Hematology Unit 9 (Bone Marrow) Flashcards
What is the purpose of bone marrow?
Hematopoiesis - makes 6 billion cells/kg/day in adults
What happens to bone marrow as we age?
At birth, nearly all bones contain red marrow.
As we age, fat cells begin to replace red marrow in the appendicular skeleton.
Red marrow is then limited to only the axial skeleton in late adolescence.
Adipocytes occupy ~____% of red marrow space in 30-70 y/o.
50%
BM puncture is PROHIBITED in patients with ___________
coagulopathies
Why would a BM exam be ordered on a patient?
- Neoplasia diagnosis/staging
- BM failure causing cytopenias
- Metabolic disorders
- Infections
- Monitoring of treatment
What are the 2 parts of a BM specimen and how are they different?
Aspirate: liquid portion; obtained by BM aspiration and allows for identification of types/proportions of cells and look for morphologic variance
Core biopsy: solid portion; obtained by trephine biopsy and demonstrates bone marrow architecture and estimates cellularity
What is the most commonly used site for a BM collection? What other sites may be collected from, for adults and why?
`Posterior superior iliac crest of the pelvis - most common
Other sites:
- Anterior superior iliac crest of the pelvis (for patients who can only lie supine)
- Sternum (aspirate only)
- Spinous process of vertebrae/ribs (rare)
What areas on the body can a BM specimen be collected in children?
Posterior superior iliac crest most common
Anterior medial surface of the tibia, can only get aspirate
BRIEFLY list the steps of a BM biopsy/aspiration procedure
- Palpate body site
- Local anesthetic injected
- Needle inserted and rotated to core through bone
- Core biopsy removed
- Touch preps of biopsy made
- Aspiration of liquid
- BM smears made
Advantages of a BM aspirate smear
Fast
No need for decalcification
Quantitation of cell type
Material for ancillary studies
Advantages of a BM core biopsy
Can analyze cells AND stroma
Represents all cells
Explains dry taps
Disadvantages of a BM aspirate smear
May not represent all cells
Dry tap may occur
Doesn’t represent architecture
Inability to analyze stroma
Disadvantages of a BM core biopsy
Slow processing
Decalcification required and preludes ancillary studies
Inability to perform quantitative diff count
What is a direct aspirate smear?
Made similarly to periph. blood smears (wedge technique)
Bony spicules = good and allows for more cells (do not crush them)
What is a crush smear?
Bony spicules placed on slide and crushed by another slide; superior to wedge prep method for morphological exam
What is a buffy coat smear? Why would you use this technique?
EDTA aspirate transferred to narrow-bore tube and centrifuged, ME and plasma layers are aspirated and smears are prepared using crush smear technique
- Useful when there is hypocellular marrow
What is a touch prep smear?
Made from core biopsy specimen; biopsy is repeatedly touched to a slide and is valuable when the specimen is clotted or there is a dry tap
What is the normal stain to use on a bone marrow specimen?
Wright or Wright-Giemsa stains
What is the purpose of the Prussian blue stain for bone marrow specimens?
Used to estimate marrow storage iron or iron metabolism abnormalities
Highlights presence of ring sideroblasts
What does MPO stain allow for detection of in BM specimens?
Detects myeloid cells
What does SBB stain allow for detection of in BM specimens?
Detects myeloid cells
What does PAS stain allow for detection of in BM specimens?
Detects lymphocytic cells and certain abnormal erythroid cells
What do esterase stains allow for detection of in BM specimens?
Distinguish myeloid from monocytic maturation stages
What does TRAP stain allow for detection of in BM specimens?
Hair cell leukemia
What can we perform under low power examination of BM?
- Assess peripheral blood dilution
- Locate bony spicules, aggregations of bone, and hematopoietic cells
- Estimate cellularity
- Search for tumor cells
- Examine and estimate megakaryocytes
What can we perform under high power examination of BM?
- Observe maturation of nucleated cells
- Perform a differential
- Calculate M:E ratio
What is the estimated cellularity of children? 30-70 y/o? How do we estimate cellularity for >70 y/o?
Children = 80%
30-70 y/o = 50%
> 70 y/o = subtract patient age from 100% and add +/- 10%
(Ex. 75 y/o = 15-35%
Osteoblast vs osteoclast
Osteoblast = bone formation
Osteoclast = bone resorption
What is the purpose of the H&E stain?
Used for the examination of core biopsy specimen when aspiration procedure yields a dry tap; assess cellularity but cannot perform a differential
What is the purpose of cytochemical studies on BM?
Diagnosis of leukemias/lymphomas
What is the purpose of cytogenetic studies on BM?
Diagnosis of acute leukemias via chromosomal abnormalities
What is the purpose of molecular studies on BM?
PCR for diagnostic point mutations
What is the purpose of FISH testing on BM?
Staining for diagnostic mutations
What is the purpose of flow cytometry testing on BM?
