abstracts from the 23rd annual meeting of the clinical cytometry society october 12–14, 2008...

CCS Abstracts

Abstracts From The 23rd Annual Meetingof the Clinical Cytometry Society

October 12–14, 2008 Portland, Oregon

1

MULTIPARAMETER FLAER-BASED WBC ASSAY VERSUS CD59EXPRESSION ON RBCS IN THE PRIMARY SCREENING OFPNH CLONES

Tanya Anderson,1 Nancy Kuek,1 David Barth,1 Michael Keeney,2 andRobert Sutherland3

1Department of Pathology University Health Network,

Toronto, ON, Canada2London Health Sciences Centre, Toronto, ON, Canada

3Department of Medical Oncology/Hematology University

Health Network, Toronto, ON, Canada

PNH is an acquired stem cell disorder caused by a somatic

mutation in the X-linked PIG-A gene. This leads to a partial/

complete loss of all GPI-linked proteins and clinical features

of chronic intravascular hemolysis, thrombosis, and marrow

failure. Diagnosis and follow-up of PNH improved with flow

cytometry-based assays that involved the analysis of CD55

and CD59, typically on red cells and neutrophils. However,

the ability to accurately detect PNH RBCs is compromised

by prior hemolysis and/or the presence of transfused RBCs.

Aplastic anemia (AA) and myelodysplastic syndrome (MDS)

patients may also show the presence of PNH clones that

are not readily detectable by RBC-based flow assays. We

recently described a sensitive multiparameter fluorescent

aerolysin (FLAER)-based flow assay containing CD45, CD33,

and CD14 that accurately identified PNH monocyte and

neutrophil clones in PNH, AA, and MDS patients. Here, we

compared the efficiency of detection of PNH WBC clones

using the FLAER assay with a CD59-based assay on RBCs

over a 4-year period. Of 415 evaluable samples, PNH clones

were detected in 58 with the FLAER assay, while PNH RBCs

were detected in only 64% of this subset, and always with a

smaller clone size. When PNH clones were detected in rare

samples with the CD59 test only, repeat analysis of both

assays demonstrated the RBC-derived data to be in error.

These results suggest that the FLAER assay on WBCs is a

much more sensitive and robust primary screening assay for

the detection of PNH clones in the clinical flow laboratory.

2

PRESERVATION OF CELL-SURFACE IMMUNOPHENOTYPE WITHINTRACELLULAR PHOSPHOPROTEIN FLOW CYTOMETRY

S.S. Belouski,1,2 K. Shults,3 C.L. Green,1,2 and J. Ferbas1,2

1Amgen, Department of Medical Sciences

2Amgen, Department of Clinical Immunology

3Esoterix Clinical Trial Services, A LabCorp Company

Introduction: Measurement of phospho-epitopes by

flow cytometry (phosphoFC) is an enabling method that

adds value to our understanding of signaling events in indi-

vidual cells. One limitation of phosphoFC is that common

commercial buffers often destroy scatter properties and epi-

topes recognized by antibodies against cell-surface recep-

tors. Method: We conducted a proof of concept study to

demonstrate that phosphoFC buffers can be formulated that

preserve cell surface markers. A repeat-measures assessment

(5 healthy donors tested 3 times over 3 weeks) was utilized

to understand whether signals from cells in stimulated (ly-

ophilized PMA/ionomycin) whole blood are reproducible in

a clinical trial setting. Results: The data show that p-Akt

and p-Erk can be reliably detected in peripheral T (CD3), B

(CD19 and/or CD20), NK (CD16 and/or CD56), and mono-

cyte (CD14) subpopulations with cell-lineage antibodies

that lose reactivity when a common commercially available

fixative is used. Because the novel buffers allowed us to uti-

lize a broader array of antibodies, combinations of CD19

and CD20 as well as CD16 and CD56 could be utilized with

the same or different fluorochromes to either provide a

high signal-to-noise ratio or study B and NK subsets, respec-

tively. Moreover, CD14 staining was retained so that mono-

cytes could be identified by this conventional marker. Con-clusion: It may be premature to conclude that all phospho-

proteins and cell-lineage markers will retain reactivity with

this approach; however, these proof of concept data point

to a path forward that will further maximize the value that

phosphoFC has added to the research and clinical arenas.

3

ALPS: THE ROLE OF FLOW CYTOMETRY

Mı́riam P. Beltrame, Ana P. Azambuja, Rosana I. Cattaneo,Orley Kantor, Ricardo Pasquini, and Mariester Malvezzi

Universidade Federal do Paran~a

Autoimmune lymphoproliferative syndrome (ALPS) is

inherited in an autosomal dominant manner, caused by defec-

tive lymphocyte homeostasis. A Brazilian boy with ALPS is

described. The clinical picture was dominated by chronic non-

infectious lymphadenopathy, splenomegaly, and recurrent bac-

terial infections. At the age of 4 months, the patient began the

following symptoms: fever and increase in bilateral cervical

region. Laboratory investigation by flow cytometry studies

showed increase of T-lymphocyte subsets (CD3þTCRabþ

Published online in Wiley InterScience (www.interscience.wiley.com).DOI: 10.1002/cyto.b.20456

Cytometry Part B (Clinical Cytometry) 74B:364–376 (2008)

q 2008 Clinical Cytometry Society

CD42CD82 T lymphocytes). This case points role out the im-

portance of flow cytometry screening becomes increasingly

important as a recent study showed that ALPS patients

showed statically changes in the immunophenotypic profile of

PB cells with a simple panel: CD3, CD4, CD8, CD45, TCRab,TCRgd, compared with other individuals.

4

EVALUATION OF AN AUTOMATED ASSAY SYSTEM FOR THEENUMERATION OF PERCENT AND ABSOLUTE CD4 T-CELLS INRESOURCES-LIMITED SETTINGS

Michele Bergeron, Tao Ding, and Nathalie Sempels

Public Health Agency of Canada

CD4 absolute count is critical in decision making for ini-tiating therapy and monitoring disease progression in HIVpatients. In infants, CD4 lymphocyte percent is the preferredmeasurement. Therefore, technologies measuring percent andabsolute CD42T cells are suitable for both adult and pediatricsettings. Guava Technologies recently introduced a new reagentcocktail integrated to an automated analysis software to simulta-neously measure percent and absolute CD42T cells. This studyevaluates the performance of the Guava Auto CD4/CD4% Sys-tem. Percent and absolute CD4 measurements from 25 adultpatients (1 HIV2 and 24 HIVþ) were determined using theGuava System and the reference method. The referencemethod was a four-color, single-platform technique performedon a FACSCalibur. Correlation and concordance between bothmethods were assessed using linear regression, Bland-Altmanand Pollock statistical analysis. Intra-assay precision was eval-uated by replicate analysis of 10 sample aliquots on a singleday. The correlation of CD4 measurements obtained by bothmethods was 0.9794 and 0.9833 for absolute and percent,respectively. Mean bias was þ0.74% [limits of agreement (LOA)22.26% to þ3.75%] for % CD4. For CD4 absolute count, themean bias was þ50 cells/mL [LOA 269 to þ170 cells/mL] orþ10.5% [LOA 210.82% to þ31.79%] as per Pollock. Measuredprecision as %CV was 3.5% and 4.2% for absolute and percent,respectively. This preliminary study showed that the new sys-tem is comparable to the reference method. The Guava micro-capillary platform presents several advantages for limited-resour-ces countries in respect to reagent cost, simplicity, and abilityto monitor adult and pediatric samples.

5

EVOLUTION OF LYMPHOCYTE SUBSETS AMONGHIV-UNINFECTED CHILDREN BORN OFHIV-POSITIVE MOTHERS IN CAMEROON

Sagnia Bertrand,1 Tetang Suzie,1,2 Torimiro Ndongo Judith,1

Meloch Bayambe Renata,1 Barbieri Ernesta,2 and Cappelli Giulia1

1Chantal BIYA International Reference Centre (CIRCB)

2Centre d’Animation Sociale et Sanitaire (CASS)

Background: The evolution of lymphocyte subsets dur-

ing infancy among HIV-uninfected children in Cameroon has

not been previously described. This study identifies these val-

ues as well as differences associated with age. Methods: Preg-nant HIV-1-infected women enrolled in the PMTC Plus Pro-

gram from May 2006 to April 2008 in Yaounde were recruited

in this study. The infants’ HIV status was assessed by serology

after age 18 months. During regular visits at 2, 6, 9, 12, and

18 months of age, blood was collected from 444 infants for

lymphocyte subset analysis using flow cytometry FACSCalibur

with Multiset software. Results: In all five age groups (0–2, 3–

6, 7–12, 13–18, and �19 months), the absolute values werehigher than adult values, while the percentages were signifi-cantly lower. There was a steady increase in the CD4 and CD8T lymphocytes levels expressed in absolute counts from birthwith a peak after 24 months, whereas CD4 percentage andCD4:CD8 ratio declined steadily from birth to 18 months (P <0.05). The most marked changes were the decrease in percent-age CD4 and increase in percentage CD8. Conclusions: Thisstudy shows that the evolution of lymphocyte subset valuesamong HIV-negative children born of HIV-positive mothers inCameroon differs from that of seropositive children. These dif-ferences observed among HIV-1-exposed but uninfected versusinfected infants could reflect a successful immune response toHIV-1 challenge. These results could be used as reference val-ues for pediatric populations with vertical exposure to HIV-1in Cameroon or the central African region.

