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Lec 25 Bone Marrow and Cytochemistry

Terms in this set (51)

Romanowsky stain
● Consists of Methylene Blue and Eosin
● Oxidized methylene blue forms azures or basic compounds that stain acidic components of cell (nucleus)
● Eosin, acidic dye, binds to basic cellular structures like cytoplasm and Hgb
● ex: Wright, Wright-Giemsa, May-Grunwald, Lishman, Jenner
How do cell components stain?
○ PMN granules, slightly basic, stain weakly with azures - lilac
○ Eos granules, basic, stain with eosin - red/orange
○ Basophils - acidic, stain w/ azure, black/dark purple
Buffy Coat
made from concentrated WBC
○ do when diff WBC <2500
○ perform diff on buffy coat
○ perform WBC estimate, RBC morphology, and platelet estimate on regular smear
Bone Marrow
○ proximal portion of long bones in adults (location of medullary hematopoiesis)
○ at birth, all bones contain hematopoietic marrow but fat replaces marrow age 5-7
Normal vs Fatty BM (bone marrow)
○ Normal BM (bone marrow) is rusty red, thick fluid consistency, with various amounts of fat and spicules (gray white marrow fragments)
○ Need spicules present to be able to analyze
○ Fatty BM is yellow and haematopoietically inactive
Bone Marrow Biopsy
○ sterile technique
○ MLS - processes specimen, determine if adequate
○ anterior or posterior iliac crest (most common)
Bone marrow biopsy needles and prep
○ Trephine needle (Jamshidi) that has cutting edge
○ Touch prep slide of bone biopsy
○ Bone biopsy goes into decalcification and is processed by histology
Bone Marrow Aspirate
○ aspirate 1.0-1.5mL
○ make slides fast to prevent clotting
○ crush prep smears of spicules
○ spun down in wintrobe tube
○ Dry Tap: myelofibrosis, packed marrow in leukemia, and aplastic anemia
What patient has a higher cellularity?
○ Infants/Children: 70-90%
○ Teens/Young Adults: 50-80%
○ Adults/Middle Age: 30-60%
○ Elderly: 20-40%

Infants/kids have most, as you get older you have less.
Fat content BM
none in infants, more in adults
Smear Examination of BM
○ Fat content
○ Cellularity = volume of hematopoietic cells
○ Total marrow space = cells + fat
Bone Marrow Smear Exam
○ Examine under low, high, oil
○ 500 cell diff under oil
○ Low power - look for predominant cell type
--look for abnormal cells or clumps
--estimate # megakaryocytes (1-3 normal)
--estimate M:E ratio
Heterogenous BM
different cell types, or one cell type looks multiple ways (ex: reg lymphs and reactive lymphs)
ME ratio
ME ratio is how many white cells to red cells

myeloid: erythroid or
myeloid: NRBC
M:E Ratio
Normal is 3:1 or 4:1
ME Ratio increased/decreased
↑↑ : Infection (leukemoid reaction), CML, erythroid hypoplasia

