8/12- Pathology of Leukemia and Lymphoma: Tools & Techniques Flashcards Preview

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Flashcards in 8/12- Pathology of Leukemia and Lymphoma: Tools & Techniques Deck (44):

Which of the following is a marker of B cells?

A. CD20

B. CD3

C. CD34

D. CD4

Which of the following is a marker of B cells?

A. CD20 ?

B. CD3

C. CD34 

D. CD4


Which of the following is a marker of T cells?

A. CD19

B. CD33

C. CD8

D. CD34

Which of the following is a marker of T cells? 

A. CD19

B. CD33 

C. CD8 ?

D. CD34


Which of the following laboratory techniques require fresh tissue?

A. Cytogenics

B. Fluorescent in situ hybridization

C. immunohistochemistry

D. Flow cytometry

E. Molecular diagnostics



What clinical clues should be considered in the presenting patient

- Age, gender

- Symptoms

- Location and extent of disease

- Rate of growth/time of onset

- Lab abnormalities


What diagnostic material may be obtained?

- Peripheral blood draw

- Bone marrow aspirate and biopsy

- Lymph node biopsy

- Other tissue biopsy, depending on affected organs


How is the CBC performed/obtained? What information is provided

- Performed on automated hematology analyzer


- Quantitation of red cells, platelets, WBCs

- Some info on characteristics of those cells

- Reference ranges for all counts and parameters vary with age


What is the peripheral blood smear used for (broadly)?

- Examined in conjunction with CBC data and clinical history

- Confirmatory of cell counts (low or high)

- Morphologic analysis of red cells, white cells, and platelets

- Look for abnormal cells


What is shown here? 

Q image thumb

Peripheral Blood Smears

Left: much red cell fragmentation Right: high RBC count; many granulocytes in many different stages of maturation (this is CML)


Where on the slide should PBS analysis be approached?

The feathered edge (the "Goldilocks")

- If red cells are too thin- all look like spherocytes

- If red cells are too thick- all look like agglutination and rouleaux

- When just right, RBCs should be well-spaced with good central pallor 

A image thumb

Approach to analyzing the PBS?

- Feathered edge

- Start with CBC (tells you what to expect)


- RBC then platelets then WBC

- Make sure you look at everything; don't miss anything

- Don't get dazzled by reactive lymphocytes and miss schistocytes!


What are the components of the bone marrow exam?

- Aspirate

- Core biopsy

- Clot section


Which part of the bone marrow exam is best to see what cells really look like and to look for abnormal cells?

Bone marrow aspirate


What is this? 

Q image thumb

Bone marrow aspirate smear

Normal bone marrow:


What are core biopsy/clot section good for?

- Best way to see marrow cellularity, architecture

- Look for things that don't belong (fibrosis, tumor cells, lymphoma)


What is this? 

Q image thumb

Bone marrow biopsy

- Pink regions = bone; use acid to decalcify so aspirate can be taken

- White spaces = adipocytes; surrounded by normal marrow cells (cellularity decreases with age)


What can be taken diagnostically for a patient with an enlarged lymph node

1. Fine needle aspiration (FNA)

2. Core needle biopsy

3. Open LN biopsy


Describe FNA?

Fine Needle Aspiration

- Suction aplpied to a small needle and cells are pulled out

- Smears are made with the aspirated material (can see morphology and cellular details, but have destroyed architecture/cellular relationships)

- Material can also be sent for other studies (culture, flow cytometry, cytogenics)


What is this? 

Q image thumb

Fine needle aspiration smeared on slide


What can be done with a fresh lymph node biopsy?

- Morphology (almost 75%)

- Cytogenics

- Flow cytometry

- Freeze

- Culture 

A image thumb

What does "submitting for morphology" mean?

- Slices of LN (or bone marrow biopsy, clot, or other tissue) are put into fixative preservation solution (formalin)

- Chemically processed overnight and embedded in paraffin wax ("block")

- Thin 4-6 um section are cut and put on a glass slide

- Slides are stained with H/E and observed under microscope


What is seen here? 

Q image thumb

Lymph Node Biopsy-

- Normal architecture replaced by sea of small lymphocytes


What is this? 

Q image thumb

(Lymph Node Biopsy)

- Monotonous small lymphocytes suspicious for non Hodgkin lymphoma


How do we evaluate the immunophenotype (pattern of antigen "marker" expression)?

- Flow cytometry

- Immunohistochemistry


What is flow cytometry? Uses?

On what is it performed? Process?

