abstracts from the 26th annual meeting of the international clinical cytometry society. october...

ICCS Abstracts

Abstracts from the 26th Annual Meeting of theInternational Clinical Cytometry SocietyOctober 14-18, 2011, Portland, Oregon

P1

WT1 RNA EXPRESSION IN DIFFERENT CELL LINEAGES INNORMAL AND LEUKEMIC BONE MARROW

Daphne C. Ang, Fei Yang, Ginny Lanier, Richard Press, and Guang Fan

OHSU, Portland, Oregon

The WT1 gene is over-expressed in the bone marrow

and peripheral blood of patients with acute myeloid leu-

kemia (AML) and high expression levels are prognostic of

poor outcomes. WT1 has been previously shown to be

expressed in normal CD34þ bone marrow cells, but at a

level about 100 to 1000 times lower than in leukemic

cells. However, WT1 expression in other cell lineages

including PMN’s, lymphocytes, and monocytes (in normal

and leukemic samples) is unknown. Bone marrows (BM)

from seven normal, eight AML, one acute promyelocytic

leukemia (APL), and four acute lymphoblastic leukemia

(ALL) patients were fluorescence-activated cell sorting

(FACS)-sorted into four lineages: CD34þ blasts, CD14þmonocytes (mono), CD45þ lymphocytes (LYMPH), and

neutrophils (PMN) sorted by characteristic side and for-

ward scatter. FACS-sorted samples were examined for

WT1 expression by quantitative reverse transcriptase-poly-

merase chain reaction and normalized to the ABL refer-

ence gene. The WT1 expression level of normal and ALL

patients were significantly lower across all cell lineages as

compared to AML and APL patients (P ¼ 0.01). Among

AML and APL samples, WT1 was highly expressed, not

only in the blast population but also in the PMN’s and

occasionally in the monocytes. (Fig. 1) These results sug-

gest that WT-1 is aberrantly overexpressed in myeloid leu-

kemic cells. WT-1 expression is not solely confined to

the blast cells, but is also seen in the maturing myeloid

and monocytic lineages. The WT-1 expressing leukemic

blasts may then ultimately mature to, neutrophils and

monocytes.

P2

ACOUSTIC CYTOMETRY FOR RARE EVENT DETECTION OFPAROXYSMAL NOCTURNAL HEMOGLOBINURIA CELLS

Jolene A. Bradford1 and Mike Suter2

1Flow Cytometry Systems, Life Technologies, Eugene,

Oregon2PeaceHealth Laboratories, Springfield, Oregon

Rare event analysis is an area of broad interest in pa-

thology. An area of recent interest is detection of small

populations of neutrophils in the blood, which lack sur-

face expression of markers due to absence of a glyco-

phosphatidylinositol (GPI)-anchor protein. In classic par-

oxysmal nocturnal hemoglobinuria (PNH), a clone of

hematopoeitic stem cells arises, which expands. However,

small PNH-like neutrophil populations have been detected

in the blood of up to 15% of patients who have myelo-

dysplasia and 24% of patients with aplastic anemia. These

patients do not progress to classic PNH, and do not have

the clinical sequelae of hemolytic anemia or thrombosis

of PNH A recent advance in cytometry instrumentation

uses acoustic focusing, in which acoustic energy is used

to align precisely cells for more sensitive detection, and

sample acquisition time can be reduced by 10-fold or

more. The detection limit with acoustic focusing cytome-

try is in the range of 0.1–0.01% of nucleated cells, accu-

mulating 1 million total events over a 5-minute interval

per sample. We present an approach for the detection of

PNH-like neutrophils using acoustic cytometry. Utilizing a

panel of fluorescent reagents including CD45, CD15,

CD16, CD24, and fluorescent aerolysin (FLAER) surface

markers, a gating strategy is employed for high-sensitivity

analysis of neutrophils using lineage markers. Results of

both normal and abnormal patient samples are shown.

Acoustic cytometry provides the ability to detect PNH

neutrophils by acquiring statistically significant cells for

rare event detection.

FIG. 1. WT-1 expression of different cell lineage in normal

and leukemia BM.

Published online 20 September 2011 in Wiley Online Library(wileyonlinelibrary.com)DOI: 10.1002/cyto.b.20618

Cytometry Part B (Clinical Cytometry) 80B:375–389 (2011)

q 2011 International Clinical Cytometry Society

P3

CRITICAL ASSESSMENT OF CELL POPULATION IDENTIFICA-TION TECHNIQUES FOR FLOW CYTOMETRY DATA: RESULTSOF FLOWCAP-1

Ryan Brinkman,1 Nima Agheepour,1 Richard Scheuermann,2 Raphael

Gottardo,3 and Tim Mosmann4

1British Columbia Cancer Agency, Vancouver, British

Columbia, Canada2University of Texas Southwestern, Dallas, Texas

3Fred Hutchinson Cancer Research Center, Seattle, Wash-

ington4Univeristy of Rochester, Rochester, New York

Traditional methods for flow cytometry (FCM) data proc-

essing have relied on subjective manual gating to define cell

populations for statistical analysis. Recently several groups

have developed computational methods for identifying cell

populations in multidimensional FCM data, potentially obviat-

ing the need for manual gating. To compare the performance

of these methods, the Flow Cytometry: Critical Assessment

of Population Identification Methods (FlowCAP) challenge

was established. Thirty-six analysis result submissions

from fourteen research groups were received for the first

FlowCAP competition (FlowCAP-1). Several parametric and

nonparametric clustering methods performed well in com-

parison with manual gating by domain experts as the

gold standard, using statistical measures of algorithm per-

formance. Combining results using a computational ensem-

ble method yielded further performance improvements.

These results suggest that automated computational methods

have reached a level of maturity and accuracy such that they

are poised to replace manual gating for routine FCM data

analysis (Fig. 1).

P4

BIOINFORMATICS TO BEDSIDE: AN AUTOMATED AND GENER-ALIZABLE MULTIDIMENSIONAL FLOW CYTOMETRY DATA ANAL-YSIS APPROACH IMPROVES DIAGNOSTIC ACCURACY BETWEENMANTLE CELL LYMPHOMA AND SMALL LYMPHOCYTICLYMPHOMA

Ryan R. Brinkman,1,2 Habil Zare,1,3 Robert Kridel,4,5

Nima Aghaepour,1 Gholamreza Haffari,6,7 Josef M. Connors,4,8

Randy D. Gascoyne,4,5 Arvind Gupta,3 Andrew P. Weng,1,5 and

Ali Bashashati6

1Terry Fox Laboratory, British Columbia Cancer Agency,

Vancouver, British Columbia, Canada2Department of Medical Genetics, University of British

Columbia, Vancouver, British Columbia, Canada3Department of Computing Science, University of British

Columbia, Vancouver, British Columbia, Canada4Center for Lymphoid Cancers, British Columbia Cancer

Agency, Vancouver, British Columbia, Canada5Department of Pathology and Laboratory Medicine,

University of British Columbia, Vancouver, British

Columbia, Canada6Department of Molecular Oncology, British Columbia

Cancer Agency, Vancouver, British Columbia, Canada7Faculty of Information Technology, Monash University,

Victoria, Australia8Faculty of Medicine, University of British Columbia,

Vancouver, British Columbia, Canada

Mantle cell lymphoma (MCL) and small lymphocytic

lymphoma (SLL) exhibit similar, but distinct immunophe-

notypic profiles. While many cases can be diagnosed with

high confidence, based on flow cytometry (FCM) results

alone, ambiguous cases are frequently encountered and ne-

cessitate additional studies. To determine if greater diag-

nostic accuracy could be achieved from flow cytometry

data alone, we developed an unbiased, machine based

algorithm and used it to automatically identify those fea-

tures within the multidimensional space that best distin-

guish between the two disease types. Data from 44 MCL

cases and 70 SLL cases were analyzed. Using conventional

diagnostic criteria, we were able to assign accurately only

64% of MCL and 69% of SLL cases. Using features identified

by our automated approach, we were able to assign 100%

of MCL and 97% of SLL cases correctly. The most discrimi-

nating feature was the ratio of mean fluorescence inten-

sities (MFI) between CD20 and CD23. Unexpectedly, we

FIG. 1. Comparison of manual gating and ensemble cluster-

ing results. Population membership assignments using man-

ual gating (a, c, e) are compared against assignments using

the ensemble approach (b, d, f) in the HSCT dataset exam-

ple. Each identified population is color-coded separately.

376 ICCS 2011 ABSTRACTS

Cytometry Part B: Clinical Cytometry

also observed that inclusion of FMC7 expression in the

diagnostic algorithm reduced its accuracy. Computational

methods allow objective assessment of the relative contri-

bution of component data features to overall diagnostic ac-

curacy, and reveal some conventional criteria can actually

compromise this accuracy. Furthermore, computational

approaches enable exploiting the full dimensionality of

FCM data and can potentially lead to discovery of novel

biomarkers relevant for clinical outcome.

