From the Diagnostic

Immunology Laboratories ISSUE 9 | SPRING 2012| PAGE 1

Spring Meetings Please stop by and say “hello” in May at this year’s American Society of Pediatric Hematology/Oncology (ASPHO) Annual Meeting in New Orleans as well as the Clinical Society (CIS) Annual Meeting in Chicago. We will be there with the Molecular Genetics Laboratory as the integrated Diagnostic Center for Heritable Immunodeficiencies.

IN THIS ISSUE: We congratulate Virgil Villanueva on his retirement and CD45 RARO...... 1-2 thank him for all his wonderful years of service! WASP Transplant Panel ...... 3 (pictured here, left to right, with coworkers, Barb Wanstrath, Lindsay Dunn, Carrie Gifford, department AVP, Sue Laupola, and Laboratory Supervisor, Darryl Hake) Bulletin Board ...... 4 Current Menu ...... 5

THIS ISSUE’S FOCUS – CD45 RARO Cincinnati Children’s Hospital Medical Center We are very pleased to officially introduce our revised CD45 Cancer & Blood Diseases Institute RARO panel. The assay was modified to include singlet gating, 3333 Burnet Avenue CD45, CD27, CCR7 and CD31 to enable identification of Cincinnati, Ohio 45229 subpopulations of Naïve and Memory/Effector cells. 513-636-4685 Naïve T are resting T cells that have not yet encountered . Naïve T cells are present in both the CD4 and Director CD8 subsets. In CD4+ T cells, the naïve cell population can be Alexandra H. Filipovich, MD further divided into those which are recent thymic emmigrants

Associate Director (CD31+) and those which proliferate post-thymically but have not 1 Jack J.H. Bleesing, MD, PhD yet encountered antigen (CD31-) . See Figure 1.

Director of Test Development Rebecca A. Marsh, MD

Laboratory Supervisor Darryl Hake

From the Diagnostic Immunology Laboratories ISSUE 9 | SPRING 2012| PAGE 2

CD4 Memory T cells are long-lived antigen-specific T A CD4 B CD4CD4 4 104 cells that have the capacity to quickly differentiate to 10 Tem Tcm Tem Tcm end stage effector cells upon re-exposure to antigen. 3 103 Memory T cells can be further divided into Central 10 Memory and Effector Memory populations. Central 2 102 Memory T cells (Tcm) express homing receptors, 10 CD45RO CD45RO such as CCR7, that allow the cells to migrate to 1 10 101 secondary lymphoid organs versus nonlymphoid Naive Naive 0 tissue. Effector Memory T cells (Tem) are 10 100 0 1 2 3 4 0 1 2 3 4 characterized by the presence of immediate effector 10 10 10 10 10 10 10 10 10 10 CCR7 CCR7 function. Central and Effector memory T cells are present in both the CD4 and the CD8 subsets2. See Figure 2. An adult (A) and a pediatric sample (B) Figure 2 for examples of Tem and Tcm populations illustrating the CD4 Tem and Tcm population changes TEMRA cells (T Effector-Memory cells with with age. Tcm cells are CCR7+. reacquired RA) are a subset of very mature effector memory cells that have reacquired RA, and carry the A CD4 B CD8CD8 4 largest amount of perforin of any of the effector 10 104 cells2. Our previous methodology would have 103 3 classified these as “Naïve,” but with our additional 10

markers, we are now able to identify the TEMRA 102 102 cells (Figure 3). Using eight-color flow cytometry, CD45RO monoclonal antibodies against characteristic CD45RO 1 1 of each of the above populations are used to 10 10 quantitate the relative proportions of each subset in 0 the peripheral blood. 100 10 100 101 102 103 104 100 101 102 103 104 CD45RA CD45RA 1. “Life after the thymus: CD31+ and CD31- human CD8 CD8 naïve CD4+ T-cell subsets” Blood, 22 January 2009, CD8 CD8 C4 D 4 Vol. 113, No. 4, pp769-774. 10 10 TEMRA TEMRA 2. “Central Memory and Effector Memory 3 3 Subsets: Function, Generation, and Maintenance.” 10 10 Annual Review of Immunology, 2004, 22:745-763 2 2 10 10 CD45RA CD45RA CD4 CD4 A CD4 B 1 1 10 10 4 4 10 10 0 0 10 3 10 3 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 CD27 CD27

2 2 Figure 3. CD8 TEMRA cells (memory cells that have 10 10 CD45RO

CD45RO reacquired RA) are usually a small proportion of total

1 memory cells. An adult control (C) is shown exhibiting 101 10 few CD8 TEMRA. Using our old methodology a patient

0 would have been classified as having a high percentage of 100 10 0 1 2 3 4 100 101 102 103 104 10 10 10 10 10 Naïve CD8 (B), but by using the new gating strategies and CD31 CD31 antibodies in the revised panel, we more accurately classify Figure 1. Typical differences in CD4 cell populations of an this patient’s CD8 population as TEMRA cells (D). Note adult (A) versus a pediatric sample (B). The the difference in this patient’s CD4 (A) and CD8 (B) CD45RO-CD31bright are recent thymic emmigrants while compartments. the CD45RO-CD31dim are cells that have proliferated but not yet encountered antigen. CD45RO+ are memory cells.