Powerful technique for analyzing markers (antigens, or "CD"s") expressed on cell surfaces or in the cytoplasm


- Examine the surface antigens present on viable cells

- Helpful in most cases of leukemia and lymphoma (identify cell lineage and normal/abnormal populations)

Performed on:

- FRESH blood, bone marrow, or tissue

- Suspension of cells prepared from specimen (then incubated with fluorescently tagged Abs)


How does flow cytometry work?

- Cell suspension

- Add tagged antibodies

- Tagged Abs attach to the cell

- Flow cytometer uses laser and detectors see what Abs are present and therefore what markers are expressed

- Final analysis presented in dot plot

A image thumb

What is the antigen expression in lymphoma?

- B cells are CD20 and CD10 positive

- Kappa light chain restricted = B cell lymphoma!!! 

A image thumb

What can be done if cells are fixed/dead (from formalin) rather than cytometry?



Describe paraffin Immunohistochemistry studies

- Similar idea as flow cytometry

- Chromagen-conjugated Ab reactions to detect cytoplasmic, nuclear, or membrane expression of proteins

- Performed on fixed (paraffin embedded) or frozen tissue: most Abs these days work in paraffin; most of the Abs we use for flow we also have for immunohistochemistry


Diagnosis of this mass staining? 

Q image thumb

Diffuse Large B cell lymphoma


What are cytochemical stains?

Used for what?

Performed on what?

Older technology for leukemias

- Special stains that highlight certain cellular enzymes or other characteristics

- May be helpful in assigning lineage in acute leukemias

- Performed on PB or BM aspirate smears


Using cytochemical stains to assign lineage in acute leukemias:

- Myeloid cells:

- Monocytic cells:

- B-lymphoblasts:

- T-lymphoblasts:

- Myeloid cells: sudan black, myeloperoxidase, specific esterase positive

- Monocytic cells: non-specific esterase positive

- B-lymphoblasts: PAS positive globules

- T-lymphoblasts: acid phosphatase positive


What is this? 

Q image thumb

PAS+ globules (B-lymphoblasts)


What is this? 

Q image thumb

Sudan black (Myeloid cells)


What is this? 

Q image thumb

Myeloperoxidase (Myeloid cells?)


What is the process of cytogenetic studies?


(fresh PB, BM, or tissue)

- Stimulate viable cells to divide in culture

- Fix them in metaphase

- Stain them with a Giemsa stain

- Examine number and structure of chromosomes


What are cytogenetic studies good for?

- Looking at whole genome

- Picking up large abnormalities

(Not good for very small lesions, can’t use for mutations, some translocations are cryptic)


What is FISH (broadly)?

A cytogenetic study

- Fluorescent in situ hybridization


Process of FISH?

- Fluorescent probes designed to detect very small genetic abnormalities

- Often used in conjunction with routine karyotyping to pick up specific abnormalities

- Can use fresh or fixed tissue

- Examines cells in interphase as well as metaphase (you don’t need dividing cells!)


- Heat DNA target to relax DNA; separate strands

- Add fluorescently tagged DNA probe

- Cool to allow annealing of probe and targer


What are the benefits of FISH?

- Great for asking a specific question (t(9;22) present or absent?)

- Won't give you all the information (only answers the question you asked, so it doesn't tell you what else may be going on in the genome)


What is this? 

Q image thumb



Characteristics of Molecular Studies?

- Usually PCR based assays

- useful for asking specific frozen questions

- use fresh, frozen, or fixed tissue (depending on the essay)

Useful for some translocations

- Similar fashion as FISH, but much more sensitive

- Looks for fusion genes

- Looks for clonality (IgH, TCR gene rearrangements)

- Mutations “Next generation” sequencing studies for mutations, other abnormalities

- Increasingly used for classification, prognosis, identification of potential drug targets


Pros and Cons of Molecular Studies?


- Very powerful, sensitive techniques

- Rapid turn around time (1-2 days)

- Doesn’t require fresh, viable tissue

- Good for following patients (minimum residual disease)


- Only asks very specific questions

- Won't tell you the whole story, chromosomally speaking (exceptions: whole genome or exome sequencing)


Putting it all together: 

Q image thumb



Questions (to be posted)

1. C (but any possible)

2. A

3. C (blasts-> acute leukemia)

4. D (flow cytometry tells lineage and age)

5. D (B lymphoblasts; 19 -> B, 34 -> immature (blasts))

6. C (IHC can be done rather than flow cytometry for markers but doesn't tell genetics... Live genetics = G-banding/cytogenics Fixed genetics = FISH Live markers = cytometry Fixed markers = IHC