Abstracts from the 28th Annual Meeting of the International Clinical Cytometry Society, October 13–15, 2013, Fort Lauderdale, Florida


1

DRIFT ANALYSIS APPROACH TO INVESTIGATE THE EFFECT OF SPECIMEN AGE AND PREPARED SAMPLE AGE ON THE STABILITY OF LYMPHOCYTE SUBSETS

Elena Afonina,1 Robert Magari,2 Jin Zhang,1 Samantha Bauchiero,1 Violetta Headley,1 and Liliana Tejidor2

1Life Science Beckman Coulter, Miami, FL
2Diagnostics Beckman Coulter, Miami, FL

A novel statistical approach was used to assess the stability of CD3+/CD4+, CD3+/CD8+, CD3−/CD56+, CD19+, and CD3+ lymphocyte subsets in CYTO-STAT® tetraCHROME™ stained samples and analyzed on FC 500™ or Navios™** (**Pending clearance by the United States Food and Drug Administration; not yet available for in vitro diagnostic use within the USA) flow cytometers. Approximately, 30 normal and clinical specimens were included in the data analysis for each study. Among them ≥ 50% had CD4+ absolute counts lower than 500 cells/ul. Specimens were stored at room temperature for the following time periods: fresh (≤8 h), 24, 48, and 72 h post-venipuncture. Additionally, samples at each time point were prepared and refrigerated up to 48 h prior to analysis. Data were modeled as linear functions of specimen age and prepared time, while sample-to-sample variability was considered as the random component of the model. The drift (change) from the reference point (0 (specimen age) to 0 (prepared sample age)) for each analyte was estimated from the model. Drifts were estimated at different points along with their 95% confidence intervals. The following graphs illustrate specimen and prepared sample stability drift for samples analyzed on Navios™ flow cytometer (Figs. 1 and 2). The most significant contributor to overall specimen stability was specimen aging in whole blood. In contrast, prepared sample storage had less impact on the cell loss. In conclusion, the derived models provide a robust method for prediction of the drift at different time points within the tested range.

Figure 1.

Specimen age. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 2.

Prepared sample. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

2

IMPACT OF MYELOID ANTIGEN EXPRESSION ON OUTCOMES OF PATIENTS WITH T-CELL ACUTE LYMPHOBLASTIC LEUKEMIA

Mohamed Al-Zaabi,1 Murtadha Al-Khabori,2 Naglaa Fawaz,2 Sulayma Al-Lamki,3 Mohammed Al-Hunieni,2 Arwa Al-Riyami,2 Sabria Al-Hashami,3 Muna Al-Riyami,3 Khalil Al-Farsi,2 Muhanna Al-Muslahi,3 and Salam Al-Kindi2

1Oman Medical Specialty Board, Muscat, Oman
2Sultan Qaboos University Hospital, Muscat, Oman
3Royal Hospital, Muscat, Oman

Prognostic value of myeloid antigen expression in T-cell acute lymphoblastic leukemia (T-ALL) remains a matter of debate. The objective of this study was to evaluate impact of this factor on complete remission (CR), event-free survival (EFS), and overall survival (OS) in patients with T-ALL treated with intensive chemotherapy. We retrospectively reviewed charts of 39 consecutive patients with T-ALL managed in two tertiary centers. The diagnosis was established by French-American-British classification or WHO criteria. Patients were considered having myeloid antigen expression if they expressed CD13, CD33, or both. Median follow-up was 12 months for all patients and 14 months for those still alive. Of 32 patients assessable for response to induction therapy, 29 (91%) achieved CR with one or two courses of chemotherapy. The difference between those with and without myeloid antigen expression was not statistically significant (P = 0.88). Twenty-five percent of patients with no myeloid markers required two courses of induction to achieve CR, whereas 58% of other group required two induction courses and this difference was statistically significant (P = 0.04). There was no significant difference in EFS and OS between the two groups in univariable and multivariable models. Our analysis suggests that patients with T-ALL and positive myeloid antigen expression required more than one cycle of induction chemotherapy to achieve CR. No significant impact on EFS or OS was seen. However, our study is limited by small sample size and short follow-up. Thus, larger prospective trials are required to confirm these findings. The authors report no conflicts of interest.

3

STABILIZATION OF DISEASE SPECIFIC ANTIGENIC EPITOPES FOR DETECTION OF MINIMAL RESIDUAL DISEASE IN PERIPHERAL WHOLE BLOOD

Jodi R. Alt and Wayne L. Ryan

Streck, Omaha, NE

Recent studies and clinical trials provide evidence for the utility of minimal residual disease (MRD) testing during treatment and post-therapy monitoring of patients with hematopoietic neoplasms. The development of standardized methods has been sought for these tests and flow cytometry is utilized by many contract research organizations and hospitals to reach these goals. The purpose of this study was to determine whether minimal residual disease cells are preserved and stable in whole blood collected in the blood collection device, Cyto-Chex® BCT. We demonstrate the stability of a tissue culture cell line, as a model for MRD, at levels as low as 0.01%, in normal peripheral whole blood for up to 7 days. Disease specific antigens (CD25, CD71, and CD117) were utilized for detection of the abnormal cells. This is stark contrast to the abnormal cells maintained in K2EDTA, which show instability by day 3. Additionally, peripheral whole blood samples containing MRD cells are also stable for up to 7 days after sample shipment. This discovery shows the valuable application of rare cell stabilization with sample stress to allow more accurate identification of patients with clinically relevant minimal residual disease which may require additional treatment for their hematopoietic neoplasm.

4

SCREENING OF CARCINOMA METASTASIS BY FLOW CYTOMETRY

Maria Arroz,1 Jose Pereira,1 Maria Acosta,1 Lylliane Luz,1 Ana Costa,1 Silvia Amaro,1 Martinha Chorao,2 Maria Brito,3 and Esmeraldina Junior1

1CHLO, Hospital S. Francisco Xavier, Lisbon, Portugal
2CHLO, Hospital Egas Moniz, Serviço Anatomia Patológica, Lisbon, Portugal
3Hospital Garcia de Orta, Serviço Anatomia Patológica, Almada, Portugal

Aim: To evaluate the efficiency of flow cytometry to detect malignant cells in different types of specimens that usually do not contain epithelial cells.

Material and methods: A total of 119 fresh samples of pleural effusions (46), peritoneal effusions (15), pericardial effusions (4), FNAs and lymph node biopsies (30), bone marrow aspirates (5), CSF (4), and other tissues (15) were analyzed by flow cytometry, using monoclonal antibodies against Ber-EP4, an epithelial antigen, CD45, a pan-leucocyte antigen, and CD33 to exclude monocyte/macrophages. Results were compared with smear and cell block morphology, as well as immunocytochemistry on paraffin wax embedded cell blocks.

Results: Cytologic and histologic results were as follows: carcinoma cells, 52 specimens; benign and non-epithelial neoplasia, 67 specimens. The sensitivity of immunophenotyping was 96% and specificity was 99%.

Conclusion: Flow cytometry is a rapid, not too expensive and highly effective method for the screening of carcinoma metastasis in different types of samples. Moreover, this is a very helpful tool to differentiate between malignant and activated mesothelial cells in effusions. As such we recommend it should be performed in parallel with cytology. In the case of biopsies the comparison with immunocytochemical results for Ber-EP4 expression showed excellent association.

5

REACTIVE OR MALIGNANT MONOCYTOSIS? THE IMPORTANCE OF DNA ANALYSIS IN DIAGNOSTIC FLOW CYTOMETRY

David Azoulay, Judith Chezar, Eti Shaul, and Andrei Braster

Immunophenotyping laboratory, Hematology and blood bank unit, Western Galille Medical Center, Naharia, Israel

Introduction: Including DNA data analysis in the routine diagnostic flow cytometry panel was shown to be helpful in differentiating between different types of lymphoma and has a prognostic value in childhood leukemia. Furthermore and as we demonstrate here, the presence of cells with an aneuploid DNA content confirms the presence of malignant clone.

Materials and Methods: A 54-years-old female was referred to our hospital due to fever of unknown origin. Total leukocyte count was 5.1 × 103/μl with absolute neutropenia. The automated differential showed an increase in the proportion of lymphocytes and monocytes. Atypical monocytes with occasional azurophilic vacuoles were seen on the blood smear. The peripheral blood sample was sent for flow cytometry and stained with 3–5 color antibody combinations. DNA cell cycle analysis was performed alone or in combination with FITC conjugated antibodies.

Results and Discussion: A myeloid blast population was identified (4%) which stained positive for CD34/CD117/CD38 and HLA-DR. In addition, 45% of the total nucleated blood cells in the sample were classifiable as monocytes expressing: CD45/CD64/CD36/CD33/CD13/CD14/CD4/CD15/CD11b/CD56/HLA-DR/cytMPO and cytCD68. It was not possible to determine whether this monocytosis was reactive or malignant. DNA cell cycle analysis demonstrated a distinct aneuploid population (DNA-index: 2) that consisted of 45% of the analyzed cells and coexpressed the monocytic markers: CD14 and CD36. The appearance of the tetraploid peak strongly suggested that the monocytosis was malignant.

Conclusion: This example emphasized the usefulness of DNA cell cycle analysis for the diagnosis of leukemia and should be taken in consideration when discussing consensus protocols.

6

EVALUATION OF CD85k IN FLOW CYTOMETRIC EVALUATION OF MONOCYTIC/MYELOMONOCYTIC LEUKEMIAS

Ila Bansal, HsuehHua Chen, Daniel M. Jones, and Charles F. Repetti

Quest Diagnostics, Chantilly, VA

The immunoglobulin-like inhibitory receptor, CD85k (ILT3), is expressed during monocytic differentiation. One flow cytometric study suggested CD85k detection may assist in distinction of acute myelomonocytic leukemia (M4) and acute monocytic leukemia (M5) from other AML types (Dobrowolska et al., Cytometry 2013;84B:21–29). Routinely used monocyte markers include CD64, which is positive throughout monocytic differentiation but also stains myeloid cells weakly; CD36, which also recognizes cytophilic platelets; and CD14 which is monocyte-specific but is expressed later in maturation. This study evaluated whether CD85k offers an advantage over CD36 and CD64 in detecting immature monocytic differentiation in AML or if CD85k aberrant expression levels are found on leukemic blasts. CD85k-APC or CD14-APC staining was combined with CD64-PE and CD36-FITC. Blood and bone marrow samples with AML-M4/M5 (n = 12) and normal samples (n = 3) were tested. Anti-CD85k (clone ZM4.1-APC, eBioscience) identified non-neoplastic monocytes similarly to CD64, and more extensively than CD36 or CD14. CD85k was detected in 11/12 cases of AML-M4/M5 at similar levels in both the CD14− and CD14+ blast populations, with CD64 detected in all cases. A minor CD64−, CD85k+ population was found in some leukemia samples, likely representing dendritic cells based on scatter properties. CD85k staining was generally dimmer in leukemic blasts than normal monocytes. In conclusion, CD85k staining has similar pan-monocytic expression to CD64 in AML-M4/M5 but lacks dim reactivity of CD64 in granulocytic cells.

7

REJECTION BIOMARKER PANEL PROFILE FOR IDENTIFICATION OF HIGH-RISK HEART TRANSPLANT RECIPIENTS

Markus J. Barten,1 Maja-Theresa Dieterlen,1 Attila Tarnok,1 Stefan Dhein,1 Friedrich W. Mohr,1 and Hartmuth B. Bittner2

1University Leipzig Heart Center, Leipzig, Germany
2Florida Hospital Orlando, Orlando, FL

Background: Identification of suitable biomarkers for detection of rejection after heart transplantation (HTx) is critical for long-term outcome. Thus, we assessed a biomarker panel profile within the first twelve months after transplantation.

Methods: Activation markers CD25 and CD95, intracellular cytokines IL-2 and IFNg, the chemokines IP10 and MIG, subsets of dendritic cells (DCs) as well as antibodies (Abs) against human leukocyte antigens (HLA), and major histocompatibility complex class I-related chain A (MICA)-antigens were analyzed at five different time points within 1st year using FACS and Luminex xMAP technology.

Results: Levels for CD25, CD95, IP10, MIG, and myeloid DCs did not change, whereas expression of IL-2, IFNg, and plasmacytoid DCs significantly increased (P < 0.01 for each). Anti-HLA Abs decreased continuously, while anti-MICA Abs showed minor increase. No differences were between recipients with mild acute cellular rejection and recipients without ACR regarding cellular parameters and cytokines. An association between percentage of plasmacytoid DCs and anti-MICA Abs positivity was proven. Furthermore, analysis revealed that plasmacytoid DCs, IFNg-producing T-cells, and IP10-concentration were associated in a stronger way with age and gender of HTx recipients than with Abs against HLA or MICA.

Conclusions: We recommend quantification of CD25, IL-2, IFNg, plasmacytoid DCs, and monitoring of anti-HLA and anti-MICA Abs after HTx to display patient-specific changes of immunological status to identify HTx recipients with high-risk profile for rejection.

8

FLOW CYTOMETRIC ASSESSMENT OF DENDRITIC TO PREDICT

Markus J. Barten,1 Maja-Theresa Dieterlen,1 Attila Tarnok,1 Stefan Dhein,1 Friedrich W. Mohr,1 and Hartmuth B. Bittner2

1University Leipzig Heart Center, Leipzig, Germany
2Florida Hospital Orlando, Orlando, FL

Background: Over the last decade many studies evaluated the potential of certain biomarkers to predict acute cellular rejection (ACR) without finding their way in the clinical routine. Thus, in our study we monitored dendritic cells (DCs) in heart transplant recipients (HTxR) treated either with a tacrolimus (TAC) or cyclosporine A (CsA)-based immunosuppressive regimen in the context of ACR.

Methods: Both groups consisted of 14 maintenance HTxR. At different study time points (0, 3, and 6 months after study start) peripheral blood from all HTxR was drawn to analyze for (1) blood CsA or TAC concentration (trough value) and (2) to analyze myeloid and plasmocytoid (m and p) DCs with FACS. Histological rejection grading was performed of endomyocardial biopsies.

Results: TAC treated HTxR had significantly higher values of pDCs (CsA-group 53.9 ± 13.0%, TAC-group 67.5 ± 8.4%, P < 0.05) and significantly lower values of mDCs than CsA treated HTxR (CsA-group 57.9 ± 19.0%, TAC-group 45.2 ± 10.7%, P < 0.05). In general, HTxR with rejection grade of ≥2 ISHLT 1990 had significant (P < 0.05) lower values of pDCs (55.1 ± 16.2%) compared to HTxR without rejection (63.6 ± 10.5%). TAC treated HTxR had significantly less rejections compared to CsA treated HTxR (CsA-group 0.86 ± 0.95%, TAC-group 0.2 ± 0.4%, P < 0.05).

Conclusions: Our results showed that HTxR with high pDCs have a lower risk of rejection and that TAC treated HTxR had higher pDCs values compared to CsA treated HTxR. Future studies are needed to confirm whether monitoring pDCs and mDCs have the potential value as biomarkers to identify HTxR at risk to develop ACR.

9

IMMUNOLOGICAL MONITORING OF EXTRACORPOREAL PHOTOPHERESIS AFTER HEART TRANSPLANTATION

Markus J. Barten,1 Maja-Theresa Dieterlen,1 Attila Tarnok,1 Stefan Dhein,1 Friedrich W. Mohr,1 and Hartmuth B. Bittner2

1University Leipzig Heart Center, Leipzig, Germany
2Florida Hospital Orlando, Orlando, FL

Objective: Extracorporeal photopheresis (ECP) has been used as a prophylactic and therapeutic option to avoid and treat rejection after heart transplantation (HTx). Tolerance-inducing effects of ECP like up-regulation of regulatory T cells (Tregs) are known, but specific effects of ECP on Treg subsets and dendritic cells (DCs) are lacking. We analyzed different subsets of Tregs and DCs as well as the immune balance status during ECP treatment after HTx.

Methods: Peripheral blood samples were collected from HTx patients treated with ECP for prophylaxis (n = 9) or from patients with histological proven acute cellular rejection (ACR) of grade ≥1B (n = 9) as well as from control patients without ECP (HTxC; n = 7). Subsets of Tregs and DCs as well as different cytokines levels were analyzed.

