Download quantitation of cd34+ cells

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1628
CORRESPONDENCE
QUANTITATION OF CD34+ CELLS
To the Editor:
The recent study by Brugger et all reflects the impact of
quantitative determinations of circulating hematopoieticcells identified as CD34+ cells. However, the goal of reliable determinations
on a parameter such as CD34 cells/pL of blood stresses the need
for proper quantitative methods.
Brugger et a1 report k mean number of 300 CD34 cells/mL of
blood in steady-state healthy adults, with levels up to 2 x 106/mL
of blood in patients after chemotherapy and treatment with
cytokines. The method used for the quantitation is reported to be
microscopical evaluation of slides after immunocytochemistry (using CD34 antibody) of peripheral blood mononuclear cells. Although they are essential for proper understanding of the data, no
mention is made of the total number of cells evaluated nor of the
percentage (“permillages”) of cells considered to be CD34+.
Therefore, we have made our own calculations. Given a mean
number of about 2,500 1ymphocyteslpL of blood (there is no
absolute leukocyte count given in the report) and the reported
mean number of 0.3 CD34 cells/pL of blood, it can be deduced
that Brugger et a1 detected 1cell considered CD34+ from approximately 7,500 cells looked at. In view of the classical reports of
Rumke2p3focussingon the 95% confidence limits when performing
microscopical differentials, one may question the reproducibility
and reliability of the data reported by Brugger et a1 observing less
than 0.01% of cells. Furthermore, it is noteworthy that Brugger et
a1 assign a sensitivityof 0.01% to their microscopical method. The
assignment of this high degree of sensitivity is directly linked to a
previous report in which Bross, one of Brugger’s coauthors,
coauthored! Interestingly, in that report,” no hint is given to
suggest such a high degree of sensitivity.
Furthermore, the authors state that the mean numbers of
colony-forming units granulocyte-macrophage(CFU-GM) per milliliter of blood and of burst-forming units-erythroid (BFU-E) per
milliliter of blood determined from steady-state healthy adults
were 130 and 85, respectively. In view of the mean number of 300
CD34 cells/mL of blood, a cloning efficiency of about 75% can be
deduced. This, in fact, is in some contrast to the clonogenities
reported by others applying flow cytometry-based determinations
of blood CD34 cell^.^-^
Major incongruities are also found in another report on quantitative determinations of blood CD34 cells published in Blood.’ Siena
et a17 stated that with single samples of 50 FL of blood they
performed the flow cytometrical analyses of CD34 cells with 10,OOO
cells per determination in each case. This volume (50 FL)of blood
was considered necessary samples with leukocyte counts down to
150/pLof blood. Perhaps we are wrong in calculating, but we think
that 150 x 50 = 7,500. So, with samples with a leukocyte count less
than 200/pL of blood there will not even be the l0,OOO cells reported
when drawing the 50 pL, not to think of cell loss associated with
centrifugationand the setting of the gate on the cytometer.
STEFAN SERKE
DIETER HUHN
UnivemitatswinikumMlf V ~ h o w - C h h ~ n b w g
Abteihg Innse Me& und Poliklinik
HamatorogLschesZ e n b u h b r
Bedit&Germany
REFERENCES
3. Riimke C L The imprecision of the ratio of two percentages
observed in differential white blood cell counts: A warning. Blood
R, Kanz L Mobilization of peripheral blood progenitor cells by
sequential administration of interleukin-3 and granulocyteCells 11:137,1985
4. Bross KJ, Pangalis GA, Staatz CG, Blume KG: Demonstramacrophage colony-stimulatingfactor following polychemotherapy
tion of cell surface antigens and their antibodies by the peroxidasewith etoposide, ifosfamide, and cisplatin. Blood 79:1193,1992
antiperoxidase method. Transplantation 25:331,1978
2. Riimke C L The statistically expected variability in differen5. Serke S, Sauberlich S, Huhn D: Multiparameter flowtial leukocyte counting, in Koepke JA (ed): Differential Leukocyte
cytometricalquantitation of circulating CD34-positivecells: ComeCounting. Skokie, IL, College of American Pathologists, 1979, p 39
1. Brugger W, Bross K, Frisch J, Dern P, Weber B, Mertelsmann
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
CORRESPONDENCE
1629
lation to the quantitation of circulating haemopoietic progenitor
cells by in vitro colony-assay. Br J Haematol77:453,1991
6. Ema H, Suda T, Miura Y, Nakauchi H: Colony-formation of
clone-sorted hematopoietic progenitors. Blood 75:1941, 1990
7. Siena S, Bregni M, Brando B, Belli N, Ravagnani F, Gandola
L, Stern AC, Lansdorp PM, Bonadonna G, Gianni AM: Flow
cytometry for clinical estimation of circulating hematopoietic
progenitors for autologous transplantation in cancer patients.
Blood 77:400,1991
RESPONSE
We would like to respond to the letter by Serke and Huhn
regarding our recent manuscript published in Blood.
Serke and Huhn comment on the identification and quantification of CD34+ cells by the immunoperoxidase slide technique used
in our study. In addition, they criticize the way Siena et a12 have
analyzed CD34+ cells by flow cytometry. We will focus on the issues
relevant to our report.
We described the differential mobilization of hematopoietic
progenitor cells into the peripheral blood after a standard-dose
chemotherapy regimen with W16, ifosfamide, and cisplatin (VIP)
and the administration of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-3 (IL-3) + GM-CSF. CD34+
cells were evaluated either by flow cytometry or by an immunoperoxidase staining method. This method was originally described by
Bross et a1 197S3; the adhesion slides are now commercially
available.