6

DEAD CELL STAINS IN FLOW CYTOMETRY: A COMPREHENSIVEANALYSIS

Jolene A. Bradford and Gayle M. Buller

Molecular Probes-Invitrogen

Loss of membrane integrity is an indicator of cell death inflow cytometric analysis. Cells that exclude a dead cell dye areconsidered viable, while cells with compromised membranesallow dye inside the cell to stain an internal component, thusidentifying the cells as dead. Antibody conjugates can nonspe-cifically bind dead cells and can potentially lead to erroneousresults, especially in rare event analysis. Many dyes for dead cellidentification are available for this across a wide range of excita-tion and emission wavelengths. A comprehensive study of 25stains that have been used for identification of dead cells basedon membrane integrity is presented: classic membrane-imper-meant nucleic acid dyes, monomeric cyanine dyes, AnnexinVdyes, and amine-reactive dyes. All dyes are tested with a mixtureof live and heat-killed cells before and after formaldehyde fixa-tion, with aged culture cells, and with apoptotic cells. Immuno-phenotyping application data showing results with and withoutdead cell gating in multicolor testing is presented. All dyes stud-ied identify dead cells in live þ heat killed samples. The amine-reactive dyes maintain dead cell distinction after formaldehydefixation; this discrimination was lost with all other dyes. Themonomeric cyanine and AnnexinV dyes identified early and lateapoptotic cells, while the DNA intercalating and amine-reactivedyes generally identified late apoptotic/necrotic cells. Excludingdead cells in immunophenotyping and functional assays is animportant aspect in experimental design to ensure the accuracyof results.

7

IDENTIFICATION OF IMMUNOPHENOTYPIC SIGNATURES BYCLUSTERING ANALYSIS IN PEDIATRIC PATIENTS WITHPHILADELPHIA CHROMOSOME-POSITIVE ACUTELYMPHOBLASTIC LEUKEMIA

365CCS ABSTRACTS

Cytometry Part B: Clinical Cytometry

Barbara Buldini, Andrea Zangrando, Barbara Michielotto,Alessandra Benetello, and Giuseppe Basso

Department of Pediatrics, University of Padua

Detection of Philadelphia chromosome t(9;22) (Phþ)

in children with precursor-B-ALL (pB-ALL) is an adverse

prognostic factor thus leading to a high-risk protocol for

treatment. RT-PCR is the gold-standard for the detection of

this abnormality, but no rapid screening method is available

for the identification of Phþ pB-ALL cases. Genetic subclasses

have recently been identified in childhood and adult ALL by

specific gene and protein expression signatures by microarray

and flow cytometric analyses, respectively. Our aim is the

immunophenotypic characterization of Phþ pB-ALL for a fast,

accurate, and cheap screening approach. Forty-one children

with Phþ and 99 Ph-pB-ALL were analyzed performing quanti-

tative multivariate analysis on immunophenotypic data. The

expression level of 16 marker proteins was monitored by five

color flow cytometry and quantified in terms of geometric

mean fluorescence (GMF). Unsupervised and supervised anal-

yses were applied to the patient cohort to identify and charac-

terize Phþ samples according to the similarity of the antigen

expression profiles. The patient cohort was then separated

into two main clusters (A and BþC) by cluster analysis on

GMF expression values. Cluster A includes the majority of

Phþ patients (35/41) and is associated with upregulation of

CD52, TdT, CD45, CD34, HLA-DR, CD33, and downregulation

of CD38, CD24, CD58, CD22, CD19. Cluster BþC gathers

most of the Ph-patients (86/99) showing the opposite tend-

ency for listed markers. The immunophenotypic method iden-

tifies Phþ cases with a comprehensive accuracy of 86% pro-

viding a valuable, rapid, and effectual screening method for

the identification of Phþ pB-ALL.

8

EVALUATION OF AN 8-COLOR FLOW CYTOMETRIC REFERENCEMETHOD FOR WHITE BLOOD CELL DIFFERENTIALENUMERATION.

Sindhu Cherian,1 Greg Levin,1 W.Y. Lo,1 Megan Mauck,1 Crystol Lee,2

Donald D. Kuhn,1 and Brent L. Wood1

1Department of Laboratory Medicine, University of

Washington2Seattle Cancer Care Alliance

Manual microscopy is the current reference method forwhite blood cell (WBC) differential counts. However, manualcounts are time and labor intensive, sometimes difficult inpatients with low WBC counts, and can misclassify cells hav-ing difficult morphology. We investigated an 8-color, single-tube, lyse no-wash flow cytometric method to perform anextended 8-part differential as a potential replacement refer-ence method for WBC differential enumeration. Whole bloodwas stained using a panel of antibodies including CD45APC-Cy7, CD16þCD19FITC, CD33þCD64 PE-Cy5, CD123PE, HLA-DR PE-Cy7, Hoechst 34580, CD34þCD117APC, andCD38A594 to generate an 8-part differential including lym-phocytes, granulocytes, immature granulocytes, monocytes,eosinophils, basophils, blasts, and nucleated RBCs. TruCountbeads were used to generate an absolute count for all WBCpopulations. Data was collected on an LSRII (minimum of100,000 events collected per sample). Manual differentialswere performed on Wright-stained smears per CLSI H20-A2.Ten random samples meeting criteria for acceptance of aninstrument differential and 88 random samples from an oncol-ogy outpatient clinic were evaluated. The correlationbetween WBC and differential using the hematology analyzer,flow cytometry, and manual differential are shown in Table 1.

Autodifferentia1 group (N 5 10)(WBC range 7.7 - 14.8 Kcells/ul)

Oncology clinic: group (N 5 88)(WBC range 0.03 - 104.9 Kcells/ul)

Flow vs. Sysmex XE2100 Flow vs. manual Flow vs. Sysmex XE2100 Flow vs. manual

WBC y 5 1.006x 2 350.34 y 5 1.0243x þ 125.15R2 5 0.9813 R2 5 0.9979

Neutrophils y 5 1.023Hx 2 250.11 y 5 1.0003x 2 334.16 y 5 1.0389x 2 9.8796 y 5 1.0364x 2 101.87

R2 5 0.9949 R2 5 0.9877 R2 5 0.9956 R2 5 0.9952

Lymphocytes y 5 0.9356x 2 7.1264 y 5 0.8941x þ 2.451 y 5 1.0476x 2 77.606 y 5 1.0333x 2 106.3

R2 5 09925 R2 5 0.9631 R2 5 0.9645 R2 5 0.9978

Monocytes y 5 O.8186x þ 59.142 y 5 1.0849x þ 124.03 y 5 0.3626x þ 367.03 y 5 0.941x þ 180.86

R2 5 0.9851 R2 5 0.77 R2 5 0.2997 R2 5 0.7513

Eosinophils y 5 0.8831x þ 2.6814 y 5 1.0556x þ 11.491 y 5 1.0294X þ 4.3795 y 5 0.9845x þ 11.614

R2 5 0.9815 R2 5 0.9509 R2 5 0.9978 R2 5 0.9667

Basophils y 5 1.4333x 2 2.4067 y 5 0.0693x þ 25.594 y 5 0.22x þ 30.012 y 5 0.6591x þ 36.397

R2 5 0.7512 R2 5 0.0031 R2 5 0.2385 R2 5 0.1585

Immature Grans N/A y 5 0.2047x þ 31.741 N/A y 5 1.9093x þ 37.702

R2 5 0.0165 R2 5 0.8944

Blasts N/A N/A N/A y 5 0.9239x 2 2.8966

R2 5 0.8473

NRBCs N/A N/A y 5 0.9745x 2 0.9656 y 5 1.4683x 2 1.8969

R2 5 1 R2 5 0.8454

366 CCS ABSTRACTS


Discordances were largely due to misclassification of blasts,monocytes, and/or basophils by Sysmex and/or morphology.This study describes a novel single-tube flow cytometricmethod for performing a WBC count and differential that per-forms well with both relatively normal and difficult patientsamples. These findings confirm the results of prior studiessupporting the use of a flow cytometric differential as animproved reference method for the WBC differential.

9

PSEUDO-DUAL SURFACE IMMUNOGLOBULIN LIGHT CHAINEXPRESSION IN CHRONIC LYMPHOCYTIC LEUKEMIA

Chrissie Dyson1 and Jacek Polski2

1Mobile Infirmary Medical Center

2University of South Alabama

Background: Surface light chain expression analysisby flow cytometry is commonly performed in clinical flowcytometry. Although rare exceptions exist, light chainrestriction usually correlates with B-cell clonality. Interpreta-tion of light chain expression is not always easy and falsenegative and false positive results are possible. Surface lightchains can be undetectable or dual surface light chainexpression can be rarely detected. Recently, we encoun-tered a case of chronic lymphocytic leukemia with a pattermimicking dual surface light chain expression with one setof antibodies but revealing a light chain restriction withanother reagent. Methods: The patient was an asymptom-atic 69-year-old male with severe leukocytosis (216,800/mL)and lymphocytosis (96%). Flow cytometry of peripheralblood was performed on the Canto II instrument. Results:The leukemic cells were positive for CD5, CD19, CD20,CD22, CD23, CD25, and CD45. Kappa and Lambda resultswith polyclonal antibodies (BD Pharmingen) showed a dualexpression pattern. However, results with monoclonal anti-bodies (BD Oncomark) showed a kappa light chain restric-tion. That was also confirmed with alternative polyclonalantibodies (DakoCytomation). Discussion: These resultssuggest that the initial dual surface light chainexpressionpattern was a laboratory artifact. This case also documentsthat repeat testing with different reagents can help resolvedifficult surface light chain expression patterns.

