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The patient was a 62-year-old Caucasian man with blood smear and flow cytometry concerning for acute promyelocytic leukemia with FISH ultimately confirming PML-RARA translocation. He had a 30-year history of employment at a nuclear power plant. He presented with diffuse intravascular coagulation, hyperleukocytosis, and quickly developed acute respiratory distress syndrome. On day four of ATRA + Hydrea, a bronchoalveolar lavage was performed and was non-bloody. On microscopic fluid review, abnormal immature cells with bilobed nuclear contours were identified, similar in morphology to those seen on the diagnostic blood smear review, amidst background alveolar-type macrophages. Subsequent flow cytometric analysis showed these cells to be abnormal promyelocytes; however, they differed from the blood flow cytometry study performed prior to initiation of ATRA by showing maturational immunophenotypic changes. While the leukemic promyelocytes on bronchoalveolar lavage were morphologically immature, these immunophenotypic changes somewhat recapitulated those seen in normal granulocyte maturation and were thus suggestive of so-called differentiation syndrome. Unfortunately, the patient passed away during induction chemotherapy due to complications from diffuse intravascular coagulation and differentiation syndrome. Important pathobiological information can be gathered from fluid review and concomitant flow cytometric analysis.

Objectives Sezary syndrome (SS) is characterized by erythroderma, generalized lymphadenopathy, and the presence of circulating atypical lymphocytes, which are difficult to identify by morphologic data. Methods We revised our series of 107 patients in an attempt to better define the phenotypic aberrancies in blood at diagnosis and the immunophenotypic stability over time detected by flow cytometry. Polymerase chain reaction assay was also used to study CD26/dipeptidyl peptidase IV (DPPIV) gene methylation. Results The most common aberrancies were represented by the lack of CD26 (96/107) or CD38 (101/107) expression and the presence of a "dim" CD3, CD4, or CD2 population. There was a high variability in CD7 expression. In total, 31% of the patients had phenotypical heterogeneity in CD26 and CD7 expression at diagnosis. The phenotype was stable over time in 73 of 95 patients with available follow-up data, while 22 of 95 patients developed changes in CD26, CD7, or CD2 expression. CD4+CD26- SS showed hypermethylation of the CpG islands for the promoter region of CD26/DPPIV. Multivariate analysis showed that CD26 expression is a favorable prognostic factor (hazard ratio, 2.94; P = .045). Conclusions We confirm the relevance of CD26 negativity in SS diagnosis and monitoring. Nevertheless, the presence of rare CD26+ cases suggests that a multiparameter flow cytometry approach should be used. Changes in methylation profile could account for phenotypical heterogeneity.

BACKGROUND: Hairy cell leukemia (HCL) is an indolent B-cell malignancy strongly expressing CD22, with high sensitivity to purine analog chemotherapy, but relapses frequently occur, associated with minimal residual disease (MRD). Targeted non-chemotherapeutic approaches have shown efficacy but leave MRD. Moxetumomab pasudotox is an anti-CD22 recombinant immunotoxin recently reported in relapsed/refractory HCL to achieve 57% and 51% complete remission (CR) rates in phase 1 and 3 testing, respectively. Eradication of MRD by bone marrow flow cytometry or immunohistochemistry was associated with improved CR duration. We have previously reported that Taqman real-time quantitative PCR (RQPCR), which amplifies patient- and HCL-specific B-cell immunoglobulin rearrangements (IGHV), can detect one HCL cell in 106 normal cells. In the present study we determined whether MRD eradication measured by RQPCR is associated with improved CR duration. METHODS: A total of 28 patients achieving CR to moxetumomab pasudotox in phase 1 (n=18) and phase 3 (n=10) trials were studied at the National Institutes of Health by multicolor flow cytometry of the bone marrow aspirate (BMA) and blood. Patients received moxetumomab pasudotox at 40-50 mcg/Kg on days 1, 3 and 5 of 2-7 cycles (median 5) at 4-week intervals. Blood and bone marrow samples were obtained to determine CR. In addition to end of treatment, bone marrow samples for MRD were generally obtained every 6-12 months for 2.5 years, then every other year. Blood flow cytometry was obtained every 6 months for 2.5 years, then yearly. Follow-up (time since treatment initiation) was 26.9-121.8 (median 84) months. For this analysis, CR for each bone marrow time point was determined by standard criteria for all patients independent of specific data-reporting requirements and data cut-off time points. RQPCR testing for each patient required cloning the immunoglobulin heavy chain variable regions and designing sequence-specific primers and probes. RNA samples were made from PAXgene tubes of blood and sodium-heparin tubes of BMA. RESULTS: We found that BMA RQPCR was most sensitive, followed by BMA flow cytometry, blood RQPCR, and blood flow cytometry. Of the 28 patients studied, 12 (43%) achieved MRD-free CR by all 4 tests and 2 of 12 patients (17%) relapsed. Seven (25%) were MRD+ by BMA RQPCR but negative by the other 3 studies, and none relapsed. One patient RQPCR+ and flow-negative in both blood and BMA relapsed. Seven patients flow-negative but RQPCR+ in blood, flow+ in BMA, and either BMA RQPCR+ or RQPCR not done, all relapsed. One patient with BMA RQPCR not done and the other 3 studies positive relapsed. With respect to RQPCR of blood as a single test, 3 of 20 negative vs 8 of 8 positive patients relapsed (p CONCLUSIONS: RQPCR negativity of blood is strongly associated with prolonged CR duration after moxetumomab pasudotox and may be a useful measure of MRD when bone marrow studies cannot be done. Additional testing will be needed to determine if blood RQPCR can be used to determine the optimal number of cycles of immunotoxin to administer. BMA RQPCR, though more sensitive than blood RQPCR, was not as useful due to fewer patients being evaluable and more time needed for BMA-RQPCR+ patients to relapse. Additional testing and follow-up may determine if BMA RQPCR negativity will correlate significantly with CR duration. Figure. Figure. Disclosures Yao: MedImmune: Employment. Marshall:MedImmume: Employment. Pastan:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox. Kreitman:NIH: Patents & Royalties: Co-inventor on the NIH patent for Moxetumomab Pasudotox.