Results: Almost 80% of the HTx patients responded to ECP treatment with an increase of Tregs and plasmacytoid DCs (pDCs). Percentage of pDCs before ECP treatment was significantly higher in non-responders (26.3% ± 5.6%) compared to patients who responded to ECP (9.8% ± 10.2%; P = 0.011). Analysis of functional subsets of CD4+CD25highCD127low Tregs showed that CD62L, CD120b, and CD147-positive Tregs did not differ between the groups. CD39-positive Tregs increased during ECP treatment compared to HTxC. ECP-treated patients showed higher levels for Th1, Th2, and Th17 cytokines. Cytokine levels were higher in HTx patients with rejection before ECP treatment compared to patients with prophylactic ECP treatment.

Conclusions: We recommend a monitoring strategy that includes the quantification and analysis of Tregs, pDCs, and the immune balance status before and up to 12 months after starting ECP.

10

IMPACT OF EXTRACORPORAL PHOTOPHERESIS IN HEART TRANSPLANT RECIPIENTS WITH DIFFERENT IMMUNE STATUS

Markus J. Barten,1 Maja-Theresa Dieterlen,1 Attila Tarnok,1 Stefan Dhein,1 Friedrich W. Mohr,1 and Hartmuth B. Bittner2

1University Leipzig Heart Center, leipzig, Germany
2Florida Hospital Orlando, Orlando, FL

Objective: Immunmodulatory effects of extracorporal photopheresis (ECP) in heart transplant recipients (HTxR) with different immune status are still missing. Thus, we measured regulatory CD4+ T cells (Tregs) and dendritic cells (DCs) in HTxR treated with ECP for prophylaxis of rejection (PRX), to treat acute rejection (AR) or to treat chronic rejection (CR).

Methods: HTxR were treated monthly three times with ECP: PRX-group (n = 5) at month 4 after HTx; AR-group (n = 7) at time of biopsy proven rejection and two times thereafter; CR-group (n = 4) monthly at time of angiographic proven diagnosis of allograft vasculopathy at two times afterwards. Peripheral blood was analyzed each time before ECP therapy to assess Tregs and myeloid and plasmocytoid (m and p) DCs by FACS. Blood samples before ECP treatment and one month after the last ECP therapy were compared (% ± SD).

Results: In the PRX-group, pDC levels decreased from 28 ± 25.4% to 15.2 ± 9.2% (P = 0.10), while mDC levels increased from 58.7 ± 26.0% to 68.7 ± 12.2%, respectively (P = 0.23). Whereas Tregs increased from 6.3 ± 3.5% to 7.2 ± 2.6% (P = 0.46). In the AR-group both m and pDCs increased from 56.6 ± 21.1% to 68.5 ± 10.4% and 4.2 ± 3.9% to 14.2 ± 5.7%, respectively (P = 0.05). Tregs did not change before and after ECP (6.8 ± 4.25% to 6.2 ± 2.0%). Whereas Tregs expression in the CR-group with 4.8 ± 0.8% prior to ECP and 7.3 ± 3.7% after ECP (P < 0.05). mDC levels in the CR-group rose from 63.2 ± 12.7% to 73.7 ± 7.7% (P = 0.48), but pDC levels declined from 24.0 ± 8.8% to 13.3 ± 9.7% (P = 0.33).

Conclusion: We showed that ECP therapy had different effects on Tregs and DCs in HTxR with different immune status. Further clinical studies are needed to identify the optimal time point and duration of ECP therapy depending on the indication after HTx.

11

ACTIVATION OF T-CELL MARKERS AFTER CARDIOPULMONARY BYPASS

Markus J. Barten,1 Maja-Theresa Dieterlen,1 Attila Tarnok,1 Stefan Dhein,1 Friedrich W. Mohr,1 and Hartmuth B. Bittner2

1University Leipzig Heart Center, Leipzig, Germany
2Florida Hospital Orlando, Orlando, FL

Background: Cardiopulmonary bypass surgery (CPBS) is associated with inflammatory immune responses that may lead to postoperative immunosuppression that is accompanied by an increased risk for infections, or to subsequent organ dysfunction. T cell activation is a central mechanism during inflammatory processes, and monitoring of T cell activation may help to identify patients at risk for postoperative complications. Therefore, we developed a whole blood assay to evaluate the T cell activation pathways in patients undergoing CPBS.

Methods: Blood was obtained from patients (n = 11) preoperatively, at postoperative days (POD)−3 and POD-7. Blood was stimulated with different concentrations of Concanavalin A (ConA). Cyclosporine (CsA) and sirolimus (SRL), inhibiting different target enzymes of the cell cycle, were added to whole blood at clinically relevant concentrations. Expression of T cell activation biomarkers CD25 and CD95 was analyzed by flow cytometry.

Results: In untreated blood, expression of CD25 and CD95 significantly increased with higher ConA concentrations (P < 0.05), but, regardless stimulation, expression of both antigens decreased over time with a maximum on POD-7 (P < 0.05). Independently from the ConA concentration, inhibition of CD25 and CD95 expression was highest preoperatively for SRL and on POD-3 for CsA. At all time points, inhibition of CD25 and CD95 expression was significantly higher after CsA compared to SRL treatment (P < 0.001).

Conclusion: For the first time, we showed that different pathways of T cell activation are impaired after CPBS over time. Future studies have to show the predictive value of T cell activation biomarkers with clinical outcome and the therapeutic consequences after CPBS.

12

DIAGNOSIS AND CLASSIFICATION OF PEDIATRIC TUMORS BY MULTIPARAMETER FLOW CYTOMETRY

Vitor D. Botafogo,1 Cristiane S. Ferreira-Facio,1 Cristiane Milito,2 Marcela Fontana,1 Leandro S. Thiago,3 Elen Oliveira,1 Ariovaldo S. da Rocha-Filho,4 Fernando Werneck,4 Danielle N. Forny,1 Samuel Dekermacher,4 Ana Paula de Azambuja,5 Sima Esther Ferman,6 Paulo Antônio Silvestre de Faria,6 Marcelo G. P. Land,1 Alberto Orfao,7 and Elaine S. Costa1

1Pediatrics Institute IPPMG, Federal University of Rio de Janeiro (UFRJ), Rio de Janeiro, Brazil
2Department of Pathology, Faculty of Medicine, UFRJ, Rio de Janeiro, Brazil
3Pediatric Hematology and Oncology Program, Cancer Research Center, Brazilian National Cancer Institute (INCa), Rio de Janeiro, Brazil
4Servidores do Estado Hospital, Rio de Janeiro, Brazil
5Clinic Hospital, Federal University of Parana (UFPR), Curitiba, Brazil
6Department of Pediatric Oncology/Brazilian National Cancer Institute (INCa), Rio de Janeiro, Brazil
7Cytometry Service, Department of Medicine and Cancer Research Center (IBMCC, University of Salamanca-CSIC and IBSAL), Salamanca, Spain

Pediatric cancer is a relatively rare and heterogeneous group of hematological and non-hematological malignancies which require multiple procedures for its diagnostic screening and classification. Until now, flow cytometry (FC) has not been systematically applied to the diagnostic work-up of such malignancies, particularly for solid tumors. Here, we evaluated a FC panel of markers for the diagnostic screening of pediatric cancer and further classification of pediatric solid tumors. The proposed strategy aims at the differential diagnosis between tumoral vs. reactive samples, and hematological vs. non-hematological malignancies, and the sub-classification of solid tumors. In total, 94 samples from 72 patients suspicious of containing tumor cells were analyzed by FC in parallel to conventional diagnostic procedures. The overall concordance rate between both approaches was of 91.5% (86/94 diagnostic samples), with 100% agreement for all reactive/inflammatory and non-infiltrated samples as well as for those corresponding to solid tumors (n = 50), with only eight false negative cases diagnosed as Hodgkin lymphoma (7) and anaplastic lymphoma (1). Moreover, clear discrimination between samples infiltrated by hematopoietic vs. non-hematopoietic tumor cells was systematically achieved. Distinct subtypes of solid tumors showed different protein expression profiles, allowing for the differential diagnosis of neuroblastoma (CD56hi/GD2+/CD81hi), primitive neuroectodermal tumors (CD271hi/CD99+), Wilms tumors (>1 cell population), rhabdomyosarcoma (nuMYOD1+/numyogenin+), carcinomas (CD45−/EpCAM+), germ cell tumors (CD56+/CD45−/NG2+/CD10+), and eventually also hemangiopericytomas (CD45−/CD34+). In summary, our results show that multiparameter FC provides fast and useful complementary data to routine histopathology for the diagnostic screening and classification of pediatric cancer.

13

NEW FLUOROPHORE FOR VIOLET LASER EXCITATION

Jolene A. Bradford, Wenjun Zhou, Bradley Dubbels, Quentin Low, Bradley Bone, and April Anderson

R&D Molecular Probes® Labeling and Detection Technologies, Eugene, OR

Flow cytometry instruments offering solid-state violet laser diodes are becoming more prevalent due to their small size, low power requirements, cost effectiveness, and reliability over long periods of time. Concurrently, the use of violet-excited fluorochromes in multiparametric flow cytometry has been essential in the expansion of multicolor flow cytometry, enabling greater numbers of markers to be detected in one sample. We have developed a novel violet-excited dye, Pacific Green™ dye, which has an excitation maximum of 411 nm and an emission maximum of 500 nm. The development of this dye addresses the need to transfer well-resolved markers off the common Blue 488 nm and Red 635 nm excitation lines and onto the Violet 405 nm excitation line, thus enabling the detection of other markers with the 488 nm and 635 nm lasers. Pacific Green™ dye can be used for three colorimmunophenotyping using violet laser excitation with Pacific Blue™ and Pacific Orange ™ dyes with minimal compensation and without 488 nm excitation. Data is shown for human lymphocytes stained with anti-CD3 complexed with Pacific Green™ Zenon® labeling reagent, Goat anti-Mouse seconday detection, and Streptavidin secondary detection. Six color immunophneotyping data is shown using direct conjugates of Pacific Green™, Alexa Fluor® 488, phycoerythrin (PE), PE-Cy7, Pacific Blue™, and Pacific Orange™ conjugates. Pacific Green™ dye can be used with the far red emitting QDot® 605, 655, and 705 conjugates to enable higher multiplexing using violet laser excitation. For Research Use Only.

14

DEMONSTRATION OF THE ANALYTICAL PERFORMANCE OF THE NAVIOS FLOW CYTOMETER IN A MULTI-CENTER STUDY

Diana B. Careaga,1 Robert Magari,1 Karen W. Lo,1 Michael Keeney,2 Janice Popma,2 Joanne Luider,3 Aito Ueno,3 Gerard Lozanski,4 Bruce Briggs,4 Elena Afonina,5 Enrique Rabellino,5 and Lilliana M. Tejidor1

1Clinical Research, Diagnostics, Beckman Coulter, Inc., Miami, FL
2London Health Sciences Centre, London, Canada
3Foothills Hospital, Calgary, Canada
4Ohio State University Medical Center, Columbus, OH
5Clinical Applications Development, Life Sciences, Beckman Coulter Inc., Miami, FL

A multi-center study evaluated the performance of the Navios™ cytometer* (*Pending clearance by the US FDA; not yet available for in vitro diagnostic use within the USA) with CYTO-STAT tetraCHROME™ reagents compared to the Cytomics FC500 for lymphocyte subset analysis in patients suspected of immunodeficiency. The Navios automated gating method (Navios tetra) allows enumeration of lymphocyte subsets and reporting of absolute counts and percent positive values for CD3+, CD3+/CD4+, CD3+/CD8+, CD19+, and CD3-/CD56+ markers. Modifications to the Navios tetra algorithm compared to the Cytomics FC500 algorithm (tetraCXP) include lymphocyte populations with altered Forward light scatter properties that maintain both Side Scatter and CD45 fluorescence intensity properties. Navios tetra was compared to both FC500 manual gating and the tetraCXP algorithm in ≥300 samples stained with CYTO-STAT tetraCHROME™ reagents covering the CD4+ medical decision levels. The comparison of Navios tetra to FC500 manual gating showed minimal positive bias (upper 95% limit of bias for CD4+ count at 50, 350, and 500 cells/µl was 18, 12, and 13 cells, respectively) and a slightly higher positive bias when compared to tetraCXP (upper 95% limits of 22, 30, and 39 cells, respectively). The Navios™ cytometer showed comparable inter- and intra-laboratory precision for both percentage and absolute counts with control materials as compared to FC500 and excellent repeatability with specimens across the CD4+ medical decision ranges. Normal adult reference intervals for all lymphocyte subsets were consistent with published values. The Navios™ flow cytometer demonstrated excellent performance in lymphocyte subset analysis with CYTO-STAT tetraCHROMETM.

15

EFFECTIVE SEPARATION OF HEMATOGONES FROM PRECURSOR B-CELL ACUTE LYMPHOBLASTIC LEUKEMIA BY 5-COLOR SINGLE TUBE ASSESSEMENT

Melissa Cervania, Matthew J. Miller, Serhan Alkan, and Raju Pillai

Cedars-Sinai Medical Center, Los Angeles, CA

Previous studies showed that CD58 is commonly expressed in pre B-ALL but not in hematogones; however, CD58 as a single marker somewhat limited due to overlapping features especially when it is expressed at lower levels. In order to improve diagnostic accuracy, we generated a five-color antibody panel including CD10, CD19, CD45, CD38, and CD58 to assess the feasibility of a single tube panel to distinguish pre B-ALL from hematogones. Design: 58 cases including 37 cases of B-ALL, 18 cases of hematogones, and 3 cases of both hematogones and B-ALL were analyzed using a single tube containing CD10-FITC/CD58-PE/CD19-ECD/CD38-PC5/CD45-PC7 simultaneously with the standard acute leukemia panels.

Results: Hematogones typically showed expression of bright CD38, moderate (mod) CD19, mod CD10, variable CD20, and no CD58; in contrast pre B-ALL showed weak to mod CD38, mod to strong CD19, mod to strong CD10, and mod CD58. This panel allowed reliable separation of leukemia from hematogones with exception of a single case of B-ALL with mod to bright CD38 with dim CD58, with overlapping features of hematogone.

Conclusions: The combination of CD38 and CD58 increases diagnostic accuracy in differentiation of hematogones from B-ALL. Without both CD38 and CD58 together, some cases would have been difficult to interpret. Based on this analysis, we recommend inclusion of these markers in the evaluation of bone marrow samples that are submitted for acute leukemia evaluation especially post-treatment samples in order to prevent misdiagnosis. Use of this antibody panel in a single tube could cut costs and improve patient care.

16

A SHORT PRIMARY IMMUNOPHENOTYPING PANEL HAS A ROLE IN THE DIAGNOSIS OF PEDIATRIC ACUTE LEUKEMIA

Amar Dasgupta, Manisha Ramani, Vishal Mehrotra, Pradnya Chaudhari, Santosh Parab, and Janmejay Yadav

SRL Limited, Mumbai, India

There is a need for a primary screening panel for acute leukemia (AL) that is both cost-effective and informative. We describe here our experience with an abridged antibody panel for pediatric acute leukemia (PAL). One hundred fourteen cases of PAL were immunophenotyped using a four-color antibody panel against CD3, 5, 10, 13, 19, 20, 45, and HLA-DR. The composition of the panel was influenced by the fact that B-ALL is the commonest PAL. Cases undiagnosed by the abridged panel were diagnosed in the next step using cytochemical MPO and additional markers (CD2, 4, 7, 8, 22, 33, 34, 41, 61, 99, 117, glycophorin, cMPO, cCD79a, and cCD3). In 74/76 (97%) cases of B-ALL the diagnosis could be made in the first instance with the help of the abridged panel; and in 19/21 cases of AML (CD13+ and HLA-DR+) and in all 14 cases of T-ALL (CD3− and HLA-DR− CD5+) a presumptive diagnosis could be made. Additional CD markers (2–9) were required in 40/114 (35%) cases (T-ALL = 14; AML = 21; B-ALL = 2; others = 3). CD13+, CD19+, and HLA-DR+ phenotype by abridged panel represented AML and not B-ALL on further study. We were able to correctly diagnose two-thirds of PAL cases by applying an abridged, low cost primary antibody panel consisting of only eight CD markers. In the remaining 35% of cases also, a presumptive diagnosis of the type of leukemia could be made which was confirmed by examination of additional markers, thereby proving the usefulness of the abridged panel.

