UPDATE ON USMUS PROGRAMMNE IN WESTMEAD

HOSPITAL AND ROYAL ALEXANDRA HOSPITAL FOR

CHILDREN

Dr Rosline HassanHematology DepartmentSchool of Medical SciencesUniversiti Sains MalaysiaKelantan

USMUS The programmne begins 1st Oct – 30th Dec 2002 Site : Sydney, NSW Summer (Temp : range 18 C – 40 C)

Sunrise : 5 am Sunset : 8 pm Environmental Hazard : Bush fire Money exchange : Aust 1 dollar : RM 2.16 Main transport : train and bus People : Multiracial Working environment : Good : helpful, caring,

well manner (5 days a week)

Programmne Weekly programmne:

Tuesday morning meeting 8am-10 am• Morphology, and journal review• Cytogenetic and FISH data presentation

Wednesday lunch hour meeting 12 noon – 2 pm

• Flow meeting

Fortnightly programmne Thursday lunch hour meeting

• Hb Electrophoresis data presentation CME lunch hour meeting

Westmead Hospital Department of Immunology

Flow unit• Leukemia & Lymphoma work-up• PNH work up• MRD (under research)- only for ALL

• A limited Ab panel can distinguish B-precursor ALL from normal B prec. With four color flow cytometry: implication for residual disease detection : Leukemia (1999) 13;558-567

BMT Transplant Unit• CD 34 + quantitation

Department of Hematology Hb electrophoresis lab Molecular Lab

Leukaemia & Lymphoma work-up Viability and manual cell count

Used Tryphan Blue Viability count = Number of viable cell x 100

Total cell Example :

• Total count = 170, no viable = 160• Viability 94%• Leucocyte count = number of cell in 2 quadrant x 106/ml

2x10• Taking 350,000 cells acquired • Vol of blood = 35 x 104 = 44 ul

8 x 106/ml

Leukemia typing Specimen collection :

Peripheral blood : 10 ml heparinised blood Bone marrow : Sodium Heparin tube Lymph node biopsy : RPMI medium (must

reach lab 1 hour- 4C) FNA : RPMI medium – 4C Pleural fluid : 10-50 ml fluid in heparin – 4C Ascitic fluid : 10-50 fluid in heparin – 4 C

Leukemia typing Specimen preparation

Lymph node• Tease cell from biopsy with forcep in RPMI

medium• Pass through nylon filter (53 nm) into a 15

ml centrifuge tube CSF, FNA & others

• Spin down cell and wash with PBS• If RBC contaminated, lyse with RBC lysing

solution

Leukemia typing Whole Blood

• Density Gradient Separation• Done in almost all cases• Two types :

• Underlay : WB < 2.5 ml• Overlay : WB >2.5 ml

• Whole Blood Lysis• If blood is old & to exclude loss of cell

Leukemia typing Flow cytometry

Instrument set up Data acquisation

• 10,000 events acquired per tube Data analysis

• Using CELLQUEST• Template based on FSC vs SSC

Leukemia typing Quality control

Daily• Flow check : to check detector for all 4 types of

fluorescence (Beckman coulter) Weekly

• Flow set : (Beckman coulter) to check cells detected by FL-1(FITC), FL-2(PE), FL-3(PerCP), FL-4(APC)

Monthly• Calibrite beads

• 3 colors(cat : 340486)• 3 colors(cat: 349502)

Leukemia typing Antibody preparation

Titration of monoclonal Ab• Used normal sample-WB (100 ul)• Monoclonal Ab 1, 5, 10 ul• Add lysing buffer• Analyse sample• Interpretation: Vol of Ab is used is

determined by highest signal•SEE EXAMPLE

Reagent Lysing solution for the detection of

intracytoplasmic and nuclear Ags Revised guideline on immunophenotyping in

acute leukaemias and chronic lymphoproliferative disorders : Clin. Lab. Haem (2002, 24; 1-13)

Used Fix & Perm (Caltag cat no : GAS-003) Preferably used FITC conjugated monoclonal

(small molecules)

Reagent Lysing Buffer

10x stock solution• 8.29gm NH4Cl• 1.0 gm KHCO3• 0.0372 gm EDTA• Make up to 100 ml with distilled water• Store 4 C• Used 1x

PBS/BSA/NaN3 or PBS/Albumex 20/NaN3 2 gm BSA from CSL (final 0.1%) or 2 mls

Albumex 20

Leukemia typing Leukemia/Lymphoma screening panel

Lymphoma in tissue, FNA & CSF• Tube 1: Dako Kappa FITC, Dako Lambda PE, Imm

CD20 PC5• Tube 2 : BD Calla FITC, BD Leu 1 PE, Imm CD19 PC5• Tube 3 : BD Leu 2a (CD8) FITC, BD Leu 3a(CD4) PE,

Imm CD3 PC5• Tube 4 : BD I Gg1 FITC, BD IgG2a PE, Imm IgG1 PC5

Leukemia typing Screening for acute leukemia

Tube 1 : BD CD10 FITC, BD CD13 PE, Imm CD19 PC5 Tube 2 : BD CD2 FITC, BD CD 33 PE, BD CD34 Per CP Tube 3 BD CD 7 FITC, BD HLA-DR PE, Imm CD 45 PC5 Tube 4 : Dako k FITC, Dako lambda PE, Imm CD20

PC5 Tube 5 : BD IgG1 FITC, BD IgG2a PE, Imm IgG1 PC5 Cytoplasmic marker is performed, when lineage

assignment is in doubt SEE EXAMPLE

Paroxysmal Nocturnal Hemoglobinuria Application of flow cytometry to the diagnosis

of paroxysmal nocturnal hemoglobinuria Stephen J. Richards, Andrew C. Rawstron, Peter