10

IN VITRO DRUG SENSITIVITY AS A PREDICTIVE TOOL OF EARLYCLINICAL RESPONSE IN CHILDHOOD ACUTE LYMPHOBLASTICLEUKEMIA

Faith Galderisi,1 Linda Stork,1 Ju Li,2 Motomi Mori,3

Solange Mongoue-Tchokote,3 and James Huang2

1Department of Pediatrics, Oregon Health and Science

University2Department of Pathology, Oregon Health and Science

University3Department of Public Health and Preventive Medicine,

Oregon Health and Science University

Residual disease or rapidity of response to induction

therapy is among the most powerful predictors of outcome

in pediatric acute lymphoblastic leukemia (ALL). We hypothe-

size that in vitro drug sensitivity at the cellular level predicts ra-

pidity of response to induction therapy in ALL.We applied amul-

tiparameter flow cytometric drug cytotoxicity assay on bone

marrow (BM) samples of 23 patients with newly diagnosed ALL.

Fourteen patients were rapid early responders (RER) and nine

were slow early responders (SER) by COG criteria at Days 15 and

29. Leukemic cell survival index (LCSI5 Average Replicate/Aver-

age Control3 100) was determined at 48 h after in vitro culture

of leukemic cells with individual standard induction agents for

pediatric ALL: vincristine, asparaginase, dexamethasone, predni-

sone, and daunomycin. For dexamethasone, a significantly lower

LCSI was seen in the RER compared with the SER cohort: RER

mean LCSI5 40.2%, SERmean LCSI5 70.1% (p5 0.01). A trend

toward a lower mean LCSI in the RER compared with the SER

group was noted for asparaginase and vincristine at individual

and averaged concentrations (p<0.1). Mean LCSI was not differ-

ent between the RER and SER groups for daunomycin and pred-

nisone. The findings indicate that flow cytometry cytotoxicity

assay is applicable to in vitro drug sensitivity evaluation of rou-

tine clinical samples of acute leukemia. In vitro drug sensitivity

response profile for dexamethasone appears to be the most

predicative of the early response to standard induction therapy

in pediatric ALL.

11

A WHOLE BLOOD ASSAY FOR EVALUATION OF NATURALKILLER CELL FUNCTIONAL ACTIVITY

Kevin Gorski,1 John Ferbas,1 and Wayne Tsuji2

1Amgen, Clinical Immunology

2Amgen, Early Development

A traditional approach for measuring NK-cell activity

has included NK-sensitive cells as targets and gradient-

enriched PBMC as effectors in a 51chromium release assay.

This method is labor-intensive and not well suited for clini-

cal trials. The ideal assay would eliminate the need for

PBMC purification from blood, which by definition

removes potentially important serum factors and the

administered therapeutic from the assay matrix. We

decided to explore the utility of flow cytometry in this set-

ting and considered measurements of viability or depletion

of fluorescently labeled target cells. In practice, however,

this is a difficult endpoint because the (target) cells change

morphology and/or scatter properties as they progress to

lysis, which is difficult to capture in an analysis gate. Our

solution to this problem leveraged recent reports of

CD107a expression as a cell surface neoantigen that

appears during NK-cell degranulation. We studied this end-

point by adding fresh and cryopreserved K562 cells to

whole blood and included CD69 expression and intracellu-

lar perforin measurements in our initial analyses. Addition

of fresh or cryopreserved K562 cells to whole blood

increased the proportion of CD32CD56þ NK-cells display-

ing cell surface CD107a and/or CD69. As expected,

CD107aþ NK-cells had reduced intracellular perforin lev-

els—consistent with degranulation. There was no evidence

for CD107a or CD69 expression on non-NK-cells (e.g.,

367CCS ABSTRACTS


CD3 T cells), speaking to assay specificity. This assay has

been validated in-house and successfully transferred to an

external laboratory for use in clinical trials where evalua-

tion of NK function is important.

12

QUALIFICATION OF A WHOLE BLOOD IMMUNOPHENOTYPINGASSAY TO MONITOR DENDRITIC CELL AND MONOCYTESUBSETS IN HEALTHY VOLUNTEERS AND PATIENTSWITH MULTIPLE SCLEROSIS

C.L. Green,1,2 S.S. Belouski,1,2 M. Vincent,1,2 B. Rees,1,2 J. Ferbas,1,2

and K. Gorski1,2



Background: Flow cytometry is an ideal platform to

investigate immune status and proof of biological activity in de-

velopment of therapeutics designed to reduce inflammation or

treat autoimmune diseases. We developed a mid-complexity

flow-cytometric assay to enumerate and measure CD86 and

GM-CSF-Receptor a expression on plasmacytoid (pDC) and my-

eloid (mDC) dendritic cells, monocyte subsets (CD162/þ), andeosinophils in HV and MS. Methods: Technical and biological

issues define the complexity of this assay. As such, development

and qualification were designed to understand and minimize

within sample and longitudinal variability. Whole blood from 10

HV and 5 MS were collected in Cyto-Chex1 BCT tubes and la-

beled with antibodies with specificity to LIN-FITC, CD64-FITC,

CD16-PE, CD86-PE, CD116-PE, HLA-DR-PerCP, CD11c-APC,

CD14-APC, and CD45-AmCyan. Precision was the primary pa-

rameter of assay qualification. Biological variability was charac-

terized by three weekly repeat collections and specimen stabil-

ity was assessed through 72 h postvenipuncture. Results: Pop-ulation % and antigen density measurements met precision and

biological variability acceptance criteria within replicates and

repeat collections for populations�200 events. Blood stabilized

using Cyto-Chex1 BCT tubes proved superior to sodium hepa-

rin, resulting in decreased variability in pDC, mDC, and mono-

cyte populations (�200 events) for up to 72 h postvenipunc-

ture. Conclusions: Precision, biological variability, and stability

analyses demonstrated the reliability of this assay to measure

DC and monocyte subsets in HV and MS. We plan to utilize this

approach in clinical trials where evaluation of DC and mono-

cyte subsets may provide useful insight to assess the impact of

candidate therapeutics.

13

QUALIFICATION OF A WHOLE BLOOD IMMUNOPHENOTYPINGASSAY TO MONITOR MHC CLASS I AND II EXPRESSION INHEALTHY VOLUNTEER AND SYSTEMIC LUPUSERYTHEMATOSUS PATIENTS

C.L. Green,1,2 J. Ferbas,1,2 and S.S. Belouski1,2



Background: Inflammatory diseases evoke cytokine

expression patterns that impact the relative number of anti-

gen presenting cells (APC) and their MHC expression levels.

Flow cytometry methods can measure analytes as bio-

markers to assess therapeutics designed to reduce inflamma-

tion or treat autoimmune diseases. We developed a flow

cytometric method to measure MHC class I and II protein

expression in HV and SLE. We have further demonstrated a

stabilization method to facilitate shipment to a central lab.

Methods: Whole blood from 18 HV and 10 SLE volunteers

were collected in sodium heparin and Cyto-Chex1 BCT

tubes and labeled with antibodies with specificity to HLA-

DR, HLA-DP, HLA-DQ, and HLA-ABC. Precision served as the

primary parameter of assay qualification. Biological variabili-

ty was characterized by three weekly repeat collections and

specimen stability was assessed through 96 hours postveni-

puncture. Results: Population % and antigen density meas-

urements demonstrated acceptable precision within repli-

cates for cell populations �5%. Biological variability of anti-

gen density measurements showed �25% CV across

3 weekly collections. Blood stabilized using Cyto-Chex1

BCT tubes resulted in decreased variability in population %

and antigen density for up to 72 h postvenipuncture. Con-clusion: Precision analyses qualify the reliability of this

assay. However, appreciable changes in antigen density and

population composition within 24 h postvenipuncture were

detected. The current Cyto-Chex1 BCT formulation success-

fully stabilized HLA class I and II antigens for up to 72 h.

Through stabilization of immediate ex vivo analytes, this

assay may more accurately measure therapeutic impact

while simultaneously simplifying implementation for multi-

site clinical studies.

14

BCR-ABL DETECTION IN CML AND ALL SAMPLES USING ANEWLY DEVELOPED FUSION PROTEIN IMMUNOASSAY

Deborah Greenberg,1 Yen-Ping Liu,2 Xiao-Yuan Liu,1 Johanna Reneke,1

Hobert Wai,2 and Charlene Bush2

1Kaiser Permanente TPMG Regional Laboratory

2BD Biosciences

Methods to subclassify CML and ALL as BCR-ABL posi-

tive include cytogenetics and molecular technologies. The

BDTM BCR-ABL Protein Kit* (BD Biosciences, San Jose, CA,

USA) is a newly developed qualitative immunoassay that

detects BCR-ABL fusion protein in blood, bone marrow, and

CSF. This study compared the performance of this immuno-

assay to cytogenetics and molecular methods in 17 blood,

five bone marrow, and one CSF specimen from 14 patients

with either CML or ALL. Specimens were tested at Kaiser

TPMG Regional Laboratory by cytogenetics, FISH, and RT-

PCR, and at BD Biosciences blindly, using the BCR-ABL Pro-

tein Kit and performing with a BD FACSCantoTM II flow cy-

tometer** (BD Biosciences). This assay provides a BCR-ABL

result in <5 h and has a limit of detection of 10 pg/mL.

