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_MS2 HemeOnc > 8/12- Pathology of Leukemia and Lymphoma: Tools & Techniques > Flashcards

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

1
Q

Which of the following is a marker of B cells?

A. CD20

B. CD3

C. CD34

D. CD4

A

Which of the following is a marker of B cells?

A. CD20 ?

B. CD3

C. CD34

D. CD4

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2
Q

Which of the following is a marker of T cells?

A. CD19

B. CD33

C. CD8

D. CD34

A

Which of the following is a marker of T cells?

A. CD19

B. CD33

C. CD8 ?

D. CD34

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3
Q

Which of the following laboratory techniques require fresh tissue?

A. Cytogenics

B. Fluorescent in situ hybridization

C. immunohistochemistry

D. Flow cytometry

E. Molecular diagnostics

A

?

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4
Q

What clinical clues should be considered in the presenting patient

A
  • Age, gender
  • Symptoms
  • Location and extent of disease
  • Rate of growth/time of onset
  • Lab abnormalities
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5
Q

What diagnostic material may be obtained?

A
  • Peripheral blood draw
  • Bone marrow aspirate and biopsy
  • Lymph node biopsy
  • Other tissue biopsy, depending on affected organs
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6
Q

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

A
  • Performed on automated hematology analyzer

Includes:

  • Quantitation of red cells, platelets, WBCs
  • Some info on characteristics of those cells
  • Reference ranges for all counts and parameters vary with age
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7
Q

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

A
  • 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
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8
Q

What is shown here?

A

Peripheral Blood Smears

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

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9
Q

Where on the slide should PBS analysis be approached?

A

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
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10
Q

Approach to analyzing the PBS?

A
  • Feathered edge
  • Start with CBC (tells you what to expect)

System:

  • 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!
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11
Q

What are the components of the bone marrow exam?

A
  • Aspirate
  • Core biopsy
  • Clot section
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12
Q

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

A

Bone marrow aspirate

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13
Q

What is this?

A

Bone marrow aspirate smear

Normal bone marrow:

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14
Q

What are core biopsy/clot section good for?

A
  • Best way to see marrow cellularity, architecture
  • Look for things that don’t belong (fibrosis, tumor cells, lymphoma)
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15
Q

What is this?

A

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)
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16
Q

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

A
  1. Fine needle aspiration (FNA)
  2. Core needle biopsy
  3. Open LN biopsy
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17
Q

Describe FNA?

A

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)
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18
Q

What is this?

A

Fine needle aspiration smeared on slide

19
Q

What can be done with a fresh lymph node biopsy?

A
  • Morphology (almost 75%)
  • Cytogenics
  • Flow cytometry
  • Freeze
  • Culture
20
Q

What does “submitting for morphology” mean?

A
  • 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
21
Q

What is seen here?

A

Lymph Node Biopsy-

  • Normal architecture replaced by sea of small lymphocytes
22
Q

What is this?

A

(Lymph Node Biopsy)

  • Monotonous small lymphocytes suspicious for non Hodgkin lymphoma
23
Q

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

A
  • Flow cytometry
  • Immunohistochemistry
24
Q

What is flow cytometry? Uses?

On what is it performed? Process?

A

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

Uses:

  • 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)
25
Q

How does flow cytometry work?

A
  • 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
26
Q

What is the antigen expression in lymphoma?

A
  • B cells are CD20 and CD10 positive
  • Kappa light chain restricted = B cell lymphoma!!!
27
Q

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

A

Immunohistochemistry

28
Q

Describe paraffin Immunohistochemistry studies

A
  • 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
29
Q

Diagnosis of this mass staining?

A

Diffuse Large B cell lymphoma

30
Q

What are cytochemical stains?

Used for what?

Performed on what?

A

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
31
Q

Using cytochemical stains to assign lineage in acute leukemias:

  • Myeloid cells:
  • Monocytic cells:
  • B-lymphoblasts:
  • T-lymphoblasts:
A

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

- Monocytic cells: non-specific esterase positive

- B-lymphoblasts: PAS positive globules

- T-lymphoblasts: acid phosphatase positive

32
Q

What is this?

A

PAS+ globules (B-lymphoblasts)

33
Q

What is this?

A

Sudan black (Myeloid cells)

34
Q

What is this?

A

Myeloperoxidase (Myeloid cells?)

35
Q

What is the process of cytogenetic studies?

A

Process:

(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
36
Q

What are cytogenetic studies good for?

A
  • Looking at whole genome
  • Picking up large abnormalities

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

37
Q

What is FISH (broadly)?

A

A cytogenetic study

  • Fluorescent in situ hybridization
38
Q

Process of FISH?

A
  • 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!)

Mechanics:

  • Heat DNA target to relax DNA; separate strands
  • Add fluorescently tagged DNA probe
  • Cool to allow annealing of probe and targer
39
Q

What are the benefits of FISH?

A
  • 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)
40
Q

What is this?

A

FISH

41
Q

Characteristics of Molecular Studies?

A
  • 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
42
Q

Pros and Cons of Molecular Studies?

A

Pros:

  • Very powerful, sensitive techniques
  • Rapid turn around time (1-2 days)
  • Doesn’t require fresh, viable tissue
  • Good for following patients (minimum residual disease)

Cons:

  • Only asks very specific questions
  • Won’t tell you the whole story, chromosomally speaking (exceptions: whole genome or exome sequencing)
43
Q

Putting it all together:

A

(:

44
Q

Questions (to be posted)

A
  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

_MS2 HemeOnc (33 decks)

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