17

CD99 EXPRESSION IS NOT USEFUL AS A MARKER OF IMMATURITY IN T-LYMPHOID NEOPLASMS

Amar Dasgupta, Manisha Ramani, Vishal Mehrotra, Pradnya Chaudhari, Santosh Parab, and Janmejay Yadav

SRL Limited, Mumbai, India

Cases of T-acute lymphoblastic leukemia (T-ALL) are morphologically highly heterogeneous and blast cells in T-ALL can be “mature-looking” and therefore, indistinguishable from the neoplastic cells in T-chronic lymphoproliferative disorders (T-CLPD). Conversely, T-lymphoma cells can have “blast-like” morphology. CD99 has been reported to be useful in distinguishing T-lymphoblasts from T-lymphoma cells by virtue of being a reliable marker of immaturity—even better than CD34 and TdT in this regard. Our experience with CD99 as a marker of immaturity in T-lymphoid neoplasms described here, however, contradicts the above view. We examined CD99 expression in 17 cases of T-ALL and in five cases of T-CLPD (PTCL-1; T-PLL-2; cutaneous T-cell lymphoma-2) that were immunophenotyped using a multicolor antibody panel against CD2, CD3, CD4, CD5, CD7, CD8, CD10, HLA-DR, CD34, and TdT (in some cases) in addition to B-lymphoid and myeloid lineage-associated antigens. While all T-ALL cases expressed variable degrees of CD99, it was strongly expressed by blast cells in only 3 of the 17 cases. Blasts in the majority of the cases expressed dim CD99. CD34 was positive in only four cases. TdT was dimly positive in all the four cases that were tested for this nuclear antigen. Interestingly, four out of five cases of T-CLPD were positive for CD99, albeit dimly. There was no correlation between CD99 expression by the neoplastic T cells and that of any other marker tested. Our observation raises a question mark against the reliability of CD99 as a marker of immaturity for distinguishing T-lymphoblasts from T-lymphoma cells.

18

CD200 EXPRESSION IN B-CHRONIC LYMPHOCYTIC LEUKEMIA—IT IS NOT AN ALL OR NONE PHENOMENON

Amar Dasgupta, Manisha Ramani, Vishal Mehrotra, Santosh Parab, Janmejay Yadav, and Pallavi Gujarathi

SRL Limited, Mumbai, India

Introduction: Immunophenotypic distinction between B-chronic lymphocytic leukemia (B-CLL) and non-CLL B-chronic lymphoproliferative disorders (B-CLPD) can be challenging due to overlapping features among these entities. However, a firm diagnosis is necessary for proper management of different types of CLPD. Recently CD200, a B-lymphoid antigen, has been shown to be useful in this regard by virtue of its close (100%) association with B-CLL and its absence in the lymphoma cells in non-CLL B-CLPD, except hairy cell leukemia and B-lymphoblastic leukemia/lymphoma. Hence CD200 has been proposed in recent studies as a diagnostic marker for B-CLL. We wanted to verify this claim by checking the specificity and sensitivity of this marker for the diagnosis of B-CLL.

Material and methods: Twenty-eight cases of B-CLL and 28 cases of other types of B-CLPD (splenic marginal zone lymphoma 7; mantle cell lymphoma 10; “hairy cell” leukemia 3; others 8) were immunophenotyped between April and August 2012 using a large panel of antibodies against CD3, 5, 10, 11c, 19, 20, 22, 23, 25, 38, 103, 200, FMC7, HLA-DR, immunoglobulin light chains, and surface immunoglobulins.

Results: Lymphoma cells in all 28 cases of B-CLL were CD200 positive (sensitivity 100%), while 9/28 cases of non-CLL B-CLPD (“hairy cell” leukemia: 3/3; mantle cell lymphoma: 2/10; others: 4/8) also were CD200 positive indicating a relatively low specificity (68%).

Conclusions: Our study suggests that although CD200 is a highly sensitive marker for B-CLL, its low specificity would limit its utility in the differential diagnosis between this entity and the other types of B-CLPD.

19

CO-LYOPHILIZATION OF PGD2 ASSAY CONTROL AND QUANTUM DOT NANOCRYSTALS TO TRACE EXPERIMENTAL VARIABLES AND ANALYTES IN A WHOLE BLOOD FLOW CYTOMETRIC ASSAY

Christine M. Evangelista,1 John K. Thomas,1 John Dunne,2 John J. Ferbas,1 and Shelley S. Belouski1

1Amgen Inc., Thousand Oaks, CA
2BD Biosciences, San Jose, CA

Custom assays can provide meaningful biomarker data to clinical studies but can be limited by logistical challenges that introduce variability. We have previously reported a flow cytometric (FCM) assay that stimulates fresh whole blood specimens with prostaglandin D2 (PGD2) to induce down-regulation of the CRTH2 receptor. The inhibitory effect of investigational CRTH2 antagonist therapies can be assessed within the method by comparing unstimulated to a predefined low and high concentration of PGD2 stimulated whole blood specimens drawn at key pharmacokinetic time points throughout the study. To decrease the variability of this method performed at the clinical sites, we implemented lyophilization and nanocrystal technologies. Plates were pre-loaded with lyophilized excipients only, low and high PGD2 to control concentration and preserve biologic activity. Unique quantum dot Qdot® nanocrystals were included in each of the lyophilized preparations to code the PGD2 dose identity of the wells. Results demonstrate our ability to trace or decode each PGD2 treated or untreated sample in whole blood throughout the analysis chain. Moreover, the addition of Qdot® nanocrystals to the wells had no impact on assay performance. Therefore, reagent co-lyophilization with pre-defined Qdot® nanocrystal combinations presents a favorable approach to maximize reagent stability and eliminate sample misidentification in clinical FCM assays.

20

DEVELOPING AND OPTIMIZING A FLOW CYTOMETRY BASED AUTOPHAGY DETECTION METHOD FOR ONCOLOGY DRUG DEVELOPMENT AND BIOMARKER IDENTIFICATION

Zheng Feng, Theodore Baginski, Michael Donio, Diane Werth, and Jacob Bode

EMD Millipore Corporation, St. Charles, MO

Background: Emerging evidence indicates that enhanced autophagy promotes tumor progression in established tumors, and inhibition of autophagy is recognized as a novel adjuvant cancer therapeutic strategy when combined with conventional chemotherapy for maximum therapeutic efficacy and overcoming chemo-resistance. PI3K/mTOR inhibitors have been developed for cancer treatment, when combined with an autophagy inhibitor; synergistically improved efficacy was observed which consequently leads to multiple clinical programs to assess combination therapy regimes. Development of a reliable readout(s) to measure autophagy modulation is critical for discovery of selective modulators, and/or identification biomarkers either for patient selection or drug combination strategy for clinical development.

Methods: Human U87MG glioblastoma and MCF-7 breast cancer cells are treated with PI3K/mTOR inhibitors—rapamycin in combination with or without the autopahgy inhibitor—hydroxychloroquine. The sequestered detection of LC3 (microtubule-associated protein1 light chain 3), which is recognized as autophagy marker, is analyzed by flow cytometry. 7-AAD staining is used for cell cycle analysis. The efficacy of individual compounds and optimal combination doses are identified with cell proliferation assay.

Results: Our preliminary results indicated that autophagy is induced by rapamycin treatment. Enhanced inhibition of cellular proliferation and increased apoptosis are observed in the MCF-7 cell in combination regime when compared to treatment with either compound alone.

Conclusions: The dynamic impacts of treatment(s) on the activation status of autophagy at various time points are currently under investigation, and we are continuing to expand flow cytometry panels by incorporating other potential markers. Studies are funded by EMD Millipore (a division of Merck KGaA, Darmstadt, Germany).

21

COMPARISON OF INVARIANT NATURAL KILLER T-CELLS IN NASAL POLYP TISSUE AND PERIPHERAL BLOOD

Mohammad Fereidouni,1 Shaghayegh Sadat Noorani Hasankiadeh,1 and Farahzad Jabbari Azad2

1Asthma, Allergy & Immunology Research Center, Birjand University of Medical Sciences, Birjand, Iran
2Immunology Department, Mashhad University of Medical Sciences, Mashhad, Iran

Introduction: Nasal polyps (NPs) are a common problem in adults especially in asthmatics but the exact etiology is not clear. Some studies showed that invariant natural killer T-cells (iNKT) cells may skew the immune response toward a Th2 profile in the lung of asthmatic patients. There are some interesting similarities between NP and asthma; therefore, iNKT cells may play a role in pathogenesis of nasal polyps. The aim of this study was to compare frequency of iNKT cells in peripheral blood and nasal polyp tissue among some patients suffering from nasal polyposis. Material and Methods: 16 patients with nasal polyposis (M/F ratio 9/7; mean age: 35 years) participated in this study and nasal polyp specimens as well as peripheral blood samples were taken during polypectomy. Blood and polyp cell suspension were immunostained for CD3, CD4, and iNKT cells. The iNKT cells were identified by immunostaining with CD3 and 6B11 antibodies.

Results: The mean percentage of T cells was approximately 6% of total cells in tissue suspension. Among the CD3+ T cells, percentage of iNKT cells varied from 0.5% to 11% (mean 4.7%) in Polyp. In the peripheral blood samples, the average number of CD3+, CD4+, iNKT cells, and iNKTCD4+ cells, were 59%, 56%, 0.52%, and 43%, respectively. The number of iNKT cells in nasal polyp was significantly higher than blood (mean 4.7 vs. 0.52, P <0.001).

Conclusions: This study shows that iNKT cells are fairly abundant in the nasal polyp and likely to play a role in pathogenesis of nasal polyps.

22

EXTENDED NK CELLS PHENOTYPING IN PATIENTS WITH ACUTE MYELOID LEUKEMIA

Emmanuel Gautherot,1 Gaelle Bouvier,1 Florence Orlanducci,2 Christine Arnoulet,2 Cyril Fauriat,2 and Daniel Olive2

1Beckman Coulter, Marseille, France
2Institut Paoli Calmettes, Marseille, France

The human lymphocyte subset of natural killer cells plays a critical role in the innate immune response, particularly in the control of tumor development and growth. The activation of NK cells is the result of a balance between inhibitory and activating signals. NK cells express activating and inhibitory receptors and the resulting effect depends on the equilibrium of these engaged receptors. The most studied inhibitory receptors are KIR receptors. KIRs recognize HLA class I molecules that prevent killing of normal cells. NKG2A is also an important inhibitory receptor that heterodimerizes with CD94. The receptors NKG2D, the natural cytotoxicity receptors NKp30, NKp44, and NKp46, the activating form of KIR known as KIR-S and CD16 provide activating signals enhancing toxicity, production of cytokines, and lysis of cancer cells. Evaluation of their expression should be of interest in NK cell-based therapy and several NK-based therapy were successful in acute myeloid leukemia (AML). We have developed three combinations to phenotype NK cells receptors by flow cytometry in order to access to inhibitory and activating molecules on different patient samples. Healthy donors and AML patients at different stages of the disease were analyzed. The three following combinations were used: -CD3-FITC/NKp30-PE/VIVID-ECD/CD56-PC5.5/CD158a, h-PC7-CD158b1/b2, j-PC7/NKG2D-APC/NKp46-APC-A700/CD45-KO/DNAM-PB. -CD3-FITC/NKp30-PE/VIVID-ECD/CD56-PC5.5/CD158a, h-PC7-CD158b1/b2, j-PC7)/CD96-APC/NKp46-APC-A700/CD45-KO/DNAM-PB. -CD3-FITC/NKp30-PE/VIVID-ECD/CD56-PC5.5/CD158a, h-PC7-CD158b1/b2, j-PC7)/CD159-APC/NKp46-APC-A700/CD45-KO/CD57-PB. Data were analyzed with Kaluza Software version 1.2. These results allowed obtaining an extended phenotype of the NK cells in healthy donor and during AML disease. These combinations will be a powerful tool to investigate the potential application (regulation mechanisms of expression and activation of these receptors) related to these diseases.

23

AN 8-COLOR PANEL FOR DETECTION OF HUMAN BLOOD DENDRITIC CELLS BY FLOW CYTOMETRY

Emmanuel Gautherot,1 Nathalie Dupas,1 Anders Kverneland,2 Mathias Streitz,2 Birgit Sawitzki,2 Felix Montero-Julian,1 and Tewfik Miloud1

1Beckman Coulter Life Sciences, Marseille, France
2Institute of Medical Immunology, Charité Universitätsmedizin, Berlin, Germany

Dendritic cells (DCs) are antigen presenting cells capable of presenting antigen and priming a T cell response. They form a heterogeneous group of cells based on phenotype, location, and function. In human blood, DCs represent less than 1% of white blood cells, and can be separated into two main cell subsets, namely the myeloid DCs (mDCs) and the plasmacytoid DCs (pDCs). Among the mDCs, three distinct cell subsets are identified: CD1c+mDCs, CD141+mDCs, and CD16+mDCs. In blood, the frequency of DCs is affected in certain pathological conditions such as HIV, diabetes, asthma, chronic viral hepatitis, and graft-versus-host-disease. Thus, the detection and enumeration of different blood DC subsets is important to understand immune regulation in pathological conditions and to guide specific patient treatments. Due to the lack of specific markers for DC definition, the combination of several markers is required to allow their identification. Based on current knowledge in human DC biology, we have evaluated the expression and association of several DC markers to design an optimized 8-color panel for flow cytometry which allows for the detection of all DCs subsets in whole blood samples or peripheral blood mononuclear cells (PBMCs). This panel (CD1c/HLA-DR/Lineage/CD11c/CD16/Clec9A/CD123/CD45) provides an easy and robust assay to study the role of DCs in healthy donors and patient samples.

24

LONG-TERM STABILITY OF MULTI-COLOR COCKTAILS: ONE STUDY CONSORTIUM PANELS AS EXAMPLES OF ROBUST FORMULATION AND TANDEM DYE STABILITY

Emmanuel Gautherot,1 Nathalie Dupas,1 Felix J. Montero,1 James W. Tung,2 and Tewfik Miloud1

1Beckman Coulter Life Sciences, Marseille, France
2Beckman Coulter Life sciences, Miami, FL

Multi-color applications for flow cytometry constitute powerful tools to monitoring immune responses. Six 7- to 9-color leukocyte profiling panels have been designed in collaboration with the ONE Study Consortium, which has used these panels to demonstrate high levels of standardization across clinical studies at multiple sites. We have investigated the stability of these panels in two configurations: (1) formulated into three-vials with at most three fluors per vial and where APC/APC tandem and PE/PE tandem conjugates do not co-exist in the same vial to prevent potential dye interaction and (2) the entire panel formulated as a single cocktail. Three-vial configurations of different ages were evaluated versus fresh reference cocktail using two primary criteria: mean fluorescence intensity and population percentage compared to predetermined ranges. Each one-vial format was evaluated in real time versus a three-vial reference. Our data show that all panels provided stable performances up to at least a year after formulation, regardless of single-vial or three-vial configuration, and all measures in at least one panel showed satisfactory performance to 18 months. PE and APC tandem dyes in these formulations showed acceptable performance beyond 12 months in cocktail, indicating that tandem dyes are suitable for cocktails that require long-term stability. This study demonstrates that high plex antibody cocktails, when well-formulated, are stable for extended periods, providing powerful tools for multi-center and longitudinal immune profiling studies.

25

FLOW CYTOMETRIC ANALYSIS OF CD200 EXPRESSION IN PULMONARY SMALL CELL CARCINOMA

Hiyab Gebru,1 Evan Davis,1 Steven J Kussick,2 and Jason E Love3

1Multicare Health Systems, Tacoma, WA
2PhenoPath Laboratories, Seattle, WA
3Western Washington Pathology, Tacoma, WA

CD200 is a membrane bound glycoprotein that is expressed by a variety of normal tissues and hematopoietic malignancies. Flow cytometric analysis of CD200 expression has proven utility in the evaluation of mature B cell neoplasms, myeloma, and acute lymphoblastic leukemia. By including CD200 in routine flow cytometry panels we incidentally discovered convincing flow cytometric evidence of CD200 expression in seven cases of pulmonary small cell carcinoma and subsequently confirmed the expression using immunohistochemistry. CD200 expression in pulmonary small cell carcinoma has not been previously reported. CD200 may play a role in the biology of pulmonary small cell carcinoma, and it could represent a target for future therapies. These findings represent another example of how flow cytometry may be used to evaluate non-hematopoietic malignancies.