Results revealed 100% concordance (11/11 positive and 12/

12 negative specimens) between cytogenetic and conven-

tional molecular methods and the immunoassay. In positive

specimens, the concentration of fusion protein ranged from

12 pg/mL to 7.6 ng/mL. We conclude that the immunoassay

368 CCS ABSTRACTS


gives concordant results as conventional methods and has

the advantage of being a rapid and standardized assay avail-

able in a kit. In addition, because this immunoassay directly

measures the BCR-ABL oncoprotein, further evaluations are

warranted to study CML and ALL biology, and disease pro-

gression, using this assay.

*For research use only. Not for diagnostic or therapeu-

tic procedures.

**ClassI (1) Laser Product.

15

CD38 EXPRESSION IN CLL CORRELATES WITH THEPROLIFERATION INDEX AND DOES NOT CORRELATE WITHCD25 AND BCL-2 EXPRESSION

Eugeny Gretsov,1 Natasha Barteneva,2 and Ivan Vorobjev1

1Hematological Research Center, Harvard Medical School

2Immune Disease Institute, Harvard Medical School

Background: The chronic lymphocytic leukemia (CLL)

is a highly heterogeneous disease. It was previously

reported that high CD38 expression is associated with CLL

cases with a worse clinical outcome. The purpose of our

study was to search for other markers which could be help-

ful in explanation why CD38high is associated with more

aggressive course of CLL. Methods: 196 patients with CLL

were analyzed for the expression of CD38, CD25, bcl-2, and

Ki-67 using multicolor flow cytometry. Results: The

patients were divided into subgroups based on the percent-

age of CD5þCD19þ cells expressing CD38 by selective gat-

ing. 47% of the patients contained less than 30% CD38-

expressing leukemic cells (CD38low), and 21% of the

patients contained 85% or more CD38-positive leukemic

cells (CD38high). The rest of cohort was considered as

CD38intermediate group. Blood and bone marrow samples

from CD38high CLL patients contained 0.89% Ki-67 express-

ing cells (0.06%-min, 5.63%-max) which was significantly

higher than percentage of B-cells from CD38low (average

0.38%; min-0.05%, max-2.07%). Biopsy specimens from

CD38high CLL patients contained 3.15% Ki-67 expressing

cells. No correlation was observed between the level of

CD38 expression and bcl-2 and CD25 expression. Conclu-

sion: We suggest analysis of Ki-67 expression in CLL may

help to explain why CD38high CLL cases are more aggres-

sive disease and have poor clinical prognosis.

16

IMMUNOPHENOTYPIC PROFILE OF ADULT PATIENTS WITHPRECURSOR B LYMPHOBLASTIC LEUKEMIA ASSOCIATEDWITH THE T(4;11)(Q21;Q23)

Inga Gurevich,1 Suyang Hao,2 and Pei Lin1

1M. D. Anderson Cancer Center

2University of Massachusetts Medical School

Background: Pre B ALL with the t(4;11)(q21;q23) is

the most common type of ALL in infants. The blasts are typ-

ically CD19þCD102CD15þ. Rare cases have been reported

to evolve to acute monoblastic leukemia after chemother-

apy. Methods: We studied 22 adult ALL with the t(4;11) by

3- or 4- color flow cytometry using a panel of monoclonal

antibodies. Expression of each antigen was recorded as pos-

itive (>50%), partial/dim (15–50%), or negative (<15%).

Results: All cases assessed were positive for CD19, CD15,

HLA-DR, and cCD79a. Other markers expressed and the fre-

quency of their expression were as follows: TdT (90%),

CD22 (65%), CD34 (45%), cIg (42%), CD13 (32%), CD33

(27%), CD10 (18%), and CD20 (5%). Of the positive cases,

partial expression was observed in a subset of cases: CD33

(67%), CD13 (57%), cIg (50%), CD34 (40%), and TdT (37%).

Follow-up studies were performed in six cases. The expres-

sion profiles remained relatively stable; only occasional anti-

gens displayed stronger or weaker expression compared to

the original analysis. No case showed a complete switch to

a monoblastic immunophenotype. Discussion: Similar to

pediatric ALL with t(4;11), blasts in adult cases are typically

CD19þCD15þCD102. However, a spectrum of immuno-

phenotype can be observed with regard to expression of

CD10, CD20, CD13, CD33, and CD34. Although most cases

express TdT, a small number of cases are CD19þCD10

2CD342TdT2. In these cases, CD22 and cCD79a are more

often positive than cIg and may aid lineage determination.

The overall immunophenotype appears to be stable over

time and supports pre-B than biphenotypic leukemia.

17

SIGNIFICANCE OF CD38 ANTIBODY CLONE IN ANALYSIS OFCHRONIC LYMPHOCYTIC LEUKEMIA

Suyang Hao1 and Pei Lin2

1UMass Memorial Medical Center

2UT M. D. Anderson Cancer Center

Background: ZAP-70 and CD38 expression are

reported to correlate with CLL with unmutated IgH and

unfavorable prognosis. Their expression can be assessed by

flow cytometry (FCM). However, concordance between

ZAP-70 and CD38 expression varies among different studies

and may depend on the type of monoclonal antibodies or

the cutoff levels employed in the analysis. Materials andMethods: 28 peripheral blood and two bone marrow speci-

mens from 30 consecutive CLL cases were analyzed by five-

color FCM using Coulter FC-500 instruments [Beckman

Coulter (BC), Hialeah, FL]. Cytoplasmic ZAP-70 and surface

CD38 expression were assessed using anti-ZAP-70-FITC

(clone 1E7.2 CALTAG Laboratories) and anti-CD38-PC5

(clone LS198, Immunotech) along with a panel of monoclo-

nal antibodies against CD3, CD4, CD5, CD7, CD8, CD10,

CD14, CD19, CD20, CD34, CD45 HLA-DR, Kappa/Lambda.

The cutoff levels for ZAP-70 and CD38 were 20 and 30%,

respectively. Results: 13 (43%) cases expressed ZAP-70

while 4 (13%) cases expressed CD38. Concordant ZAP-70

and CD38 expression was observed in 17 (57%) cases

including 2 (7%) double positive (ZAP-70þ/CD38þ) and 15

(50%) double negative (ZAP-702/CD382). Discordant

expression was observed in 13 (43%) cases: 11 (37%) were

ZAP-70þ/CD382 and 2 (7%) were ZAP-702/CD38þ. Con-clusions: The frequency of ZAP-70 expression in this study

is similar to what is reported in the literature (43 vs. 46%),

369CCS ABSTRACTS


however, the frequency of CD38 expression is much lower

(13 vs. 29%), suggesting different CD38 antibody clones

may affect the sensitivity of detection and the concordance

rate between ZAP-70 and CD38 expression.

LITERATURE CITED1. Schroers R, Griesinger F, Tramper L, Haase D, Kulle

B, Klein-Hitpass L, Sellman L, Dahrsen U, Darig J. Combined

analysis of ZAP-70 and CD38 expression as a predictor of

disease progression in B-cell chronic lymphocytic leukemia.

Leukemia 2005;19:750–758.

18

OPPOSING PATTERNS OF CD123/CD34 EXPRESSION INHEMATOGONES AND PRECURSOR B-ALL BLASTS.

Nagwa M. Hassanein, Felisa Alcancia, Patrick J. Buckley,and Anand S. Lagoo

Department of Pathology, Duke University Medical Center

We compared the expression of interleukin-3 receptor

alpha (CD123) on normal B-cell precursors in bone marrow

(‘‘hematogones’’) and on leukemic blasts in newly diagnosed

precursor B acute lymphoblastic leukemia. Hematogones in

50 bone marrows obtained from patients without a diagno-

sis of precursor B-ALL were first identified by their charac-

teristic side scatter, CD45, CD19, and CD10 expression pat-

terns. Expression of CD34 and HLA-DR was also analyzed

and the hematogones were separated into 2 groups—less

mature (dimmer CD45, CD34þ, DRþ) and more mature

(moderate CD45, CD342, DRþ). In every case studied, we

observed that CD123 was expressed on a majority of more

mature hematogones which lacked CD34. In contrast, less

mature, CD34þ hematogones did not express CD123 on a

majority of cells in any case. The expression of CD34 and

CD123 on hematogones was thus discordant. In contrast,

concordant expression of CD34 and CD123 was seen in

93% of 35 cases of precursor B-ALL studied. We found that

27/35 (79%) of cases expressed both antigens while 5/35

(14%) expressed neither antigen. Only 3/35 (7%) cases of

precursor B-ALL expressed CD34 and CD123 differentially.

These opposing patterns of CD34/CD123 expression on

hematogones and precursor B-ALL blasts would be useful in

identifying blasts in recurrent precursor B-ALL where an

increased number of hematogones may also be present.

