12. Laboratory Investigation Protocols

12.1 Flow Cytometry Panels

Screen for suspected lymphoproliferative disorder

Numbers of CD3⁺ T-cells, CD19⁺ B-cells and CD3^-CD16⁺ NK cells will be determined.

Assessment of light chain restriction on CD19 gated B-cells and CD4, CD8 & HLA-DR on CD3 gated T-cells.

Phenotype	Abs. Count	Request ExT	Request PCR	Diagnosis	Comments
CD3⁺8⁺16⁺DR⁺	> Normal	No	Yes	T- or NK cell lymphocytosis	Suggest 6Mo FBC
CD3⁺8⁺16^-DR⁺	>5 x 10⁹/L	Yes	Yes	Depends on ExT
CD3⁺8⁺16^-DR⁺	<5 x 10⁹/L	No	No	Reactive Changes	Suggest 6Mo FBC
CD3⁺8⁺DR^-	> Normal	Yes	Yes	Depends on ExT
CD3^-CD16⁺	>5 x 10⁹/L	Yes	No	T- or NK cell lymphocytosis or LGL leukaemia
CD3^-CD16⁺	<5 x 10⁹/L	No	No	T- or NK cell lymphocytosis	Suggest 6Mo FBC
CD3⁺4⁺	>5 x 10⁹/L	Yes	Yes	Depends on ExT
CD3⁺4⁺	<5 x 10⁹/L	No	No	Reactive Changes	Suggest 6Mo FBC

Diagnostic panel for primary diagnosis of B-cell lymphoproliferative disorders

The following antibodies are assessed using 3 colour flow cytometry on CD19 gated B-cells: CD5/CD20, CD38/CD10, lambda/kappa, CD22/FMC7, CD23/CD11a, IgD/IgM, CD3/CD3 and CD79b/IgG

Additional markers

Suspected Hairy cell leukaemia

CD25/CD103 and CD62L/CD11c

For patients who may be treated with Alemtuzumab:

CD52/CD59

ZAP-70 to be done in CLL on trephine/node.

Follow up of patients with B-CLL

The following antibodies are assessed using 4 colour flow cytometry on CD19 gated B-cells: CD5/CD22/CD81, CD5/CD38/CD20, CD5/lambda/kappa, CD3/CD56/CD45, CD5/CD43/CD79b

Diagnosis and follow up of myeloma and MGUS

3 colour flow cytometry on plasma cells identified by CD38/CD45 expression assessing CD3, CD19, CD56, CD138

Diagnosis of T-cell lymphoproliferative disorders

3 colour flow cytometry on T-cells gated on CD3 or CD4 or CD8 and light scatter using: CD1a/CD34, CD2/CD3, CD5/CD7, CD3/CD8, TCR_αβ/CD38, CD57/CD56, HLA-DR/CD16, CD45RO/CD62L, TCR_γδ/CD25, CD52/CD21.

Diagnosis of acute lymphoblastic leukaemia

B-cell

The following antibodies are assessed using 4 colour flow cytometry on blasts using CD19 and light scatter: CD20/CD10/CD34, CD45/CD38/CD34, Tdt/IgM/CD20 and myeloid antigens CD13,CD33 and CD15.

T-cell

The following antibodies are assessed using 4 colour flow cytometry on blasts using CD7 and light scatter; CD1a/CD4/CD8, TCR_αβ/CD38/CD34, CD2/TCR_γδ/sCD3, Tdt/cCD3/CD5.

Diagnosis of acute myeloid leukaemia/MDS with >10% blasts

The following antibodies are assessed using 4 colour flow cytometry on blasts identified using CD34/CD45 and light scatter: CD15/CD117, CD7/CD33, HLA-DR/CD13, CD14/CD64, CD56/CD19 and cytoplasmic MPO, CD3 and CD79a.

Diagnosis of MDS

The following antibodies are assessed using 4 colour flow cytometry on blasts identified using CD34/CD45 and light scatter: CD15/CD117.

