAGE protocol can be adapted to include a third antibody conjugate so that the qualitative composition of bona fide CD34+ cells can be assessed (for example see Experimental Hematology 23:1619-1627, 1995). Alternatively, in an allotransplant setting, we can incorporate CD3 in a three-colour analysis so that we can enumerate CD34+ cells and contaminating lymphocytes and T lymphocytes in for example, Cellpro selected CD34+ cell suspensions. We can also add fluorescent microspheres to the basic two/three colour protocol to generate an absolute CD34+ cell count from a single instrument platform, without having to use an automated hematology analyser. Thus this protocol is very flexible and can be used in a number of different clinical and research laboratory settings on both normal and abnormal hematologic samples. D. Robert Sutherland ISHAGE Stem Cell Enumeration Committee, Oncology Research, Toronto Hospital, 67 College Street, Ontario M5G 2M1, Canada.
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