Figure 1.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Figure 1.

FIG. 1.

26

A NEW APPROACH FOR RAPID AND RELIABLE ENUMERATION OF CIRCULATING ENDOTHELIAL CELLS IN PATIENTS

Jan W. Gratama, Stefan Sleijfer, John Foekens, and Jaco Kraan

Department of Medical Oncology, Erasmus MC, Rotterdam, The Netherlands

Introduction: Mature circulating endothelial cells (CEC) are surrogate markers of endothelial damage. As many diseases are associated with vascular damage, enumeration of CEC is considered a promising tool to monitor disease activity with potential to assess prognosis and response to treatment. Several methods have been described to enumerate CEC. Lack of standardized assays and consensus on CEC phenotype has resulted in a wide variation of reported CEC numbers. Here, we present a novel approach to enumerate CEC using multi-parameter FCM method without immunological pre-enrichment.

Methods: CEC were defined as CD34+, CD45neg, CD146+, and DNA+ events based on the immunophenotype of endothelial cells from vein-wall dissections. As the vast majority of CEC express high levels of CD34, we based our assay on absolute CD34 counts after analyzing all CD34 positive events in a total blood volume of 4 ml. This relatively large volume is needed for a precise enumeration of CEC at a frequency of <1 cell per microliter. Part of the samples was tested with the CEC count kit (IQ Products, Groningen, The Netherlands), which is based on our assay.

Results: The endothelial origin of sorted CEC was confirmed by immunophenotyping (co-expression of endothelial specific/associated markers; CD31, CD105, CD141, and CD144), morphology, immunohistochemistry (vWF and CD31 expression), and gene expression of endothelial specific genes (CDH5, MCAm, and vWF). The new FCM assay was tested in parallel with a validated assay (i.e., CellSearch®, Veridex [Raritan, NJ]) and resulted in a high level of agreement (N = 28, R2 = 0.96). CEC levels ranged from 4 to 79 CEC/4 ml in healthy individuals (N = 30, median 17) and were significantly higher in patients with advanced solid malignancies (N = 55, median 38, P < 0.005) and in patients with hematological malignancies in remission prior to stem cell transplantation (SCT) (N = 60, median 38, P < 0.0005) (Fig. 1).

Conclusion: This CD34 based FCM assay and kit are able to enumerate a well-defined CEC population in peripheral blood. We envisage that this flow cytometric method can be used as a relatively cheap, high-throughput assay to accurately enumerate and further explore the characteristics of CEC. This will be particularly attractive for large-scale clinical studies to establish the clinical value of CEC in disease with endothelial damage or to monitor angiogenesis-inhibiting therapies.

27

FLOW CYTOMETRIC EVALUATION OF CD200 IN HEMATOLOGIC MALIGNANCIES AND ITS POTENTIAL ROLE IN THE DIAGNOSIS OF HAIRY CELL LEUKEMIA

Diana M. Haninger,1 Samuel R. Wellhausen,2 James A. Hoyer,2 Julianne Hubert,2 Marsha L. Jackson,2 Alvin W. Martin,2 and Sameer S. Talwalkar2

1Department of Pathology and Laboratory Medicine, University of Louisville, Louisville, KY
2CPA Laboratory, Norton Healthcare, Louisville, KY

Context: CD200 has been speculated to be a useful marker in differentiating chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). We investigate the utility of CD200 expression by flow cytometry (FC) in differentiating typical and atypical CLL (aCLL) from MCL, and its expression patterns in hematologic malignancies including hairy cell leukemia (HCL). Design: Blood, bone marrow, and fresh tissues were analyzed for CD200 expression using FC on 53 B-cell leukemias/non-Hodgkin lymphomas: 24 CLL, 5 aCLL, 6 MCL, 7 follicular lymphomas, 5 diffuse large B-cell lymphomas, 2 extranodal marginal zone lymphomas, and 4 HCL. Eleven multiple myelomas, 10 B-acute lymphoblastic leukemias, 7 acute myelogenous leukemias, 4 cases with hematogones (CD10+/CD20+), and 9 with benign plasma cells were also included. Expression based on mean fluorescence intensity (MFI) was categorized as negative/weak (<2.5 × 103), moderate (2.5 × 103 to 7.5 × 103) and bright (>7.5 × 103). Average MFI was calculated for each group and results plotted.

Figure 1.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Results: 96 cases were analyzed (Fig. 1). CD200 expression in CLL was bright (average MFI 1.15 × 104), moderate in aCLL (average MFI 5.68 × 103), and negative/weak in MCL (average MFI 9.66 × 102). All four cases of HCL showed bright and uniform expression (average MFI 2.19 × 104).

Conclusions: CD200 is expressed in a variety of hematologic malignancies and also in non-neoplastic cells. Its expression intensity can help differentiate CLL and aCLL from MCL. Due to its bright expression in HCL, it may be a potentially useful marker in the diagnosis, monitoring of residual disease, and targeted therapy with monoclonal antibody in HCL cases.

28

MIXED LYMPHOCYTE RESPONSE TO EXOGENOUS IL-15 IN GULF WAR ILLNESS AND MYALGIC ENCEPHALOMYELITIS/CHRONIC FATIGUE SYNDROME

Jeanna M. Harvey,1 Zachary M. Barnes1, Timothy Plitnik,2 Gordon Broderick,3,4 Xiao R. Zeng,1 Nancy G. Klimas,4,5 and Mary Ann Fletcher1

1Department of Medicine, University of Miami, Miami, FL
2Department of Microbiology/Immunology, University of Miami, Miami, FL
3Department of Medicine, University of Alberta, Edmonton, Canada
4Institute for Neuro Immune Medicine, Nova Southeastern University, Fort Lauderdale, FL
5Miami Veterans Affairs Healthcare System, Miami, FL

Background: After returning from Operation Desert Storm, an alarming number of veterans presented with a constellation of symptoms eventually labeled Gulf War Illness (GWI). This complex disorder affects nervous, endocrine, and immune regulation. Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), a complex illness affecting approximately 1 million people in the US alone, also impacts multiple systems and presents a significant clinical overlap with GWI. Both illnesses exhibit dysregulation of cytokines, specifically the observation of low IL-15. A profound impairment of NK cell function has been observed in both illnesses. We propose that stimulation with IL-15 may restore NK cell function in these patients.

Methods: Whole blood samples collected from GWI (n = 7), ME/CFS (n = 9), and healthy control (HC) (n = 10) subjects were recovered from cryopreservation in DMSO. Viable samples were incubated in growth medium for 18 h with and without 25 ng/ml of exogenous IL-15. NK cell cytotoxic function was assessed prior to cryopreservation and after stimulation using 51Cr labeled K562 target cells and lymphocyte abundance profiled with a fluorochrome multi-parameter cytometer.

Results: In vitro treatment with IL-15 improved NK cell function roughly 3-fold in both illnesses (ME/CFS P < 0.02; GWI P < 0.01), while CD3−CD56+ NK lymphocyte enumeration remained unchanged. Flow cytometry data exhibited significant increases in CD2+CD26+ and CD4+CD26+ abundance in ME/CFS (P < 0.01) and significant increases in CD8+CD26+ abundance in GWI (P < 0.01); no other significant cell population changes were observed. The increase of CD26+ lymphocyte expression and repair of NK cell cytotoxicity indicates IL-15 has a positive effect on immune function in GWI and ME/CFS.

29

ENUMERATING RETICULATED PLATELETS IN PATIENTS

Benjamin Hedley, Cyrus Hsia, and Mike Keeney

London Health Sciences Centre, London, Canada

Thrombocytopenia is a common indication for hematology consultation. The underlying causes of thrombocytopenia are numerous and bone marrow aspiration is often required but invasive. Previous methods of enumerating rPLTs initially relied upon light scatter characteristics to identify platelets. This is of limited utility as in several conditions “giant” platelets may be present which overlap with RBCs and may be excluded from the analyses. This study builds on flow cytometric analysis of rPLTs using Thiazole Orange (TO) fluorescent dye first described in 1990 by Kienast and Schmitz. In our laboratory, we have experience with the ISLH immunological platelet enumeration method and combined this with the TO RNA dye method outlined by Kienast and Schmitz. To evaluate the performance other nuclear dyes to TO we performed experiments as first described by Matic et al. (1998). Hypothesis: Platelet enumeration combined with a nucleic acid dye with low non-specific staining gives a more accurate assessment of rPLTs in patient with thrombocytopenia. Platelets were identified using CD41/CD61 APC (Beckman Coulter and Ebiosciences, respectively). Nuclei acid stains were used at various concentrations to determine optimal staining and stability. Normal donors, spent clinical samples from patients with thrombocytopenia, as well commercially available control material with published rPLT values (Abbott Laboratories©) Platelet combined with rPLT enumeration is possible using an amalgamation of the ISLH platelet method and previously published work. A careful gating strategy that includes immunological staining of platelets and discrimination of immature platelets by TO or other nuclear dyes allows for accurate rPLTs percentage to be determined.

30

EVALUATION OF THE PERFORMANCE CHARACTERISTICS OF A CD4 APTAMER FOR MULTICOLOR FLOW CYTOMETRIC ANALYSIS OF CD4+ T-CELL SUBSETS

Hsin-Hsuan Huang,1 Jonathan Fromm,1 Sheela Waugh,2 Urs A. Ochsner,2 Brent Wood,1 Geoffrey Baird,1 and David Wu1

1The Department of Laboratory Medicine, University of Washington, Seattle, WA
2SomaLogic, Boulder, CO

Introduction: Aptamers are engineered oligonucleotides that can be selected to bind with high specificity and affinity. As aptamers are cost-effective (estimated to be ∼1,000-fold cheaper than antibodies) and temperature-stable, aptamers may be useful in resource-limited settings for T-cell subset quantification.

Methods: A CD4-biotinylated-streptavidin-PE aptamer provided by SomaLogic, Boulder, CO was combined with CD3-PC5, CD8-ECD, and CD45-FITC antibodies in the multicolor flow cytometric assay; a CD4-A594 antibody was also added as an internal control for the CD4 aptamer. Residual patient samples were evaluated using the aptamer versus antibody reagents. Both the proportion and absolute count of CD4 T cells were analyzed. Results and Conclusion: The CD4 aptamer showed comparable results to the established method, which uses CYTO-STAT reagent and an FC-500 flow cytometry system, with r2 > 0.92 of CD4 T-cell% measurement. Further, both the percentage and absolute count of CD4 T cells showed excellent agreement over the range of T cell proportions (8.6%–50%) and absolute T-cell counts (150 cells/μl to 1,342 cells/μl) as assessed by the CD4 aptamer as compared to an internal CD4-A594 antibody control, with r2 > 0.99 and 9 cells /μl in mean difference. Our results suggest that a CD4 aptamer can perform comparably to an established CD4 antibody for CD4 T-cell quantitation and support further development of aptamers as cost-effective reagents for resource-limited regions. Support: This work was supported by the UW Hemapathology Laboratory, University of Washington. The aptamer reagent was provided by SomaLogic INC.

31

IMMUNOPHENOTYPIC PATTERN OF LEUKEMIC PRESENTATION OF BLASTIC PLASMACYTOID DENDRITIC CELL NEOPLASM BY 10-COLOR FLOW CYTOMETRY

Elizabeth Hyjek, Amr Rajab, Brian Sheridan, Emina Torlokovic, and Anna Porwit

Department of Laboratory Hematology, Laboratory Medicine Program, University Health Network, Toronto, Canada

Although classified by WHO 2008 as belonging to category “acute myeloid leukemia and related precursor neoplasms,” blastic plasmacytoid dendritic cell neoplasm (BPDCN) presents as acute leukemia (AL) only in a minority of cases. We have studied five BPDCN patients with AL bone marrow morphology (4 at diagnosis, 1 at relapse, 4/5 with pancytopenia).

Methods: 4-tube 10-color flow cytometry was applied (AML1: CD65 FITC, CD13 PE, CD14 ECD, CD33 PC5.5, CD34 PC7, CD117 APC, CD7 A700, CD11b A750, CD16 PB, AML2: CD36 FITC, CD64 PE, CD56 ECD, CD33 PC5.5, CD34 PC7, CD123 APC, CD19 A700, CD38 A750, HLA-DR PB, CD45 KO, AML3: CD71 FITC, CD11c PE, CD4 ECD, CD33 PC5.5, CD34 PC7, CD2 APC, CD10 A700, CD235a A750, CD15 PB, CD45 KO, cytoplasmic tube: nTdT FITC, cyt.MPO PE, CD14 ECD, CD33 PC5.5, CD34 PC7, cyt.CD79a APC, cyt.CD22 A700, CD19 A750, cyt.CD3 PB, CD45 KO with NaviosTM flow cytometer and KaluzaTM analysis software (Beckman Coulter).

Results: Leukemic cells were in the blast gate (CD45dim/low SSC) and were positive for CD4(bright), CD33(dim), CD56(heterogenous), CD123(bright), CD36, CD38, HLA-DR, CD71, but negative for CD10, CD11b, CD13, CD14, CD15, CD16, CD19, CD34, CD64, CD65, CD235a. Cyt.MPO, cyt.CD3, cyt.CD22 and nTdT were negative while dim cyt.CD79a was seen in three cases. CD2, CD7 and CD117 were found in single cases only.

Conclusion: We have determined characteristic BPDCN flow cytometry pattern of antigen expression in recently established 10-color AL panel, which will allow correct diagnosis in BPDCN patients presenting as acute leukemia with or without skin changes.

32

BICLONAL LYMPHOPLASMACYTIC LYMPHOMA MIMICKING BENIGN CD5(−)/CD10(−) POLYTYPIC B-CELLS BY FLOW CYTOMETRY. A CASE REPORT

Afshan Idrees,1 Jeffrey E. Lancet,2 and Pedro Horna2

1University of South Florida, Tampa, FL
2H. Lee Moffitt Cancer Center, Tampa, FL

Biclonal B-cell lymphomas are characterized by the presence of both kappa and lambda-expressing tumor sub-populations, which can be misinterpreted as benign polytypic B-cells. A 79-year-old asymptomatic male with a history of seizures was referred for hematological evaluation due to worsening thrombocytopenia and lymphocytosis. There was no palpable lymphadenopathy or visceromegaly on clinical examination. A complete blood count showed: WBC = 19,760/μl, hemoglobin = 13.7 g/dl, and platelets = 103,000/μl. A white blood cell differential count yielded 88% lymphocytes. A peripheral blood smear showed abundant small mature lymphoid cells with slightly eccentric nuclei and moderate amounts of pale blue cytoplasm containing occasional eosinophilic granules. Serum protein electrophoresis revealed an IgM lambda monoclonal protein of 0.9 g/dl. A bone marrow biopsy showed a diffuse infiltrate of atypical B-lymphoid cells, similar to those on peripheral blood, accounting for 70% of the marrow cellularity. Surprisingly, flow cytometric analysis of the bone marrow aspirate showed CD5(−)/CD10(−) small B-cells with a kappa:lambda ratio of 3.7:1 and homogeneous expression of all other surface antigens analyzed. CD25 was notably overexpressed on both kappa and lambda-restricted subpopulations. Increased staining intensity for surface immunoglobulin was also noted. Given the above findings, a diagnosis of biclonal lymphoplasmacytic lymphoma was rendered. Biclonal B-lymphomas can mimic benign polytypic B-cells, especially when lacking CD10 and CD5 expression. The presence of subtle immunophenotypic aberrancies and/or slight shifts in the kappa:lambda ratio should alert the cytometrist for a possible biclonal B-cell neoplasm.