P5

A NOVEL FLOW CYTOMETRY STIMULATION ASSAY USINGCYTO-CHEX1 BCT TUBES FOR USE IN CLINICAL TRIALS

Lynette M. Brown and Jennifer J. Stewart

Flow Contract Site Laboratory, LLC, Kirkland, Washington

The Cyto-Chex1 BCT tubes from Streck Laboratories con-

tain a priority fixative that stabilizes the blood at room tem-

perature making flow cytometry analysis possible for up to

1–2 weeks after collection. This application has been advan-

tageous for doctors and scientists in remote locations being

able to transport specimens back to a testing laboratory that

is not in close proximity to the draw site. These BCT tubes

have also become very popular for use in clinical research

trials for flow cytometry testing since humans are drawn

from multiple sites and then the specimens are shipped to a

central laboratory. One disadvantage of the tube is that since

it contains a fixative, flow cytometry applications, which

require stimulation, cannot be performed once the specimen

is exposed to the fixative. In our laboratory, we are working

on ways to stimulate the specimen prior to placing in the

Cyto-Chex1 BCT tube. In one such method, we can measure

the intracellular cytokine IL-17A expressed in T cells up to 5

days post-stimulation. This method can also be used to mea-

sure many phosphorylated proteins. These methods will

greatly benefit the pharmaceutical and biotechnology indus-

try in that whole blood specimen can be stimulated immedi-

ately after the draw, fixed, and then shipped to a central test-

ing facility.

P6

EVALUATION OF LYMPHOCYTE SUBSET ENUMERATION INTRANSPLANT PATIENTS USING A NOVEL FLOW CYTOMETERTHAT INTEGRATES SAMPLE PREPARATION AND ANALYSIS INONE SYSTEM

Raquel Cabana,1 Alexandra Amador,2 Michael Brochu Jr.,1

and Phillip Ruiz2

1Blue Ocean Biomedical, Pembroke Pines, Florida

2Immune Monitoring Laboratory, University of Miami

Miller School of Medicine, Miami, Florida

The objective of the study was to compare the process

from sample collection to result when enumerating lym-

phocyte subsets by a novel versus conventional flow

cytometry technology. The innovative system, the Blue

OceanTM CR300, a fully integrated cell analyzer that auto-

matically processes barcoded primary patient tubes and

performs sample preparation and analysis simultaneously

in one instrument without operator intervention was com-

pared to the manual conventional clinical cytometry sys-

tem. The study was performed in the Immune Monitoring

Laboratory at the University of Miami. One hundred sev-

enty-one samples from patients in an organ transplant pro-

gram were run in parallel using a CR300 System and BD

FACSCaliburTM. The samples were 95% from post trans-

plant patients within 3 years of transplantation. The mean

age of patients was 49-years-old with different solid organ

transplant. Percentages and absolute counts were obtained

for lymphocyte subpopulations in peripheral blood. Blue

Ocean panels LSA-1(CD45/CD4/CD8/CD3) and LSA-2

(CD45/CD56þ16/CD19/CD3) were compared with BD

Multitest 4-color panels. Absolute counts were obtained by

single platform (volumetric) on the CR300 and dual plat-

form on the FACSCaliburs. The results show strong correla-

tion for percentages and absolute counts for all the lym-

phocyte subpopulations tested. Workflow improvements

were significant, specifically in reducing user intervention

and minimizing both the number and skill level of required

flow operators. In addition, potential sample identification

errors were virtually eliminated. Finally, turnaround time

was reduced while maximizing sample throughput.

Although this study focused on lymphocyte subset analysis,

the CR 300 System is well suited for other applications

that require repetitive testing.

P7

HEMATOGONE LIGHT CHAIN BIAS IN LYMPHOMA STAGINGBONE MARROWS CAN MIMIC INVOLVEMENT BY NONHODGKINLYMPHOMA

Pramoda Challagundla, Sa A. Wang, Cynthia J. Swett,

Linda C. Powers, and Jeffrey L. Jorgensen

UT MD Anderson Cancer Center, Houston, Texas

Background: Various studies have described light chain

restriction in germinal center B cells in reactive lymphoid

hyperplasia, autoimmune diseases, and HIV. We have

observed occasional cases with light chain bias in CD10þ

377ICCS 2011 ABSTRACTS


B-cells, in bone marrow (BM) submitted for staging of non-

Hodgkin lymphoma (NHL), suggestive of involvement by

lymphoma. However, detailed characterization using multi-

ple additional markers showed a phenotype consistent with

benign B cell precursors (hematogones, HGs).

Methods: Study group includes 11 BMs with a his-

tory of various B-cell neoplasms and was categorized into

two types: with and without concurrent residual lym-

phoma, as shown in Table 1. The staining panel included

monoclonal antibodies against CD5, CD10, CD19, CD20,

CD22, CD38, CD43, CD45, CD200, Kappa, and Lambda

(BD Biosciences, San Jose, CA). Data were acquired on

FACS Canto II cytometers (BD Biosciences), and analyzed

using FlowJo (Treestar, Ashland, OR). Kappa/Lambda (K/

L) ratios were determined for HGs (CD19þ/CD10þ), and

mature B cells (CD19þ/CD10). The remaining markers

were used to characterize HGs, and mature benign and

neoplastic B-cells.

Results: HGs in all cases showed a characteristic phe-

notype: CD5 negative, positive for CD10, CD19, and CD20

variable, CD22 dim, CD38 bright, CD43, CD45 dim, and

CD200. All NHL but one was CD5þCD10 or CD5CD10

(Table 1). Four of 11 cases showed the opposite light chain

expression in NHL versus hematogones.

Conclusions: In rare cases, HGs can show a marked

bias in light chain expression. Familiarity with the character-

istic pattern of antigen expression on HGs can help to avoid

false positive diagnoses of NHL.

P8

‘‘INDOLENT’’ T-LYMPHOBLASTIC PROLIFERATION INVOLVINGBONE MARROW? A DIFFICULT DIAGNOSTIC DILEMMA

Jennifer Crow,1 Patrick Buckley,1 Jon Gockerman,2 and

Anand Lagoo1

1Department of Pathology, Duke University Medical Cen-

ter, Durham, North Carolina2Department of Medicine, Duke University Medical Center,

Durham, North Carolina

A 63-year-old man, previously in perfect health, developed

severe anemia and mild thrombocytopenia following a linger-

ing ‘‘cold’’ and upper respiratory symptoms lasting several

weeks. Joint and muscle pains had developed during this

time, but fever, chills, or weight loss were absent. Chest, ab-

domen, and pelvic CT scan was negative. Flow cytometric ex-

amination of the bone marrow at another institution showed

10% ‘‘blasts’’ based on CD45 and side scatter features and

expression of CD5, CD7, CD10, CD13, CD34, and TdT, lead-

ing to a diagnosis of T-Acute Lymphoblastic Leukemia. The

patient declined chemotherapy and was treated with blood

transfusion, antibiotics, and steroids (prednisone 100 mg).

Over the next 4 weeks, his blood counts improved to just

below normal values and steroids were tapered off. Repeat

bone marrow examination showed a distinct population of

blasts, constituting 17% of nucleated cells (by flow cytometry)

and expressing cytoplasmic CD3 and surface CD5, CD7,

CD13, CD34, CD38, CD117 (partial), CD123, HLA-DR, and

TdT. No dysplasia was seen in the normocellular marrow. Mo-

lecular studies did not show clonal IgH or T-cell receptor gene

rearrangement and there was no evidence of EBV infection.

The patient remained clinically well without further treat-

ment. This is the first description of an indolent T-lymphoblas-

tic proliferation involving the bone marrow, although similar

proliferations forming mediastinal masses have been rarely

described in the literature. Awareness of this possibility draws

attention to a difficult diagnostic dilemma and emphasizes the

need for careful clinical correlation.

P9

A SCORING SYSTEM FOR IMMUNOPHENOTYPIC DISTINCTIONBETWEEN ACUTE MYELOGENOUS LEUKEMIA WITH THYMICMARKERS [AML(T)] AND T-ACUTE LYMPHOBLASTIC LEUKEMIAWITH MYELOID MARKERS [T-ALL(M)] USING A PRIMARYANTIBODY PANEL

Amar Dasgupta, Vishal Mehrotra, Archana Vazifdar, and

Manisha Ramani

Sections of Hematology and Flow Cytometry, Super Reli-

gare Laboratories, Mumbai, India

Table 1Comparison of NHL and HG Phenotypes

Case number NHL history NHL phenotype

Residuallymphomaidentified

Light chain inHGs (K/L ratio)

1 MCL CD5,CD10, Lambdaþ No Kappa(7.0)2 MCL CD5þ,CD10, Kappaþ No Lambda(0.25)3 MCL CD5þ,CD10a No Kappa(17)4 MCL CD5þ,CD10, Kappaþ No Kappa(43)5 Diffuse large B cell

lymphoma (DLBCL)CD5,CD10, Lambdaþ No Lambda(0)

6 DLBCL CD5þ,CD10, Kappaþ No Kappa(12)7 DLBCL CD10,Kappaþ No Kappa(56)8 Follicular lymphoma CD5,CD10þ, Kappaþ No Kappa(4.0)9 MCL CD5þ,CD10, Lambdaþ Yes Kappa(16)10 MCL CD5,CD10, Kappaþ Yes Kappa(7.5)11 MCL CD5þ,CD10, Kappaþ Yes Lambda(0)

aImmunohistochemical phenotype.



Blasts in 20–40% cases of AML and in 20–30% cases of

T-ALL express T-lymphoid and myeloid antigens, respec-

tively, causing diagnostic dilemma. The high cost of a step-

wise immunophenotyping of hematolymphoid neoplasms

prompted us to develop a scoring system for clear distinc-

tion between these two entities based on the antigenic

features of these cells using a primary antibody panel.