12.2 Immunocytochemistry Panels

All suspected B-cell malignancies where only fixed tissue is available

CD20, CD79, CD3, CD10, CD21, CD5, bcl-2, Mib1, p53, p21, bcl-6, MUM-1, FOXP1

Additional markers

All small cell diffuse B-cell lymphomas or CD5⁺ large cell lymphoma

Bcl-1, CD23

ZAP-70

Suspected or follow up of Hairy cell leukaemia

DBA44

Large B-cell lymphoma presenting in the mediastinum

CD30

Suspected plasmacytoma where only fixed tissue is available

CD138, CD56, Pax-5, MUM1(IRF4), BCL1, kappa & lambda

Suspected Hodgkin lymphoma

CD30, CD15, BOB-1, Oct-2, CD20, CD3, MUM-1

Suspected Lymphocyte predominant Hodgkin lymphoma

Hodgkin lymphoma panel + Bcl-6, CD57, CD79

Suspected precursor leukaemia/lymphoma where only fixed tissue is available

B-ALL

CD20, CD34, Pax5, CD79, Tdt, CD10

T-ALL

CD34, Tdt, CD1a, CD7, CD3

Suspected T-cell lymphoma where only fixed tissue is available

1. All T-cell lymphomas including mycosis fungoides

CD2, CD3, CD4, CD5, CD7, CD8

Additional markers

CD30, Alk-1 for cutaneous large T-cell lymphoma

2. All non-skin T-cell lymphoma

Common panel + CD20, CD10, CD68, Mib1, CD30

Additional markers

All CD30⁺ tumours

Alk-1

Suspected angioimmunoblastic lymphoma

CD23, EBV-associated protein (LMP-1), EBER in situ

Suspected NK cell tumours

CD56, CD57, granzyme B

Suspected myeloid malignancies where only fixed tissue is available

CD34, CD20, CD3, GlyC, MPO, CD117, Factor 8, Lysozyme

Suspected non-haematological paediatric malignancy in bone marrow or other tissue

Myogenin, MyoD1, CD56, CD99, pgp9.5, Desmin

12.3 Interpretation of Immunocytochemistry

Positivity Score	Definition
+	Majority / all tumour cells positive
+/-	all antibodies except P53 and BCL6: >50% of tumour cells positive ¹P53 and BCL6: >15% of tumour cells positive
-/+	0-15% or 0-50% tumour cells (see above) or Complete absence of staining in tumour cells
-	Completely negative or only occasional tumour cells positive

p53 should be classified in association with p21 expression. This will be interpreted automatically by HILIS from entries in the histology results database to appear on the report as follows:
- p53⁺p21^- = p53 deregulation
- p53⁺p21⁺ = p53 normal
- p53^+/-p21^+/- = p53 normal
- p53^- = p53 negative
- FOXP1 = pos ONLY when 100% of tumour cells have strong nuclear staining, otherwise +/-

12.4 Fluorescent in situ Hybridisation

In all cases control probes will be included where available

CD5+ B-cell lymphoproliferative disorders

Trisomy 12, t(11;14), deletion 13q14, deletion of 17p(p53), deletion of 11q

Diffuse large B-cell lymphoma

3q27 rearrangement, t(14;18), deletion of 17p(p53), t(8;14) or cMYC breakapart

Suspected Burkitt lymphoma

t(8;14) or cMYC breakapaprt and rest of DLBCL panel in adults.

Suspected follicular lymphoma or CD5- B-cell lymphoproliferative disorder in bone marrow

t(14;18), deletion of 17p(p53)

Extranodal Marginal Zone lymphoma (presentation samples only)

MALT rearrangement

Follicular Lymphoma; BCL2 negative

3q27 rearrangement

12.5 PCR and RT-PCR based investigations

Suspected acute myeloid leukaemia in patients aged 60 or less

RT-PCR for t(8;21) and inv16

Additional Test

Suspected APML by morphology or phenotype

RT-PCR for t(15;17), PML protein immunofluorescence will be done in all cases

Suspected acute lymphoblastic leukaemia

RT-PCR for t(9;22) (p190 and p210); [t(12;21) in children only]

Suspected chronic myeloid leukaemia

RT-PCR for t(9;22) (p210)

Additional Test

Quantitative PCR for t(9;22) following allogeneic bone marrow transplantation

Diagnosis and follow up of follicular lymphoma

Multiplex PCR using mbr,mcr,5'mcr and 3'mbr with JH consensus primers
Follicular Lymphoma; BCL2 negative: IgH PCR

Identification of monoclonality in lymphoproliferative disorders

B-cell

Multiplex IgH PCR using multiple target to FR1/FR2/FR3 and JH

T-cell

Multiplex TCRγ PCR using consensus and Vγ9,10,11 primers with a set of 4 J region primers. If this is polyclonal, TCRβ PCR should be carried out

12.6 Summary of Laboratory Investigations

Fresh Tissue: Precursor Cell and Myeloproliferative Disorders

Panel:	Cytogenetics	Cytometry		PCR			Cytochemistry			Other
Panel:	Cytogenetics	Acute	CD34	ALL	AML	CML	CEst	MPO	Perls	Other
ALL	+	+		+						cIg-µ
B-ALL follow-up										Tdt-B
T-ALL follow-up		+								Tdt-T
AML presentation <60yr	+	+			+		+	+
AML presentation >60yr	+	+					+	+
AML follow-up			P^$
AML relapse		+					+	+
APML	+	+			+		+	+		PML
APML follow-up					+
CML presentation	+		P^$			+
CML follow-up post-BMT						+
CML transformation	+	+
CML STI/IFN follow-up	+		P^$
MDS low blast count	+^�		P^*						+
MDS raised blast count	+^�		+						+
Aplastic anaemia			+
Stem-cell harvest										Stem cell panel

^$ Partial panel: CD34/CD45
^* Partial panel: CD34/CD45, CD34/CD14, CD34/CD117
^� Cytogenetics only requested when definitive diagnosis is made