33

IDENTIFICATION OF TWO DISTINCT SEZARY CELL SUBPOPULATIONS BY FLOW CYTOMETRY. A REPORT OF FIVE CASES

Afshan Idrees,1 Lubomir Sokol,2 and Pedro Horna2

1University of South Florida, Tampa, FL
2H. Lee Moffitt Cancer Center, Tampa, FL

Flow cytometry is commonly used to quantify Sezary cells (SCs) in peripheral blood, playing a critical role in the staging of mycosis fungoides and Sezary syndrome. Most analytical approaches rely on identifying a single population of CD4-T-cells with aberrant loss of CD26 and/or CD7 expression. We hereby report the presence of two markedly distinct clusters of SCs in peripheral blood specimens from five patients with Sezary syndrome. The median age was 72 years old (range: 70–84 years) with a male to female ratio of 3:2. Two clusters of SCs were identified by their remarkably different fluorescence intensity for CD7 (three cases), CD26 (two cases), CD2 (two cases), forward scatter (two cases), CD3 (one case), and CD4 (one case). Some subpopulations of SCs exhibited normal levels of expression of CD7 (three cases), CD26 (two cases), or both (one case). The ratio between the most and least abundant SC cluster ranged from 1.4:1 to 6.9:1 (median 2.2:1). A single small cluster of residual benign CD4 T-cells was also identified in three of five patients. Available follow-up studies showed persistence of the two SC subpopulations in one patient, and the disappearance of one of the SC clusters in another. The presence of two clusters of SCs with distinct immunophenotype is not a rare occurrence. Thus, gating and analytical strategies should consider this possibility when quantifying peripheral blood tumor burden by flow cytometry.

34

IMMUNOPHENOTYPIC AND MORPHOLOGIC SPECTRUM OF T-CELL LYMPHOMA IN SEROUS EFFUSIONS AND CORRELATION WITH TISSUE BIOPSY

Laura, J. Johnson, Wilhelmina A. Robetorye, and Katalin Kelemen

Department of Laboratory Medicine and Pathology, Mayo Clinic Arizona, Phoenix, AZ

T-cell lymphomas are uncommon in serous effusions. The aim of this study is to evaluate the morphology and immunophenotype of T-cell neoplasms found in serous effusions and to compare those with tissue biopsy results. Materials and methods: We performed retrospective search of our flow cytometry (FC) database to identify all serous effusions that were evaluated by FC and selected all cases of T-cell lymphoma for further study. Cytomorphologic and immunophenotypic features were reviewed and compared to tissue biopsy findings. Four-color FC was performed on a BD FACSCanto-II flow cytometer. Data analysis was performed by FACSDiva.

Results: Of seventy-nine serous effusions analyzed between February 2008 and June 2013, five T-cell lymphomas were diagnosed: one anaplastic large cell lymphoma (ALCL), one T-lymphoblastic leukemia/lymphoma (T-LBL), and three peripheral-T-cell lymphomas (PTCL). All five patients had a history of lymphoma. ALCL exhibited large anaplastic cells in cerebrospinal fluid and T-LBL presented with blasts in pleural fluid. PTCLs showed highly variable cytomorphology in cerebrospinal fluid with the following variations: (a) large, anaplastic cells, resembling ALCL; (b) heterogenous small lymphocytes, suggestive of reactive infiltrate; (c) monotonous small cells with moderate cytoplasm and mature chromatin, resembling a low-grade B-cell lymphoma. The FCIP was similar to tissue IP results in T-LBL and all PTCL cases.

Conclusions: ALCL and T-LBL retain their classic morphologic features in serous effusions. In contrast, PTCLs exhibit highly variable cytology that is commonly discordant with tissue findings and the diagnosis cannot be established without FC. The immunophenotype appears stable between tissue and serous fluid findings.

35

CORRELATION OF FLOW CYTOMETRIC AND CYTOLOGICAL ANALYSIS OF FINE-NEEDLE ASPIRATES

Aruna Kodituwakku,1,2 Thomas Fuller,1 Sandy Smith1, Peter Earls,3 and William Sewell1,4

1Immunology Department, SydPath, St Vincent's Hospital, Sydney, NSW 2010, Australia
2Immunology Department, SA Pathology, Royal Adelaide Hospital, Adelaide, SA 5000, Australia
3Anatomical Pathology Department, SydPath, St Vincent's Hospital, Sydney NSW 2010, Australia
4St Vincent's Clinical School, University of NSW, Sydney NSW 2052, Australia

Fine-needle aspirate (FNA) samples for the diagnosis of hematological malignancies are analyzed by cytology, usually in conjunction with flow cytometry. We assessed the relative contribution of each modality in the assessment of tissue FNAs. We reviewed 612 FNA samples that had been analyzed by both cytology and 8-color flow cytometry. Samples were included if results were abnormal by either or both modalities. However, samples with discordant results with no further diagnostic information were excluded. Of the 612 samples, there were 155 samples with abnormal results by either or both modalities, for which further information was available. Review of the medical records revealed that 136 of these 155 samples were true positive. Of the 136 cases, the majority (71%) were positive by both cytology and flow cytometry; however, a substantial proportion were detected by cytology alone (20%) or flow cytometry alone (9%). Cytology was more sensitive for diffuse large B-cell lymphomas. Hodgkin lymphomas (13%) were detected only by cytology. By contrast, flow cytometry detected numerous lymphomas that were missed by cytology, particularly low grade lymphomas such as follicular lymphomas. Flow cytometry had a greater PPV (93%), than cytology (89%). Combining cytology with flow cytometry increased the PPV to 96%. The data support the inclusion of flow cytometry in conjunction with cytology for FNA samples in the diagnosis of hematological malignancies. The results highlight the relative strengths and weaknesses of these modalities. (First two authors contributed equally. Work was carried out at, and supported by, St Vincent's Hospital, Sydney, Australia.)

36

VALIDATION OF A LABORATORY-DEVELOPED MULTI-PARAMETRIC FLOW CYTOMETRY ASSAY FOR DIFFERENTIATION BETWEEN INFECTIOUS AND NON-INFECTIOUS SYSTEMIC INFLAMMATORY RESPONSE SYNDROME IN CRITICALLY ILL PATIENTS

Stacy C. League,1 Matthew Smith,1 Amanda Martinez,1 Sarah M. Jenkins,2 and Roshini S Abraham1

1Department of Laboratory Medicine and Pathology, Mayo Clinic, Rochester, NY
2Department of Health Sciences Research, Mayo Clinic, Rochester, NY

Sepsis, systemic inflammatory response syndrome (SIRS) plus documented or suspected infection, is a leading cause of morbidity and mortality. Diagnosis is heavily reliant on microbial culture findings. Assessment of parameters, such as expansion of CD15− neutrophils and gain of CD64 expression on this subset and loss/decrease of HLA DR expression on monocytes have been proposed as alternatives for early diagnosis of sepsis. Flow cytometric evaluation in lieu of blood culture offers significant advantages such as lower blood volume requirements, decreased analytical time, and higher analytical sensitivity. A two-part multi-color flow cytometry assay was developed in our lab to characterize HLA DR molecules/cell on monocytes, percent of CD64+ neutrophils, CD64+ molecules/cell on neutrophils, and the ratio of CD15+/CD15− neutrophils in whole blood. A novel feature of this assay is the ability to quantify CD64 and HLA DR molecules per cell using Quantibrite™ flow reagents. There are minimum six parameters required for analytical validation of a laboratory-developed test: precision, accuracy, stability, reportable range, reference range, analytical sensitivity/specificity. We have presently obtained data on stability and precision and developed a pediatric reference range with samples from 116 infants/children. The expression of these markers was found to be stable for 48 h based on changes in MFI between fresh samples and subsequent time-points. Intra- and inter-assay precision were well within standard laboratory acceptance criteria of 20% CV. Additional analytical and clinical validation studies are under way. In summary, this assay has demonstrated the potential to be clinically relevant in the rapid diagnosis of sepsis in critically ill patients.

37

DEVELOPING APTAMER PROBES FOR DETECTION OF ACUTE MYELOGENOUS LEUKEMIA AND FOR BIOMARKER DISCOVERY

Ying Li, Guohua Jiang, Minli Yang, Wenjing Li, Samer Z. Al-Quran, and Christopher Carter

University of Florida, Gainesville, FL

In order to improve survival rate of patients with AML, we need to develop new strategies to discover biomarkers for detection and target therapy of AML. One of the advantages of the aptamer-based technology is the unique cell-based selection process, which allows us to efficiently select for cell-specific aptamers without knowing which target molecules are present on the cell surface. Using NB4 acute leukemia cell line as target cells, we selected and characterized a group of new DNA aptamers.

Figure 1.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Methods: Aptamers were selected from a single stranded DNA library containing more than 1 × 1014 sequences. The biotin-labeled aptamer sequences and PE-labeled streptavidin (SA) were used for the binding assay by flow cytometry. Aptamer-binding proteins were extracted by SA-magnetic beads, separated by SDS-PAGE and identified by mass spectrum analysis.

Results: The selected aptamers have high affinity and can be used to phenotyping AML in clinical specimens with the aid of cluster analysis (Fig. 1). Then, by using biotin-labeled aptamers, we identified target surface membrane proteins in low abundance, including CD64, Siglec-5, and a receptor-type tyrosine-protein phosphatase.

Conclusion: We have demonstrated: a pipeline approach for biomarker discovery: (1) to employ the Cell-SELEX technique to select for DNA aptamer probes against live leukemia cells; (2) to test selected aptamers in clinical specimens; and (3) to identify target proteins on leukemia cell surfaces.

38

USE OF PRECISION PROFILES TO ESTIMATE PRECISION OF LYMPHOCYTE SUBSET ANALYSIS BY FLOW CYTOMETRY

Robert Magari,1 Diana B. Careaga,1 Karen W. Lo,1 Xiangyang Dong,2 Elena Afonina,2 and Liliana M. Tejidor1

1Clinical Research, Diagnostics, Beckman Coulter, Inc., Miami, FL
2Clinical Applications, Life Sciences, Beckman Coluter, Inc., Miami, FL

A method to estimate precision based on profiles is presented. Precision of measuring the lymphocyte subsets depends on the range of measurement. Measurement of CD4+ T-cells occurs at various medical decisions levels, making it necessary to characterize precision throughout the range of measurements. Blood samples from >600 samples, covering the relevant measuring ranges, were stained with CYTO-STAT tetraCHROME™ reagents and tested in duplicate on the Navios*** (***Pending clearance by the US FDA; not yet available for in vitro diagnostic use within the USA) flow cytometer. Precision profiles were based on the relationship between the mean for each sample and the coefficient of variation (CV%). Mean represented the measuring range while CV% represented the precision performance as, CV = α Meanβ + ε, where α and β were the parameters of the model and ε was the random error. SAS 9.3 statistical software was used for analysis. Precision profiles were calculated for Total CD3+, CD3+/CD4+, CD8+, CD19+, and CD3-/CD56+ markers. Precision (CV%) at three percentiles of the measuring range and at medical decision points (for CD3+/CD4+) were estimated from the profiles. Upper 95% confidence limits for all precision estimates were also calculated. The upper 95% limits for CD3+/CD4+ repeatability were 4.9, 4.1, 3.0, and 2.7 CV% for levels of 50, 100, 350, and 500 CD3+/CD4+cells/µl, respectively. Precision profiles represent an improved approach for estimating precision throughout the measuring range. Estimations in this study represent typical responses, actual performance will depend on the capability of the instrument as well as laboratory conditions.

39

FREQUENCY OF LYMPHOPROLIFERATIVE DISORDERS WITH MORE THAN ONE MALIGNANT CELL POPULATION AS DETECTED BY 10-COLOR FLOW CYTOMETRY

Talal Mahdi, Amr Rajab, and Anna Porwit

Department of Laboratory Hematology, Laboratory Medicine Program, University Health Network, Toronto, Canada

We have evaluated the frequency of lymphoproliferative disorders (LPD) with more than one aberrant population detected during routine hematopathological diagnostics, using our newly introduced 10-color flow cytometry panels.

Materials and Methods: B-cell panel: Kappa-FITC/lambda-PE/CD19-ECD/CD38-PC5.5/CD20-PC7/CD34− APC/CD23 APC-AF700/CD10 APC− AF750/CD5− PB/CD45− KO. T-cell panel: CD57− FITC/CD11c− PE/CD8-ECD/CD3− PC5.5/CD2− PC7/CD56− APC/CD7− APC-AF700/CD4− APC− AF750/CD5− PB/CD45− KO. Plasma cell (PC) panel: cyt.kappa FITC/cyt lambdaPE/CD56ECD/CD138 PC5.5/CD34PC7/CD117APC/CD19A700/CD38 A750/CD20PB/CD45KO. Acquisition: NaviosTM flow cytometer. Analysis: KaluzaTM software (Beckman Coulter).

Results: Between May 2012 and January 2013, 2,600 blood, bone marrow, FNA, lymph node, and pleural fluid cell suspensions were analyzed. Seven hundred ninety samples showed at least one aberrant B-cell population and 27 showed at least one aberrant T-cell population. Among 41 samples with two B-cell populations, there were 13 with two co-existing aberrant B-cell and/or plasma cell populations and different sIg (one kappa+ and one lambda+) most with B-CLL-related phenotype. Seven cases showed two B-cell populations with the same light chain but distinctly different immunophenotypes. In 21 cases, two populations had the same light chain and differed by expression of one or two markers, thus, a possibility of intraclonal differentiation could not be excluded. In only two samples one population with B-cell LPD and one clonal T-cell population with LGL-related phenotype were found.

Conclusion: Using our panels, 5.2% of cases with LPD-associated aberrant findings show two aberrant lymphoid and/or plasma cell populations.

40

FOXP3+ Tregs CELLS SENSITIVITY TO LOW DOSES OF IL-2 CAN BE RAPIDLY AND EASILY MONITORED BY P-STAT-5 DETECTION IN EX VIVO WHOLE BLOOD SAMPLES

Fabrice Malergue, Valerie Mallet, Caroline Scifo, Tewfik Miloud, and Felix Montero

Beckman Coulter Life Science, Marseille, France

Introduction: Phospho-epitopes are difficult to detect by flow cytometry, currently requiring dedicated techniques and buffers, a total work time of 2–4 h, 37°C, and ice incubations, and 4–8 wash steps. The reference methods also use methanol, which is toxic for the user and harmful for the other cell markers, compromising some multi-parametric studies. The FOXP3 marker is incompatible with “harsh” phospho-epitope detection methods, rendering even more challenging the studies of regulatory T cell (Treg) functions.

Methods: We have used a new reagent kit, PerFix EXPOSE (Phospho Epitopes Exposure Kit), which uses multiple innovations in the permeabilization, staining, and wash steps required for intracellular staining. It is devoid of methanol and supports staining of all common phospho-epitopes with one single procedure of about 1 h. Since many extra-cellular markers can be combined together with the anti-phospho markers, we evaluated also various FoxP3 clones and conjugates, and tried some procedure optimizations. The best conditions were then used to analyze the ex vivo functionality of FoxP3+ cells, including IL-2 pathway signaling.

Results: PerFix EXPOSE allows for the simultaneous detection of some antigens that are otherwise incompatible, such as FOXP3 and phospho-STAT epitopes. An IL-2 dose-response curve could be easily generated directly from fresh whole blood that confirmed on normal donors a 10- to 100-fold difference in sensitivity to IL-2 between Tregs and other T cells.

41

SIMPLE AND ROBUST WHOLE BLOOD STAINING PROCEDURE USING PerFix-nc REAGENTS GENERATES UNPRECEDENTED FOXP3 SIGNAL/NOISE RATIO

Fabrice Malergue,1 Laeticia Khemici,1 Carlos Garcia,2 Tewfik Miloud,1 and Felix Montero1

1Beckman Coulter Life Science, Marseille, France
2Beckman Coulter Life Science, Miami, FL

FOXP3 is a nuclear antigen expressed specifically in regulatory T cells (Tregs). Tregs play a critical role in the maintenance of dominant self-tolerance and have been subject of extensive research effort for the last two decades. Today the detection of FOXP3 is laborious, time consuming (3–4 h) and poorly reproducible. A new commercially available method, PerFix-nc, allows simultaneous intra- and extra-cellular staining without any wash step. This new kit allows for the reduction of the staining workflows to 45 min. In this study, we optimized the PerFix-nc protocol for the detection of FOXP3. We evaluated antibody clones from various vendors, optimizing titration and incubation time, and adding some extra washing steps. The FOXP3 signal-to-noise ratio obtained with PerFix-nc was at least 40% better than the ratio obtained for the same samples with a reference procedure. Improved cell scatter separation compared to reference method was revealed. Purity and recovery were also significantly increased. Furthermore, the method was robust, demonstrating excellent repeatability with respect to percentages of FOXP3-positive cells and signal-to-noise ratios. The total workflow was reduced from 3 to 4 h using the reference method to about 95 min using PerFix-nc. In conclusion, the novel PerFix-nc reagent significantly improves FOXP3 detection in several different ways: it simplifies the FOXP3 intracellular staining procedure, generates robust and highly reproducible results, and provides unprecedented signal-to-noise ratios.

