CBC
Stain and Testing
Leukocytosis
chronic myeloid leukemia (CML)
Treatment Regimen
100
What readings are present in CBC with differential but not a normal CBC
Answer : Neutrophils, lymphocytes, monocytes, eosinophils, basophils.
CBC with differential is performed by automated hematology analyzers, which enumerate and classify leukocytes into major categories: neutrophils, lymphocytes, monocytes, eosinophils, basophils and, in some systems, can flag or quantify immature granulocytic precursors bands and blasts.
100
What is the full form of FISH?
What is it used to detect?
Fluorescence in situ hybridization
Used to detect genetic abnormalities such as: Chromosomal abnormalities, Gene duplications or deletions, Translocations, etc.
FISH can be performed on both bone marrow and peripheral blood samples. The test is highly sensitive and specific for detecting both classic and cryptic BCR-ABL1 rearrangements
(Cryptic just means the rearrangement is hidden and therefore is not seen on a regular karyotype. In this case FISH would be necessary)
100
What on your CBC would indicate leukocytosis?
high WBC (>10K/ul)
but normal hemoglobin and platelets.
100
What translocation results in CML?
CML is associated with a specific genetic abnormality called the Philadelphia chromosome, which results from a translocation (exchange of genetic material) between chromosomes 9 and 22. This leads to the formation of a new gene called BCR-ABL, which produces an abnormal protein that drives the development of CML.
100
What type of receptor do Imatinib and Dasatinib inhibit? Is this inhibition competitive or noncompetitive?
Tyrosine Kinase via competitive inhibition.
Competitive inhibitors reversibly bind to the tyrosine kinase via the active site, but can be overcome by increased concentration of the substrate.
Vmax remains unchanged, meaning the total efficacy (degree to which the drug produces the desired outcome) remains constant. Km increases, meaning the potency (dose required to produce a pharmacologic response) decreases.
In competitive inhibition, there is no sacrifice to the efficiency of the drug, while also lowering the dosage required to produce the response.
200
What readings are in a peripheral blood smear and not in a differential CBC
Answer: bands, metamyelocytes, myelocytes, promyelocytes and blasts
A peripheral blood smear involves manual microscopic examination of a stained blood film by a trained technologist or hematologist. This method allows for direct visualization, identification, and enumeration of all leukocyte types, including subtle or rare immature granulocytic precursors (bands, metamyelocytes, myelocytes, promyelocytes) and blasts, as well as assessment of cell morphology.
200
We performed fish on T(9;22). Which American city is this chromosome named after?
Answer: Philadelphia
Fluorescence in situ hybridization (FISH) for the t(9;22) translocation is a molecular cytogenetic technique that uses fluorescently labeled DNA probes to detect the fusion of the BCR gene on chromosome 22 and the ABL1 gene on chromosome 9, which together form the Philadelphia (Ph) chromosome.
200
What causes leukocytosis?
Leukocytosis forms as part of the body’s natural response to stress, infection, inflammation, or disease.
It stems from a pathologic insult that stimulates a systemic inflammatory reaction.
Cells in the body (like macrophages or endothelial cells) release cytokines such as:
Interleukin-1 (IL-1)
Interleukin-6 (IL-6)
Tumor necrosis factor (TNF)
Granulocyte colony-stimulating factor (G-CSF)
Stimulate the bone marrow to produce and release more white blood cells (WBCs) into the bloodstream.
200
what is the early “chronic phase” v.s “acute blast” phase?
chronic phase: normal bone marrow function retained, WBCs differentiate, does not behave like a malignant disease
acute blast phase: added chromosomal defects. symptoms: fatigue, malaise, loss of appetite, abdominal fullness
200
What site on the Tyrosine Kinase do Imatinib/Dasatinib block? What specific pathway in CML is disrupted by this?
ATP-binding site. BCR-ABL1 pathway
The mechanism of these drugs begins by binding the ATP-binding site of the specific tyrosine kinase in CML (BCR-ABL1), which inhibits tyrosine phosphorylation of downstream signaling proteins. This then inhibits proliferation and induces apoptosis in BCR-ABL1 positive leukemic cells.
BCR-ABL1 is produced by the Philadelphia t(9;22) chromosome in CML patients.
300
In the lineage of myeloid cells, which comes first: Bands or Blasts.
Should both of them be present in the peripheral blood?
If not, where should they be found?
Blasts are a PRECURSOR to Bands in the myeloid lineage.
Blasts therefore should not be present in peripheral blood and this can be a sign of? Bands can be present in small amounts in the peripheral blood.
Blasts should be present in bone marrow. (Somewhat a hint for next question)
300
Name the stain used in Mondays case?
What color did our target structures appear in the stain?
Bonus: Name 2 of the cells that were colored in the stain.
Wright-Giemsa Stain
The stains conventionally used for peripheral blood smears are the Romanowsky-type stains, most commonly Wright, Giemsa, Wright-Giemsa, and May-Grünwald-Giemsa stains. These are considered routine stains for hematology, not special stains, and are standard for blood smear evaluation in clinical laboratories.
Our targets appear purple in the Wright Giemsa Stain.
Peripheral smear (Wright-Giemsa, ×1000). Noted is the spectrum of maturation within the myeloid cells, including immature forms (blast, red arrow), mid-stage myeloids (blue arrows), and late-stage myeloids (neutrophils and band forms, black arrows). A basophil is also present (asterisk).
The nuclei of all white blood cells (due to the Romanowsky-Giemsa effect on DNA/chromatin).This purple coloration results from the interaction of eosin and methylene blue dyes with nucleic acids.