For the studies performed by our group, the use of the
immunoperoxidase staining technique offers several advantages
when compared with flow cytometry:
(1) It can be easily applied at very low numbers of absolute
white blood cell counts in the peripheral blood, drawing only 2 mL
of blood. Because kinetic analyses for the recruitment of peripheral blood progenitor cells were one of the major objectives of our
study, it was important to detect the increase of progenitor cells as
early as possible. Those cells started to increase at day 7, when
peripheral blood white blood cell counts still were less than
1,000/KL.
(2) In addition to the specific staining with the monoclonal
antibody, all cells present on the adhesion slide can be analyzed
with respect to their morphology. Thus, there are no false positive
results due to the unspecific binding of anti-CD34 antibodies to
immature granulocytes or monocytes. It is well established that
there are overlapping gates for hematopoietic progenitors and
monocytic cells as analyzed by flow cytometry.
(3) At least 10,000 cells (as enumerated with the help of a grid)
are attached on each spot of the slides and at least two separate
spots were analyzed for CD34 expression. All positive cells can be
easily detected and identified by the brown membrane staining and
the typical morphology. The assay is reproducible and reliable.
(4) The percentage of CD34+ cells at the day of maximal
numbers of progenitor cells was up to 42%, with a median value of
18% (range, 5% to 42%), as measured by the immunoperoxidase
method. Therefore, we are not looking at cells below the percentage of 0.01, as mentioned by Serke and Huhn.
In our study, the number of CD34+ cells was considerably low
only under steady-state conditions (0 to 30 cells per spot). To
calculate a cloning efficiency of CD34+ cells under steady-state
hematopoiesis by dividing the median number of CD34+ cells by
the median number of clonogenic progenitors (as suggested by
Serke and Huhn) is very errorsome, because under baseline
conditions the numbers of CD34+ cells, as well as clonogenic
progenitor cells (as shown in our report), are very low and, thus,
the variation might be very high. Due to this fact, the calculated
cloning efficiencies from different investigators might be different.
Serke and Huhn should be aware that the only way to reliably
determine the cloning efficiency in this setting is by sorting CD34+
cells and to subsequently determine their clonogenic capacity.
(5) The sensitivity of flow cytometry is approximately 0.5% to
1%; cell populations of less than 0.5% are inadequately estimated
by flow cytometry as to draw major conclusions from whether a
population comprises 0.1% or 0.3%, for example.
Interestingly, using flow cytometry, Serke and Huhn report a
nearly identical percentage of CD34+ cells in normal volunteers
when compared with our study using the immunoperoxidase
technique!
In our lab, we routinely use flow cytometry to determine the
timing for the routine harvest of peripheral blood progenitor cells
in our patients. Moreover, we apply this simple technique for dualand triple-color analyses of CD34+ cells.
We have studied in detail both methods in comparison for
CD34+ cell populations greater than 0.5% and found that the
results are nearly identical at percentages greater than 1% CD34+
cells.
In summary, we have used a very sensitive, reliable and reproducible method to detect circulating CD34+ cells. The method and its
application has been published in numerous international journals
since its original publication by Bross and B l ~ m e . ~ - ~
WOLFRAM BRUGGER
KLAUS BROSS
ROLAND M E R T E L S M A "
LOTHAR KANZ
Klinikum der Albert-Ludwigs- UniversitatFreiberg
Medizinische Universitatsklinikund Poliklinik
Abteilung Innerre Medizin I
Hamatologie, Onkologie
Freiberg, Germany
REFERENCES
cytometry for clinical estimation of circulating hematopoietic
1. Brugger W, Bross K, Frisch J, Dern P, Weber B, Mertelsmann
R, Kanz L Mobilization of peripheral blood progenitor cells by
progenitors for autologous transplantation in cancer patients.
Blood 77400,1991
sequential administration of IL-3 and GM-CSF following polychemotherapy with etoposide, ifosfamide, and cisplatin. Blood
3. Bross KJ, Pangalis GA, Staatz CF, Blume KG: Demonstra791193,1992
tion of cell surface antigens and their antibodies by the peroxidase2. Siena S, Bregin M, Brando B, Belli N, Ravagnani F, Gondola
antiperoxidase method. Transplantation 25:331, 1978
L, Stern AC, Landsdorp PM, Bonadonna G, Gianni AM: Flow
4. Fauser AA, Kanz L, Bross KJ, Llihr GW: T cells and probably
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
1630
B cells arise from the malignant clone in chronic myelogenous
leukemia. J Clin Invest 75:1080,1985
5. Morich FJ, Momburg F, Moldenhauer G, Hartmann KU,
Bross KJ: Immunoperoxidase slide assay (IPSA)-A new screening method for hybridoma supernatants directed against cell
CORRESPONDENCE
surface antibodies compared to other binding assays. Immunobiology 164:192,1983
6. Kanz L, Bross KJ, Mielke R, Liihr GW, Fauser AA: Fluorescence-activated sorting of individual cells onto poly-L-lysinecoated slide areas. Cytometry 7:491,1986
From www.bloodjournal.org by guest on August 3, 2017. For personal use only.
1992 80: 1628-1630
Quantitation of CD34+ cells [letter; comment]
S Serke and D Huhn
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