Erythrocyte-based micronucleus tests have traditionally been performed with bone marrow specimens, since, in most preclinical animal models, the spleen can efficiently remove aberrant erythrocytes from the circulation. Even so, evidence is mounting that by examining tens of thousands of young (CD71-positive) circulating reticulocytes for the presence of micronuclei via flow cytometry, a sensitive assay of cytogenetic damage is realized. The work described herein was designed to test this hypothesis further, using an important preclinical toxicology model, the beagle dog. In these experiments, purebred male beagles were treated for five consecutive days with cyclophosphamide (0, 6.25, 12.5 or 25 mg/m 2 /day) or for two consecutive days with etoposide (0, 1.56, 6.25 or 12.5 mg/m 2 /day). Before treatment, and on each day of administration, blood specimens were collected and processed for flow cytometric scoring of micronucleated reticulocyte (MN-RET) frequency. Twenty-four hours after the final administration, blood MN-RET frequencies were determined via flow cytometry, and frequencies of micronucleated bone marrow polychromatic erythrocytes (MN-PCE) were determined using acridine orange and May-Grunwald Giemsa staining. In the case of cyclophosphamide, elevated blood MN-RET frequencies were observed 2 days after treatment began, and the maximal frequency was achieved 1 day later. Similarly, etoposide-induced blood MN-RET were not evident 1 day after administration began, but a robust effect was apparent 2 days after treatments were initiated. Twenty-four hours after the final administrations, dose-related micronucleus responses were evident for both agents and in both blood and bone marrow compartments. Good overall agreement between MN-RET and MN-PCE frequencies was evidenced by high Spearman's correlation coefficients—0.89 for blood flow cytometry versus bone marrow acridine orange staining and 0.83 for blood flow cytometry versus bone marrow May-Grunwald Giemsa staining. Taken together, these results provide further support for the cross-species utility of flow cytometry-based blood MN-RET measurements.

The purpose of this study was to evaluate the quantification of fetomaternal hemorrhage by the manual and automated microscopic analysis of Kleihauer-Betke stained slides and by flow cytometry.Blood smears were stained and evaluated manually according to the Kleihauer-Betke test. The same slides were used for automated microscopy. In addition, blood flow cytometry was performed by anti-hemaglobin F immunostaining.Fetomaternal hemorrhage0.1% was detected in 4 patients by manual and automated Kleihauer-Betke test and by blood flow cytometry. Fetomaternal hemorrhage was absent according to all 3 methods in 13 patients; fetomaternal hemorrhage0.1% was detected in 27 patients by either manual or automated Kleihauer-Betke test or both. Moderate agreement was observed between the manual and automated Kleihauer-Betke test (weighted kappa, 0.56; 95% CI, 0.33-0.78). Agreement between the manual Kleihauer-Betke test and blood flow cytometry was fair (weighted kappa, 0.40; 95% CI, 0.15-0.66).Automated microscopic detection of fetal blood cells in clinical samples provides accurate quantification that is comparable to the manual Kleihauer-Betke test in both small and large fetomaternal hemorrhage. Blood flow cytometry is capable only of quantifying fetomaternal hemorrhage of0.1%.