One thousand eight hundred seventy-four cases of acute

leukemia were immunophenotyped using a comprehensive

panel of antibodies. Cytochemical MPO was tested in all

cases expressing translineage markers while cytoplasmic

(c) CD3 and cMPO were checked in 40/187 (21%) these

cases for confirmation of T-lymphoid and/or myeloid phe-

notype, respectively One hundred forty-four out of 791

cases of AML (18%) and 43 out of 128 cases of T-ALL

(34%) showed the presence of T-lymphoid and myeloid

markers, respectively. ‘‘Myeloid’’ phenotype of blasts in

AML(T) was suggested by HLA-DR positivity [(135/144;

94%); (Score 1)]; CD117 expression [(127/144; 88%);

(Score 0.5)], CD13 [(131/144; 91%); (Score 1)], and ab-

sence of CD5 [(140/144; 99%); (Score 1)], and of CD10

[(142/144; 99%); (Score 1)]. In contrast, ‘‘T-lymphoid’’ phe-

notype in T-ALL(M) was indicated by normal intensity

CD45 expression (100%), HLA-DR negativity (33/43; 77%),

CD5 expression (28/43; 65%), and a high incidence of CD

13 negativity (35/43; 81%). All cases of AML(T) yielded a

score of ¼/>4.5 while it was ¼/< 3.5 for T-ALL(M) cases.

Cases of true mixed lineage (myeloid/T-lymphoid) leukemia

had a score between 3.5 and 4.5. The above approach

represents a more objective and accurate system for distin-

guishing AML(T) from T-ALL(M).

P10

REFERENCE VALUES OF LYMPHOCYTE SUBSETS IN NORMALHEALTHY ADULT POPULATION IN MIAMI, FLORIDA, USA

Khaled Deeb,1,2 Ranjini Valiathan,1 Deshratn Asthana,1

Ashman Margarita,1 and Sachdeva N.1

1University of Miami, Miami, Florida

2Barry University, Miami Shores, Florida

Background: Although Florida State was ranked the

third highest in reported AIDS cases, there is no data avail-

able on normal ranges of lymphocyte subsets. Information

regarding lymphocyte reference ranges is essential for

assessing human immunodeficiency virus staging and mak-

ing decisions regarding opportunistic infection prophylaxis.

Methods: Lymphocyte counts were determined by

Coulter AcT5 5-part differential hematology analyzer and

Flow cytometry was performed on FACS Caliber and ana-

lyzed using the Cell Quest Pro software. Data were analyzed

using PASW Statistics.

Results: The mean percentage of CD4þ cells was

�47% in both men and women. The mean absolute CD4þcounts in the total population was 1,003 cells/�l (921

cells/�l in men and 1,041 cells/�l in women). The mean

percentage of CD8þ cells was 28% in both men and

women. The mean absolute CD8þ counts in total popula-

tion was 590 cells/�l (560 cells/�l in men and 603 cells/�lin women). Men tend to have lower CD4þ and CD8þabsolute counts than women do. The mean CD4:CD8 ratio

was 1.82, which was significant for women (P ¼ 0.049).

No age-specific differences were observed for any of the

parameters.

Conclusions: Our study confirms that the gender dif-

ference plays a significant role when calculating the CD4/

CD8 ratios. Women were significantly younger than men

were (P ¼ 0.002) and the age distribution curve was

skewed to the right. Women had a significantly higher val-

ues of CD8þ and CD4þ cells (P > 0.05). Our subpopula-

tion had less CD4% but higher NK cell% compared to the

previous study indicating that a difference exists within dif-

ferent geographical regions.

P11

BIMODAL BCL-2 EXPRESSION IN CHRONIC LYMPHOCYTIC LEU-KEMIA: INTERCLONAL AND INTRACLONAL HETEROGENEITY

Heba A. Degheidy, Shahinaz Gadalla, Mohammed Z. H. Farooqui,

Fatima Abbasi, Steven Bauer, Adrian Wiestner, Stetler-Stevenson

M. A., and Marti Gerald

Bethesda, Maryland

Introduction: Bimodal distributions of bcl-2 in deletion

13q14.3 chronic lymphocytic leukemia (CLL) cases were

examined.

Material and Methods: Multicolor flow cytometry

panel of CD3/CD5/CD19/CD20/anti-lambda/anti-kappa and

anti-bcl-2 was used. Bcl-2 index (MFI of the CLL clone/MFI

of residual T-cell) was evaluated in 54 untreated CLL/MBL

cases. Cell sorting and Interphase FISH were performed on

selected cases.

Results: Of the 54 cases, 33 cases had 13q14.3 dele-

tion, and 21 cases were designated as non 13q14.3 deletion.

Bcl-2 index of �1.75 strongly predicts 13q14.3 deletion sta-

tus (OR ¼ 16.75 and P ¼ 0.0002). Detailed analysis of bcl-2

expression of del 13q14.3 cases revealed that 7/33 cases

showed bimodal bcl-2 expression. Group I (n ¼ 3; Intraclo-

nal bcl-2 heterogeneity) cases showed a difference in bcl-2



expression within their CLL clonal cells. Group II (n ¼ 4;

interclonal bcl-2 heterogeneity) cases showed difference in

bcl-2 expression based on CD20 expression [bright (br) and

dim] subpopulations. In Group I, cases with a mixture of

hemizygous and diploid CLL cells, sorting showed enrich-

ment of hemizygous cells in bcl-2 br population. In Group I

case that showed a mixture of hemizygous and homozygous

del 13q14.3 in the CLL clone, sorting led to enrichment of

homozygous cells in bcl-2 br population. Interphase FISH

analysis on Group II sorted cells showed enrichment of ei-

ther hemizygous or homozygous in one of the CLL subpo-

pulations.

Conclusion: Intraclonal and interclonal bcl-2 bimodal

expression profiles identify subpopulations with varying

amounts of either hemizygous or homozygous del 13q14.3.

Further studies are required to isolate pure hemizygous or

homozygous, from diploid CLL subpopulations.

P12

EVALUATION OF FIXATIVES COMPATIBILITY WITH CD4 T CELLTECHNOLOGIES

Tao Ding, Peggy Seely, Christian Chabot, Alexandre Martel, Xuefen

Yang, Dragica Bogdanovic, and Michele Bergeron

National Laboratory for HIV Immunology, Public Health

Agency of Canada, Ottawa, Ontario, Canada

Background/Objective: CD4 T-cell enumeration

remains the best surrogate marker for staging and moni-

toring the immune status of HIV patients under treat-

ment in resource-limited countries. QASI, an interna-

tional external Quality Assessment Program (EQAP),

evaluates the performance of A 600 clinical laboratories

and assists national reference laboratory with implemen-

tation process of independent national EQAP. The selec-

tion of adequate testing material is critical to that

process. This preliminary study is evaluating the com-

patibility of commercial fixatives with various CD4

counting platforms.

Method: Compatibility was assessed on the capacity of

each platform: (1) to measure CD4 in fixed blood; (2) to

generate an accurate measurement and; (3) the degree of

similarity between fixed and unfixed samples (cluster reso-

lution). ‘‘Streck Cell Preservative’’ and ‘‘TransFix’’ fixatives

solution were used as recommended. One-day-old fixed

HIV-specimens were prepared in triplicate according to

manufacturers’ instruction and tested on their respective

platforms: FACSCalibur, Epic-XL, FACSCount, Guava, CyFlow

Counter, PointCare Now, and PIMA. The reference method

was based on 4-color reagent using FACSCalibur with Flow-

count beads.

Results: All platforms tested could measure CD4

except PointCare. Accuracy (% difference) was < 15% as

compared to reference method. Fixed preparations had vari-

ous degree of similarity.

Conclusion: The results showed that fixatives are not

fully compatible across the whole array of CD4 technologies

and EQA provider must be aware of these limitations. The

next phase of the study will involve fixed HIVþ blood prep-

arations and the evaluation of their stability in suboptimal

environmental condition.

P13

USING RPMI TRANSPORT MEDIA INCREASES RECOVERY OFPAUCICELLULAR SPECIMENS SUBMITTED FOR FLOWCYTOMETRY TESTING

Bruce W. Greig1 and Maryalice Stetler-Stevenson2

1Vanderbilt University Medical Center, Nashville, Tennes-

see2NIH/NCI, Bethesda, Maryland

Introduction: Flow cytometry testing of paucicellular

specimens such as CSF has historically been challenging

due primarily to lack of sufficient cells for adequate testing

purposes. Several flow cytometric protocols designed to

increase cellular yield have been reported, including imme-

diate stabilization of cells in TransfixTM or RPMI with fetal

calf serum at the time of specimen collection. On average,

�200 CSF samples are received annually in our laboratory

for flow cytometric testing. In many cases, these specimens

are collected from pediatric patients with possible CNS

involvement with leukemia. We report on preanalytical

steps that improve recovery and results in a series of CSF

specimens.

Methods: One hundred five CSF specimens were

studied and results compared to 217 consecutive CSF

specimens previously received in the laboratory. Previous

CSF specimens were not placed in media and were proc-

essed using published CLSI guidelines. Preanalytical pro-

cedures were modified in that CSF specimens were imme-

diately placed in RPMI upon receipt in the laboratory and

setup procedures were altered to minimize specimen

manipulation on the premise that ‘‘less equals more

cells.’’

Results: Adding RPMI to CSF specimens and limiting

centrifugation to a single step has greatly increased recov-

ery of viable intact cells for testing purposes. Using this

method on 105 specimens, 98 (94%) had adequate numbers

of viable cells (>100) suitable for performing flow cytome-

try testing.

Conclusions: Adding RMPI as a stabilization media

and reducing specimen manipulation results in increased

cell yield and improved flow cytometric testing results.