42

DETECTION OF IgG, IgM, IgA, AND C3d ATTACHED TO ERYTHROCYTES IN AUTOIMMUNE HEMOLYTIC ANEMIA DETECTED BY FLOW CYTOMETRY

Luis Guillermo Manrique,1 Maria Alejandra Alzate,1 Natalia Bolaños,1 Monica Duarte,2 Paola Ximena Coral,3 and John Mario Gonzalez1

1Grupo de Ciencias Básicas Médicas, School of Medicine. Universidad de los Andes, BOGOTA, Columbia
2Hematology section, Fundación Santa Fe de Bogotá, BOGOTA, Columbia
3Rheumatology section, Fundación Santa Fe de Bogotá, BOGOTA, Columbia

Introduction: Immunoglobulin complexes (ICs) attached to the erythrocytes' membrane are involved in the pathogenesis of autoimmune hemolytic anemia (AIHA). Currently, the method for ICs detection is the direct Coombs test (DAT), whose sensitivity and specificity are considered low. Objective: To design a flow cytometry protocol for simultaneous detection of IgG, IgM, IgA complexes and C3d attached to the erythrocytes' membrane. Methodology: Positive in vitro controls were designed for each ICs using anti-Rh or anti AB antibodies for IgG and IgM, respectively. For IgA, erythrocytes treated with acid tannic plus IgA were used. A technique for simultaneous detection of IgG, IgM, IgA complexes, and c3d immune was standardized with fluorochrome conjugated anti-human antibodies. The protocol was applied to capillary blood samples of patients with AIHA, patients at risk of developing the disease and healthy controls.

Results: We evaluated 24 blood samples, 9 patients with AIHA, 5 people at risk of AIHA, and 10 healthy controls. Cut-off points were calculated using the mean and standard deviation of each individual result. In the AIHA positive group, eight patients (88.9%) were positive for C3d, six (66.7%) for IgG, five (55.6%) for IgA, and two (22.2%) for IgM. Three AIHA patients were negative for IgG immune complexes, but they were highly positive for C3d.

Conclusions: Flow cytometry can be used to identify IgG, IgM, IgA complexes, and C3d attached to the erythrocytes' membrane. This simultaneous detection could help to dissect the physiopathology mechanisms of lysis in AHAI patients.

43

TIME TO REMOVE SPURIOUS EVENTS

Zhengwei J. Mao,1 Andinet Tefera,1 Charanjeet Singh,2 Bin Yin,3 and Timothy P. Singleton2

1University of Minnesota Medical Center, Fairview, Minneapolis, MN
2University of Minnesota, Minneapolis, MN
3Cyrus Tang Hematology Center, Jiangsu Institute of Hematology, the First Affiliated Hospital, Soochow University, Suzhou, China

Background: As minimal residual disease (MRD) detected by flow cytometry is widely used to guide therapy for leukemias and lymphomas, flow cytometry assays with high sensitivity have become clinically important. Time can be displayed to remove artifacts from these high-sensitivity assays. Our objective is to categorize spurious events that were observed during routine analysis of specimens for leukemias and lymphomas and that could be removed by gating with time.

Methods: We analyzed 540 cases in a clinical flow cytometry laboratory over three different time periods: the beginning of 2009 and in June and July 2012. Scatterplots were analyzed for forward scatter versus time, side scatter versus time, side versus forward scatter, and side scatter versus CD45. Patterns were categorized and evaluated for possible etiologies.

Results: Five abnormal patterns were observed with plots of light scatter versus time: (1) unchanged median of light scatter but with different densities of events, (2) increased or decreased median of light scatter, (3) events across the entire spectrum of light scatter, (4) one or more gaps with rare-to-absent events, and (5) a mixed pattern. Each pattern could be correlated with possible etiologies: change in flow rate, fluidic clog, air bubbles, etc. The incidence of these artifacts decreased as the laboratory became aware of the problems.

Conclusions: Artifacts are seen during the routine analysis of patient specimens in clinical flow cytometry laboratories. It is important to examine light scatter versus time to remove spurious events.

44

THE PRESENCE OF IMMUNOPHENOTYPIC ALTERATIONS IN BONE MARROW CELLS OTHER THAN PLASMA CELLS FROM MULTIPLE MYELOMA PATIENTS PREDICTS FOR MYELODYSPLASIA-ASSOCIATED CYTOGENETIC ABNORMALITIES

Sergio Matarraz,1 Bruno Paiva,2 María Jara-Acevedo,1 Jose Maria Sayagues,1 Jesus Fernando San Miguel,2 and Alberto Orfao1

1Centro de Investigación del Cáncer and IBSAL, Cytometry Service and Medicine Department, Salamanca, Spain
2Servicio de Hematología, Hospital Universitario de Salamanca and IBSAL, Salamanca, Spain

The progress achieved in treatment of multiple myeloma (MM) has been accompanied by a concern for therapy-related secondary primary malignancies (SPMs). High-dose melphalan (HDM) and also lenalidomide have been associated with higher risk of SPMs development, particularly myelodisplastic syndromes (MDS) and/or acute myeloid leukemia. Consequently, it is of major relevance to identify MM patients at higher risk of developing SPMs. We investigated whether the presence of MDS-associated phenotypic alterations (MDS-FC) by 8-color flow cytometry predicted for MDS-associated cytogenetic abnormalities in a total of 257 unselected MM patients. The presence of MDS-FC was detectable in 21 MM cases (8%). In these patients, FACS purified CD34+ hematopoietic stem cells (HSC), neutrophils, monocytes, and erythroblasts were analyzed by FISH (in male patients, n = 12) for screening of MDS recurrent cytogenetic abnormalities. Conversely, in female patients (n = 9) these cell populations were screened for clonality using the human androgen receptor X-chromosome inactivation test (HUMARA). Five male patients (42%) showed cytogenetic abnormalities; three cases with del(5q31), one with –Y, and one with trisomy 8 in all purified BM cell populations, while six of seven (86%) female patients depicted clonality by HUMARA. The presence vs. absence of MDS-FC predicted for a clonal HUMARA test in HSC (100% vs. 11%; P = 0.005) and neutrophils (67% vs. 0%; P = 0.05), with a similar trend being also found in erythroblasts (80% vs. 29%; P = 0.08). Our results show that immunophenotypic dysplastic features are present in one-third of MM patients at diagnosis and that in around half of them these alterations are associated with clonality.

45

ZAP-70 IS PREDOMINANTLY NUCLEAR IN CLL CELLS: AN ANALYSIS BY IMAGE CYTOMETRY

Howard Meyerson,1 Georgetta Blidaru,1 Alison Edinger,1 Ebenezer Osei,1 Karen Schweitzer,1 and Amy Graham2

1University Hosp Case Medical Centre, Cleveland, OH
2Case Western Reserve University, Cleveland, OH

ZAP-70 is a T cell receptor-associated signaling molecule. In CLL, ZAP-70 expression enhances signaling through the B-cell receptor potentially accounting for the more aggressive behavior of ZAP-70(+) CLL. The sub-cellular location of ZAP-70 in CLL cells has not been evaluated. Therefore, we examined the intracellular distribution of ZAP-70 in CLL cells using the Amnis ImageStream X imaging cytometer. The expression level of ZAP-70 assessed on the imaging cytometer was comparable to the level on the clinical laboratory FACSCanto flow cytometry (R2 = 0.58). ZAP-70 expression was primarily nuclear and heterogeneous in CLL cells with little cytoplasmic or membranous signal. The mean nuclear Similarity score (a logarithmic transformation of the Pearson correlation coefficient) of ZAP-70 was 1.93 ± 0.55. There was no relationship between Similarity and percentage of ZAP-70(+) cells. ZAP-70 distribution in normal peripheral blood T cells was also primarily nuclear (Similarity = 1.82 ± 0.42). Nuclear specific fluorescence accounted for 82.3% ± 7.2 of total cellular ZAP-70 fluorescence in CLL. Since the mean nuclear/cytoplasmic area of CLL cells was 32.0% ± 8.2%, ZAP-70 was, on average, 11.3 ± 2.8 times more concentrated in the nucleus than the remainder of the cell in CLL. Findings were similar for T cells where ZAP-70 was 10.6 ± 4.4 fold more concentrated in the nucleus. We conclude ZAP-70 expression is primarily nuclear in CLL and peripheral blood T cells. Image cytometry may uncover unexpected cellular distributions of marker molecules potentially of biologic and clinical significance. The role of nuclear ZAP-70 is unexplained.

Figure 1.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

46

CORRELATION BETWEEN FLOW CYTOMETRY AND HISTOLOGY FOR LYMPHOMA DETECTION IN BIOPSY AND FINE-NEEDLE CYTOLOGY SAMPLES

K. C. Ng, S. F. Tan, J. Li, S. Y. Lee, S. B. Ng, S. Wang, and T. C. Liu

Department of Laboratory Medicine, National University Hospital, Singapore, Singapore

Objective: Flow cytometry (FC) is increasingly being requested for patients who are suspected to have a lymphoma (NHL). Its clinical utility, however, remains uncertain.

Figure 1.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Methods: We reviewed the results of FC screening and the eventual histology (HI) diagnosis on 427 patient samples. The samples included 68% lymph node biopsies, 17% organ biopsies, and 7% needle aspirates. FC screening for lymphoma was performed using a one-tube antibody combination, either FITC(CD8+Kappa)/PE(CD56+Lambda)/ PerCP-Cy5.5(CD4+CD19)/APC(CD38)/APC-H7(CD20)/V450 (CD14)/PE-Cy7(CD3)/AmCyan(CD45) or FITC(CD8+Lambda)/ PE(CD56+Kappa)/PerCP-Cy5.5(CD5)/APC(CD3)/PE-Cy(CD19+ TCRδγ)/APC-H7(CD38)/Pacific Blue(CD4+CD20)/V500(CD45). Follow-up information from patients with discordant results was retrieved to ascertain the eventual diagnosis.

Results: Histology reported 212 samples as lymphoma, 22 metastatic disease, 22 Hodgkin's lymphoma, 163 reactive, and 2 inconclusive samples. Concordant FC results were reported for 323 samples (75.6%). There were six FC+/HI− (1.4%) samples. FC called a B-NHL on these six samples. Of these, three were negative on IgH rearrangement studies and only one patient was eventually confirmed to have a B-NHL. The other five FC+/HI− patients remained well without treatment. Ninety-six samples (22.5%) were FC−/HI+. This included all the patients with metastatic disease and Hodgkin's lymphoma. Twenty-six of the remaining 52 samples received for FC were deemed sub-optimal for meaningful analysis. FC analysis was attempted and NHL was not detected on the other 26 samples (6.1%). The assessed cell viability was <50% on 10 of these 26 samples.

Conclusion: Discordant FC and HI results are present on 22.5% of samples. The majority of these correspond to samples with low cell yield/viability submitted for FC. Histology remains a better overall diagnostic tool for the assessment of lymphoma in biopsy specimens.

47

DETECTIONOF LEF1 BY FLOW CYTOMETRY: APPLICATION IN DIAGNOSIS OF CHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTIC LYMPHOMA AND MONOCLONAL B-LYMPHOCYTOSIS

Sarah L. Ondrejka, Juraj Bodo, Betty Gay, Deborah Katanik, Finbar Emanus, Florence Namiotka, Linda Henrich, Cynthia Polz, and Eric D. Hsi

Department of Clinical Pathology, Cleveland Clinic, Cleveland, OH

Mature B-cell lymphoproliferative disorders (LPDs) have overlapping morphologic/immunophenotypic features. Accurate diagnosis by flow cytometry requires a panel of antibodies. Recently, overexpression of lymphoid-enhancer-binding-factor-1 (LEF1) showed high sensitivity/specificity for identification of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL) in FFPE tissue by immunohistochemistry. We studied LEF1 expression to determine its efficacy in identifying CLL/SLL and its precursor state, MBL. Peripheral blood (PB) or bone marrow (BM) samples were analyzed: 25 CLL, 13 MBL, 2 CD5+ LPD, not otherwise specified (NOS), and 2 healthy controls. Tissue samples included: three SLL, three mantle cell lymphoma, three follicular lymphoma, and one marginal zone lymphoma. Antibodies used were: CD5, CD19, CD20, CD22, CD23, CD45, FMC7, kappa/lambda light chain, and LEF1 (Alexafluor 488- conjugated, Cell Signaling Technologies, Danvers MA). FACS Lysing Solution (BD Bioscience) was used to permeabilize samples. Background normal B-cells were an internal negative control for LEF1. LEF1 was positive when expressed by ≥ 20% of cells. For PB/BM samples, LEF1 was expressed in 21/25 CLL (84%), 9/13 MBL (69%), 0/2 CD5+ LPD, NOS, and 0/2 normal patients. For tissue samples, CLL/SLL cases were positive (3/3), while all other lymphoma subtypes were negative. Matutes scores, for comparison, were 4–5 in 22/25 CLLs and 11/13 MBLs. LEF1 was not detected in granulocytes and non-neoplastic B-lymphocytes, and stained neoplastic B-cells with a similar intensity as background T-lymphocytes. LEF1 may be helpful in identifying cases of CLL/SLL when used in conjunction with a panel of other antibodies, but is not likely to be reliable as an individual marker. Additional cases are being studied.

48

POTENTIAL OF NEUTROPHILIC CD64 AND MONOCYTIC HLA-DR IN MONITORING OF NEONATAL SEPSIS

Richeek Pradhan,1 Paresh Jain,2 Anshuman Paria,1 Anindya Saha,1 Jagdish Sahoo,1 Noel Warner,3 Arun Singh,1 and Mitali Chatterjee1

1Institute of Post-Graduate Medical Education and Research, Kolkata, India
2BD Biosciences, Gurgaon, India
3BD Biosciences, San Jose, CA

Background: Neutrophilic CD64 (nCD64) is an established frontrunner for diagnosis of neonatal sepsis while monocytic HLA-DR (mHLA-DR) has prognostic potential. This prospective study aimed to evaluate the monitoring potential and survival prognostication of nCD64 and mHLA-DR in neonatal sepsis.

Methods: The study included 100 neonates with suspected sepsis and 29 controls. Of the cases, 21 were culture positive while 42 met European Medical Agency clinical sepsis criteria. Expression of nCD64 and mHLA-DR was evaluated by flow-cytometry and a sepsis index (SI) was derived (nCD64/mHLA-DR × 100). In cases, blood was collected at day 1 based on clinical suspicion (Sample 1), day 2 (Sample 2), days 4–6 (Sample 3); serial monitoring (Samples 1, 2, and 3) was done in 46 cases.

Results: In sepsis cases, nCD64 and SI were significantly up-regulated while mHLA-DR was significantly down-regulated as compared to controls (P < 0.001). Sensitivity and specificity to detect sepsis at day 1 for nCD64 were 73.01% and 89.18%, for mHLA-DR were 19.35% and 71.42% while for SI, 73.01% and 72.22%. Sensitivity improved to 86.53% (nCD64) and 82.69% (SI) with information from Sample 2. In sepsis cases, where Sample 3 was > cut-off, the odds ratio (OR) for change of antimicrobials on/beyond day 4 for nCD64 and SI was 5.92 (P = 0.021, 95% CI: 1.22–32.22) and 6.33 (P = 0.007, 95% CI: 1.52–26.34) respectively; with Sample 3 lesser than cut-off for mHLA-DR, OR was 1.86 (P = 0.318, 95% CI: 0.54–6.04). In a receiver operated characteristic curve to detect 30 day mortality, SI showed greatest area under curve (0.736) > mHLA-DR (0.720) > nCD64 (0.640).

Conclusion: nCD64 alone had the best diagnostic efficacy while SI correlated best with change of antimicrobials and survival. Funding source: The project was part of a collaborative research agreement between IPGME&R and BD Biosciences. As a part of this agreement, flow-cytometry related reagents were supplied by BD. No other financial support was received.