Immunophenotyping of malignant hematopoietic cells
What information may be included on a bone marrow examination report?
- Patient medical history and CBC results
- Diagnostic narrative:
Summary of the recorded BM findings and additional lab chemical, microbiologic, and immunoassay tests
Each tech counts _____ cells during a BM exam at RH.
250
What is meant by the term “bone marrow failure”?
Reduction/cessation of blood cell production affecting one or more cell lines (PLTs/WBCs/RBCs)
What is the result of bone marrow failure?
Pancytopenia
Name 6 potential causes of bone marrow failure
- Destruction of HSCs through drugs/chemicals/radiation etc.
- Premature senescence and apoptosis of HSCs due to genetic mutations
- Ineffective hematopoiesis
- Disruption of BM microenvironment
- Decreased production of hematopoietic growth factors
- Loss of normal
hematopoietic tissue due to infiltration of marrow space with abnormal cells
What is the major form of BM failure?
Aplastic anemia
What are the characteristic features of aplastic anemia?
Pancytopenia
Reticulocytopenia
BM hypocellularity
Depletion of HSCs
Name a few associations/causes of aplastic anemia
Idiopathic –> unknown
Secondary –> drugs, chemicals, radiation (benzene/cytotoxic drugs)
Viral infections –> EBV, HIV, parvovirus B19
Most cases of aplastic anemia are _________ (85%) and few are ________ (15%).
acquired; inherited/congenital
What is the PRIMARY cause of aplastic anemia?
Quantitative and qualitative deficiency of HSCs
What are some laboratory findings associated with aplastic anemia?
Decreased ANC
HGB <10
Decreased retics
Decreased neuts/monos/PLTs
Macrocytic/normocytic RBCs
Toxic granulation
Increased iron and %TSAT
Decreased CD55 and CD59
Decreased CD24 and CD14
Severe hypocellularity in BM
What is the most common of the inherited bone marrow failure syndromes?
Fanconi anemia
What is Fanconi anemia?
Inherited chromosome instability disorder characterized by aplastic anemia, physical abnormalities, and cancer susceptibility
What population is most affected by Fanconi anemia?
Ashkenazi Jewish and South African Afrikaner populations
What are the clinical findings of those with Fanconi anemia?
Thumb malformations
Cafe-au-lait lesions on skin
Short stature
Increased cancer risk
What is the diagnostic test for Fanconi anemia?
Chromosomal breakage analysis; FA cells have increased fragility
Lab results of those with Fanconi anemia
Pancytopenia
Reticulocytopenia
Hypocellular bone marrow
What is the prognosis of someone with Fanconi anemia?
1/3 develop MDS and/or AML by 14 y/o
1/4 develop solid tumors by 26 y/o
> 90% develop BM failure by 40 y/o
If it goes untreated, death by 20 y/o due to BM failure.
Treatment for Fanconi anemia
HSCT
What are the clinical findings of someone with Dyskeratosis Congenita?
- Mucocutaneous abnormalities (abnormal skin pigmentation, dystrophic nails, oral leukoplakia)
- Increased cancer risk
- Multisystem abnormalities
- BM failure/pancytopenia
Genetics and pathophysiology of Dyskeratosis Congenita
DC chromosomes have very short telomeres leading to premature cell death and predisposition to cancer
Mutation in one of at least 11 genes, most commonly a mutation of the DKCI gene on the X chromosome
How to diagnose/detect Dyskeratosis Congenita?
Flow cytometry FISH test for detection of short telomeres
Pancytopenia and macrocytic RBCs
Treatment/prognosis of someone with Dyskeratosis Congenita?
Median survival = 42 yrs
Androgen therapy treats, but does not halt progression of BM failure
What is Shwachman-Bodian-Diamond syndrome (SBDS)?
Multisystem disorder characterized by pancreatic insufficiency, cytopenias, skeletal abnormalities, and a predisposition to hematologic malignancies
Clinical findings of SBDS?
- Peripheral blood cytopenia
- Decreased pancreatic enzyme secretion
- Immune dysfunction
- Short stature
Genetics/pathophysiology of SBDS
Biallelic mutations in the SBDS gene
Deficiencies in CD34+ cells
Increased apoptosis in HSCs
Short telomeres in granulocytes
Laboratory findings in SBDS
Neutropenia
Anemia/thrombocytopenia
Hypocellular bone marrow
Increased fecal fat
Treatment for SBDS
- G-CSF for neutropenia
- Transfusion support for anemia/thrombocytopenia
- Enzyme replacement for pancreatic insufficiency
- Stem cell transplant if AML/MDS develops
What is PRCA and how does it differ from the other types of BM failure disorders discussed?
