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How to Establish the Cut-off Values when Estimating Fragmented RBCs by Flow Cytometry

Address correspondence to Kyungja Han, M.D., Department of Clinical Pathology, Catholic University Medical College, St. Mary’s Hospital, Youngdeungpo-gu, Youido-dong 62, Seoul, 150-713 Korea (south); tel: 82 2 3779 1297; fax 82 2 783 6648; e-mail hankja{at}catholic.ac.kr.

Keywords: fragmented RBC, anti-hemoglobin antibody, flow cytometry

There have been several recent case reports of thrombotic thrombocytopenic purpura and hemolytic uremic syndrome (TTP/HUS), associated with drugs, cancer, and other diseases[1–3]. To facilitate the diagnosis of TTP/HUS, we described a new, simple flow cytometric method for detecting fragmented RBCs using anti-hemoglobin antibody (anti-Hb) in hypotonic solution [4].

Several scientists have complained that it is difficult to establish cut-off values accurately when flow cytometry results are read after staining RBCs with anti-Hb antibody. We found that staining the RBCs with anti-Hb in normal saline as a control is better than staining them with isotypic control antibody to establish the cut-off values. The intensities of fluorescent dyes conjugated with anti-Hb and isotypic control antibody may be different. This may explain why using normal saline and the same anti-Hb antibody is a better negative control than using isotype-matched antibody. Because the fragmented RBCs are stained with anti-Hb antibody only in hypotonic solution, and not in normal saline, it is easy to establish cut-off values using the same antibody in normal saline. The difference between the positive signals in normal saline and in 0.6% NaCl is due to fragmented RBCs.

We studied 150 peripheral blood samples, 50 samples showing fragmented RBCs and 100 samples from normal people. Twenty thousand cells in the preset gate (Fig. 1) were analyzed by flow cytometry. Because some fragmented RBCs were expected to be smaller than intact RBCs, a large gate that included all intact RBCs and fragmented RBCs was preset. This method could eliminate the small number of analyzed cells when there were many debris signals. The cut-off values were established using anti-Hb and normal saline, where 0.5 to 1.0% of cells were included as positive. Using anti-Hb in 0.6% NaCl and same cut-off values, the percentage of positive cells was read on a dot-plot (Fig. 1). Although only one reagent (phycoerythrin conjugated anti-Hb) was used, a dot-plot was better than a histogram to rule out false positive signals by debris, which usually distributed in a diagonal line on a dot-plot.

View larger version (17K): [in this window] [in a new window]   Fig. 1. Dot-plots of RBCs from a patient after allogeneic bone marrow transplantation. The RBCs were stained with phycoerythrin conjugated anti-Hb antibody. The left dot-plot was obtained with a preset large gate; the center dot-plot shows a few positive signals with anti-Hb in normal saline; the right dot-plot shows many positive signals with anti-Hb in 0.6% NaCl. The difference in this case was 1.70%.

In summary, establishment of cut-off values using anti-Hb antibody in normal saline is recommended for estimating fragmented RBCs by flow cytometry. The difference between the positive percentages using 0.6% NaCl and normal saline is reported as the percentage of fragmented RBCs.

References

1. Gordon LI, Kwaan HC. Cancer- and drug-associated thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. Semin Hematol 1997;34: 140–147.[Medline]
2. Porta C, Danova M, Riccardi A, Bobbio-Pallavicini E, Ascari E. Cancer chemotherapy-related thrombotic thrombocytopenic purpura: biological evidence of increased nitric oxide production. Mayo Clin Proc 1999;74:570–574.[Abstract/Free Full Text]
3. Bennett CL, Davidson CJ, Raisch DW, Weinberg PD, Bennett RH, Feldman MC. Thrombotic thrombocytopenic purpura associated with ticlopidine in the setting of coronary artery stents and stroke prevention. Arch Intern Med. 1999;159: 2524–2528.[Abstract/Free Full Text]
4. Lee W, Kim Y, Lim J, Kim M, Lee EJ, Lee A, Lee KY, Kang CS, Kim SY, Han K, Pai SH. Rapid, sensitive diagnosis of hemolytic anemia using antihemoglobin antibody in hypotonic solution. Ann Clin Lab Sci 2002;32:37–43.[Abstract/Free Full Text]

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