19

SIMULTANEOUS ANALYSIS OF ZAP70 AND CD38 EXPRESSIONIN CLL PATIENTS AND COMPARISON TO IMMUNOGLOBULINHEAVY CHAIN (IGH) GENE MUTATION STATUS

Nagwa M. Hassanein,1 Kathryn Perkinson,1 J. Brice Weinberg,2,3 andAnand S. Lagoo1

1Department of Pathology, Duke University Medical Center

2Department of Medicine, Duke University Medical Center

3Veterans Administration Hospital

Using a four-color, single tube assay, we analyzed ZAP70

and CD38 expression in 111 patients with CLL and examined

the IgH gene mutation status in a subset. Gating on CD19þ/

CD5þ, CD19þ/CD52, and CD192/CD5þ populations, we

identified leukemia cells, normal B-cells, and normal T-cells,

respectively. We assigned ZAP70 positivity by two criteria:

�1.9 MFI ratio comparing leukemia cells to normal B-cells

and �40% leukemia cells beyond the isotype control thresh-

old. When 0, 1, or 2 criteria were met, the case was inter-

preted as ZAP70 negative, indeterminate, or positive, respec-

tively. Of the 111 cases, 30% were ZAP70 positive, 14% were

indeterminate, and 56% were negative. For CD38 expression,

36/111 (32%) cases were positive using a 30% cutoff. Among

these, ZAP70 was positive in 15/36 (41%), negative in 16/36

(44%), and indeterminate in 5/36 (15%). Among the 75 cases

that were negative for CD38, 46/75 (61%) were also negative

for ZAP70, 18/75 (24%) were positive for ZAP70, and 11/75

(15%) were indeterminate. There was significant correlation

between cases testing either positive or negative for both tests

(P 5 0.05, Pearson chi square), but not if the indeterminate

ZAP70 cases were included in the positive category (P 50.09, Pearson). In 29 cases, IgH gene mutation status was ana-

lyzed: among the ZAP70 positive group 12/15 were unmu-

tated, in the indeterminate group 2/5 were unmutated, and in

the negative group 1/9 was unmutated (P 5 0.004, Pearson).

Thus, our method provides a simple and robust test for evalu-

ating two prognostic markers in CLL.

20

ABNORMAL IMMUNOPHENOTYPIC PROFILE OF REACTIVET CELLS IN CHRONIC LYMPHOCYTIC LEUKEMIA

S. David Hudnall, Jeremy Crim, Jyoti Patel, Karen Giddings,and Joe Martinez

UTMB Department of Pathology (Hematopathology)

Chronic lymphocytic leukemia (CLL) is the most common

adult leukemia in North America. There is growing recognition

that the host mounts an often ineffective immune response to

cancer. We have examined the flow immunophenotype of cir-

culating T and NK cells in seven untreated cases of CLL to gain

insight into the immune defects in CLL. We compared results

with those from 10 normal adult controls to identify statisti-

cally significant differences. No difference in CD4/CD8 ratio is

detected in CLL versus normal. In comparison with normal

controls, CLL is characterized by an increased proportion of

memory CD4þ T cells that exhibit immunophenotypic fea-

tures of poor functionality (CD28 and CD38 negative). No dif-

ference in numbers of specific CD4þ subsets (CCR5þ TH1,

CCR4þ TH2, or FoxP3þ regulatory T cells) is identified. In

contrast to CD4þ T cells, no difference in proportions of naive

and memory CD8þ T cells is noted in CLL versus normal. How-

ever, as with CD4þ T cells, CD28 expression is significantly

decreased in CD8þ T cells, suggesting poor functionality.

CD8þ T cells also exhibit reduced CD62L positivity, suggesting

poor lymph node homing ability. HLA-DR expression on CD4þand CD8þ T cells is increased in CLL as compared with normal

controls, but this difference did not reach statistical signifi-

cance. Lastly, CLL is characterized by decreased numbers of

natural killer (NK) cells, with no difference in number of NK-T

cells. These preliminary results indicate that the circulating

370 CCS ABSTRACTS


lymphocytes in CLL exhibit immunophenotypic features con-

sistent with immune suppression.

21

CELL LABELING CRITERIA FOR LARGE NUMBERS OF CELLS INCLINICAL CYTOMETRY

Ruud Hulspas and Lydia Villa-Komaroff

Cytonome, Inc

The increasing demand for purified cell subpopulations

in cellular therapy relies on a range of cell-selection strat-

egies. Many selection strategies target specific subpopulations

of cells by using one or more cell surface antigen-specific

antibodies. Regardless of the selection strategy, clinical appli-

cations typically require processing significantly more cells

than research applications. As a result, procedures used for

research are often scaled up for clinical samples. In particu-

lar, the amount of antibody necessary to saturate antigens on

a large number of cells is frequently extrapolated in a simple

linear fashion; a practice that is implicitly supported by most

labeling protocols provided by antibody manufacturers. How-

ever, the amount of antibody used to label a relatively small

number of cells is commonly sufficient to label many more

cells. Here, we report optimal conditions for labeling large

numbers of human hematopoietic cells with a minimum

amount of fluorochrome-conjugated antibody. The lowest

amount of antibody used to optimally label a suspension of

108 normal human peripheral blood mononuclear cells was

as low as 60 ng (or �2.4 3 1011 molecules) of anti-human

CD3. To properly label an equally large cell suspension with

anti-human CD4 or anti-human CD38, 250 ng of antibody

was required. In addition, these experiments revealed that

the level of unwanted antibody binding (in particular of low

affinity antibodies) is reduced significantly by allowing bind-

ing of antibodies while cells are resuspended at the highest

cell concentration possible (�109 cells/mL).

22

DOES ERYTHROBLASTIC RECOVERY REFLECT ABSENCE OFMINIMAL RESIDUAL DISEASE (MRD) IN ACUTELYMPHOBLASTIC LEUKEMIA (ALL)?

Maura R.V. Ikoma, Marina M.O. Souza, Marcimara Penitenti,Mair P. Souza, Vergilio A.R. Colturato, Claudia T. Oliveira,Andrea M. Pedro, Ederson R. Mattos, and Marcos A. Mauad

Hospital Amaral Carvalho

During the therapeutics of ALL, hematopoiesis recovery

seems to occur concomitantly with the leukemia cells

decrease. We noticed that most patients showed erythroblas-

tosis in bone marrow during ALL treatment. Our objective is

to correlate the quantity of erythroblasts with the amount of

MRD to know if the erythroblastic recovery reflects the elim-

ination of leukemia cells. We analyzed 328 samples of ALL-B

lineage and 54 samples of ALL-T lineage from 144 patients,

treated according BFM protocol. They were between

3 months old and 54 years old. The samples analyses were

done in different phases of therapeutics: induction, intensifi-

cation and maintenance. MRD was researched by flow

cytometry. Panels of 3 or 4 colors of monoclonal antibodies

were chosen according to the appropriate indication. Chi-

square test was used to evaluate the statistical significance of

the influence of MRD in erythropoiesis. Numeric data were

calculated using the SPSS 15.0 software. The results showed

that in all samples analyzed there was inverse relation of the

erythroblasts number and the presence of MRD only in B lin-

eage ALL during the maintenance phase of treatment (P <0,003). The groups were not separate by treatment phases

and age, but there was no statistical significance in median

of all patients’ age and erythroblast amount, with or without

presence of MRD. These data do not permit to conclude that

erythropoiesis recovery occurs only when the leukemia

clone is eliminated. Factors as intensity of chemotherapy,

inhibitors of erythropoiesis, age, and other than the leukemia

activity may influence the hematopoiesis.

23

A COMPARATIVE STUDY OF 25 DIFFERENT CD4 CONJUGATES

Chris L. Langsdorf, Yu-Zhong Zhang, and Jolene A. Bradford

Molecular Probes-Invitrogen

Regular advances in reagents and instrumentation for

flow cytometry allow the researcher to run increasingly com-

plex multicolor experiments. This increased complexity

requires a more strategic approach to experiment design,

especially when choosing which fluorochome to pair with

each antibody. With dozens of fluorochromes to choose from,

it is easy to find one that excites and emits in any segment of

the spectrum. The current study evaluates the performance of

25 dyes each conjugated to CD4, including organic dyes and

recently developed quantum dot nanocrystals. To illustrate

the relative brightness of each conjugate, we calculated the

staining index. The data indicates that some dyes have a low

staining index and should not be used with low abundance

markers. However, we demonstrate that even a dim dye can

be an effective part of a panel, as long as it is carefully matched

to an appropriate antibody. Most of the organic dyes have

been well characterized in terms of fixation and permeabiliza-

tion, but little is known about the quantum dot nanocrystals

with these treatments. We have included a study of quantum

dot nanocrystal surface staining followed by common fixation

and permeabilization methods. Understanding the relative

brightness of commonly used dyes and a knowledge of antigen

expression are crucial elements to ensure accuracy of results.