Fresh Tissue: Lymphoproliferative Disorders

	Cytometry
Panel:	CD34	B-cell	Lymph screen	MRD	FISH	Other
B-LPD presentation; PB		+
B-LPD presentation; BM		+			+	ZAP-70
B-LPD presentation; tissue		+			+	ZAP-70
B-LPD - follow-up		P^$
Hairy cell		+				CD11c,CD25,CD62L,CD103
HIV						HIV panel
Lymphocytosis NOS			+
MRD B-CLL				+
MRD Campath - B				+
MRD Campath - T						T-PLL panel
Myeloma/MGUS						Myeloma panel
PNH						PNH screen
Staging marrow - FCL		P^$				t(14;18) PCR
Staging marrow - LPD		P^$
T-cell LPD			+			TcR PCR
Tissue biopsy			+

^$ Partial panel: CD5/CD20, CD38/CD10, κ/λ

Fixed Tissue Panels

Panel:	Common-B	Common-T	Nodal-T	HD Common	PCR	Other
Angio-immunoblastic TCL�		+	+		TCR	CD23,LMP-1
B-ALL						B-ALL panel
DLBCL	+
Suspected Burkitt	+					FISH for cMYC rearrangement
Extranodal small B-cell	+					Bcl-1
Follicular lymphoma	+
Follicular Lymphoma; bcl-2 negative	+				IgH, t(14;18)	3q27 FISH
Hodgkin Lymphoma; Classical				+
Hodgkin Lymphoma; LP Nodular				+		CD57,CD79,Bcl-6
Mediastinal large B-cell	+					CD21,CD30,p65
Metastatic carcinoma						Adult non-haem panel
Mycosis fungoides		+			TCR
Myeloid						Myeloid panel
Myeloma/MGUS/plasmacytoma						Myeloma panel + BCL1, Kappa & lambda
NK cell		+	+		TCR	CD56,CD57, Granzyme B
Nodal small B-cell	+					Bcl-1, CD23
Other T-cell lymphoma		+	+		TCR
Paediatric						Paediatric panel
T-ALL					TCR	T-ALL panel

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[URL: www.hmds.org.uk/investigation.html]

12. Laboratory Investigation Protocols

12.1 Flow Cytometry Panels

Screen for suspected lymphoproliferative disorder

Diagnostic panel for primary diagnosis of B-cell lymphoproliferative disorders

Additional markers

Suspected Hairy cell leukaemia

For patients who may be treated with Alemtuzumab:

Follow up of patients with B-CLL

Diagnosis and follow up of myeloma and MGUS

Diagnosis of T-cell lymphoproliferative disorders

Diagnosis of acute lymphoblastic leukaemia

B-cell

T-cell

Diagnosis of acute myeloid leukaemia/MDS with >10% blasts

Diagnosis of MDS

12.2 Immunocytochemistry Panels

All suspected B-cell malignancies where only fixed tissue is available

Additional markers

All small cell diffuse B-cell lymphomas or CD5+ large cell lymphoma

Suspected or follow up of Hairy cell leukaemia

Large B-cell lymphoma presenting in the mediastinum

Suspected plasmacytoma where only fixed tissue is available

Suspected Hodgkin lymphoma

Suspected Lymphocyte predominant Hodgkin lymphoma

Suspected precursor leukaemia/lymphoma where only fixed tissue is available

B-ALL

T-ALL

Suspected T-cell lymphoma where only fixed tissue is available

1. All T-cell lymphomas including mycosis fungoides

Additional markers

2. All non-skin T-cell lymphoma

Additional markers

All CD30+ tumours

Suspected angioimmunoblastic lymphoma

Suspected NK cell tumours

Suspected myeloid malignancies where only fixed tissue is available

Suspected non-haematological paediatric malignancy in bone marrow or other tissue

12.3 Interpretation of Immunocytochemistry

12.4 Fluorescent in situ Hybridisation

CD5+ B-cell lymphoproliferative disorders

Diffuse large B-cell lymphoma

Suspected Burkitt lymphoma

Suspected follicular lymphoma or CD5- B-cell lymphoproliferative disorder in bone marrow

Extranodal Marginal Zone lymphoma (presentation samples only)

Follicular Lymphoma; BCL2 negative

12.5 PCR and RT-PCR based investigations

Suspected acute myeloid leukaemia in patients aged 60 or less

Additional Test

Suspected APML by morphology or phenotype

Suspected acute lymphoblastic leukaemia

Suspected chronic myeloid leukaemia

Additional Test

Diagnosis and follow up of follicular lymphoma

Identification of monoclonality in lymphoproliferative disorders

B-cell

T-cell

12.6 Summary of Laboratory Investigations

Fresh Tissue: Precursor Cell and Myeloproliferative Disorders

Fresh Tissue: Lymphoproliferative Disorders

Fixed Tissue Panels

All small cell diffuse B-cell lymphomas or CD5⁺ large cell lymphoma

All CD30⁺ tumours