300
Can Leukemia be present without physical symptoms? How long until its onset?
Leukemia can be present without physical symptoms, especially in chronic forms, and the time from disease onset to symptom development ranges from weeks (in acute leukemias) to months or years (in chronic leukemias).
300
Which percentage of leukemia cases are CML and does it have a male or female predominance?
accounts for 15% of all cases of leukemia with slight male predominance.
300
Imatinib and Dasatinib are theoretically prescribed for how long?
Indefinitely.
Studies have shown efficacy of Imatinib at 400 mg daily beyond 10 years in patients, with about 10-20% of patients discontinuing the drug without remission. In 100 mg daily dasatinib treatment, about 40-50% of patients are able to discontinue the drug without remission.
In addition, dasatinib induces faster and deeper molecular and cytogenetic responses than imatinib, but is typically only used in the case of imatinib resistance due to imatinib's longer term established efficacy, safety, and cost profile. There are no researched agonists or antagonists of either drug.
An example of this reduced safety is the presence of common side effects of dasatinib in our case this week, including the low platelet and leukocyte counts evident in the virtual disclosure.
400
Rank these 3 immature granulocytic precursors in order of maturation: myelocytes, metamyelocytes, and promyelocytes.
Bonus: Tell me what cells they originate from
The spectrum of myeloid maturation is as follows: myeloblasts (large, high nuclear-to-cytoplasmic ratio, fine chromatin, nucleoli) → promyelocytes (primary granules, slightly more cytoplasm) → myelocytes (secondary granules, round/oval nucleus) → metamyelocytes (indented/kidney-shaped nucleus) → bands (horseshoe-shaped nucleus) → segmented neutrophils (multi-lobed nucleus).
To note: These precursors appeared in the labs through peripheral blood smear. They would not be present in peripheral blood of a healthy individual and therefore are indicative of leukemia as discussed.
400
Why did we do a bone marrow biopsy?
Hypercellularity: The biopsy shows if the bone marrow is "hypercellular," meaning it's packed with an abnormally high number of blood-forming cells, a classic feature of CML.
Cell Morphology: A pathologist can examine the different types of cells, their maturation stages, and their overall appearance. This helps distinguish CML from other conditions that might present with similar blood test results. For instance, the biopsy can reveal the characteristic "dwarf" megakaryocytes and other specific cellular features of CML.
Cytogenetic Analysis: The bone marrow sample is used for karyotyping, a test that looks at the chromosomes. This is how the classic Philadelphia (Ph) chromosome—the shortened chromosome 22 resulting from the BCR-ABL1 translocation—is definitively identified.
400
what is Neutrophilia?
An absolute increase in the number of neutrophils, which are a subtype of WBCs, in the peripheral blood, typically above 7.5 × 10⁹/L in adults. It is a type of leukocytosis.
400
How do you get CML? When is it usually diagnosed and how do you diagnose it?
How to get it?
Exposure to high doses of ionizing radiation, disease is acquired (somatic mutation). Not a germline one (not passed down from parents).
When is it diagnosed?
Incidence increases with age, median age at diagnosis is 55-65 years.
How to diagnose?
CBC: high white blood cell count
Genetic testing (FISH or PCR) to confirm the Philadelphia chromosome or BCR-ABL1 fusion gene.
400
What is the most common mechanism of resistance to Imatinib in CML patients?
Kinase binding domain mutations.
Other options would be gene amplification or alterations in the drug transport system.
Most commonly, a point mutation occurs in BCR-ABL1 kinase binding domain, which impairs TKI binding. This reduces the effect of imatinib to induce apoptosis in BCR-ABL1 positive leukemic cells.
Gene amplification would contribute to resistance by promoting production of BCR-ABL1 positive leukemic cells.
Alterations in the drug transport system would affect resistance by reducing presence of the transporter utilized by imatinib for cellular uptake. This would reduce the intracellular levels of the drug, therefore reducing the therapeutic response.
500
Neutrophils, lymphocytes, monocytes, eosinophils, and basophils are types of what?
Rank them in order of abundance in the body
Answer:
Neutrophils 50–70%
Lymphocytes 20–40%
Monocytes 2–8%
Eosinophils 1–4%
Basophils<1%
500
Which test is used to identify and quantify the BCR-ABL1 fusion transcript
Answer: Reverse transcription polymerase chain reaction (RT-PCR)
RT-PCR is highly sensitive and specific, making it the method of choice for gene expression analysis, detection of viral RNA, and diagnosis of genetic diseases.
The main purpose of RT-PCR is to take an RNA sample, (in this case BCR-ABL1 mRNA from leukocytes in peripheral blood) convert it into DNA, and then make a large number of copies of that DNA to see if the original RNA sequence was present.
500
Unlike leukocytosis, this blood cancer often shows immature, non-functional white blood cells called blasts.
leukemia
500
What is the new gene that forms due to the CML translocation?
BCR-ABL:
The BCR-ABL protein is a fusion protein produced by the abnormal BCR-ABL gene, primarily found in chronic myeloid leukemia (CML). This protein has an abnormal tyrosine kinase activity, meaning it can cause cells to grow and divide uncontrollably, leading to leukemia. It's a key target for treatment in CML, with drugs called tyrosine kinase inhibitors (TKIs) that block its activity.
500
What family of kinases does dasatinib target that imatinib does not?
SRC family.
The mechanism of actions for imatinib and dasatinib are very similar, except for the binding of dasatinib to BCR-ABL1 when it is in both the inactive or active conformation, while imatinib only binds it in the inactive conformation. Dasatinib is also able to have a broader spectrum of inhibition of CML due to its additional kinase target of the SRC family.