49

DETECTION OF ANAPLASTIC LARGE CELL LYMPHOMA IN A PATIENT WITH HEMOPHAGOCYTIC LYMPHOHISTIOCYTOSIS: A CASE SHOWING USEFULNESS OF FLOW CYTOMETRY

Jyotinder N. Punia,1 Amos S. Gaikwad,2 M. Tarek Elghetany,1 and Andrea M. Sheehan1

1Department of Pathology and Immunology, Baylor College of Medicine and Texas Children's Hospital, Houston, TX
2Texas Children's Cancer and Hematology Centers, Texas Children's Hospital, Houston, TX

Secondary hemophagocytic lymphohistiocytosis (HLH) is known to be due to underlying conditions like overwhelming infection, autoimmune disorder, or malignancy. We present a case of HLH where flow cytometry proved useful in identifying an unsuspected underlying hematologic malignancy. A previously healthy 17-year-old boy presented in respiratory distress following two weeks of fever, fatigue, chills, night sweats, significant weight loss, inguinal lymphadenopathy, hepatosplenomegaly, and diffuse bilateral lower lobe infiltrates. He met six out of eight criteria for a diagnosis of HLH. Bone marrow biopsy showed increased hemophagocytosis as well as a population of atypical medium to large cells with open chromatin, irregular nuclear contours, moderate basophilic cytoplasm with vacuoles and cytoplasmic blebs. These cells were also noted in the peripheral blood. Morphologic differential included acute myeloid leukemia (AML) or lymphoma. Flow cytometry showed expression of CD45, CD7, CD2, CD25 (very bright), CD13, CD33 (dim), HLA-DR, CD16+56 (dim), and CD58. Subsequent immunohistochemical stains showed expression of CD30 and anaplastic lymphoma kinase (ALK). Corresponding cytogenetic study revealed der(2)t(1;2) and fluorescence in situ hybridization confirmed ALK gene rearrangement, establishing a diagnosis of ALK positive anaplastic large cell lymphoma (ALK+ ALCL). In conclusion, flow cytometry is a very useful tool in identifying an underlying hematologic malignancy in cases of secondary HLH, and ALK+ ALCL should be considered in the differential diagnosis. The expression of myeloid markers can be a pitfall, causing confusion with AML, and awareness is needed to arrive at the correct diagnosis.

50

TEN-COLOR 14 ANTIBODY FLOW CYTOMETRY PANEL FOR IMMUNOPHENOTYPING OF PAUCICELLULAR CYTOLOGIC SPECIMENS

Amr Rajab, Scott Boerner, Gilda da Cunha Santos, William Geddie, Hyang Mi Ko, and Anna Porwit

Department of Laboratory Hematology and department of Cytology Laboratory Medicine Program, University Health Network, Toronto, Canada

Introduction: Flow cytometry (FCM) has an important role in the evaluation of lymphoid cell populations identified in cytologic specimens (CS). Small quantity of cells or low sample volume often poses a challenge for FCM analyses of CS. We have developed a single tube panel to provide maximum immunophenotyping information applicable to paucicellular CS.

Methods: 14 monoclonal antibody/10 fluorochrome panel was applied: kappa+CD4 FITC, Lambda+CD8 PE, CD3 + CD14 ECD, CD34 APC, CD20+CD56 PC7, CD10 APC-A750, CD19 APC-A700, CD33 PC5.5, CD5 PB, and CD45KO. As many as possible events (on average 5,000) were analyzed using NaviosTM flow cytometer and KaluzaTM software (Beckman Coulter). Results were correlated with cytomorphological diagnosis.

Results: Between August 2012 and June 2013, 56 paucicellular CS were analyzed (25 CSF, 18 FNA, 7 bronchoalveolar lavage, and 6 vitreous fluid). Hematologic neoplasm was detected by FC/CC in 15 of 56 cases (27%). 11/15 (73%) CS showed lymphoma involvement based on expression of CD10 on B-cells (n = 5, 45%), aberrant expression of CD5 on CD19+ cells (n = 3, 27%) and/or the presence of κ/λ restriction (n = 8, 72%). 4/15 CS (26%) showed acute leukemia involvement [three AML (CD33+, CD34+, CD45dim) and 1 T-cell ALL (CD3−, CD5+, CD4+, CD8+, CD45dim)]. One sample showed 2% CD5+/CD19+/Kappa B-cells by FC but no cytological evidence for lymphoma. Two FNA samples negative by FC showed invasive carcinoma and epithelioid sarcoma.

Conclusions: Developed 14 MAb 10-color FCM panel is effective and reliable to identify hematopoietic neoplasms in paucicellular cytologic specimens.

51

FLOW CYTOMETRIC CLL MRD TESTING: IMPACT OF PANEL DESIGN, AND AUTOMATED CELL PROCESSING ON SENSITIVITY

Margo Santiago, Dalia A. Salem, Mary M. Addison, Constance M. Yuan, Gregory A. Jasper, and Maryalice Stetler-Stevenson

NCI, NIH, Bethesda, MD

Flow cytometric minimal residual disease (FC-MRD) is an independent predictor of progression-free and overall survival. Furthermore, the FDA is considering FC-MRD as a surrogate endpoint in clinical trials. A recent FDA workshop emphasized that multiple strategies are utilized in CLL FC-MRD, from the European Research Initiative in CLL (ERIC) panel (analyzing for abnormal expression of CD19, CD5, CD20, CD22, CD79b, CD43, CD81, and CD3 without surface immunoglobulin analysis) to strategies utilizing abnormal expression of antigens such as CD19, CD5, CD20, and CD22 with surface immunoglobulin analysis. We created serial dilutions of CLL cells in peripheral blood to systematically compare the sensitivity of FC-MRD detection using the following panels:

PanelFITCPEPerCPPC7APCAH7v450v500
ERIC8179b2219432053
LC 1KappaLambda19200CD53845
LC 2KappaLambda20195
LC 3Lambda22519Kappa-2011c45

Furthermore, specimens were split and processed manually and with the BD-Lyse Wash Assistant. There was no difference in the sensitivity of CLL FC-MRD detection between specimens processed by either method. The LC 1 panel was inferior, showing a 10-fold lesser sensitivity of CLL FC-MRD detection. The ERIC, LC 2, and LC 3 panels showed comparable CLL FC-MRD sensitivity (0.02–0.002% CLL). The results indicate that multiple successful approaches exist for CLL FC-MRD.

52

MULTICHANNEL FLUORESCENCE OF DRAQ7 MAY ALLOW ACCURATE EXCLUSION OF DEAD CELLS WITHOUT THE USE OF A DEDICATED CHANNEL

Richard D. Schretzenmair, Andrew D. Bantly, Lifeng Zhang, and Jonni S. Moore

University of Pennsylvania, Philadelphia, PA

Goal: Discrimination of live cells is critical for accurate flow cytometric evaluation of clinical samples. Often this consumes a precious extra parameter that could be used for phenotypic evaluation. Here, we attempt to use the broad excitation and emission spectra of DRAQ7 and DAPI to eliminate the need for a dedicated live/dead channel. Method: The DRAQ7 fluorescence is very bright and overlaps stoichiometrically into four channels on a standard FACSCanto. DAPI, also bright, spills into two channels but is less stoichiometric than DRAQ7. Thus we sought to create gating strategies based on the relative fluorescence of all four DRAQ7 channels that would allow the co-determination of live/dead without the need for a dedicated channel.

Results: Generally, DRAQ7 in a channel concurrent with a marker that is tightly regulated, such as CD45, may be adequate to exclude the majority of dead cells. If it is determined that all dead cells must be identified, markers that are mutually exclusive (e.g., Kappa, Lambda) may be included in the four channels into which DRAQ7 bleeds, the double positives being dead. However, in a worse case scenario, when a single cell population has intense antigen expression in all four DRAQ7 bleed channels, full identification of all dead cells would not be possible. This method may be used if care is taken to ensure appropriate panel construction, or if rare dead cells are not a problem. Far from being a liability, multi-channel fluorescence of dead cell markers may be beneficial.

53

B-ALL MINIMAL RESIDUAL DISEASE FLOW CYTOMETRY: CRITICAL EVALUATION OF HISTORICAL DATA AND VALIDATION OF SINGLE-TUBE MODEL

Aaron C. Shaver,1 Bruce Greig,1 Claudio A. Mosse,1,2 and Adam C. Seegmiller1

1Vanderbilt University Medical Center, Nashville, TN
2VA Tennessee Valley Healthcare System, Nashville, TN

Detection of minimal residual disease (MRD) is one of the most potent applications of clinical flow cytometry, and B lymphoblastic leukemia (B-ALL) is the most well established application of flow MRD. As more markers have been introduced and testing panels have grown larger, the technical complexity and cost associated with B-ALL MRD testing has increased. We describe our experience with a three tube, 7-color B-ALL MRD panel used in our clinical flow cytometry laboratory over the course of one year, and we analyze the relative contribution that each marker and marker pair makes to identifying MRD on a case-by-case basis. This novel technique highlights the relative contribution each marker makes toward diagnosing MRD, and weights the contribution more heavily toward markers that are useful in difficult cases (i.e., those with fewer abnormal markers). Based on this analysis, we present the validation of a low-cost one tube, 8-color panel and describe the relative utility of each marker and marker pair, resulting in a significant decrease in technical complexity and material cost with no loss of diagnostic utility in a retrospective analysis. While the technique of MRD analysis for B-ALL MRD is a mature one, being performed already in many clinical flow labs, we suggest that these results may serve as a model for reducing complexity in existing testing strategies, as well as increasing the ease of adoption of testing for labs that have not yet implemented B-ALL MRD.

54

FLOW CYTOMETRIC CD103+CD4+/CD4+ RATIO IN BRONCHOALVEOLAR LAVAGE FLUID: A TOOL FOR DIAGNOSING SARCOIDOSIS

Jagmohan Sidhu,1 Sonia Brar,2 Christopher Remakus,2 Julia Miller,2 and Edward Santelli3

1Department of Pathology and Laboratory Medicine, UHS Hospitals, Johnson City, NY
2Department of Internal Medicine, UHS Hospitals, Johnson City, NY
3Department of Radiology, UHS Hospitals, Johnson City, NY

Context: Three CD103+CD4+ to CD4+ ratios in the bronchoalveolar lavage fluid (BALF) [<0.2, <0.31, and <0.45]), two CD4+ to CD8+ ratios [>3 and >2.5]) and a ratio of BALF CD4+ CD8+ to peripheral blood (PB) CD4+ CD8+ [>2]) have been used by various investigators as tools for discriminating pulmonary sarcoidosis from other interstitial lung diseases. We investigated the role of these flow cytometric analysis (FCA) ratios in the diagnosis of pulmonary sarcoidosis at various radiologic stages. Design: FCA ratios in 12 cases of suspected pulmonary sarcoidosis with >10% lymphocytes in the BALF were correlated with clinicoradiologic and histolopathologic diagnosis. FCA of BALF and PB (when available) utilized CD45/CD3/CD19/CD4/CD8/CD103 antibody panel in all cases.

Results: Eleven of 12 cases had CD103+CD4+ to CD4+ ratio at <0.45. Nine of these 11 cases had sarcoidal granulomas (with negative stains for acid fast organisms and fungi) in the lung biopsy and/or hilar lymph node specimens. Only the criterion of BALF CD103+CD4+ to CD4+ ratio was met in all nine sarcoidosis cases. In one of two cases without granulomas, idiopathic pulmonary fibrosis (IPF) was reported in the biopsy that was performed about 4 years after the FCA of BALF and, therefore, this case could be a burnt-out sarcoidosis. Other case without granulomas and CD103+CD4+ to CD4+ ratio at <0.45 had respiratory bronchiolitis-associated interstitial lung disease.

Conclusions: Flow cytometric CD103+CD4+ to CD4+ ratio at <0.45 in BALF is quite reliable for diagnosing sarcoidosis. The other two above-mentioned ratios in BALF are not useful for this purpose.

55

PRE-VALIDATION STUDY OF NUCLEATED RED BLOOD CELL ENUMERATION BY FLOW CYTOMETRY

Carl Simard,1 Marc Cloutier,1 and Sonia Néron1,2

1Hema-Québec, Québec, Canada
2Université Laval, Québec, Canada

The enumeration of nucleated red blood cells (NRBC) in cord blood units is a required criterion for QA in cord blood banks. Although specialized hematological analyzers exist for NRBC enumeration, manual count on blood film is still considered the gold standard. Because QA labs already have flow cytometers for CD34 enumeration, the same instrument could be used for NRBC enumeration. Based on the method developed by Tsuji at al. (Cytometry 1999;37:291), we have performed a pre-validation study comparing NRBC enumeration from 34 samples by flow cytometry on a Accuri C6 and blood film. Enumerations showed good Pearson's correlation (r = 0.83, two-tailed P < 0.0001). Bland–Altman analysis also showed good agreement between the methods, although we observed a slight but clinically irrelevant bias of 1.4% NRBC between the two methods. We conclude that NRBC enumeration by flow cytometry could efficiently replace manual count. A validation study is now in progress to incorporate NRBC enumeration by flow cytometry as a routine test in our cord blood bank QA facility.

56

INCREASED REGENERATING MYELOID BLASTS DURING TREATMENT OF B-LYMPHOBLASTIC LEUKEMIA

Charanjeet Singh,1 Zhengwei J. Mao,2 and Timothy P. Singleton1

1University of Minnesota, Minneapolis, MN
2University of Minnesota Medical Center, Fairview, Minneapolis, MN

Background: The presence of residual neoplastic blasts guides therapy for acute leukemias. Although increased myeloid blasts have been described after treatment with growth factors, most of the published reports occurred in patients who had or developed myeloid neoplasms. We report patients who had increased myeloid blasts during treatment for B-lymphoblastic leukemia.

Materials and Methods: All cases of B-lymphoblastic leukemia diagnosed between 2011 March and 2013 March were reviewed. Cases with greater than 5% myeloid blasts in the bone marrow or greater than 1% myeloid blasts in the blood were selected. Of 439 biopsies from 119 patients, 6 had increased myeloid blasts. The patients ranged from 1 to 48 years in age. These biopsies were obtained at day 14 to day 44 from the most recent chemotherapy. Three patients (3 of 3) received growth factors at 2 to 11 days prior to the biopsies.

Results: Four peripheral bloods had 2–9% CD34-positive myeloid blasts, and none of these had neoplastic B lymphoblasts. The blood smears had 2–4% blasts. Two marrow biopsies had 5–9% CD34-postive myeloid blasts, and one of these had 0.1% neoplastic B lymphoblasts. By morphology, the marrow biopsies had 5–13% blasts. With flow cytometry, the myeloid blasts expressed CD13, CD33, CD34, CD45, and HLA-DR but lacked CD10 and CD19. None of the patients developed a myeloid neoplasm.

Conclusions: Increased CD34-postive regenerating myeloid blasts may be seen during chemotherapy for B-lymphoblastic leukemia. Flow cytometry is a useful technique to distinguish these from neoplastic B lymphoblasts.

57

ALTERED IMMUNE CORRELATES OF DISEASE STATUS IN HUMAN VISCERAL LEISHMANIASIS AS DEFINED BY CYTOKINE RELEASE IN WHOLE BLOOD

Om Prakash Singh,1 Kamlesh Gidwani,1 Rajiv Kumar,1 Marleen Boelaert,2 David Sacks,3 and Shyam Sundar1

1Infectious Disease Research Laboratory, Department of Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India
2Department of Public Health, Institute of Tropical Medicine, Antwerp, Belgium
3Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases, National Institute of Health, Bethesda, MD

Visceral leishmaniasis is associated with a marked depression of T-cell responses, which has been characterized by the absence of IL-2 and IFN-γ production by PBMCs on in vitro stimulation with leishmania antigen, and is thought to underlie the progressive nature of this disease. We have recently reported the surprising results that patients with active VL maintain the capacity to secrete significant levels of antigen driven IFN-γ and IL-10. We aimed to extend these findings to a larger cohort of subjects, and to employ the whole blood assay (WBA) to detect additional released cytokines that might better correlate with the disease status of infected individuals. We used WBA to evaluate IFN-γ, TNF-α and IL-10 production in 35 patients with active VL, 54 cured VL, 27 patients with other diseases, and 52 Non-Endemic Healthy Controls (NEHC). We also tested the assay on 147 Endemic Healthy Controls (EHC), all close contacts of VL cases. The whole blood cells from the majority of both active (80%) and cured (85%) VL patients, as well as 24% of EHCs with presumed sub-clinical infections, showed significantly elevated levels of antigen specific IFN-γ. Importantly, only the active VL patients also produced IL-10, which in conjunction with IFN-γ better reflects the immune responses that distinguish active cases from clinically exposed, immune individuals. CD4+ cells were found to be the main source of antigen driven IFN-γ in these cells and antileishmanial antibodies in whole blood do not contribute in antigen specific cytokines production.