Rare disorder of erythropoiesis characterized by severe decrease in erythroid precursors ONLY in an otherwise normal bone marrow
**Difference is that this disorder has normal WBC/PLT counts
What are the two types of PCRA and describe them
Acquired PRCA: aka transient erythroblastopenia of childhood, many patients have a history of viral infection
Congenital PCRA: Diamond-Blackfan Anemia - mutations in the RPS19 gene with craniofacial dysmorphisms, short stature, neck and thumb malformations
What are CDAs?
Congenital dyserythropoietic anemias characterized by refractory anemia, reticulocytopenia, hypocellular bone marrow with markedly ineffective erythropoiesis and distinctive dysplastic changes in BM erythroblasts
Briefly describe CDA I
Macrocytic RBCs
Megaloblastoid erythroblasts with internuclear chromatin bridges or nuclear stranding; spongy heterochromatin with a swiss cheese appearance
Briefly describe CDA II
The most common subtype
Normocytic RBCs
Normoblastic erythroblasts with some binucleated forms
Briefly describe CDA III
Least common subtype
Macrocytic RBCs
Megaloblastic erythroblsats with up to 12 nuclei
Describe myelophthisic anemia
Due to infiltration of abnormal cells into the BM and subsequent destruction and replacement of normal hematopoietic cells
- Leukoerythroblastic
What is the primary cause of anemia in chronic kidney disease?
Inadequate renal production of EPO
Define cytogenetics
The study of chromosomes, their structure, and their inheritance
What is the purpose of cytogenetic testing?
To analyze chromosome disorders
Why might cytogenetic testing be ordered on a patient?
Intellectual disability
Infertility
Ambiguous genitalia
Short stature
Fetal loss
Risk of genetic or chromosomal disease
Cancer
What is metaphase and why are metaphase chromosomes important in cytogenetic testing?
Metaphase is the stage in mitosis in which chromosomes align on the equatorial plate. This is important because they are highly condensed and have a high mitotic rate
Define haploid. What is the haploid number in humans?
Haploid represents gametes and have half the diploid number (23 chromosomes in these cells) it is represented as “n”.
Define diploid. What is the diploid number in humans?
Diploid is the number in dividing somatic cells and is 46 in humans. It is represented as “2n”.
Nucleosome
DNA looped around a core of histones
Solenoid
Nucleosomes are coiled into twisted forms
Metacentric centromeres
Centromere located in the middle of the chromosome
Submetacentric centromeres
Off centered centromeres
Acrocentric centromeres
Located at one end of the chromosome
Mitogens
Stimulate division of specific cell types
p arm vs q arm
p arm = short arm of chromosome
q arm = long arm of chromosome
What is Q banding?
Differentiates chromosomes into bands of differing widths and relative brightnesses
Utilizes a dye called quinacrine mustard which binds to A-T rich areas
What is G banding?
The most common method used for staining chromosomes
Utilizes Giemsa stain, stains A-T rich areas
What is C banding?
Stains the centromere and the surrounding condensed heterochromatin; Giemsa stain used
Karyotype
Summary of the chromosome analysis, resulted into a karyogram (picture of all the chromosomes aligned from 1-22)
Describe FISH. What information can FISH results provide?
A molecular technique used to detect and locate a specific DNA sequence on a chromosome
- Can provide information on structural/numeric abnormalities of chromosomes, Ability to diagnose
How would the karyotype nomenclature look like for a patient with a Trisomy of chromosome 8 in a male?
47,XY,+8
What would the karyotype nomenclature for a normal male and female look like?
Male: 46,XY
Female: 46,XX
Translocation
Designated “t”, gene has moved from one area to another
Deletion
Designated “del”, gene has been deleted from a chromosome, loss of the segment
Aneuploidy
ANY abnormal number of chromosomes that is NOT a multiple of the haploid number (23)
Trisomy
Presence of an extra chromosome
Monosomy
Absence of a single chromosome
Polyploidy
Chromosome number higher than 46, but is ALWAYS an exact multiple of the haploid chromosome number (23)
Ex. Triploidy and Tetraploidy
Triploidy
Karyotype with 69 chromosomes (3n)
Tetraploidy
Karyotype with 92 chromosomes (4n)
Hypodiploid
Cell has fewer than 45 chromosomes
Hyperdiploid
Cell has more than 46 chromosomes
High hyperdiploidy
Cell has more than 50 chromosomes
Inversions
One or two breaks in a single chromosome, no loss or gain of material
Insertion
Movement of a segment of a chromosome from one location to another
Duplication
Partial trisomy for part of a chromosome
What would Down syndrome nomenclature look like for a male?
46,XY,21+
What is the tissue most frequently used to study the cytogenetics of a hematologic malignancy?
Bone marrow
What abnormal chromosome is associated with CML and how is it treated? How is the treatment monitored?
Philadelphia chromosome
Treated with imatinib mesylate
Pt response is monitored by cytogenetic analysis and FISH
What is the amplification of HER2 gene on chromosome 17 associated with?
Aggressive form of invasive breast cancer
What is a good prognosis for childhood ALL?
> 50 chromosomes (high hyperdiploidy)