P14

USING GEMSTONETM IN THE ROUTINE ANALYSIS OF CLINICALSTEM CELL LIST-MODE DATA

Bruce W. Greig,1 David Miller,2 Don Herbert,3 and C. Bruce

Bagwell3

1Vanderbilt University Medical Center, Nashville, Tennes-

see2U S Labs, Brentwood, Tennessee

3Verity Software House, Topsham, Maine

Background: Given the subjectivity of human-derived

analysis, there has long been a desire in clinical flow



cytometry to automate and thus standardize the analysis of

list mode data. This holds true for stem cell analysis. Since

the stem cell count is predictive for engraftment, accurate

results are imperative. Preliminary findings show consider-

able potential for performing this analysis successfully using

Gemstone software. The primary goal of this project is to

test the feasibility of using GemStone in automating and

standardizing the evaluation of the level of CD34þ stem

cells in clinical samples from stem cell transplantation

programs.

Methods: This study evaluates results from two institu-

tions, one using a single-platform method (flow cytometry

results only; n ¼ 58), and the other a dual-platform (flow

cytometry plus hematology results; n ¼ 50. All data were

compared WinListTM (Verity Software House) or FACSDivaTM

(Becton Dickinson) manually versus GemStoneTM (auto-

mated) analysis. Specimens for single-platform method are

cord-blood and bone marrow. Specimens for dual-platform

method are peripheral blood, apheresis products, and bone

marrow. The scope of the study is limited to identifying the

extent that GemStone analysis can be as good as or better

than current manual gating approaches for analysis of stem

cell specimens.

Results: Initial results show that automated detection

of viable CD34þ stem cells by GemStone is in excellent cor-

relation with manual gating methods.

Conclusions: GemStone has the potential to answer

this need with high correlation to current methods. Addi-

tional model development and studies to confirm fully auto-

mated processing are warranted.

P15

PERFORMANCE EVALUATION OF FMH QUIKQUANTTM, ANEW ASSAY FOR FETOMATERNAL HEMORRHAGE ONMULTIPARAMETRIC FLOW CYTOMETERS AND HEMATOLOGYANALYZERS

Irina Grigorieva, Teresa Casas, and Susan Osier

Northside Hospital, Atlanta, Georgia

Detection of fetal RBCs is used to estimate a degree of

FMH in suspected placental injury or Rhesus-D incompati-

bility. The Kleihauer-Betke (K-B) assay for FMH is a mi-

croscopy-based test built on differential resistance of fetal

hemoglobin to acid elution. Despite the availability of

more precise and reproducible methods for measurement

of FMH, K-B assay is mostly used in clinical laboratories,

performed on 24/7 basis. The Trillium Diagnostic FMH

QuikQuantTM is an innovative flow cytometric assay,

based on binding of fluorescently labeled mAB to fetal he-

moglobin. Elimination of repeated centrifugal washes

makes this assay simplified and rapid relative to existing

methods. Bringing of Propidium Iodide fluorescent dye

for positive identification and exclusion of nucleated cells

increases the test precision. This assay is proposed for

use on multiparametric flow cytometers and on Cell-Dyn

Sapphire Hematology analyzer, which allows performing

the test on 24/7 basis. We evaluated the performance of

FMH QuikQuantTM assay on flow cytometer FACSCanto

(BDBioSciences) and hematology analyzer Cell-Dyn Sap-

phire (Abbott) in comparison with Caltag Fetal Hemoglo-

binTM and K-B tests. Assessment of accuracy, precision,

linearity, assay stability, and carryover was performed on

clinical specimens submitted for K-B analysis and spiked

samples. Replicate analysis for flow cytometry method

shows CV < 5%, for hematology method CV < 10%.

Linearity was shown over the range of 0.05–6%. Correla-

tion between Caltag and QuikQuant assays on both plat-

forms was excellent (r2 ¼ 0.9951 for flow cytometer; r2

¼ 0.9953 for hematology analyzer). We have concluded

that FMH QuikQuantTM assay demonstrates excellent per-

formance on both instruments–flow cytometer and hema-

tology analyzer.

P16

RARE EVENT ANALYSIS USING ACOUSTIC CYTOMETRY

Kristi L. Haataja and Jolene A. Bradford

Life Technologies, Eugene, Oregon

Analysis of rare cell populations by flow cytometry

requires the collection of high numbers of events to attain a

reliable measure of accuracy, which leads to long acquisition

times using traditional hydrodynamic focusing. An accurate

measurement of a population with a 1/1,000 probability

requires the collection of 1.6 � 106 events to detect the

population with a CV of 2.5%. When the probability of event

decreases to 1/10,000 the number of events needed for a CV

of 2.5% increases to 1.6 � 107. The ability of acoustic cytom-

etry to run samples at up to 1,000 �l/min while maintaining

precision and accuracy provides a distinct advantage over

typical hydrodynamic focusing cytometers for collecting such

large samples. We present two panels that show the power

of acoustic cytometry to make sensitive and accurate meas-

urements while greatly increasing the speed to acquire very

large sample volumes. First, a panel was developed to detect

mouse plasmacytoid dendritic cells (pDCs). The specialized

cell population that produces large amounts of type I inter-

ferons in response to viruses, typically comprises < 0.5% of

the total splenocyte population in naıve mice. A second

panel was developed to detect extremely rare circulating en-

dothelial cells (CECs), which have a typical range for healthy

individuals of 1 � 107 to 1 � 105 CEC per leukocyte (1–20

cells/ml of venous blood). To reduce the risk of loss of CECs

in processing, a no-lyse, no-wash procedure was developed,

which requires the speed and accuracy of acoustic focusing

to process such dilute samples.

P17

DETECTION OF AUTOANTIBODIES AGAINST PLATELET GLYCO-PROTEINS IN PATIENTS WITH IDIOPATHIC THROMBOCYTO-PENIC PURPURA BY FLOW CYTOMETRY

Yang He and Mingqing Zhu

The First Affiliated Hospital of Soochow University,

Jiangsu Institute of Hematology, Suzhou, China



Objective: Autoantibodies against platelet glycoproteins

(GPs) exist in patients with idiopathic thrombocytopenic

purpura (ITP), contributing to the disease mechanism. The

aim of this study is to establish a flow cytometric assay to

detect autoantibodies against platelets.

Methods: Polystyrene beads were coated with mono-

clonal antibodies SZ2, SZ22, SZ21, and 7E3 against plate-

let GPs Ib, IIb, IIIa, and IIb/IIIa, respectively, and incu-

bated with platelet lysate. Captured platelet GP/antibody

complexes were detected by FITC-labeled anti-human Ig

antibodies by flow cytometry. The mean fluorescence in-

tensity (MFI) in samples from 85 patients with ITP, 17

patients with non-ITP (NITP) and 50 healthy controls was

measured. The sensitivity and specificity of these antibod-

ies for the diagnosis of ITP were analyzed with the ROC

curve, and compared with those of the ELISA-based

monoclonal antibody immobilization of platelet antigen

assay (MAIPA).

Results: The MFI of SZ2 in ITP group was signifi-

cantly higher than that in NITP and healthy groups [1.49

(0.88–16.24) vs. 1.12 (1.0–1.33) and 1.01 (0.83–1.37), P

< 0.01]. Similarly, the MFI values of SZ22, SZ21, and 7E3

were all higher in ITP group than those in NITP and

healthy groups [SZ22: 1.55 (0.84–11.30) vs. 1.13 (1.0–

1.29) and 0.98 (0.85–1.24), P < 0.01; SZ21: 1.50 (0.87–

11.04) vs. 1.13 (0.97–1.32) and 1.01 (0.85–1.48), P <

0.01; and 7E3: 1.51 (0.84–9.81) vs. 1.05 (0.86–1.13) and

1.03 (0.74–1.28), P < 0.01]. In ROC analysis, the area

under the curve was 0.86, 0.9, 0.87, and 0.84, and the

sensitivity was 58.8, 52.9, 52.9, and 51.8%, respectively,

for SZ2, SZ22, SZ21, and 7E3. When all four antibodies

were used, the sensitivity for the diagnosis of ITP was

increased to 74.1%, significantly higher than that of MAIPA

(50.6%, �2 ¼ 6.78, and P < 0.05).

Conclusions: Flow cytometric assays with multiple

antibodies detected platelet-associate autoantibodies in

patients with ITP and increase the diagnostic sensitivity.

P18

FLOW CYTOMETRIC ANALYSIS OF LYMPHOMA CELL SIZE OFDIFFUSE LARGE B-CELL LYMPHOMA

Michelle Huang,1 Farzaneh Sayedian,1 Aaron Cotrell,1

Thomas Fennell,1 Alaa Muslimani,1 Xia Chen,2 Smith Marc,1 and

James Huang1

1William Beaumont School of Medicine, William Beau-

mont Hospital, Royal Oak, Michigan2William Beaumont School of Medicine, William Beau-

mont Hospital, Troy, Michigan

Introduction: Diffuse large B-cell lymphoma (DLBCL) is

a tumor of diffuse growth pattern of large B-cells. The size

of lymphoma cells is typically evaluated based on micro-

scopic comparison between neoplastic B-cells and normal

macrophage or normal lymphocyte on a hematoxylin and

eosin stained section. It is not clear if the size of lymphoma

cells in DLBCL can be objectively measured by flow cytome-

try based on forward scatter (FSC). This study seeks to

determine if FSC is different between DLBCL and follicular

lymphoma grade 3 (FL3) and if it is potentially helpful in

lymphoma classification.

Materials and Methods: We retrospectively analyzed

the flow cytometric listmode data of 26 cases of DLBCL and

26 cases of FL3. The mean channels of FSC of intact neo-

plastic B-cells and internal reactive T-cells were collected

and compared with Student’s t-test.

Results: The mean FSC of internal reactive T-cells

within the lymphoma tissue was about the same (425 vs.

420; P ¼ 0.145) between DLBCL and FL3. The mean FSC of

neoplastic B-cells of DLBCL was significantly higher than

that of FL3 (589 vs. 491; P < 0.001). The difference of

FSC between lymphoma cells of DLBCL and internal reac-

tive T-cells was 164 (95% confidence limits: 125 and 203).