From the Diagnostic Immunology Laboratories ISSUE 9 | SPRING 2012| PAGE 3

WASP Transplant Panel CD3+/CD4+ 69 Many may already be familiar with our M2: 22% M1: 78% Wiskott-Aldrich Syndrome Protein (WASP) 52

Screening Assay, in which we compare WASP 35 expression on patient lymphocytes against normal peripheral blood (PB) control lymphocytes. In 17 this assay we report a ratio of the mean channels 0 (MC, or intensity of staining). It is a very useful 0 1 2 3 4 10 10 10 10 10 assay to serve as a screen for potential disease WASP state (see Figure 4). Figure 5. WASP expression on CD4+ T cells on a healthy control (left) and a WAS Patient Post- Transplant with mixed donor chimerism (right). Note the 78% donor cells in Marker 1 (M1) and 22% recipient in Marker 2 (M2).

Patient Vignette: An infant male, who presented at another institution with petechiae at birth as well as occasional eczema and blood in stool, was diagnosed with WAS at four months of age. He underwent a preparative regimen of Bu/Cy/ATG and was transplanted with a 10/10 cord blood (male donor). He had minimal Figure 4. Histogram of a WAS patient overlaid with problems during his transplant hospital course. the PB control showing very descreased WASP At Day +28 he was found to be only 30% expression. donor. Immune suppression (cyclosporine and MMF) was rapidly weaned off, and he has since continued to have increasing engraftment, with The WASP Transplant Panel is intended to total engraftment now 90% donor. A WASP monitor the absence or otherwise atypical staining transplant panel was performed and expression pattern of intracellular WASP after Bone Marrow was very encouraging with a good percentage of Transplantation (BMT). Success of engraftment all T, Myeloid, and NK cells expressing WASP after BMT can be evaluated by performing multi- (Figure 5). The current plan is to continue color flow cytometric analysis using monoclonal monitoring and comparing total engraftment and antibodies to cell surface antigens to characterize WASP Transplant expression monthly until the hematopoetic cell populations of interest stable. Subsets, CD45 RARO, and before the intracellular staining for WASP. We Mitogen Stimulation are also regularly ordered to can, therefore, indicate the disease, carrier state, or observe T cell reconstitution. With evidence of mixed chimerism after BMT. increasing engraftment, the family was reminded to monitor for signs of GVHD. We look at the WASP expression on CD4+ and CD8+ T cells, NK cells, B cells, Monocytes, and Granulocytes. If we note chimeric expression, we include the percent of each cell lineage expressing or not expressing the WASP.

From the Diagnostic Immunology Laboratories ISSUE 9 | SPRING 2012| PAGE 4

New Tests Now Available:

The Diagnostic Immunology • Panel, now available on Laboratories, consisting of the Clinical peripheral blood AND CSF Immunology Laboratory and the Research Immunology Laboratory, are included are: committed to providing the highest - IL-1B - IL-2 quality, comprehensive clinical testing available to aid in the detection, - IL-4 - IL-5 diagnosis and treatment of pediatric - IL-6 - IL-8 immunologic, as well as oncologic and hematologic, disorders. We're committed - IL-10 - IFN-g to applying scientific advances to - TNF-a - GM-CSF promote efficiency, enhance patient care and improve clinical utility. • Neopterin, peripheral blood and CSF

The clinical diagnostic laboratories are in compliance with all major regulatory New Tests Down The Pipeline: agencies including CLIA (Clinical • Laboratory Improvement Amendments), Panel (new markers!) CAP (College of American Pathologists), • Campath – Plasma Levels HCFA (Health Care Financing Administration), HIPAA (Health Insurance • Extended Mitogen Panel (PHA, PMA Calcium Portability and Accountability Act) and Ionophore at three concentrations, IL-2, JCAHO (Joint Commission on Accreditation of Healthcare CD3/CD28 Organizations). • Restimulation Induced Cell Death (RICD),

The current menu of immunologic assays complements Fas-mediated assay and information regarding shipping instructions is published on the last page of this Newsletter. The accompanying Feedback: Test Requisition Form can be obtained through our website. Previous editions of We would like to hear from our Clients. We invite you to the Newsletter can also be found at this share your questions and comments with us. Feel free to website: www.cchmc.org/DIL send/fax/email your comments to us: Fax 513-636-3861;

Email: [email protected]

CONTACT US

Please visit our website or call us with any inquiries: Ph: 513-636-4685 Fax: 513-636-3861 www.cchmc.org/DIL

Diagnostic Immunology Cincinnati Children’s Hospital Laboratories New Liberty Campus Location

From the Diagnostic Immunology Laboratories ISSUE 9 | SPRING 2012| PAGE 5

Current Menu of Available Tests:

IN THE UPCOMING ISSUE:

• Apoptosis – Restimulation Induced Cell Death (RICD)