24

OPERATING A CLINICAL FLOW CYTOMETRY LABORATORYWITHIN A QUALITY MANAGEMENT SYSTEM

Sarah A. Lee

Vancouver Island Health Authority

A Quality Management System is a tried and tested

framework for taking a systematic approach to manage an

organization’s processes so that they consistently turn out

product that satisfies customer’s expectations. Business

371CCS ABSTRACTS


activities are described in a set of policies, processes, and

procedures required for planning and execution of the core

business. Quality management systems enable the medical

laboratory to identify, measure, control, and improve the

processes that will ultimately lead to improved perform-

ance. This equates to patient safety. Right results at the

right time! The International Organization for Standardiza-

tion (ISO) document ‘‘ISO 15189 (2007) Medical laborato-

ries-Particular requirements for quality and competence’’

and several Clinical and Laboratory Standards Institute

(CLSI) documents provide guidelines for the laboratory to

create a quality management system. The CLSI documents

integrate the Path of Workflow and the12 Quality Systems

Essentials (QSE) to assist the laboratory in defining and

improving its services. The quality systems essentials are

addressed in a quality manual created by the laboratory and

used by accreditation bodies to confirm competence of a

medical laboratory. In the flow cytometry lab, consideration

and implementation of the 12 QSE ensures the laboratory

addresses and documents every aspect of a successfully run

business. Particular attention is given to process maps and

supporting documents defining workflow, training and com-

petency material, and process control.

25

CAN CYTOMETRY DIFFERENTIAL REPLACE MORPHOLOGYDIFFERENTIAL IN BLAST ENUMERATION IN THE DIAGNOSISAND CLASSIFICATION OF ACUTE LEUKEMIA?

Karen M. Li, Nicholas Mei, Guang Fan, and James Huang

Department of Pathology, Oregon Health and Science

University

The differential cell count is significant for leukemia

classification in the current WHO classification scheme. The

morphology differential is labor intensive and difficult due

to atypical cytological features or suboptimal preservation,

whereas the flow cytometry differential is more reproduci-

ble and efficient. Because they have not been carefully com-

pared, it is unclear whether flow cytometric differentials

can replace morphological differentials for diagnosis and

classification of acute leukemia. We retrospectively studied

49 randomly selected routine clinical samples, including

nine peripheral blood samples and 40 bone marrow sam-

ples from patients who had acute leukemia with leukemic

blasts ranging from 0.3% to 91%. The morphology and flow

cytometric differentials were compared with Student’s t-

tests. A remarkable correlation (r > 0.8) was found in the

percentages of major cell populations between the two dif-

ferentials. With peripheral blood samples, no significant sta-

tistical differences were detected. With bone marrow aspi-

rates, the mature lymphocyte percentage was significantly

higher (p 5 0.006) and the nucleated red cell percentage

was significantly lower (p 5 0.00005) in the flow cytomet-

ric differentials. The average differences were about twofold

for lymphocyte percentages and fivefold for nucleated red

cell percentages. The percentages of blasts, maturing mye-

loid cells, and monocytes were not statistically different (P

> 0.5). However, the differences in blast percentages were

more than 20% in six samples. Flow cytometric differentials

may replace manual morphology differentials in leukemic

blast enumeration with peripheral blood samples. However,

further optimization and standardization are needed to es-

tablish appropriate diagnostic criteria for bone marrow aspi-

rates based on flow cytometry differentials.

26

FLOW CYTOMETRIC EVALUATION OF CD38 EXPRESSION ISABLE TO DISTINGUISH FOLLICULAR HYPERPLASIA FROMFOLLICULAR LYMPHOMA

Kristin M. Mantei1,2 and Brent L. Wood3

1University of Washington

2Cellnetix Pathology and Laboratory

3University of Washington

The distinction of follicular lymphoma (FL) from reac-

tive follicular hyperplasia (FH) is a diagnostic challenge in

flow cytometry. Immunophenotypic features commonly

used to differentiate FL from FH such as surface light chain

restriction and relative changes in intensity in antigens such

as CD19 and CD10 cannot always be relied upon to make

an accurate distinction. In this study, the median fluorescent

intensity (MFI) of CD38 as assessed by flow cytometry on B

and T cell subpopulations in 102 lymph node specimens

with histopathologically confirmed FL was compared with

55 cases of FH. The MFI of CD38 was highly significantly

reduced in the neoplastic B cells in FL as compared with

the reactive germinal center B cells in FH (P < 1.0E-16).

The MFI of CD38 did not differ significantly between the

non-neoplastic B-cells in FL and nongerminal center B-cells

in FH (P 5 0.14) or between T-cells and non-neoplastic B-

cells in FL (P 5 0.63). A marginally significant difference in

the MFI of CD38 was seen for T cells between FL and FH

(P 5 0.04). A significant difference in the MFI of CD38 was

identified between T-cells and nongerminal center B-cells in

FH (P 5 0.005). No significant difference in CD38 expres-

sion was seen between Grades I, II, or III FL. This study

confirms decreased CD38 as a common finding in FL in

comparison with FH and provides an additional tool to help

differentiate FL from FH by flow cytometry.

27

IMPLEMENTING A FLOW CYTOMETRY WIKI—A WEB-BASEDCOLLABORATIVE SYSTEM TO SHARE INFORMATION, CASEEXAMPLES AND ENHANCE TRAINING AND EDUCATION

Teri Oldaker,1 Tanya Tolmachoff,2 Daniel Zimmerman,2

and David Novo2

1Consultant

2De Novo Software

Training and education for clinical flow cytometry pri-

marily involves books and live courses. These methods suf-

fer many well-known drawbacks, including lack of space,

time, and expertise as well as high cost. To overcome these

limitations, we have created the Clinical Flow Wiki (http://

wiki.clinicalflow.com), based on collaborative ‘‘wiki’’ tech-

372 CCS ABSTRACTS


nology popularized by Wikipedia. Anyone can contribute to

the site by adding or editing articles. We provided prelimi-

nary content: descriptions of 28 hematolymphoid neopla-

sias as well as descriptions of normal lymph node and pe-

ripheral blood analyses. All descriptions included back-

ground information about each classification, expected flow

cytometric marking, morphology, additional testing, and

references. In addition, there are 33 annotated sample cases

describing suggested analysis strategies for a wide variety of

neoplasias. Sample cases were included in the FCS

ExpressTM published format, which allows visitors to access

the analyzed data and adjust the gates using the free FCS

Express Reader. Addition and modification of pages are per-

formed directly on the page using a simple WYSIWYG

word processor. A sandbox and on-line instructions were

provided for ease of use. Three months after release, the

wiki hosts an average of 30 visits per day and 4.39 pages

per visit. The majority of visitors are from the United States,

with other visitors coming from 63 different countries. We

believe the Clinical Flow Wiki is a significant asset to the

industry, both as a resource and an avenue to share informa-

tion and interesting cases.

28

A SIMPLIFIED METHOD TO OPTIMIZE REAGENTS FOR AN11-COLOR (13-MARKER) POLYCHROMATIC INTRACELLULARSTAINING (ICS) PANEL

Janet S. Ottinger, Nadia Mensali, Enzor Jennifer, Andrew K. Weinhold,and Kent J. Weinhold

Center for AIDS Research, Duke University Medical Center

Background: Development of polychromatic flow

cytometry (PFC) panels is limited by optical configuration,

expertise, and reagent availability. Previously published

methods for developing PFC panels empirically test multiple

reagent combinations. Testing all possible reagent combina-

tions is laborious, costly, and time-consuming. Recently, we

developed and optimized an 11-color (13-marker) PFC ICS

panel using a simplified method to evaluate spillover with

single-stained data files obtained during reagent titrations.

Method: Twenty-three antibody conjugates for a total of 12

ICS panels were tested: CD3 AmCyan, CD4 (PE-Cy5.5,

PerCP-Cy5.5), CD8 (Pacific Orange, Alexa700, APC-Cy7,

FITC), CD19 and CD14 Pacific Blue, LIVE/DEAD fixable vio-

let dead cell stain, CD45RO ECD, CD27 (FITC, APC-

Alexa750, APC), CD57 (Alexa647, FITC), IFN-g (PE-Cy7,

Alexa700), TNF-a (APC, Alexa700, PE-Cy7), IL2 PE, and

CD107 PE-Cy5. Each conjugated mAb was titered and the

optimal protein concentrations were selected using fre-

quency, separating titer, and minimal negative spread. After

determining optimal reagent concentrations and optimal

compensation values, spillover/spreading error was eval-

uated using the single-stained titration data files. To select

the ICS final panel, sensitivity and variability were com-

pared for the remaining panels. Results: Of 12 original ICS

panels, 1 failed titration, 3 failed spillover, and 6 failed due

to a combination of unacceptable titration and spillover fail-

ures. Of the two remaining panels, one was clearly more

sensitive for detecting functional responses to CMV peptide

stimulation. Conclusion: Our simplified method of reagent

selection and qualification saves money, time, and is an ac-

ceptable method for developing PFC panels.

29

CD38 EXPRESSION IN B-CELL DIFFERENTIATION:APPLICATION IN LYMPHOPROLIFERATIVEDISEASE CLASSIFICATION.

Samuel J. Pirruccello

University of Nebraska Medical Center

CD38 is a bifunctional ecto-enzyme that mediates leukocyte

biology by both enzymatic and nonenzymatic mechanisms. Anti-

body mediated cross-linking of CD38 on the surface of B-lineage

lymphocytes can mediate opposing effects depending on the

stage of B-cell differentiation. Likewise, the surface density of

CD38 varies predictably across B-cell differentiation states. In

flow cytometric analysis, logrithmic display of CD20 by CD38

expression of CD19 gated B-cells allows discrimination of the

major differentiation subsets. This includes B-cell precursors,

transitional B-cells, and plasma cells in bone marrow, naı̈ve, and

memory B-cells in bone marrow, blood and lymph node, and fol-

licle center B-cells in lymph node. A single, five antibody cocktail,

CD19-FITC/Lambda-PE/CD20-ECD/CD38-PC5/CD45-PC7, is suf-

ficient to identify aberrant and(or) clonal B-cell proliferations at

all stages of B-cell differentiation. Further, the density patterns of

CD20 and CD38 expression, by comparison to normal B-cell sub-

sets, are useful in classification of these B-cell lymphoprolifera-

tive disorders. Examples of CD20 by CD38 expression patterns

representing normal B-cell differentiation stages and correspond-

ing B-cell lymphoproliferative disorderswill be demonstrated.

