Unit 2: Bone Marrow & Stains Flashcards
Fat cell conversion (yellow marrow) occurs at what age?
- 4 years of age
- 18 years of age
-sternum, skull,
Proximal end of large bones,
vertebrae, Iliac Crest
- Spoke-like pattern of venous sinuses and cords of
hematopoietic tissue - Contains all the developing blood cell lines
Red bone marrow?
the process of replacing the active marrow by fat tissue
during development; results in restrictive active marrow sites
Retrogression
Major function of bone marrow?
production and
proliferation of blood
cells
Minor function of bone marrow?
antigen processing of
cellular and humoral
immunity
What are the three main reasons for preforming a bone marrow evaluation?
- In pts. with solid malignant tumors (Ex., lymphomas, carcinomas & sarcomas, with
possible “mets” to bone marrow) - As part of initial workup of unexplained ↑ or ↓ in RBCs, WBCs, &/or plts.
- As part of differential diagnosis workup for
infections that manifest clinically as “fevers of unknown origin ”.
Three findings used to verify bone marrow has been obtained (rather than p.b.)?
Presence of:
* Fat droplets
* Bone spicules
* Very immature hematopoietic cells
Four Preferred Locations for bone marrow tap (in order of preference)
- Posterior iliac crest (adults & children
- Sternum (adults)
- Vertebrae (in adults)
- Tibia (children < 1 yr. only)
Bone Marrow Tap” Procedure:
Less than 24 hrs before procedure, a CBC and manual differential are performed.
1. The MLS usually brings the biopsy kit to the patient’s
room.
2. Light general sedation is usually administered.
3. Area is washed with soap, antiseptic is applied, & site
is draped with sterile towels.
4. Local anesthetic (typically 2% lidocaine) is injected
into the skin over the intended site.
glass slide. The bone marrow is seen as gray particles
floating in among blood & fat droplets.“Bone Marrow Tap” Procedure:
5. Once skin is numb, local anesthetic is injected into the bone surface at the selected site.
6. Skin incision is made over the bony site, & the doctor inserts a needle into the bone marrow cavity.
7. The inner needle (obturator) is removed.
8. Vacuum is applied by pulling a syringe; 1st the bone marrow is aspirated, & then the trephine (core) biopsy is removed.
9. Smears are made from this aspirate by pouring a drop onto a glass slide. The bone marrow is seen
10. The marrow pieces are removed gently with a forceps,
placed between 2 clean glass slides, & the slides pulled in
opposite directions (“pull smears”).
11. Direct smears are made from drops left in the syringe.
12. Core biopsies are removed from the special syringe
attachment with forceps & drained of blood by placing
against sterile gauze. The core is touched lightly to 2-3 clean glass slides to make “touch preps” or “imprint films”, which are air-dried.
13. Remaining aspirate is used for cytogenetic workups, stains (Giemsa, Hematoxylin-Eosin, & Prussian Blue are standard),
What are the two needles used for bone marrow taps?
-Jamshidi
-Westerman-Jensen
needle
What are the five types of NORMAL bone marrow cells?
- Developing hematopoietic cells
- Macrophages or Histiocytes
More rarely: - Megakaryocytes
- Osteoblasts
- Osteoclasts
Normal bone marrow cells:
Developing hematopoietic cells (blasts of all types,
normally at overall ___% cellularity)
5
large cells, with
abundant cytoplasm & debris-filled vacuoles, &
irregular, “spreading” shape.
- Will be ↑ in disorders with rapid cell turnover (such as
leukemias & leukemoid reactions.)
Macrophages or Histiocytes
involved in platelet formation through endomitosis.
Megakaryocytes
part of bone marrow stroma; specialized bone matrix-__________cells.
Oval, elongated cells with eccentric nuclei & cometary-appearing cytoplasm.
Osteoblasts
Synthesizing
Is it common to see Osteoblasts and osteoclasts in bone marrow samples?
no, rare in normal adult bone marrow!
-huge (>100 u), multi-nucleated cells with ruffled border; formed from fusion of monos & macro-phages!
-Responsible for bone demineralization & resorption, thus ↑ whenever
bone destruction occurs.
Osteoclasts
What are the 7 aspects of routine bone marrow evaluation?
-Cellularity
-Differential cell count
-Type & concentration of abnormal aggregates
-Number & morphology of megakaryocytes
-Presence & degree of fibrosis (if any)
-Presence of abnormal intra- or extra-cellular material (if any).