The difference of FSC between lymphoma cells of FL3 and

T-cells was 72 (95% confidence limits: 40 and 104).

Conclusion: Our findings show that FSC of neoplastic B-

cells is significantly different between DLBCL and FL3. Such dif-

ference can be objectively measured using internal reactive T-

cells as a reference and applied in classification of B-lymphoma.

P19

RELIABLE FLOW CYTOMETRIC DETECTION OF TARGET POPU-LATIONS AT EXTREME LOW FREQUENCIES

Ruud Hulspas, Don F. Perrault, Emanuel T. M. Machado, and John C.

Sharpe

Cytonom/ST, LLC, Boston, Massachusetts

With acquisition speeds of 50,000–100,000 events per sec-

ond, high-speed analysis with flow cytometers has made a sig-

nificant contribution to assays that depend on rare-event

detection. As a result, enumeration of various stem cell phe-

notypes and nucleated red blood cells is practical and precise

in most routine analysis methods. Nevertheless, cross-contami-

nation and acquisition time (i.e., sample throughput) limit the

ability to perform reliable measurements on samples that con-

tain target populations at frequencies lower than 1 in

100,000. A practical approach to create a cross-contamination

free analysis is disposable microfluidic chip technology allow-

ing each sample to be run through a newly replaced fluidic

system. Sample throughput can be increased by parallel proc-

essing. We designed and tested a disposable microfluidic chip

that provides increased throughput over conventional flow

cytometers by splitting the fluid path into parallel flow chan-

nels. For analysis, we built a system capable of parallel detec-

tion and processing of event rates exceeding 200,000 events

per second. A disposable fluidic system incorporating the

chip was used to allow samples to be analyzed without cross

contamination, thus permitting statistically reliable rare event

analysis within short acquisition times. Using mixtures of fluo-

rescent and nonfluorescent particles, we were able to reliably

detect a 0.001% target population measured within a three

minutes acquisition period. As such, this parallel technology

can be useful in studies where reliable measurements of

extremely rare target populations (e.g., endothelial cells and

circulating tumor or fetal cells) are important.



P20

VALIDATION OF AN ASSAY TO DETECT PAROXYSMAL NOCTUR-NAL HEMATURIA CELL POPULATIONS WITH 0.01% LIMIT OFDETECTION: PRACTICAL AND STATISTICAL CONSIDERATIONS

Deborah A. Katanik,1 Joshua Coleman,1 James F. Bena,2 Jaroslaw P.

Maciejewski,3 and Eric D. Hsi1

1Department of Clinical Pathology, Cleveland Clinic, Cleve-

land, Ohio2Quantitative Health Sciences, Cleveland Clinic, Cleveland,

Ohio3Taussig Cancer Center, Cleveland Clinic, Cleveland, Ohio

Introduction: Recent guidelines for PNH Flow cytome-

try (FC) assays recommend a lower limit of detection (LOD)

of 0.01% PNH cells. Performance of such an assay is prob-

lematic for many laboratories due to rarity of PNH samples

and lack of familiarity with validation requirements for rare

event analyses.

Methods: We developed a FC assay to detect granu-

locytes via successive doublet discrimination, CD45-light

scatter and CD15/CD33 gating. Erythrocytes were gated

using doublet-discrimination and Glycophorin-A/light scat-

ter. Using a statistical model to validate a 0.01% LOD

with 95% confidence, we created a LOD validation sam-

ple set from 10 known PNH patients that consisted of 34

separate samples (3–5 replicates/patient) diluted to 0.01%

PNH cells using normal blood diluent. PNH-type granulo-

cytes were defined by lack of both FLAER and CD24 reac-

tivity while PNH-type erythrocytes were defined by lack

of CD59.

Results: Collecting sufficient gated events (157,000)

proved problematic, and thus a lower event threshold was

used in practice. Ninety-five% confidence of a positive result

at the 0.01% LOD level was evaluated for each sample using

the number of gated events. The number of samples

required was based on power analysis. Of the 34 samples

with between 75,000 and 157,000 events, 33 (97%) met the

threshold for positivity at 0.01% LOD, thus validating the

sensitivity of the assay.

Conclusion: High sensitivity assays are capable of

detecting 0.01% PNH cells, but our validation required bio-

statistical support to validate LOD claims and easy access to

PNH samples.

P21

NO-LYSE NO-WASH IMMUNOPHENOTYPING USING ACOUSTICCYTOMETRY

Rick Kerndt and Jolene A. Bradford

Flow Cytometry Systems, Life Technologies, Eugene, Oregon

Immunophenotyping whole blood is a primary applica-

tion in the study of white blood cell populations and their

function. Red blood cells (RBCs) traditionally have been

removed during the preparation step by lysis methods

before or after antibody staining, or by removal using den-

sity-gradient selection followed by washing steps to

remove red blood cell fragments and platelets. This proc-

essing of whole blood has its draw backs due to the poten-

tial loss of cells of interest and the difficulty in automating

cell washing. Methods have been previously described

using a no-lyse, no-wash staining protocol with the sacri-

fice of light scatter resolution. Accurate identification of

some cell populations depends on high-resolution scatter

data, so previous no-lyse, no-wash protocols require high

sample dilutions to reduce coincidence with red blood

cells. We present a no-lyse, no-wash method which takes

advantage of acoustic focusing technology to provide, fast,

sensitive, and accurate measurements of dilute samples. A

five-color panel is presented which uses a membrane per-

meable nucleic acid dye (Vybrant1 DyeCycleTM Ruby

stain) to identify nucleated cells from the whole blood

core stream. The remaining panel comprises a basic immu-

nophenotyping using mouse anti-Human CD19, CD3, CD4,

CD8, and CD56. A total of 10 ml whole blood is stained in

a 50 ml labeling volume and then diluted 1:400 fold (4 ml)

in PBS prior to acquisition on an acoustic focusing cytome-

ter. Light scatter properties and subpopulation percentages

are equivalent to lyse wash immunophenotyping protocols

with comparable acquisition times.

P22

SEQUENTIAL ANALYSIS OF CD38 EXPRESSION BY FLOWCYTOMETRY IN CHRONIC LYMPHOCYTIC LEUKEMIA

Virginia Lanier, Guang Fan, and Katalin Kelemen

OHSU, Portland, Oregon

Background: Expression of CD38 by flow cytometry

(FC) is a useful prognostic marker in chronic lymphocytic

leukemia (CLL). Our aims are to evaluate CD38 expression

in sequential samples of CLL patients and to correlate CD38

expression with cytogenetic findings.

Methods: The Pathology Archives of OHSU were

searched for CLL patients who underwent sequential FC anal-

yses of CD38. The CBC, FC results including CD38 and Zap-

70 expression, and cytogenetic findings were reviewed. The

cutoff for positive CD38 result is 30% or more CLL cells.

Results: Nineteen CLL patients had sequential FC anal-

yses of CD38 (median number of tests/patient 3; range 2–

9). Forty-five blood and 41 bone marrow FC analyses were

reviewed. Excellent correlation was found between CD38

results of concurrently involved peripheral blood and bone

marrow samples. Eight patients were CD38 (42%), eight

patients were CD38þ (42%), and three patients changed

over time from negative to positive (16%). Change of posi-

tive result into negative did not occur. No difference was

observed between the CD38 and CD38þ groups regarding

sex, age, lymphocyte count, or expression of Zap-70.

CD38 expression did not correlate with complex aberrant

karyotype, deletion of 13q34, 11q, 17p, or trisomy 12 by

FISH.

Conclusions: Expression of CD38 by FC may convert

from negative to positive over time in CLL, therefore, se-

quential FC testing of CD38 is indicated. CD38 expression

might not be linked to the pathways represented by current



cytogenetic markers. To predict prognosis, a combination of

cytogenetic, FISH and flow cytometric studies is necessary.

P23

IMPROVEMENT IN ACCURACY OF CLINICAL LYMPHOCYTE SUB-SET QUANTITATION BY FLOW CYTOMETRY USING A 7-COLORASSAY INCORPORATING CD14

Stacy C. League, Xiangyang Dong, Prabin Thapa, and

Roshini S. Abraham

Mayo Clinic, Rochester, Minnesota

Introduction: Flow cytometry is considered the gold

standard method for detailed lymphocyte subset quantita-

tion (T, B, and NK cells). Previously, we observed that the

absolute CD45 lymphocyte counts were consistently

greater than the lymphosum of individual T, B, and NK cell

populations due to the unintentional inclusion of mono-

cytes in the lymphocyte gate (Block et al., 2010). Addition-

ally, over a 3-year time period, the median difference of

observed CD3, CD4, and CD8 counts (T-Sum) went from

being less than the calculated value to approaching zero,

indicating improved concordance between the observed

and calculated results and the interquartile ranges also

decreased suggesting reduced analytical variability over

time due to operator training and experience. An alterna-

tive assay was developed which included CD14, optimized

fluorochrome choices, and simplified gating strategies with

the goal of improving the accuracy of clinical flow cyto-

metric lymphocyte subset quantitation.

Materials and Methods: Flow cytometry analysis for

TBNK quantitation was performed on 213 samples using two

different flow cytometers and analytical software: Current assay

(BD 6-color Multitest1, BD FACS Canto instruments with Canto

software v2.4) and the newly developed assay (7-color, Gallios

instrument, Beckman Coulter, Kaluza v1.2 software).

