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In this issue, in addition to the exciting review article by Dr. Thomas Spitzer and colleagues that was highlighted by both the Editor-in-Chief and Dr. H. Lazarus, there are four articles focused on how flow cytometry (FC) can improve treating patients with leukemia and lymphoma. They include a Brief Communication about a novel flow cytometric assay to verify liposomal cytarabine treatment of lymphoma beyond the blood–brain barrier. There are three original articles describing new FC-based methods for chemotherapy efficacy assessments. One describes monitoring mitoxantrone efflux in acute myeloid leukemia (AML). Myeloid nuclear differentiation antigen (MNDA) expression in a large patient population using FC is described in the second original article. The third describes a sensitive technique to detect leukemia and lymphoma in bronchoalveolar lavage specimens. This issue also features two original articles dealing with new multiparametric automated immunophenotyping software. The introduction of automated software in clinical cytometry has been relatively slow. Automated standardized performance evaluation with multivariate classification would go a long way to assure consistent quality data generation in clinical immunology. It is encouraging to see software experts take on two tedious FC-based protocols to make such assays more reliable, reproducible. Although these two articles are promising in the simplicity, reliability, speed and objectivity of their approaches, it is difficult to say how soon they will be integrated into routine clinical application. Both software systems were developed for commercial distribution and rely on a newly patented technology utilizing probability state modeling (PSM) (1, 2).

THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE

  1. Top of page
  2. THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE
  3. A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT
  4. ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL
  5. FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE
  6. AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS
  7. A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA
  8. ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY
  9. LITERATURE CITED

Central nervous system (CNS) is known to be a sanctuary for leukemic cells as cytostatic drugs usually ineffective at crossing the blood–brain barrier. Oncological patients at high risk of CNS complications are frequently administered cytarabine (DepoCytes™) intrathecally. In this issue, Stacchini et al. [(3); this issue, page 280] report a study where cytarabine is administered in encapsulated in spherical liposomal formulation of 3–30 microns. This method has the advantage of delivering the cytarabine where it is required and prolonging its half-life. Until now, the liposomes containing cytarabine were undetected in cerebrospinal fluid (CSF) specimens. This occurred because the cytospin staining procedure includes washing with alcohol, which dissolves all remaining liposome spherules. It is possible to identify residual liposomes in CSF with standard dual light scatter plot. Thanks to the introduction of flow cytometric analysis of CFS to identify neoplastic involvement, an additional issue is resolved. This discovery also requires that clinicians be warned of the possibility of falsely elevated leukocyte absolute counts with some automated cell counters in CFS of patents receiving DepoCyte™ liposomes, as the spherules may look similar to leukocytes.

A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT

  1. Top of page
  2. THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE
  3. A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT
  4. ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL
  5. FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE
  6. AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS
  7. A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA
  8. ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY
  9. LITERATURE CITED

An article by Kim et al. [(4); this issue, page 283] entitled: “flow cytometry-based assessment of mitoxantrone efflux from leukemic blast varies with response to induction chemotherapy in acute myeloid leukemia” deals with a FC-based assay that has some predictive value. It alerts physicians to possible chemotherapeutic drug resistance during treatment of AML. The assay measures median fluorescent intensity of AML blast using mitoxantone with and without cyclosporine A incubation, an inhibitor of ABC transporter. The new assay is broad in range and thus covers most of the ABC transporter-related therapy failures. The authors outline the universal nature of their approach as compared to the limited practical value of ABC transporter dysfunction detection using gene-product-specific functional assays. They suggest that such quick assay should be included in the work-up on all newly diagnosed AML patients. The MFRI-based FC test could be useful for selecting therapy options for individuals where toxicity of induction chemotherapy is predictable. Such intervention will avoid chemotherapy that will turn out to be refractory (5, 6).

ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL

  1. Top of page
  2. THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE
  3. A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT
  4. ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL
  5. FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE
  6. AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS
  7. A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA
  8. ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY
  9. LITERATURE CITED

MNDA is expressed at the highest levels in mature granulocytes and monocytes. It is believed that the MNDA expression is lower in patients with myelodysplastic syndromes (MDS) (7). In this issue, Bellos et al. [(8); this issue, page 295] explored this possibility in the article with title: “evaluation of flow cytometric assessment of myeloid nuclear differentiation antigen (MNDA) expression as a diagnostic marker for myelodysplastic syndromes in a series of 269 patients.” The authors set out to answer a clinically relevant question. Can MNDA expression in bone marrow (BM) cells help with a more reliable assessment of dyspoiesis? The answer is not absolutely clear while the addition of FC data increased the rate of correct identification by 13% and it also increased false-positive diagnosis by 14%. It is clear from this report that more similarly meticulous and comprehensive studies are required to fine-tune future MDS panel(s).

FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE

  1. Top of page
  2. THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE
  3. A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT
  4. ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL
  5. FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE
  6. AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS
  7. A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA
  8. ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY
  9. LITERATURE CITED

There is continued interest to improve assay sensitivity to detect hematolymphoid neoplasms (HNL). For almost a decade, it has been know that FC exceeds the sensitivity of cytomorphology (CM) to detect HNL (9). It is less known how effective FC is with specimens obtained with the frequently used bronchoalverol lavage technique. Song et al. [(10); this issue, page 305] communicate their results in this issue with the title: “flow cytometry increases sensitivity of detection of leukemia and lymphoma cells in bronchoalveor lavage specimens.” The premise was that FC is more sensitive at detecting disease in blood specimens; perhaps, it will also do the same with other body fluids. They conclude that FC is more sensitive method when compared to CM. However, when combining FC and CM, a dual method has superior sensitivity that exceeds either assay when used alone.

AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS

  1. Top of page
  2. THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE
  3. A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT
  4. ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL
  5. FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE
  6. AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS
  7. A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA
  8. ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY
  9. LITERATURE CITED

The first ISHAGE protocol was published 14 years ago (11). It is exciting to see a new automated approach to stem cell analysis. This article is offering a PSM strategy using list mode data to replace traditional single platform gating protocol that does require considerable operator experience. “Beyond gating-,” is a concept introduced by Finn in 2009 (12). In 2010, Bagwell embraced this concept and pushed it to a practical plateau by introducing PSM (13). In this issue, Herbert et al. [(14); this issue, page 313] describe new automated software in: “Automated analysis of flow cytometric data for CD34+ stem cell enumeration using a probability state model.” The strategy is to catalog all events into four categories: (A) beads, (B) debris, (C) intact dead, and (D) intact viable stem cells. If an event does not fit in the above-mentioned PSM classification system, it remains an unclassified event. The authors define cell types with expression profiles. They collect list mode file data until at least 500 stem cells are detected or 7 min elapsed. The flow cytometer is adjusted to a low CD45 signal threshold and no threshold limitations for FSC. Herbert et al. claim that PSM provides reproducibility, objectivity, speed, and accuracy higher or at least as good as ISHAGE. The bulk of their analyzed data is from cord blood. The resulting concordance analysis is impressive. It will be interesting to guess how soon PSM analysis will be integrated into quality clinical laboratory management practices.

A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA

  1. Top of page
  2. THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE
  3. A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT
  4. ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL
  5. FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE
  6. AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS
  7. A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA
  8. ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY
  9. LITERATURE CITED