B-cell Precursor Transitional Naive Follicle Memory Plasma

CD38 *þþþþþþto þþ þ

þþto þþþ 2 þþþþ

CD20 2 þ to þ þþþ

to þþþ þ to þþ 2*Fluorescence intensity

30

DISTINCT SEPARATION OF ZAP-70 POSITIVE FROM NEGATIVECLL CASES USING FLOW CYTOMETRIC ASSAY WITH OPTIMIZEDCONCENTRATION OF ISOTYPE ANTIBODY

Sergey N. Preobrazhensky,1 Philippe Szankasi,1 and David W. Bahler2

1ARUP Laboratories

2Department of Pathology, University of Utah

Measurement of zeta-associated protein of 70 KD (ZAP-

70) in chronic lymphocytic leukemia (CLL) cells is a valuable

marker for predicting disease aggressiveness. The distribution

of percentage positive CLL cells showing ZAP-70 expression

among different cases is thought to be continuous. We were

interested in determining whether improvement of flow cyto-

metric detection of ZAP-70 and use of an isotype antibody

would allow better separation between positive and negative

cases. ZAP-70 expression was measured using an optimized

staining procedure (Cytometry 74B: 118, 2008). The percent-

age of ZAP-70 positive cells was determined using two negative

373CCS ABSTRACTS


threshold markers, normal B-cells stained with anti-ZAP-70 anti-

body, and CLL cells stained with an isotype antibody. The

appropriate concentration of isotype antibody was determined

by staining normal B- and T-cells with sequentially diluted anti-

bodies so that normal B-cells were ZAP-70 negative and T-cells

strongly positive. The percentage of ZAP-70 positive cells was

evaluated in 60 clinical CLL samples. The normal B-cell thresh-

old gave a continuous distribution of percent positive ZAP-70

values, while using the isotype threshold, the CLL cases sepa-

rated into two distinct groups with low and high percentages

of ZAP-70 positive cells. All samples in the low ZAP-70 group

had mutated IgVH genes, and 94% of the samples in the group

with high expression were unmutated. Our results indicate

that measuring ZAP-70 in CLL cells with optimized concentra-

tion of isotype antibody allows clear separation of ZAP-70 posi-

tive and negative CLL cases and gives excellent correlation

with IgVH gene mutational status.

31

ELECTRONIC VOLUME AND ALDH EXPRESSION IN STEM CELLSFROM HUMAN APHERESIS SAMPLES

Siddharth Sharma, Sherry Shariatmadar, Raquel Cabana,and Awtar Krishan

Pathology Miller School of Medicine

In previous studies, we have described cell volume and

marker expression of stem cells from human apheresis sam-

ples. In the present study, we have analyzed mononuclear

cells with ALDHbright/SSClow characteristics from peripheral

blood apheresis (HPC-A) samples of 44 patients mobilized

with granulocyte colony stimulating factor (G-CSF). The

mean electronic volume of the ALDHbright/SSClow cells was

286mm3 (SD 6 27). The mean percentage of ALDHbright/

SSClow cells was 0.51% (SD 6 0.004). CD34 expression was

seen in 0.13% (SD 6 0.001) of the ALDHbright/SSClow cells.

The mean electronic volume of the ALDHbright/SSClow/

CD34positive cells was 270 mm3 (SD 6 33). CD133 expression

was seen in 0.28% (SD 6 0.12) of the ALDHbright/SSClow

cells. The mean electronic volume of the ALDHbright/SSClow/

CD133positive cells was 275 mm3 (SD 6 29). 0.17 and 0.04%

of the ALDHbright/SSClow cells had CD117 and CD90 positive

expression, respectively. The mean electronic volume of the

ALDHbright/SSClow cells with CD117 and CD90 expression

was 284 mm3 (SD 6 32) and 265 mm3 (SD 6 34), respec-

tively. The mean electronic volume of the ALDHdim/SSClow/

CD90positive cells was 223 mm3; on the other hand, the mean

electronic volume of the ALDHdim/SSClow cells with CD133,

CD34, and CD117 positive expression was 228, 230, and

234 mm3, respectively. The use of electronic cell volume in

conjunction with side scatter might provide a useful method

for characterizing stem/progenitor cell populations with

ALDH expression.

CD MARKER ALDHbright/SSClow ALDHdimSSClow

CD34 270 mm3 (SD 6 33) . 230 mm3 (SD 6 31)CD90 265 mm3 (SD 6 34) 223 mm3 (SD 6 25)CD117 284 mm3 (SD 6 32) 234 mm3 (SD 6 28)CD133 275 mm3 (SD 6 29) 228 mm3 (SD 6 29)

32

COMPARISON OF TWO SINGLE PLATFORM ISHAGE-BASEDCD34 ENUMERATION PROTOCOLS ON BD FACSCALIBURTM

AND BD FACSCANTOTM FLOW CYTOMETERS

Robert Sutherland,1 Rakesh Nayyar,1 Erica Acton,1 Sue Dean,2 andViki Mosiman2

1Department of Pathology, University Health Network

2Becton Dickinson Biosciences

Enumeration of viable CD34þ cells provides essential in-

formation to the bone marrow transplant physician. Previous

studies have shown that 5 CD34þ cells per microlitre of blood

predicts the collection of at least 0.53 106 CD34þ cells per kg

patient weight by apheresis. Furthermore, from the apheresis

product, the infusion of 2.53 106 CD34þ cells (measured pre-

cryopreservation) per kg will reliably permit engraftment of

the hematopoietic system (as measured by the time to 20,000

platelets/microliter), by Days 12–14 postinfusion. The single

platform variant of the ISHAGE protocol is the most reliable

method currently available to accurately quantitate this impor-

tant subset of cells. In this study, we compared the CD34þ cell

numbers derived from Flow-CountTM-based (Stem-KitTM

Reagents, Beckman Coulter) and TrucountTM tube-based

(Becton Dickinson Biosciences) ISHAGE templates on the BD

FACSCaliburTM and BD FACSCantoTM flow cytometers. Com-

parison of results showed that there was no statistical differ-

ence between samples run with Stem-Kit on the Calibur ver-

sus either Trucount-based assays on the Calibur or the Canto.

Mean (median) results for the Stem-Kit/Calibur combination

were 137 (67),, for Trucount/Calibur 140 (73) and Trucount/

Canto137 (68). Pairwise comparison of data based on rank

order showed no statistically significant difference and all cor-

relation coefficients had an R2> 0.98.

33

ABERRANT EXPRESSION OF LYMPHOCYTE CYTOTOXICPROTEINS IN CHRONIC FATIGUE SYNDROME

David L. Tamang,1 Vinnie Lombardi,1 Dan Peterson,1 John Diamond,1

Ken Hunter,2 Dorothy Hudig,2 Judy Mikovits,1 and Doug Redelman2

1Whittemore Peterson Institute

2University of Nevada, Reno

Chronic fatigue syndrome (CFS) is a debilitating disease

that afflicts more than 1 million people in the United States.

People that are afflicted suffer from flu-like symptoms such

as pain, severe and persistent fatigue, and degraded mental

facilities for years or even decades. Although there are no

unique diagnostic tests for CFS, there have been numerous

reports over the last 20þ years that patients with CFS have

decreased natural killer (NK) cell activity even though the

number of NK cells may not be decreased. The concept of

NK cells with decreased activity was supported by the

recent report from the Klimas laboratory that perforin

(PRF1) was lower in the NK cells from patients with CFS

than those from healthy controls. We have now confirmed

that NK cells from CFS patients have lower levels of PRF1

and have demonstrated that the shift is not due to increased

374 CCS ABSTRACTS


numbers of CD56Bright that normally have lower levels of

PRF1. Furthermore, the abnormality in PRF1 content was

not restricted to NK cells since the T cells from these

patients had marked reductions in the numbers of PRF1þ

cells and in the amount of PRF1 per cell. In addition to

PRF1, we also examined granzyme B (GZMB) which is

another granule-associated protein in NK and cytotoxic T

cells. In cells from healthy subjects, GZMB and PRF1

occurred together whereas we noted PRF1(2)GZMBþ T cells

in the patients with CFS. Therefore, CFS patients have alter-

ations in cytotoxic molecules in both NK and T cells.