Results: Linear regression analysis was performed for

absolute CD45, CD3, CD4, CD8, CD4þCD8þ, CD19þ,

CD16þ56þ, lymphosum, and T-Sum with the following

results: 0.96, 0.96, 0.96, 0.57, 0.65, 0.97, 0.96, 0.55, and

0.67, respectively. Additionally, paired t-test analysis

revealed significant instrument and software-related differen-

ces with a P < 0.0001 for each of these values.

Conclusions: Inclusion of CD14 as an exclusionary

marker, use of optimized fluorochromes, choice of flow

cytometry instrumentation and simplified gating strategies

significantly increased the accuracy of clinical lymphocyte

subset quantitation.

P24

DETECTION OF RARE PAROXYSOMAL NOCTURNAL HEMOGLO-BINURIA CLONES IN NORMAL HUMAN BLOOD BY FLOWCYTOMETRY

Michael A. Liew,1 Marjorie Farley,1 John Andreasen,1 and

Carl T. Wittwer1,2

1ARUP Laboratories, Salt Lake City, Utah

2University of Utah, Salt Lake City, Utah

Introduction: Paroxysomal nocturnal hemoglobinuria

(PNH) is a rare acquired disorder that affects �1.3 in

1,000,000 individuals. It is characterized by increased com-

plement-mediated lysis of RBC secondary to absent GPI

anchors of several cell surface proteins. The aim of this

study was to determine the amount of PNHþ RBC are pres-

ent in normal human blood.

Methods: RBC were assayed by washing 30 ml of

blood with PBS, followed by staining with Glycophorin

A FITC and CD59 PE. Routine testing involves counting

250,000 events, but in an attempt to establish more

accurately, the range of PNHþ RBC in normal samples,

1 million RBC were counted in 102 asymptomatic indi-

viduals, and 10 million RBC were counted in 12 individ-

uals. Results were acquired on a FACS Canto II flow

cytometer.

Results: Analysis of the normal individuals by the RBC

assay did demonstrate the presence of PNH cells. Each sam-

ple contained a range of type III PNH cells (0–8 PNH cells

per 1 million RBC). Results for the 10 million cells were

similar. Two PNH samples measured by the RBC assay had

coefficients of variation < 5%, while two normal samples

were around 100%.

Conclusions: Type III PNH RBC can be visualized

and quantified with this RBC PNH assay. These results

help to define what the cutoffs should be to define

subclinical PNH so that therapy can be imple-

mented in the appropriate patients with myelodisplastic

syndromes.

P25

STANDARDIZATION OF MULTIPLE CYTOMETERS USING BDTM

CYTOMETER SETUP AND TRACKING BEADS FOR CONSISTENTRESULTS

Ellen M. Meinelt, Mervi Reunanen, Maria C. Jaimes, and Mark

Edinger

BD Biosciences, San Jose, California

Fluorescent intensity of populations is monitored in

immunophenotypic assays. Changes in the intensity may

be due to treatment the patient is receiving or variation

in instrument settings. To generate high quality, repro-

ducible data and maintain consistency or time or across

multiple cytometers, a process has been created. This

process includes using the BD FACSDivaTM software,

which contains the Cytometer Setup and Tracking (CS &

T) module for: (1) Defining a Cytometer Baseline, (2)

Establishing 2.5 times the SD of the Electronic Noise for

each fluorescent parameter, (3) Creating Application Set-

tings, (4) Verifying the voltages with positively stained

cells, (5) Creating Target values using the BDTM CS & T

beads, and (6) Transferring the cytometer configuration,

experiment, and bright bead target values to other

cytometers. Digital flow cytometers with the same lasers

and optical configurations can be standardized with the

process presented here. Flow cytometers with different

lasers and optical configurations can also be standardized

with slight modifications to the process. Data will be



shown to demonstrate the outcome of this process using

stained samples.

P26

BLASTS WITH MEGAKARYOCYTIC DIFFERENTIATION IN MYELO-DYSPLASIA BY FLOW CYTOMETRY: PHENOTYPIC COMPARISONWITH MYELOID PROLIFERATION IN DOWN SYNDROME

Mariela Monreal,1 Valeria Cismondi,1 Olga Spinelli,1 Graciela

Geraghty,1 Marina Narvaitz,1 Silvina Palmer,2 Alejandro Flores,3 Luis

Aversa,4 Cristian Sanchez La Rosa,5 Isabel Giere,1 and Miguel A.

Pavlovsky1

1Fundaleu, Buenos Aires, Argentina

2Hospital Britanico de Buenos Aires, Buenos Aires, Argen-

tina3Sanatoria de la Trinidad Palermo, Buenos Aires, Argen-

tina4Hospital de Ninos Ricardo Gutierrez, Buenos Aires, Argen-

tina5Hospital Universitario CEMIC Saavedra, Buenos Aires, Ar-

gentina

Immunophenotyping by flow cytometry (FC) has a

known diagnostic usefulness in myelodysplasia (MDS) and

related myeloid neoplasms. Since detailed knowledge of

normal maturation patterns is needed for recognition of

abnormalities, myelomonocytic lineages have been the most

extensively evaluated. We report three adult patients (age

64–72; two males and one female) that met WHO diagnos-

tic criteria for MDS: two refractory anemia with excess

blasts (RAEB) and 1 refractory anemia (RA), with < 10%

blasts with clear phenotypic evidence of megakaryocytic

(MGK) differentiation. Bone marrow specimens were tested

with 4–8 color FC combinations; FACSCanto II and Infinicyt

software used for acquisition and analysis, respectively.

The expression patterns on blasts was highly

reproducible:CD45þ brCD36þ brCD38þ HLA DR CD71þbrCD7þ CD56 dCD4þ CD13þ CD33þ CD41þ CD117þ/

CD34/þ. Phenotypic evidence of dysplasia was detected on

granulocytes and erythroid populations as well. All patients

presented with thrombocytopenia (platelets < 30 � 109/l)

and anemia (hemoglobin 7 g/l) in 2/3. RAEB patients died

within 7 months from FC evaluation (disease progression);

RA patient was initially transfusion dependent, currently (8

months from FC) responding to treatment with 5-azacyti-

dine. In addition, FC laboratory database was searched for

cases tested with the same panel from January 2007 to

March 2011, to assess the prevalence of this atypical pheno-

type. Of a total 308 cases, eight showed MGK phenotype.

Interestingly, while five of eight cases were children classi-

fied as myeloid proliferation in Down Syndrome (MP-DS)

with the same distinct phenotype as our MDS group, the

other three cases were adult megakaryocytic leukemia, with

differences in CD38, CD7, CD36, CD4, and CD56 expres-

sion. MP-DS is an entity with unique features, frequently

presenting as MDS with thrombocytopenia and/or anemia.

However, little information is available addressing blasts

with MGK commitment in MDS. Our findings contribute to

recognition of informative antibody combinations that

should be considered in the characterization of myeloid

stem cell disorders.

P27

ANALYSIS OF THE IMMUNOPHENOTYPIC ABERRANCIES INTHE MATURING MYELOID AND MONOCYTIC COMPARTMENT INACUTE MYELOID LEUKEMIA BY FLOW CYTOMETRY

Ying Pei, Jason Schallheim, and Guang Fan

Oregon Health Science University, Portland, Oregon

Dysplastic morphologic features are commonly present

in the maturing granulocytes and monocytes in acute mye-

loid leukemia (AML). So far, no study has been reported to

investigate the immunophenotypic characters in these

maturing compartments in AML comparing to the normal

individuals by our knowledge. The purpose of this study is

to investigate the immunophenotypic aberrancies in the

maturing myeloid and monocytic compartments in AML.

Twenty cases of AML diagnosed in OHSU between 2008

and 2011 were reviewed. The flow cytometry results of pe-

ripheral blood and bone marrow aspiration were analyzed.

Flow cytometry antibody panels included CD71/CD117/

CD33/CD13/CD34/HLA-DR/CD16/CD45; CD15/CD64/

CD123/CD56/CD34/CD14/CD11b/CD45; CD5/CD10/CD34/

CD20/CD19/CD45; CD2/CD7/CD4/CD56/CD5/CD8/CD3/

CD45. Overall 85% of the cases showed immunopheno-

typic aberrancies in the maturing myeloid and monocytic

compartments. In the myeloid compartment, the most

common finding was decreased or lack of expression of

CD10 (50%), which was followed by decreased expres-

sion of CD16 (20%) and aberrant expression of CD56

(15%), CD123 (15%), and HLA-DR (10%). In the maturing

monocytic compartment, abnormal immunophenotypic

findings included aberrant expression of CD 123 (25%)

and CD 56 (20%) and decreased expression of CD14

(20%). In conclusion, immunophenotypic aberrancies are

commonly present in the maturing myeloid and mono-

cytic compartments in AML. Most of these abnormal

immunophenotypic features have also been reported in

the cases of myelodysplastic syndrome, which suggests

that maturing compartments in most of AML has immuno-

logic features of dysplastic maturation. Future studies will

expand the case numbers, focus on the correlation of

immunophenotypic aberrancies with the molecular stud-

ies, and explore prognostic implications.