-Presence of abnormal changes in bony ultrastructure (if any)
Where is routine bone marrow evaluation usually done?
usually done in the Pathology department, but can be done in the Hematology department of Oncology centers.
Aspects of Routine Bone Marrow Evaluation:
judged as normal, ↑ (hyperplastic) or ↓
(aplastic/hypoplastic); all evaluated on 10X.
Also reflected in ratio of fat cells to hematopoietic cells (which is normally 1:2 in adults).
Cellularity
Aspects of Routine Bone Marrow Evaluation:
Differential cell count (cellular distribution) – evaluated on 100X oil immersion. Requires counting _________ cells! Results highly variable.
-After count, M:E ratio is calculated
500-1000
Aspects of Routine Bone Marrow Evaluation:
Type & concentration of abnormal aggregates – especially estimation of storage Fe (essential in severe anemia diagnosis).
Requires____________ stain.
Prussian Blue
Aspects of Routine Bone Marrow Evaluation:
Fe stores reported as…
absent, ↓, adequate, mod. ↑ or mkd.
Infant bone marrow has little to no _____.
fat
normally M:E ratio ranges from _______, & is slightly higher in infants
2:1 - 4:1
What are the largest cells in normal bone marrow?
megakaryocytes
Bone marrow aspirate microscopic examination:
What is done on low power: 10x? (100x total magnification)?
-assess peripheral blood dilution
-find bony spicules and areas of clear cell morphology
-observe fat-to-marrow ratio, estimate cellularity
-search for tumor cells in clusters
-examine and estimate megakaryocytes
Bone marrow aspirate microscopic examination:
What is done on high power: 50x and 100x (500 and 1000x total magnification)?
-observe myelocytic and erythrocytic maturation
-distinguish abnormal distribution of cells or cell maturation stages
-perform differential count on 300 to 1000 cells
-compute myeloid-to-erythroid ratio
Normal Adult Bone Marrow Diffs in Concentrated Smears (1000-Cell Counts):
which single cell line
is most abundant in adult bone marrow? 2nd?
Neutrophils (various stages)
RBCs & their precursors
*Adult M:E ratio is range of 2:1 – 4:1
(Infant M:E ratio ranges from 5:1 – 6:1)
Myeloperoxidase (MPO or MPX) is pos in which AMLs?
M1 – M4
Myeloperoxidase (MPO or MPX) positive shows what color cytoplasmic granules?
gray-black or red-brown
Myeloperoxidase (MPO or MPX) stain reacts with what in monocytic cells?
w/ lysosomal enzyme in 1o granules of myeloid &
(to lesser extent)
Myeloperoxidase (MPO or MPX):
What gives the strongest reaction? What shoes less + (scattered pattern)?
Mature granulocytes give strongest + rxn.; monos & immature granulocytes show less + (scattered pattern)
Since MPX is NOT found in lymphoid cells, it is best used for differentiating between…
AML and ALL***
Sudan Black B (BB) is positive in which AMLs?
M1-M4
SBB stains what components of cells? and where is it found?
-lipoproteins & phospholipids.
-Found in 1o (azurophilic) & 2o granules of mature & immature neutrophils (& in eos, & slightly in monos & monoblasts.)
Sudan Black B (SBB) is negative for…
lymphs, megakaryocytes & erythroid precursors
SBB results parallel those for…
MPO*
-FYI: MPO faster, but SBB more stable & can be run on older specimens
SBB is best used for differentiating…
AML from ALL
Specific Esterase (SE, or Napthol AS-D Chloroacetate Esterase, NASD) is positive in which AMLs?
M1 – M4
SE/NASD:
positive cells show ________ staining of cytoplasmic granules.
reddish
SE/NASD stain is useful in separating what precursors?
monocyte precursors from granulocyte precursors
SE present in primary granules of ___________ but shows a negative or weak positive reaction in ______________.
-neutrophils (& mast cells)
-monocytes or lymphocytes
SE/NASD stain:
-Auer rods of AML myelocytes strongly +, Why?
bc. they’re fused primary granules & contain SE
Nonspecific Esterase (Alpha-Napthyl Butyrate Esterase, NBE):
positive cells show ___________ appearance.
reddish
NBE + in …
NBE – in…
monocytic cells, and T-cells
myeloid cells and megakaryocytes
The NSEs primarily are used to differentiate…
myeloid leukemias from monocytic ones!***
NAE & NBE frequently done together: just called
“______________”. (They can be done along with the Specific Esterase stain, as well.)