Hillmen Cytometry 42; 223-233 (2000)

Flow cytometry analysis of glycosylphosphatidyl-inositol-anchored proteins to assess paroxysmal nocturnal hemoglobinuria clone size Josefa Piedras, Xavier Lopez-Karpovitch Cytometry 42; 234-238 (2000)

Paroxysmal Nocturnal Hemoglobinuria Antibody Panel

CD 55 PE (Pharmigen), CD59 FITC (Pharmigen), CD45 PerCP (BD)

Collection :• 5 ul in EDTA fresh

Method :• Used Wintrobe tube• Repeat procedure with control sample

Paroxysmal Nocturnal Hemoglobinuria

Analysis:• WBC analysis : obtain FSC vs SSC dot plot• Monocyte analysis• RBC analysis

Data acquisation• Acquire 30, 000 events• Histogram & dot plot•SEE EXAMPLE

CD34 Quantitation The ISHAGE guidelines for CD34+

cell determination by flow cytometry D.Robert Sutherland et al Journal of Hematotherapy 5; 213-226

(1996)

CD34 Quantitation CD34+ cell in marrow 1-3% In blood – 0.01-0.1% Useful to mobilize from marrow to

peripheral blood by chemo or hemopoietic cytokines or both

Adv. Using PBSC Less tumour contamination Availability of cytokines to mobilize CD34+

cell Time of engraftment is shorter

CD34 Quantitation Antibody panel

CD34 PE (BD) &CD45FITC (BD) Method :

ISHAGE protocol In house Acquisation : 75,000 events with min of 100 CD

34+ Require CD34+ cord blood control (Wak-

Chemie Medical GMBH and baseline CD34+ count in normal bone marrow

SEE EXAMPLE

Hb Electrophoresis Stability testing

Isopropanol stability test• Tested by stresses that weaken the subunit bonds

by temperature or changed in solvent polarity• In this test, the solvent polarity of Hb is reduced

by addition of 18.5% isopropanol• Prepared carban tetrachloride hemolysates (10gm

%)• Results :

• Precipitation at 5 mins interval• Interpretation based on normal control neg after

30 mins and heterozygous Hb E hemolysates should be positive after 10-15 mins

Hb Electrophoresis Preparation of hemolysate

Using tetrachloroethylene (TCE) Hb of hemolysate should be between

10-12gm%

Molecular lab Tests done :

bcl-1 gene rearrangement t(11;14) bcl-2 gene rearrangement t(14;18) Ig gene rearrangement T-cell receptor gene rearrangement bcr-abl for ALL &CML E2A-PBX1 t(1;19), MLL-AF4 t(4;11) etc F V Leiden mutation, prothrombin gene

mutation MTHFR gene mutation

ROYAL ALEXANDRA HOSPITAL FOR CHILDREN

Oncology Cytogenetic Total tests : 19305 (UK 27,500) yrly Children : 1600/yr

• 20% new cases• 80% f/up cases

In Australia : CML higher freq : 12/100,00/yr FISH

From cultured cell or touch imprint or blood smear

Cytogenetic Requirement

Equipment• Incubator 37C• Biohazard hood• Sucker (high & low pressure)• Water bath• Centrifuge• Oven 90C, 60C and non heated • Hot plate• Microscope• Dryer• Fridge 8C

Cytogenetic Materials

• Flasks • Nunc tube/graduated tube 100-500 ml• Graduated pippetes• Slide frosted end (Menzel-Glaser)• Coplin jar

Cytogenetic Reagents

• Karyomax colcemid sol, liquid (10ug/ml) in PBS • Hypotonic 5.592g KCL (0.075M)• BrdU (2 x 10-3 M) Sigma cat : B5002 1g• Uridine 4 x 10-4M Sigma cat : U3750 5 g• FdU Sigma cat F0503 10 g• Fixative (3:1, methanol : glacial acetic acid)• 60% acetic acid• RPMI1640 culture medium, FCS, Hepes buffer, L-

Glutamine, Penicillin/streptomycin 5000ug/ml• BM Transport medium : HANKS buffer to Lithium

heparin

Cytogenetic Procedure

Day 1 : Synchronised BM Culture• Sample preparation :

• Discard supernatant• Drop into flasks of medium• Synchronised cultures

• Cell cycle is blocked at S phase (DNA synt phase) by Ur and FrdU

Day 2 : Harvest• Block is released by BrdU : cell at S phase move to

G2 phase and mitosis. Cells are harvested at metaphase

Cytogenetic Day 3 : Drop and Banding

• After drop on slide, require ageing unless banding is urgent E.g : newly diagnosed ac. Leukaemia

• G-banding : produce horizontal dark and light bands

• Used double strength Trypsin Sol. Analysis

• Microscope counting for 20 spreads (10 spreads/staff)

• Results are captured by cytovision and print for filing

• SEE EXAMPLES

Fluorescence insitu hybridization

Standardization criteria for the detection of BCR/ABL fusion in interphase nuclei of chronic myelogenous leukemia patients by fluorescence insitu hybridization

• Ninette Cohen et al• Cancer genetics and cytogenetic 123 (2000) 102-108

Application of interphase FISH on direct bone marrow smears for evidence of chimerism in pediatric sex mismatched bone marrow transplant

• Arabella Smith et al• Pathology (1999) 31; 25-28

Fluorescence insitu hybridization Sample

Cultured Non cultured: touch imprint & marrow smears

Slide preparation Insitu hybridization

Pretreatment Slide denaturation Probe preparation

THANK YOU :School of Medical SciencesEmeritus Prof LawrenceWestmead Hospital and

Children Hospital WestmeadAssoc Prof Dr Normah

JamaludinColleaguesMMed studentsStaffFamily