P28

EXPERIENCE WITH A HIGH SENSITIVITY FLOW CYTOMETRICASSAY FOR DETECTION OF PAROXYSMAL NOCTURNALHEMOGLOBINURIA CLONAL POPULATIONS

Charles F. Repetti, Vanessa A. Jones, and Daniel M. Jones

Quest Diagnostics Nichols Inst, Chantilly, Virginia

New guidelines for the diagnosis and monitoring of PNH

have been recently promulgated that recommend use of high

sensitivity testing of erythroid, granulocytic or monocytic line-

ages (Borowitz et al., Cytometry 2010; 78B 211:230). We



compare here the detection rate of PNH clones using

such a high sensitivity assay (0.01%) with an older assay

with lower sensitivity (1%). Erythrocytes are stained with

Glycophorin A-FITC þ CD59-PE and 250,000 cells are col-

lected. WBCs are stained with a six-color panel, including

FLAER, CD24, CD16, CD33, CD14, and CD45. Samples

from 16 patients being evaluated from PNH were tested

with the old assay compared to samples from 40 patients

with the new assay. Patient ages range from 11 to 90

years, with a male/female ratio ¼ 0.86. Using the new

assay, 10 patients (25%) showed minor PNH clones, which

were below the sensitivity of the older assay with clonal

size for neutrophils ranging from 0.05 to 98.8% (mean

44.89%). Clonal types found were: Type III only: n ¼ 35;

Type II only: n ¼ 6; Type III þ Type II: n ¼ 14; single

Type III þ double Type II: 1. No relation between age

and clonal size was evident. The ratio of clonal sizes for

neutrophils versus monocytes within a patient was similar

(r2 ¼ 1.13), suggesting that monocytes can be of value in

confirming status in cases with MDS-associated (or other)

aberrant granulocyte immunophenotypes. Higher sensitivity

rates for PNH diagnosis and monitoring will reveal a signif-

icant number of low-level PNH clones in routine clinical

samples.

P29

CD117 EXPRESSION IN ADULT T-CELL LEUKEMIA/LYMPHOMA

Margo Santiago, Prashant Tembhare, Constance M. Yuan, and

Maryalice Stetler-Stevenson

NCI, NIH, Bethesda, Maryland

Adult T-cell Leukemia/Lymphoma (ATLL) is a rare mature T-

cell neoplasm believed to be caused by human T-cell leukemia/

lymphotropic virus type-1. Using flow cytometric immunophe-

notyping (FCI), ATLL can be diagnosed and monitored in

patients. The characteristic FCI phenotype is a T cell popula-

tion that is CD3 dim, CD4 positive, CD7 negative, CD25 bright,

and CD26 negative. An outside case was referred to the Labora-

tory of Pathology, NCI, NIH with reported immunophenotypic

studies demonstrating CD117 positivity in the malignant T-cells.

CD117 is not normally expressed by mature T-cells. If CD117

positivity is a characteristic feature of ATLL it could provide a

sensitive marker for diagnosis and monitoring of residual dis-

ease. Therefore, a series of seven specimens from patients with

ATLL (six peripheral blood and one bone marrow) were exam-

ined for the expression of CD117 by ATLL cells. In all seven

cases, the malignant ATLL cells and normal T-cells were CD117

negative. We conclude CD117 is not a characteristic feature of

ATLL and cannot provide a sensitive test for ATLL disease.

Intramural Research Program of the NIH, NCI.

P30

T CELL RECEPTOR GENE REARRANGEMENT IN FLOW-SORTEDNATURAL KILLER (NK) CELLS IN CHRONIC LYMPHOPROLIFER-ATIVE DISORDERS OF NK CELLS

Jason M. Schallheim, Ying Pei, Guang Fan, and Rita M. Braziel

Oregon Health Science University, Portland, Oregon

Chronic lymphoproliferative disorders of NK-cells (NK-

CLDs) are heterogeneous disorders characterized by persis-

tently increased peripheral blood NK-cells. Rare cases of

NK-CLD have shown clonal T-cell receptor (TCR) gene

rearrangements, but specific cell subpopulations have not

been analyzed. Here, we report two cases of NK-CLD with

TCR clonality. Case 1 is an 81-year-old female with normal

peripheral blood lymphocytes (3.18 � 103/�l). Flow

cytometry identified increased NK-cells (24% CD2þ, CD3,

CD16þ, and CD56þ), and also increased NK/T-cells (13%

CD2þ, CD3þ, CD16þ, and CD56þ). Different TCR clones

were identified in the flow-sorted NK-cells (CD3/CD56þ)

and the NK/T-cells (CD3þ/CD56þ). The T-cell population

(CD3þ/CD56) was polyclonal. Case 2 is a 49-year-old

female with persistent lymphocytosis (9.47 � 103/�l).Flow cytometry identified increased abnormal NK-cells

(40% CD2þ, CD3, CD16þ, CD56, CD57þ, CD94þ, and

CD158aþ/CD158cþ killer cell immunoglobulin-like recep-

tor antigens). T-cells (59%) showed no evidence of abnor-

mal antigenic expression except a decreased CD4:CD8 ra-

tio of 0.8:1. TCR clonality was identified in the nonsorted

peripheral blood. Additional molecular analysis of flow-

sorted NK-cells and T-cells is pending, but the lack of aber-

rant antigen expression in the T-cell population suggests

that the clonality is likely in the abnormal NK-cells. In

summary, there is increasing evidence that NK-cells in

some NK-CLDs harbor clonal TCR gene rearrangements.

Molecular analysis of flow-sorted subpopulations of NK

and T-cells in NK-CLD may provide important new insights

into the complex relationship of these cells. In addition,

TCR clonality may be useful in a subset of cases as a diag-

nostic marker to differentiate NK-cell malignancy from re-

active NK-lymphocytosis.

P31

AUTOMATED CLASSIFICATION OF LEUKEMIA USING CYTOMET-RIC FINGERPRINTING

Richard D. Schretzenmair, Nicholas Kurtzman, Neisan Sabet,

Jonni S. Moore, and Wade T. Rogers

Department Pathology and Laboratory Medicine, Univer-

sity of Pennsylvania, Philadelphia, Pennsylvania

Introduction: Flow cytometry is a critical tool in the

primary diagnosis of hematologic malignancies. To discrimi-

nate better subtle differences in malignancies, panels are

evolving towards more parameters in fewer tubes to sim-

plify data acquisition and elucidate marker correlations that

are difficult to discern otherwise. However, higher dimen-

sional measurement increases the complexity of data analy-

sis, lengthening the time to results. We aim to develop com-

putational methods, amenable to high throughput technolo-

gist independent analysis, that mine such high-dimensional

datasets for patterns that discriminate hematologic malig-

nancies.

Materials and Methods: Patient samples (blood and/

or bone marrow) sent for routine diagnosis to the Clinical

FlowCytometry laboratory, were prepared according to



standard procedures. Routine clinical panels were run and

diagnoses confirmed by hematopathologists. Concurrently,

cells were stained with a second single tube panel consist-

ing of CD3, CD10, CD13, CD15, CD19, CD33, CD34, CD45,

Hla-Dr, and DAPI for viability and data was acquired on a

Beckman Coulter Gallios flow cytometer. Data analysis was

carried out with the R/Bioconductor package flowFP. After

automated gating to exclude debris and nonviable cells, fin-

gerprints were computed for each sample. Fingerprint-based

classification of AML, ALL, MDS, and normal blood/marrow

was evaluated using leave-one-out cross validation by meas-

uring a distance metric of a test sample fingerprint to mod-

els of each class constructed from all fingerprints excluding

the test sample.

Results: Accurate classification (88% sensitivity; n ¼24) of AML versus ALL and Normal was achieved. Classifica-

tion of ALL bone marrow samples was perfect but only

involved limited numbers (N ¼ 2). The authors are grateful

to Beckman Coulter for support.

P32

USE OF CD157 IN FLAER-BASED ASSAYS FOR HIGH-SENSITIV-ITY PNH GRANULOCYTE AND PNH MONOCYTE DETECTION

D. Robert Sutherland,1 Michael Keeney,2 Erica Acton,1 Bruce H.

Davis,3 and Andrea Illingworth4

1University Health Network, Toronto, Ontario, Canada

2London Health Sciences, London, Ontario, Canada

3Trillium Diagnostics LLC, Bangor, Maine

4Dahl-Chase Diagnostics, Bangor, Maine

PNH is a rare acquired disease of hematopoietic cells

characterized by loss of glyco-phosphatidyl-inositol (GPI)

linked structures. The ability to diagnose rapidly PNH by

flow cytometry has led to improved diagnosis, patient

management, and prognosis. The International Clinical

Cytometry Society Guidelines for the Diagnosis and Moni-

toring of PNH and related disorders recommended cock-

tails based on FLAER and GPI-linked protein expression

for leukocyte analysis. We developed a 4-color combina-

tion for high-resolution detection of PNH neutrophils

using FLAER, CD24, CD15, and CD45 that is highly sensi-

tive and usable on a variety of clinical cytometers. Simi-

larly, a 4-color combination of FLAER, CD14, CD64, and

CD45 was developed for high-resolution detection of

PNH monocytes. Both WBC assays are capable of detect-

ing PNH cells at < 0.05% with very low background

rates on normal samples ( < 2–4/100,000 granulocytes).

CD157 is another GPI-linked structure expressed on both

granulocytes and monocytes raising the possibility that

this single reagent might replace the GPI-linked structures

CD24 (in the granulocyte assay) and CD14 (monocyte

assay). We have found in preliminary studies that CD157

generates the same results for PNH clone size when sub-

stituted for CD24 and CD14 in the granulocyte and

monocyte assays, respectively and has similar background

staining levels in normal blood samples as the current

predicate assays. We conclude CD157 may be a more ad-

vantageous GPI-linked protein target for use in PNH diag-

nostic assays, with the potential to reduce both reagent

use and technical time required to detect PNH clones.

Studies of a wider variety of samples are ongoing.