34

DETECTION OF CD96 AS LEUKEMIC STEM CELL-SPECIFICMARKER ON THE SURFACE OF CD34þCD38– CELLS INMYELODYSPLASTIC SYNDROMES

Xingbing Wang,1 Xin Chen,1 Jun Liu,2 Wen Du,2 Jine Zheng,2

Huilan Liu,1 Zimin Sun,1 and Shiang Huang2

1Department of Hematology, Anhui Provincial Hospital,

Anhui Medical University,2Stem Cell Research and Application Center,

Union Hospital, Tongji Medical College, Huazhong

University of Science and Technology

Background: Myelodysplastic syndromes (MDSs) are

clonal, hematopoietic stem cell disorders, and frequent pro-

gression to acute myeloid leukemia (AML). Although multi-

potential precursor/stem cells in AML have been character-

ized based on immunophenotype, little information is avail-

able for the MDS counterpart in terms of immuno-

phenotype or comparison to normal or AML hematopoietic

stem cells. Recent data suggest that CD96 is a cell surface

marker present on AML stem cells. The purpose of this

study was to describe the incidence and significance of

CD96 expression in MDS ‘‘stem/precursor’’ cells. Methods:We analyzed the expression of CD96 on CD34þCD382 cells

by flow cytometry in 32 patients with MDS, as well as in

healthy donors (n 5 14) and patients with other hematolog-

ical disorders: aplastic anemia (AA, n 5 11), acute lympho-

blastic leukemia (ALL, n 5 17), and AML (n 522) at diagno-

sis. Results: Compared to normal BM, the fraction of

CD96þ on CD34þCD382 cells was significantly increased in

MDS and AML, but not in AA and ALL. Higher positivity rate

of CD96 was preferentially seen in refractory anemia with

excess blasts (RAEB) and RAEB in transformation (RAEB-T),

compared with refractory anemia (RA) and RA with ringed

sideroblasts (RARS). Conclusion: Our study confirms that

CD96 expression pattern on AML stem cells is shared by

MDS ‘‘stem’’ cells. The study is required to further test

whether CD96 is a novel marker present on truly MDS-stem

cells and may predict progression and prognosis of MDS.

35

THE ISHAGE PROTOCOL: ARE WE DOING IT CORRECTLY?

Alison J. Whitby, Liam B. Whitby, John T. Reilly, and David Barnett

UKNEQAS for Leucocyte Immunophenotyping

Flow cytometric CD34þ Stem cell enumeration is a rou-

tine pre-stem cell harvest procedure with variety of gating

protocols having been described. UK NEQAS CD34þStemCell Enumeration program has 258 international partici-

pants with 201 (80%) routinely using the ISHAGE proto-

col (Sutherland et al., J Hematotherapy 1996;5:213–

226). Recently, two laboratories were identified as poor

performers, the result of which was attributed to incor-

rect ISHAGE protocol usage/setup. This prompted us to

question if others were also making similar errors and

how these would impact on individual EQA perform-

ance. Retrospective EQA and gating strategy analysis

was undertaken over a six sample window. 81/201 labo-

ratories that stated they were using the ISHAGE protocol

submitted their dot plots to the study. Of these, 36/81

were found to be using an incorrect setup when com-

pared with the original. Furthermore, when consensus

target values were calculated from single platform ISH-

AGE protocol data (defined as the predicate method),

the overall coefficient of variation (CV) was slightly

lower when using single platform ISHAGE correctly

(Overall six sample mean CV 20.4%; range 11–31.5%)

(Keeney et al., Cytometry 1998;34:61–70) compared to

those using single platform ISHAGE incorrectly (Overall

6 samples mean CV 22.6%; range 12.6–35.1%). However,

when individual performance was assessed (using single

platform ISHAGE gating as the predicate method) those

using ISHAGE incorrectly had a 20% increased chance

they would fail an EQA exercise. These findings may

have clinical implications and thus we intend to reissue

the ISHAGE protocol for guidance and will monitor the

situation.

36

PREDICTIVE VALUE FOR LYMPHOMA OF SURFACEIMMUNOGLOBULIN-NEGATIVE B-CELLS

Amanda L. Wilson, Mohamed Eldibany, and Irene J. Check

Department of Pathology, Evanston Northwestern

Healthcare

Background: Monoclonal surface immunoglobulin

(SIg) is characteristic of non-Hodgkin B cell lymphoma,

but its absence can also be a diagnostic feature. We exam-

ined a spectrum of cases with SIg-negative B cells (SIgNB)

to assess which elements predicted a final diagnosis of B

cell lymphoma. Design: The flow cytometry database was

queried from 10/2001–12/2007 for cases with �15% B

cells and �50% SIgNB, yielding 215 of 6182 (3.4%) cases

(blood samples and acute leukemias were excluded).

Results: 57.2% had a final diagnosis of lymphoma, of

which 7.8% were mediastinal large B cell, 19.1% follicular,

22.6% small lymphocytic, and 25.2% diffuse large B cell.

Mediastinal lymphomas were 3.2 times more prevalent

than expected (WHO). Lymphomas had, on average,

higher % B cells than did reactive samples (54% vs. 28%, P

375CCS ABSTRACTS


< 0.0001), with a likelihood ratio for lymphoma of 8.3 for

cases with >65% B cells, 2.1 for those with 46–65% B

cells, and 0.48 for those with <45% B cells. All 30 cases

with >75% B cells were diagnosed as lymphoma. Conclu-sion: Approximately 10% of cases were clearly monoclo-

nal despite having a high % SIgNB. Since dim or partial

expression of SIg as well as non-specific staining can

increase % SIgNB, increased SIgNB is clearly not commen-

surate with clonality. In these cases, the percentage of B

cells helps to distinguish lymphomas from reactive speci-

mens.

37

ESTABLISHING LINEARITY OF FLOW CYTOMETERS: A NOVELAPPROACH USING STABILIZED WHOLE BLOOD

John Y. Wong,1 Liam B. Whitby,2 Nathan A. Vandergrift,1

Raul Louzao,1 Thomas N. Denny,1 and David Barnett2

1Immunology Quality Assessment, Duke University

Medical Center2UK NEQAS for Leucocyte Immunophenotyping, Royal

Hallamshire Hospital

Over recent years, there has been significant interest in

the use of stabilized blood products for implementing

quality control protocols in flow cytometry. One of the

main areas of interest has been the development of stabi-

lized whole blood with varying levels of the target cell

populations. Recently, UK NEQAS and IQA have reported

the introduction of VERIQASTM, a CD4þ T lymphocyte

training panel using stabilized whole blood specimens

that provides immediate feedback via an interactive web

site. However, while there is a need to provide operator

training, current accrediting agencies advice is that the

linearity of instruments over the reportable range should

also be established. Establishing the linearity of the test

is an important adjunct to staff training and clinical

drugs trials because this will provide confidence in both

the operator and the test. This in turn will further help

when defining drug regimens. Thus, we produced a

panel of 11 stabilized specimens with CD4þ T lympho-

cyte target levels between 100 and 600 cells/mL (50

cells/mL increments). Each specimen was assayed

10 times at the assigned value and the mean (þ2 SD) cal-

culated to define the level of acceptable variance and es-

tablish a linearity curve. An interesting outcome of this

study has been that it is potentially possible to define

the sensitivity of counting at predefined clinical levels

using flow cytometry and establish the linearity of the

flow cytometer using stabilized whole blood samples.

We are intending to roll this out to international usage

shortly. See figure.

38

CD123 DIAGNOSTIC UTILITY IN ACUTE MYELOID LEUKEMIAAND ACUTE LYMPHOBLASTIC LEUKEMIA

Wayne Yang, Marc Loriaux, Ken Gatter, Rita Braziel, James Huang,Katalin Kelemen, and Guang Fan

Dept of Clinical Pathology, Oregon Health and Science

University

Background: CD123 is IL-3 receptor a-chain, which is

highly expressed in hematopoietic stem cells and some leu-

kemia. IL-3 receptor abnormality is frequently seen in acute

myeloid leukemia (AML). The differences in CD123 expres-

sion between leukemic cells and normal hematopoietic pro-

genitors may be used in diagnosing and monitoring acute

leukemia. We screened CD123 expression in AML, acute

lymphoblastic leukemia (ALL), and postchemotherapy re-

generative marrow to establish immunophenotypic pattern

of normal regenerative progenitor cells and leukemic blasts.

Materials and methods: Retrospectively analyzed expres-

sion of CD123 by flow cytometry from 31 initial acute leu-

kemia diagnostic blood/bone marrow samples (21 AML, 8

B-ALL, and 2 T-ALL) and 21 post-treatment bone marrow

samples. Results: CD123 is positive in 76% of AML, includ-

ing AML-M1, AML-M2, AML-M5, recurrent AML, chemoresist-

ant AML, and AML arising from CMML. CD123 is seen in all

AML-M5 cases which are CD34 negative. CD123 positivity is

observed in 75% of B-ALL and 50% of T-ALL. 9/21 AML and

2/8 B-ALL cases have multiple bone marrow biopsies avail-

able for flow cytometry analysis. Comparing initial diagnos-

tic marrow with post-treatment marrows, CD123 expression

pattern is the same in persistent/residual/recurrent AML

and B-ALL. No CD123 activity is seen in hematogones.

CD123 positivity is frequently present (90%) in regenerative

CD34 positive blasts. Conclusion: CD123 is often

expressed in AML and B-ALL, and expressed in lower fre-

quency for T-ALL. CD123 is shown to be a useful marker for

both AML and ALL follow-ups, especially for CD342/

CD123þ AML, CD34þ/CD1232 AML, and CD123þ B-ALL.

376 CCS ABSTRACTS


abstracts from the 23rd annual meeting of the clinical cytometry society october 12–14, 2008...

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