8/12- Pathology of Leukemia and Lymphoma: Tools & Techniques Flashcards
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
Includes:
- 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?
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
Approach to analyzing the PBS?
- 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!
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?
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?
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
- Fine needle aspiration (FNA)
- Core needle biopsy
- 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?
Fine needle aspiration smeared on slide
What can be done with a fresh lymph node biopsy?
- Morphology (almost 75%)
- Cytogenics
- Flow cytometry
- Freeze
- Culture
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?
Lymph Node Biopsy-
- Normal architecture replaced by sea of small lymphocytes
What is this?
(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
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)
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
What is the antigen expression in lymphoma?
- B cells are CD20 and CD10 positive
- Kappa light chain restricted = B cell lymphoma!!!
What can be done if cells are fixed/dead (from formalin) rather than cytometry?
Immunohistochemistry
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?
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?
PAS+ globules (B-lymphoblasts)
What is this?
Sudan black (Myeloid cells)
What is this?
Myeloperoxidase (Myeloid cells?)
What is the process of cytogenetic studies?
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
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!)
Mechanics:
- 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?
FISH
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?
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)
Putting it all together:
(:
Questions (to be posted)
- C (but any possible)
- A
- C (blasts-> acute leukemia)
- D (flow cytometry tells lineage and age)
- D (B lymphoblasts; 19 -> B, 34 -> immature (blasts))
- 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