P33

FLOW CYTOMETRIC IMMUNOPHENOTYPIC ASSESSMENT OF T-CELL CLONALITY IN CEREBROSPINAL FLUID AND FINE NEE-DLE ASPIRATE SPECIMENS USING LIMITED V BETA REPER-TOIRE ANALYSIS

Prashant Tembhare, Constance Yuan, Armando Filie, and Maryalice

Stetler-Stevenson

Laboratory of Pathology, National Cancer Institute, NIH,

Bethesda, Maryland

Introduction: Flow cytometric (FC) TCR-V� repertoire

analysis (TCR-V�-R) is a sensitive method to detect T-cell

clonality; however, its implementation in low cellularity

specimens has not been established. We developed a strat-

egy to use TCR-V�-R in cerebrospinal fluid (CSF) and fine

needle aspirate (FNA) specimens.

Method: Comprehensive FC panel with complete TCR-

V�-R was used for initial FC evaluation in diagnostic screen-

ing specimens from 10 patients with T-cell neoplasia to

determine tumor specific TCR-V� protein expression.

Abbreviated, patient specific TCR-V�-R evaluation was per-

formed in 21 subsequent paucicellular specimens (12 CSF

and 9 FNA) using a single cocktail containing three anti-V�antibodies (one tumor specific and two negative controls)

in combination with other antibodies chosen to help gate

on atypical T-cells. The results of flow cytometry were cor-

related with cytomorphological results.

Results: TCR-V�-R demonstrated T-cell clonality in all

initial screening peripheral blood (PB) specimens from 10

patients. Clonal T-cells were detected in all FNA specimens

and in 9 of the 12 CSF specimens. The percent of lymphoid

cells that were clonal T cells ranged from 1.75 to 85% in

FNA specimens and from 1.4 to 100% in CSF specimens.

Conclusions: The V�-specific-single-cocktail TCR-V�-Rstrategy is highly sensitive and specific in evaluating T-cell

clonality in precious low cellularity specimens like CSF and

FNA. This strategy can be used to detect minimal involve-

ment despite low cellularity.

P34

PHOSPHO-FLOW CYTOMETRY TO ASSESS BIOMARKERS FOR ATHERAPEUTIC MONITORING OF IMMUNSUPPRESSIVE DRUGSAFTER ORGAN TRANSPLANTATION

Sandy von Salisch, Maja-Theresa Dieterlen, Sara Klein, Katja Eber-

hardt, Hartmuth Bittner, Stefan Dhein, Friedrich Mohr, and Markus

Barten

Heart Center Leipzig, Leipzig, Germany

Backgrounds: Therapeutic drug monitoring (TDM) of

immunosuppressive drugs after heart transplantation like

the mTOR-inhibitors sirolimus (SRL) or everolimus (ERL) is

based on measuring serum levels alone, but often results in



under- or over-immunosuppression. Earlier studies have

shown the capability of measuring pharmacodynamic drug

effects for TDM. Since in combination therapies the assess-

ment of biomarkers for individual drugs is still not clinical

routine, we used phospho-flow cytometry to assess the

downstream effects of drug-induced target inhibition.

Methods: Different clinical relevant concentrations of

SRL (0.9–91.4 �g/l), cyclosporine A (CsA, 75.1–1,202 �g/l),mycophenolate acid (MPA, 0.08–3.2 mg/l), or dexamethasone

(DEX, 0.5–200 ng/ml) were added to 200 �l of whole blood

from eight healthy volunteers, respectively. Whole blood was

mitogen-stimulated to activate the mTOR pathway, stained for

phospho-S6 in T-cells and analyzed by flow cytometry. For the

validation, we determined coefficient of inter-assay variability

for 3 different samples and 15 measurements.

Results: Phospho-flow analysis revealed that addition

of SRL suppressed phosphorylation of ribosomal protein S6

in human T-cells, whereas CsA, MPA or DEX did not inhibit

mTOR-related S6-phosphorylation. We determined the assay-

specific IC50 for sirolimus at 23.5 nM. Between-run assay

coefficients of variation ranged from 0.12 to 0.49 and

showed the robustness of the assay.

Conclusions: In this study, we established a phospho-

flow cytometry assay for biomarker assessment, which spe-

cifically measures the SRL effect on mTOR inhibition. Future

studies in organ transplanted human recipients will show if

such an assay has the potential to dose SRL in combination

with either CsA or MPA more safely without loosing efficacy.

P35

IMPROVED MONOCHROMATIC METHOD FOR HIGH PURITYMONOCYTE GATING BY FLOW CYTOMETRY

Linda Wong and Bruce H. Davis

Trillium Diagnostics, LLC, Bangor, Maine

Assays of antigen expression on myeloid cells have an

underlying premise that the assay integrates high purity gat-

ing of the leukocyte subpopulation in question. CD45/side

scatter gating provides sufficient gating purity for qualitative

assays of antigen expression; it is unsuitable for quantitative

assays of antigen changes, especially monocytes. We have

validated a monochromatic gating approach, combining

CD45 and CD64 labeled with the same fluorochrome,

which allows for high purity monocyte gating. Twenty-five

blood samples were stained using three different antibody

combinations (CD45 FITC þ CD163 PE; CD45 FITC þCD64 PE; and CD45 FITC þ CD64 FITC). Data analysis

focused on the percentage of ‘‘monocytes’’ defined by the

various antibody and side scatter gating combinations. Per-

cent monocytes recovered by monochromatic CD64 gating

was not statistically different from two color CD45 þ CD64

or CD45 þ CD163 gating. All three methods of immuno-

logic monocyte identification yielded a 12–24% reduction in

the monocyte percentage compared to CD45/side scatter

gating. Interobserver imprecision of monocyte percent

decreased (CV of > 7.7% to a CV of < 4.3%) with mono-

chromatic CD64 þ CD45 gating over standard CD45/side

scatter gating. A monochromatic combination of CD45 and

CD64 antibodies with scatter signals allows higher purity

monocyte gating by FC compared to CD45/side scatter gat-

ing. This approach allows for the development of a high re-

solution 4 color assay for detection of PNH whereby a sin-

gle 4 color tube will allow simultaneous high purity mono-

cyte (CD64þ) and neutrophil (CD15þ) analysis of both PI

linked protein expression and FLAER binding.

P36

THE UTILITY OF CD200 AND CD23 EXPRESSION IN CHRONICLYMPHOCYTIC LEUKEMIA AND MANTLE CELL LYMPHOMA

Sue Wong, Mary Sartor, Zarah Timbol, Ken Bradstock, and David

Fulcher

Flow cytometry unit, ICPMR, Westmead Hospital, West-

mead, Australia

CD200 (OX-2 antigen) is a type 1 transmembrane immuno-

globulin (Ig) superfamily glycoprotein that is expressed on

normal B- and T-cells, dendritic, endothelial, and neuronal

cells. In mice, the binding of CD200 to its receptor, CD200R1,

expressed by myeloid-derived antigen-presenting cells and

subsets of T cells, promotes a suppressive effect on T-cell

responses. CD200 is highly expressed on clonal cells in CLL,

plasma cell myeloma and hairy cell leukemia, and its expres-

sion has been linked to a poorer prognosis, consistent with

the suggestion that CD200 has a protective survival effect on

CLL cells. Based on these findings, a humanized monoclonal

antibody against CD200 is available as an anti-tumor treat-

ment. For the purposes of immunophenotyping, the distinc-

tion between CLL and MCL, both CD5-positive lymphoproli-

ferative disorders, typically rests on CD23 expression, usually

absent in MCL but expressed by CLL cells. However, in atypi-

cal cases of CLL, CD23 may be negative. Since CD200 expres-

sion is also reported to be absent in MCL, we examined its

utility in distinguishing these disorders in a routine diagnostic

laboratory. We will present our detailed analysis of this study,

although preliminary data suggests that some cases of CLL

may be CD200-negative. As anti-CD200 therapy becomes

more common in the treatment for CLL as well as other B-cell

lymphoproliferative disorders, determining the expression

patterns for CD200 may have important implications for treat-

ment strategies and minimal residual disease monitoring.

P37

CHANGES IN ANTIGEN EXPRESSION IN A FOLLOW-UP OFCHRONIC LYMPHOCYTIC LEUKEMIA/SMALL LYMPHOCYTICLYMPHOMA

Jiehao Zhou, Anupama Tewari, and Magdalena Czader

Indiana University, Indianapolis, Indiana

Diagnosis of chronic lymphocytic leukemia/small lym-

phocytic lymphoma (CLL/SLL) depends on the clinical and

laboratory features including a specific immunophenotype.

Specific antigens not only distinguish CLL/SLL from other B-

cell lymphomas, but are also increasingly used as therapeu-

tic targets. Patients with CLL/SLL undergo repeat flow cyto-

metric testing throughout the course of the disease, how-

ever the reports detailing how the expression of specific



antigens changes over time are sparse. We have evaluated

the variability of CLL/SLL immunophenotype in paired fol-

low-up samples from the 32 patients. Our results show a

significant variability in the expression levels of CD20, CD5,

CD23, surface immunoglobulin light chain, and HLA-DR in

the initial diagnostic samples. During the disease course, a

systematic statistically significant decrease in the density

and percentage of cells positive for CD23 and a significant

increase in the intensity of HLA-DR were observed. The

changes in CD5 were variable with a proportion of samples

showing decrease in CD5 intensity over time. However, no

statistic significance is observed. The number of positive

cells and fluorescence intensity did not change significantly

for CD19, FMC7, and CD22 antigen. Flow cytometric immu-

nophenotypes of CLL/SLL vary in the course of the disease

with a systematic decrease in the expression of CD23 and

increase in HLA-DR. These alterations can potentially influ-

ence the responsiveness of the disease to the monoclonal

antibody therapy and should be evaluated systematically.

Despite the changes in antigen expression, in the majority

of cases, the diagnosis of CLL/SLL could be rendered based

on the follow-up immunophenotype.



abstracts from the 26th annual meeting of the international clinical cytometry society. october...

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