Nonspecific Esterases [NSEs]
Nonspecific Esterase (Alpha-Napthyl Butyrate Esterase, NBE)
* the “_____________” makes the nonspecific esterase, more specific
* NaFl inhibits the enzymatic activity in monocytes….other cells are unaffected
fluoride inhibition step
- More sensitive than NBE.
- Pos. cells show brownish appearance.
Nonspecific Esterase (Alpha-Napthyl Acetate Esterase, NAE)
NAE strongly + in…
monocytes, T-cells, &
megakaryocytes
FYI: If you add NaFl, this reaction goes away
Periodic Acid Schiff (PAS) stains glycogen.
Many cell types stain positive… it’s the _______ that’s unique.
pattern
(Ex., plts. can be 4+, monos can be 1+.)
PAS positive cells show ____ color.
red
Periodic Acid Schiff (PAS):
- Staining intensity & pattern varies with cell type/maturity.
- Most common reaction patterns are…
diffuse, granu-lar, & mixed.
Normal RBC precursors are NOT PAS + ! This is important in identifying which of the AMLs?
?
(slide 49)
PAS pos. in 4 groups of disease states:
*80% ALL (chunky or block pattern)
*CLL
*Gaucher’s disease
*Some AML (AMoL, AEL, AMegL)
(Warning: degree of positivity can vary from cell to cell within same specimen from same patient!)
Used to identify T-cell subsets of ALL & lymphomas.
Acid Phosphatase (ACP)
Acid Phosphatase (ACP):
granules stain _____.
red
ACP + in…
monocytes, neutrophils, and T-cells
Acid Phosphatase (ACP) positive in which AMLs?
AMegL
Tartrate Resistant Acid Phosphatase (TRAP) Principle?
Add L-Tartaric acid, then stain. Normal cells will not retain acid phos. activity; however, HCL cells do retain this activity, bc. they have
a different acid phos. isoenzyme: thus called “TRAP +”.
Tartrate Resistant Acid Phosphatase (TRAP):
Used to diagnose ___________ bc. only these cells strongly + with this modified stain.
Hairy Cell Leukemia (HCL)
Only neutrophils contain this enzyme (in varying
amounts) in their secondary granules.
Leukocyte Alkaline Phosphatase (LAP)
Leukocyte Alkaline Phosphatase (LAP):
FYI: Pos. cells show _____ ppt. with fast red violet stain;
show _____ ppt. with fast blue violet stain.
red, black
Used to help differentiate early CML from other
conditions like leukemoid rxn. or PV (at a screening
level only.)
Leukocyte Alkaline Phosphatase (LAP)
LAP scoring:
- Count only segs & bands!
- how many cells do you count?
100
LAP scoring:
What is a normal sore?
~ 20-100 (varies by institution)
LAP scoring:
0 =
no granules
LAP scoring:
1+ =
very few granules (< 50% of cytoplasm)
LAP scoring:
2+ =
mod. granules scattered throughout (50 – 80%
of cytoplasm)
LAP scoring:
3+ =
numerous granules starting to coalesce (80 –100% of cytoplasm)
LAP scoring:
4+ =
cytoplasm packed with granules (only nucleus
remains visible)
How do you calculate LAP score?
Take # cells seen in each grade & multiply by that grade, then add products of
each grade together for final score. (NO UNITS!)
Example:
* 31 cells received grade 2, so 31 X 2 = 62
* 7 cells received grade 1, so 7 X 1 = 7
* 31 cells received grade 0, so 31 X 0 = 0
* 62 + 7 + 0 = 69 total score
An increased LAP score of >110 is associated with…
*polycythemia vera
*leukemoid reaction
*bacterial infections
-3rd trimester pregnancy
-steroid therapy
-chronic granulocytic leukemias
-blast crises
-chronic neutrophilic leukemias
-CML with infections
-Myelofibrosis
Normal Lab scores are associated with…
-late CML
-CML in remission
-secondary erythrocytosis
A decreased LAP score of <15 is associated with…
*early CML
-PNH
-sideroblastic anemia
-marked eosinophilia
-sickle cell anemia
-improper technique
-Myelodysplastic disorders
-viral infections
(Perl’s) Prussian Blue Iron Stain principle?
Fe 3+ (ferric) + potassium cyanide –>
ferricyanide (blue-green ppt.)
(Perl’s) Prussian Blue Iron Stain is used to evaluate RBC ____ stores: reported semi-quantitatively.
Fe
3 conditions with increased bone marrow [Fe] are:
ACD
Hemochromatosis
Sideroblastic anemias.