Figure 1.

Table 1

58

RECOMMENDED ANTIBODY CLONES AND CONJUGATES SUITABLE FOR HIGH-SENSITIVITY FLOW CYTOMETRIC DETECTION OF PNH RED (RBC) AND WHITE (WBC) BLOOD CELLS

Robert Sutherland,1 Erica Acton,1 Michael Keeney,2 Michael Borowitz,3 Stephen Richards,4 Matthew Fletcher,5 Liam Whitby,5 David Barnett,5 and Andrea Illingworth6

1Laboratory Medicine Program, University Health Network, Toronto, Canada
2Hematology and Flow Cytometry, London Health Sciences Centre, London, Canada
3Department of Pathology and Oncology, Johns Hopkins Medical Institutions, Baltimore, MD
4HMDS, St James University Hospital, Leeds, United Kingdom
5UK NEQAS for Leucocyte Immunophenotyping, Sheffield, United Kingdom
6Dahl-Chase Diagnostic Services, Bangor, ME

The ICCS Guidelines recommended the use of CD235a (for “gating”) together with GPI-linked CD59 for high-sensitivity detection of PNH RBCs. For WBCs, the use of a lineage-specific gating approach together with FLAER and one other independent GPI-linked marker were recommended for high-sensitivity detection of PNH WBCs. In the follow-up “Practical Guidelines,” we identified a small number of clones/conjugates to CD235aFITC and CD59PE, and extensively validated them on a variety of instrument platforms. Optimal clones and conjugates for high-sensitivity PNH WBC detection were also identified and validated. Two 4-color WBC assays were developed, one for granulocytes that utilized FLAER, CD24, CD15, and CD45 and one for monocytes in which CD24/CD15 were replaced by CD14 and CD64. Variants of the two cocktails were verified for FC500/Navios and FACSCalibur/Canto platforms. Instrument-specific cocktails were subsequently distributed for use in a UK NEQAS study in which stabilized whole blood samples were distributed to participants with similar data generated regardless of instrument platform used. Most recently, we have shown that CD157PE can replace both CD24 and CD14 in a single tube 5-color cocktail for the high-sensitivity detection of both PNH granulocytes and monocytes. The following conjugates performed well in our standardized cocktails: For RBCs, CD235FITC (KC16, 10F7MN, JC159), CD59PE (OV9A2, MEM43); For WBCs, CD15 (80H5PC5, HI98APC), CD64 (22PC5, 22ECD, 10.1APC), CD24PE (SN3, ALB9, ML5), CD14PE (61D3, RMO52, MOP9), and CD157PE (SY11B5). These studies were supported by Alexion Pharmaceuticals, BD Biosciences, Beckman Coulter and eBioscience.

59

DETAILED IMMUNOPHENOTYPING OF B-CELL SUBSETS IN DIFFUSE LARGE B-CELL LYMPHOMA

Dipti Talaulikar,1,2 Andrew Ziolkowski,1 Sanjiv Jain,1 Harpreet Vohra,2 Josh Tobin,2 and Matthew Cook1,2

1Canberra Hospital, Canberra, Australia
2College of Medicine, Biology and Environment, Australian National University, Canberra, Australia

Introduction: Despite the molecular classification of diffuse large B cell lymphoma (DLBCL) [i.e., the germinal centre (GC), and the activated B cell (ABC) subtypes], the disease is associated with a high level of clinical, morphological, and immunophenotypic heterogeneity, which is reflected in variable clinical outcomes with immunochemotherapy regimens. This study aims to resolve normal B cell subsets in the peripheral blood and bone marrow and determine the heterogeneity of differentiation within malignant cells in DLBCL.

Figure 1.

FIG. 1.

Methods: Ten color flow cytometry panels were used to determine the frequency of 18 B cell subsets in 42 cryopreserved peripheral blood mononuclear cells (PBMC) and/or bone marrow (BM) aspirate samples. Involvement with lymphoma was defined by light chain restriction and the detailed immunophenotype of the clonal B cell population was described.

Results: The major B cell subsets in PBMC and BM were: memory (27.0%, 29.6%); naive (22.2%, 19.2%); plasmablasts (22.2%, 17.0%); exhausted (4.7%, 7.5%); and transitional (1.7%, 3.0%). A light chain restricted clonal B cell population in the BM and/or PB was noted in 9/42 (21%) patients. All cases expressed IgM, and one case also expressed CD10 [CD19+/CD10+/CD21/CD38+]. The rest were CD10. Five cases were similar [CD27neg/CD21+/IgM+/IgD+] and another four were CD27 int/+ with variable expression of IgD.

Conclusions: The major B cell subpopulations in PB and BM are plasmablasts, naïve and memory cells. Clonal populations in DLBCL demonstrate heterogeneity of differentiation. This methodology enables clonal populations in DLBCL to be identified and isolated for further studies.

60

EVALUATION OF CD4+ AND CD4− CIRCULATING NATURAL KILLER T-CELLS (VΑ24+CD161+) AND NK CELLS FREQUENCY IN WHOLE BLOOD OF HIV PATIENTS BY FLOW CYTOMETRY

Ranjini Valiathan and Deshratn Asthana

University of Miami, Miami, FL

Background: Natural killers T-cells (NKT) are a heterogeneous group of T-cells that share properties of both T-cells and natural killer (NK) cells. NKT and NK cells are thought to be involved in innate responses against infection. We investigated NKT cells (CD4+ and CD4−) and NK cells in two different groups of HIV patients and compared it with normal controls using flow cytometry.

Methods: NKT and NK cells from virologically controlled HIV patients with mean absolute CD4 numbers (AbsCD4: 254 ± 142) (discordant, n = 15) and (AbsCD4: 853 ± 229) (concordant, n = 15) were compared with six normal controls (NC). Whole blood was stained for NKT and NK cells (CD3, CD4, CD16, CD56, Vα24, and CD161), acquired on FACSCanto and analyzed using Diva software.

Results: The NC had significantly higher NKT (CD3+ Vα24+CD161+) cell frequencies than both discordant and concordant HIV positive patients (P = 0.0002 and 0.0006). While CD4+VA24+CD161+ was significantly higher in NC than discordant patients (P = 0.018), CD4−VA24+CD161+ were significantly higher in NC than both discordant (P = 0.004) and concordant (P = 0.008) patients. Discordant patients showed significantly higher NK cells (CD3−CD16+CD56+) compared to concordant patients (P = 0.031).

Conclusion: Increased frequency of NKT cells in NC suggests that there might be continuous activation and subsequent apoptosis which are responsible for the decrease in frequencies of these cells in HIV positive individuals. Higher frequency of NK cells in discordant patients needs to be analyzed further, especially function of these cells. Discordant patients have low number of AbsCD4 T-cells and NK cells might be helping to keep the viral load in check.

61

LYMPHOCYTE POPULATION DYNAMICS IN GULF WAR ILLNESS DURING EXERCISE CHALLENGE: A NETWORK ANALYSIS

Saurabh Vashishtha,1 Zachary M. Barnes,2 Gordon Broderick,1,3 Xiao R. Zeng,1 Nancy G. Klimas,3,4 and Mary Ann Fletcher2

1Department of Medicine, University of Alberta, Alberta, Canada
2Department of Medicine, University of Miami, Miami, FL
3Institute for Neuro Immune Medicine, Nova Southeastern University, Fort Lauderdale, FL
4Miami Veterans Affairs Healthcare System, Miami, FL

Background: Within months of returning from Operation Desert Storm an alarming number of veterans began a constellation of symptoms that would eventually be called Gulf War Illness (GWI). This complex disorder affecting nervous, endocrine, and immune regulation may be mediated by the basic response to stressors whether physiological, chemical, or other. Accordingly, we propose that the structure of the immune signaling network normally expressed in response to exercise will be significantly altered in GWI subjects.

Methods: Blood samples were collected at nine time points spanning 24 h across a maximum VO2 Graded eXercise Test (GXT) from n = 23 GWI subjects and 18 healthy controls. Flow cytometry was performed on each sample to determine lymphocyte subset abundance using a Beckman/Coulter FC500. Using a linear rate equation model, a projection-based parameter estimation technique was applied to each time course to infer a directed immune response network describing the coordinated dynamics of 12 lymphocyte subpopulations. Aggregating across individual time courses, a single consensus network for GWI and one for healthy controls were obtained using specialized voting scheme. These consensus networks were analyzed for differences in structure and information flow using the principles of graph theory.

Results: Analysis of the changes in the patterns of information flow showed that all cell sub-populations gained in information throughput in GWI compared to healthy control, with one exception. In GWI, the CD3−/CD56+ NK cell fraction exhibited a dramatic decrease in information throughput, based on changes in “betweeness” centrality in GWI (P = 0.01), indicating a potential target for treatment.

62

ASSESSMENT OF LYMPHOCYTE FUNCTION USING A NOVEL NON-RADIOACTIVE FLOWCYTOMETRIC ASSAY

Suzanne Vercauteren,1,2,3 Angela Tsang,1 Peter van den Elzen,1,2,3 Jason Ford,1,3 and Nicholas Au1,3

1BC Children's Hospital, Vancouver, Canada
2Child and Family Research Institute, Vancouver, Canada
3University of British Columbia, Vancouver, Canada

The gold standard to assess lymphocyte function is the 3H-thymidine incorporation assay (3H-TdR), with the major drawback being its reliance on radioactive substances. We developed a novel non-radioactive quantitative flow cytometric assay to assess proliferation of T- and B-lymphocytes in response to mitogens. Peripheral blood mononuclear cells (PBMCs) of 43 controls as well as 30 patients with suspected immunodeficiency were stimulated for 4 or 5 days with Phytohemagglutinin, a stimulant for B- and T-cell populations; Pokeweed Mitogen, a stimulant for T-cells as well as T-cell dependent B-cell responses, and Staphylococcus Aureus Cells, which selectively stimulate cell division of B-lymphocytes. The 3H-TdR assay was performed as previously described with 3H-Thymidine pulsing 4–6 h before harvesting total PBMCs. PBMCs in the flow cytometry arm were harvested and stained with CD19, CD3, Ki-67 (cellular marker for proliferation) and proliferation cell nuclear antigen (PCNA, expressed during DNA synthesis). A quantitative measure of proliferation to specifically assess T and B cell proliferation was obtained by measuring the number of events in a fixed acquisition time by flow cytometry. CD3+/Ki-67+/PCNA+ mean event count (T-cells) as well as CD19+/Ki-67+/PCNA+ mean event count (B-cells) correlated well with the mean counts per minute of the 3H-TdR assay after 4–5 days of culture when set-up simultaneously with the mitogens. The coefficient of determination (R2) was >0.85 for 95% of controls and 87% of patients. In conclusion, this novel non-radioactive flow cytometric assay assesses lymphocyte function in a specific and reliable manner and correlates well with the gold standard.

63

SPECIFIC FEATURES OF IMMUNOPHENOTYPE OF AML LEUKEMIC CELLS WITH t(8; 21)

Jolanta J. Wozniak

Institute Hematology and Transfusion Medicine, Warsaw, Poland

Background: WHO classifies AML t(8; 21) to the subgroup of AML with recurrent genetic abnormalities. Final diagnosis is based on cytogenetic and molecular analysis. The number of blasts may be lower than 20%. Maturity of myeloid cells shows signs of dysplasia. AML t(8; 21) is a promising subtype. Our aim was to verify whether the characteristics of this leukemia immunophenotype justify good prognosis. Materials and methods: Bone marrow from 15 AML patients with t(8; 21) confirmed in RT-PCR. The control group consisted of bone marrow from 43 patients with AML M2. Data acquisition was performed on a FACS Canto cytometer.

Figure 1.

[Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

Results: The number of myeloblasts varied from 3% to 86%. CD56 expression was observed in 85% of AML patients with t(8; 21); on blasts and maturing myeloid cells. In AML M2 patients, the expression of this antigen was found in only 5%. CD38 and CD58 expression intensity were significantly higher in AML patients with t(8; 21) while the expression intensity of CD123 was significantly lower than in patients with AML M2 (Fig. 1). The CD38lowCD123 ++ phenotype of CD34 + leukemia cells is considered a bad prognosis and indicates rapid recurrence of disease. CD58 adhesion molecule is involved in the immune response related to leukemia cells.

Conclusions: The striking feature of the immunophenotype of AML leukemia cells with t (8; 21) is CD56 expression on both myeloblasts and maturing myeloid cells. CD38 ++ CD123lowCD58++ myeloblast phenotype in AML t(8; 21) may explain better prognosis for this AML subtype patients.

64

CORRECT UNDERSTANDING THE VALUE AND ROLE OF FLOW CYTOMETRY IN HEMATOLOGICAL NEOPLASMS DIAGNOSIS

Baohong Yue,1 Xiaoli Sun,2 Yu Jia,1 Yuanyu Wei,1 and Shuai Liu1

1Department of Clinical Laboratory, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, China
2Department of Clinical Laboratory, The First Affiliated Hospital of Henan University of TCM, Zhengzhou, China

Flow cytometry (FCM) plays an important role in hematological neoplasms diagnosis and prognosis, monitoring minimal residual disease (MRD). Thus, it requires the personnel engaged in this work to have an in-depth understanding of FCM technology: 1. Reasonable combination of antibodies is conducive to find hematological tumor cells and abnormalities. (1) Highly expressed antigens match weak fluorescein, and weak antigens match strong fluorescein; (2) When assessing and analyzing certain complex cells, it may need multiple assaying tubes, the antibodies combination in each tube should at least contain one common antigen in order to trace the identified cell population between different tubes. (3) Combinate cell subset antigens and series associated antigens in the same tube, in order to identify the subsets in specific cell series; (4) Combinate antigens expressed in different differentiation stages in one tube, so that we could analyze the cell maturity. 2. Correctly using FCM technology is helpful to monitor hematological neoplasms MRD. (1) The “leukemia associated immune phenotype” (LAIP) is screened out by FCM, later the residual leukemia cells could be detected according to this. Common LAIP include antigens expressed asynchronously, cross expression antigens, antigen phenotype deletion, and change of antigen expression intensity. (2) Detecting specimens abnormal phenotype characteristics which cannot appear in normal condition. 3. Correctly using FCM technology to detect intracellular component. Cytoplasm and nucleus component usually reflect the changes of series characteristics and function much earlier and more specifically. 4. Correctly understanding the influence on FCM data of specimens before experiment. Before experiment, some measures should be taken to fully guarantee cell viability in specimens.

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THE DISTINCT ROLE OF FLOW CYTOMETRY IN DIAGNOSING DIFFERENT TYPES OF T-CELL LYMPHOMAS

Xiaohong (Mary) Zhang

Geisinger Health System, Wilkes-Barre, PA

Flow cytometry (FC) analysis is very important in diagnosis of lymphomas. T-cell lymphoma is detected by FC as loss of some pan-T cell antigens or abnormal CD4/CD8. The role of FC in diagnosis of different types of T-cell lymphomas, however, has not been well studied. Total of twenty cases of T-cell lymphomas with FC studies was retrieved. Among the 20 cases, seven cases were peripheral T-cell lymphoma, NOS (PTCL), five cases were T-lymphoblastic lymphoma/leukemia (T-LBL), five cases were anaplastic large cell lymphoma (ALCL), and three cases were angioimmunoblastic T cell lymphoma (AITL). FC panels included T cell markers and B-cell markers. Immunohistochemistry (IHC) of T-cell markers were performed by using commercially available antibodies. FC provided the diagnosis for all five cases of T-LBL (100%) and six out of seven cases (86%) of PTCL. FC showed abnormal T cell markers in two out of five cases (40%) of ALCL and IHC highlighted large tumor cells in all cases. Three cases of AITL demonstrated no significantly abnormality except one case showing two subpopulations of CD2+/CD4+ T cells with CD3 high and CD3 intermediate intensity in FC. The AITL was diagnosed with IHC and confirmed by TCR gene rearrangement. The role of FC varies in diagnosing different types of T-cell lymphoma. FC is sensitive in diagnosis of T-LBL and PTCL. The combination of FC and IHC yields more accurate results for the diagnosis of ALCL. For AITL, FC has limited value and IHC plus TCR gene rearrangement offers more insights for its diagnosis.

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