In 2007, Richards et al. introduced a FC-based assay for the detection of glycosylphosphatidylinositol-deficient (GPI) clones in paroxysmal noctural hemoglobinuria (15). More recently, in 2010, Borowitz el al. published guidelines for this assay (16). Currently, a manually managed FC protocol is the standard method for GPI detection. It is clear that automated software driven analysis would simplify and speed-up a complex time-consuming assay. In this issue, Miller et al. [(17); this issue, page 319] have evaluated just such strategy in an article titled: “Automated analysis of GPI-deficient leukocytes flow cytometric data using GemStone™.” The premise is quite simple; all observed cells are classified into five possible categories. (B) normal myeloid, (C) GPI-deficient myeloid, (D) normal monocyte, (E) GPI-deficient monocyte, and (A) all remaining unclassified cells. The four classified cell categories are based on the presence of markers: CD15, CD45, CD64, SSC, FLAER, and CD24. Based on expression levels of the above-mentioned surface, markers are further subdivided into C, D and E categories. Final assignment to the four categories of cells is based on stochastic variability calculations (13). A side-by-side automated versus manual gated (predicate method) evaluation was conducted using over 500 samples with 10% having GPI-deficient lymphocytes. The results were highly correlated. This automated software strategy looks promising. Readers of the Journal may also access an exciting online link to a video provided by these authors that demonstrate the PSM software in action analyzing a PNH case. Additionally, the data file used for this analysis may also be downloaded from the article's Supporting Information on the Wiley Online Library website. Available at www.wileyonlinelibrary. com

ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY

  1. Top of page
  2. THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE
  3. A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT
  4. ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL
  5. FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE
  6. AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS
  7. A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA
  8. ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY
  9. LITERATURE CITED

Noted in the journal's previous issue were the abstracts from the International School on Practical Cytometry (ISPC) workshop (18). The second ISPC was held in Moscow from August 26–30 in 2011. It was organized under the auspices of the Russian Scientific Hematology Center, and the Russian Academy of Medical Sciences. ISPC was established in 2009 to provide every 2 years a comprehensive overview of technologies and applications of flow and imaging cytometry and a forum to encourage stimulating and practical discussions. The workshop had about 60 registered scientists from Russia and other Commonwealth of Independent States (CIS) countries. The ISPC workshop covered a wide selection of state-of-the-art lectures and seminars with invited speakers who are internationally recognized. About half of the faculty was from the USA. Dr. Lieberman (Boston) delivered the keynote lecture. She presented an up-to-date account on how cytotoxic cells are effective as killer cells. There were 22 lectures covering a spectacular variety of topics all related to cytometry. The lectures included both commercial and academic presentations. Speakers from the Boston area represented about half of the US academic delegation. Beside the keynote address, Dr. Barteneva gave two seminal presentations. Dr. Ponomarev covered a relatively new application for cytometry, microRNA (miRNA) studies and Dr. Preffer provided a contemporary perspective on leukemia and lymphoma diagnostics. There were two speakers from multilingual countries: Drs. Volvokov and Faster-Kan from Canada and Switzerland, respectively. The balance of the speakers was mostly from Moscow. There were 11 posters displayed. The 2011 poster award winner was A. Yakimenko from Hematology Corp. Inc., Moscow with “Analysis of the proaggregation capabilities of activated thrombocytes from different populations”. The overall scientific standard throughout the workshop was extremely high. These abstracts may be viewed in www.wileyonlinelibrary.com; in volume 82B, number 4, 2012 [DOI: 10.1002/cyto.b.21021] pages o1–o17.

