Diagnostic Techniques Flashcards
flow cytometry
- usually performed on BM, peripheral blood, or lymphoid tissues
- measures cellular characteristics based on their ability to scatter light (optical scatter)
forward scatter
cell volume/size
side scatter
cellular complexity
- nucleus, is the cell granulated?
immunophenotyping
phenotyping cell surface markers through Abs interacting w antigens (fluorescently-labeled monoclonal Abs)
useful in diagnosing hematological malignancies
cell markers
- neoplastic cells will stop at a certain point in maturation and will display aberrant antigenic patterns
CD
cluster of differentiation
- cell markers specific for those types of cells like CD4, CD8, etc.
CD8
suppressor T cell
CD45
leukocyte common antigen
CD34
immature hematopoietic cell
CD19, CD20
precursor and mature B cells
molecule diagnostics
- used to detect specific sequences of DNA/RNA in nucleated blood or BM cells, tissues, or body fluids
- PCR is used to amplify DNA, followed by gel or cap electrophoresis for the detection of amplified DNA
- DNA/RNA in a sample can also be measured quantitatively
Reagents needed in a PCR
- primers
- DNA polymerase (Taq)
- nucleotides (dATP, dCTP, dGTP, dTTP)
- Mg buffer
- thermocycler
enzyme-based method for amplifying a specific DNA target sequence
Polymerase Chain Reaction
Reverse Transcriptase PCR (RT-PCR)
- detects RNA expression
- mRNA used as the starting material
- reverse transcriptase produces complementary DNA (cDNA) from mRNA
- cDNA amplified using specific primers
- method can be paired with fluorescent probes for quantitatively measurement of DNA amplification
study of chromosome structure and inheritance
cytogenetics
chromosomes arranged and displayed in order of numbered pairs
Karyogram
- chrom 1 longest and shorter and shorter until chrom 22.. then chrom 23 = sex chromosomes
human somatic cells have __ chromosomes
46 (diploid or 2n)
**gametes have 23 chromosomes (haploid or n)
genes arranged on the chromosomes in humans
25 000
3 shapes of chromosomes
- metacentric
- submetacentric
- acrocentric
4 stages in the cell cycle
G1 = growth period S = DNA synthesis G2 = post DNA synthesis M = mitosis want to stop cell division here bc chromatin is thickest and best visualized ; specifically at metaphase (can chemically arrest cells here)
G banding
- routinely performed using Giemsa stain
- stains A and T rich areas and do not stain C and G
R banding
highlights telomeres
Q banding
rapid identification of the Y chromosome
C banding
stains the centromeres and condensed heterochromatin
Fluorescence In Situ Hybridization (FISH)
- couples molecular method with karyotypes to increase detection of mutations
- target DNA is denatured into ssDNA
- fluorescent-labeled DNA or RNA probe is used
> hybridize to chromatin cells on a glass slide
> red and green coloured probes to correspond to different targets on a chromosome - allows for the detection of fusion vs. break-apart signals
Acute promyelocytic leukemia
- abnormal proliferation of blasts; very very granulated like promyelocytes
- t(15;17)(q24;q21)
Acute myeloid leukemia with eosinophilia
inv(16)(p13.2q22)
Cytochemistry
- older method
- staining of unique cellular components to aid in cell identification
- can differentiate cells based on lineage and maturation
- cytochemical stain examples:
> Myeloperoxidase, esterases, Periodic Acid-Schiff, acid phosphatase
Periodic Acid Schiff
- reacts with glycogen, mucoproteins, and high molecular weight carbs
- positive reactivity:
> leukemic lymphoblasts (block positivity)
> leukemic erythroblasts (coarse and granular)
> granulocytes (+ increases w maturity)
> megakaryocytes (diffuse positivity)
Acid Phosphatase
- various isoenzymes of acid phosphatase are found in all hematopoietic cells
- lymphocytes in hairy cell leukemia produces an isoenzyme resistant to tartrate
> tartrate-resistant acid phosphatase (TRAP stain)
Acid Phosphatase
- various enzymes of acid phosphatase are found in all hematopoietic cells
- lymphocytes in hairy cell leukemia produces an isoenzyme resistant to tartrate
> tartrate-resistant acid phosphatase (TRAP stain)
gel electrophoresis
- detection of DNA
- DNA fragments migrate through gel
- rate of migration based on size and weight
- agarose or polyacrylamide gel used
- smaller fragments move the farthest
- many different methods of measuring DNA fragments
- fluorescent dyes used to visualize fragments under UV light
- qualitative measurement of DNA
The chromatin condenses during _______ to form ___________
mitosis; chromosomes
aneuploidy
- numerical chromosomal abnormality
- not a multiple of 23
polyploidy
- numerical chromosomal abnormality
- multiple of 23, greater than 46
chromosomal structural abnormalities
- rearrangements of genetic material
- may be balanced or unbalanced
- due to inversions, deletions, translocations, or duplications
myeloperoxidase
- usuall found in primary granules of neuts and eos; basos have decreased + with maturity; monocyte variable
- not present in lymphs
- leukemic myeloblasts are positive (differentiate from lymphoblasts)
esterases
- differentiates leukemic cells that are granulocytic vs monocytic
- substrates: alpha-naphthyl acetate (NSE) = monos are positive; positivity inhibited after adding NaF
- naphthol AS-D chloroacetate (SE) = granulocytes stain positive; monocytes stain weak to negative