LITERATURE CITED

  1. Top of page
  2. THANKS TO A NOVEL CLINICAL FC APPLICATION: LIPOSOME ENCAPSULATED DRUGS IN CNS SPECIMENS ARE NOW VISIBLE
  3. A POSSIBLE NEW TOOL TO PREDICT DRUG RESISTANCE TO AML TREATMENT
  4. ASSESSMENT OF MNDA EXPRESSSION IN BM CELLS WITH FC HELPS WITH THE EVALUATION OF DYSPLASIA WHEN IT IS PART OF AN MYELODYSPLASTIC SYNDROMES PANEL
  5. FC IMPROVES LUKEMIA AND LYMPHOMA DETECTION WHEN SPECIMENS ARE FROM BRONCHOALVEOR LAVAGE
  6. AUTOMATED STATISTICAL MODEL VERSUS ISHAGE SINGLE PLATFORM GATING METHOD TO ENUMERATE STEM CELLS
  7. A STANDARDIZED AUTOMATED METHOD TO DETECT PAROXYSMAL NOCTURAL HEMOGLOBINURIA
  8. ABSTRACTS FROM THE INTERNATIONAL SCHOOL ON PRACTICAL CYTOMETRY
  9. LITERATURE CITED
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    Stacchini A, Demurtas A, Aliberti S. Flow cytometric detection of liposomal cytarabine in cerebrospinal fluid of patients treated with intrathecal chemotherapy. Cytometry B Clin Cytom 2012; 82B: 280282.
  • 4
    Kim HP, Bernard L, Berkowitz J, Nitta J, Hogge DE. Flow cytometry-based assessment of mitoxantrone efflux from leukemic blasts varies with response to induction chemotherapy in acute myeloid leukemia. Cytometry B Clin Cytom 2012; 82B: 283294.
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    McClintock-Treep SA, Briggs RC, Shults KE, Flye-Blakemore LA, Mosse CA, Jagasia MH, Shinar AA, Dupont WD, Stelzer GT, Head DR. Quantitative assessment of myeloid nuclear differentiation antigen distinguishes myelodysplastic syndrome from normal bone marrow. Am J Clin Pathol 2011; 135: 380385.
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    Bellos F, Alpermann T, Gouberman E, Haferlach C, Schnittger S, Haferlach T, Kern W. Evaluation of flow cytometric differentiation antigen expression as a diagnostic marker for myelodysplastic syndromes in a series of 269 patients. Cytometry B Clin Cytom 2012; 82B: 295304.
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    Davis BH, Holden JT, Bene MC, Borowitz MJ, Braylan RC, Cornfield D, et al. 2006 Bethesda International Consensus recommendations on the flow cytometric immunophenotypic analysis of hematolymphoidneoplasia: Medical indications. Cytometry B Clin Cytom 2007; 72B( Suppl 1): S5S13.
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    Song JY, Filie AC, Venzon D, Stetler-Stevenson M, Yuan CM. Flow cytometry increases the sensitivity of detection of leukemia and lymphoma cells in bronchoalveolar lavage specimens. Cytometry B Clin Cytom 2012; 82B: 305312.
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    Keeney M, Chin-Yee I, Weir K, Popma J, Nayar R, Sutherland DR. Single platform flow cytometric absolute CD34+ Cell counts based on the Ishage guidelines. Cytometry (Communications in Clinical Cytometry) 1998; 34: 6170.
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    Finn WG. Beyond gating—capturing the power of flow cytometry. Am J Clin Path 2009; 131: 313314.
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    Bagwell CB. Breaking the dimensionality barrier. In: Kottke-Marchant K, Davis B, editors. Laboratory Hematology Practice, Chapter 11. Hoboken, NJ: John Wiley & Sons, Inc.; 2012.
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    Herbert DJ, Miller DT, Bagwell CB. Automated analysis of flow cytometric data for CD34+ stem cell enumeration using a probability state model. Cytometry B Clin Cytom 2012; 82B: 313318.
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    Richards SJ, Hill A, Hillmen P, Recent advances in the diagnosis, monitoring and management of patients with praxymal nocturnal hemoglobinuria. Cytometry B Clin Cytom 2007; 72B: 291298.
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    Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ; Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow. Cytometry B Clin Cytom 2010; 78B: 211230.
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    Miller DT, Hunsberger BC, Bagwell CB. Automated analysis of GPI-deficient leukocyte flow cytometric data using GemStone™. Cytometry B Clin Cytom 2012; 82B: 319324.
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    ISPC Abstracts from the 2nd biannual International Practical Cytometry Workshop. Cytometry B Clin Cytom 2012; 82B: o1o17. doi: 10.1002/cyto.b.21022.