↓↓ : depressed leukopoiesis, normoblastic hyperplasia
BM - Erythroblastic island
○ Erythroblasts are 25-30% of marrow cells
○ single macrophage surrounded by erythroblasts
○ macrophage cytoplasm extends out to surround the erythroblasts
○ Erythroblasts acquired Fe from macrophage
○ Young cells in center vs mature cells at periphery
Tools necessary for classification of acute leukemia (important)
○ Wright stain morphology of PB and/or BM
○ Immunophenotyping
○ Cytogenetics
○ Cytochemistry
Cytochemistry (important!)
○ Identify malignant cells in leukemia and lymphomas
○ Sudan Black B
○ Myeloperoxidase
○ Specific esterases - present in myeloid cells
○ Nonspecific esterases - monocytes
Sudan Black B (SBB) (important)
○ stains ( + ) cells brown black
○ stains fat particles
○ Myelocytic, monocytic granules, Auer rod membranes are rich in phospholipids ( + )
Sudan Black B (SBB) common results
(important)
Myeloblasts +
Monoblasts +/-
Lymphoblasts --
Control: Segs +
Myeloperoxidase
(important)
○ stains reddish brown
○ stains myeloperoxidase enzyme found in granules of neutrophils, eos, and monos
○ myeloperoxidase enzyme catalyzes oxidation of substances by H2O2
○ Benzidine stain as the color reagent with Giemsa counterstain
Myeloperoxidase results
(important)
( + ) in myelocytes and monocytes
MPO and SBB
(important)
○ MPO and SBB are performed first in lab testing
○ Myeloblasts are ( + )
○ Lymphoid blasts are ( - )
Periodic Acid Schiff Stain (PAS)
○ stains cytoplasm pink
○ 1,2-Glycol groups in presence of periodic acid form Aldehydes in presence of Schiff Reagent form pink color
○ cellular components containing glycogen will stain
PAS Stain results
Lymphoblasts and erythroblasts in Acute Erythrocytic Leukemia (DiGuglielmo's) stain ( + )
○ Lympholasts in ALL ( + )
○ Myeloblasts ( - )
○ Control: PMNS strong (+ )
PAS More results
Erythroid leukemia erythroblasts - strong ( + )
○ Normal erythroblasts ( - )
○ Lymphoblasts in ALL ( + )
What other condition causes an increase of Erythroblasts?

Would these erythroblasts stain PAS+ ?
○ Could see an increase when there's an increase in RPI (certain anemias)
○ These erythroblasts stain PAS+
Erythroblast
immature RBC with a nucleus
Specific esterase
Chloroacetate Esterase
Positive (+) for myeloblasts
Non specific esterase
Alpha-naphthyl acetate (ANAE)
Positive (+) with monoblasts
Specific Esterases
Chloroacetate Esterase
stains cytoplasm RED
○ Isoenzymes (1, 2, 7, 8, 9) found in myeloblasts stain -- specific for granulocytic series and mast cells
○ Less sensitive, but parallels MPO and SSB
Non Specific Esterases
stains cytoplasm dark blue/black
○ enzymes that hydrolyze esters found in monocytes and macrophages
○ Also megakaryocytes, hairy cells, T-lymphs
○ Isoenzymes (3, 4, 5, 6) found in monocytes
○ Used to diagnose Acute Monocytic Leukemia
Specific esterase (Chloroacetate)
Auer rods (+)
Leukocyte Alkaline Phosphatase (LAP)
○ Enzyme in secondary granules of maturing neutrophils
○ Only seen in reacting neutrophils NOT in neoplasms
○ LAP would be high in a leukemoid reaction
○ LAP is low in chronic myelocytic leukemia
Leukemoid Reactions vs CML
Acid phosphatase
○ present in lysosomes of all WBC
○ especially characteristic in T-cell ALL - see focal polarization
Hairy cells (important!)
○ In presence of tartaric acid, Hairy cells are still positive (TRAP+)
○ all other WBC become negative
TRAP
TRAP = tartrate resistant acid phosphatase

○ Naphthol phosphate substrate is hydrolyzed by acid
○ ○ Phosphatase to napthol + diazo amine → red color (all WBC + hairy cells)
○ Napthol + diazo amine + tartate → red color (Hairy Cells only)
TdT (Terminal Deoxynucleotidyl Transferase)
○ DNA Polymerase
○ Found in ALL and malignant lymphoma
○ 20% AML show (+)
○ 90% of the minimally differentiated AML subgroup can be TdT (+)
Toluidine Blue
○ (+) for basophils and mast cells
○ especially AML with basophilic differentiation
○ stains acid mucopolysaccharides
○ mast cells found in tissue
H&E (Hematoxylin and Eosin Stain)
○ Hematoxylin is a dark blue or violet stain that is basic/positive.
○ It binds to DNA/RNA -which are acidic and neg(-) charged and stain them violet

○ Eosin is a red or pink stain that is acidic. It binds to acidophilic substances, and stains them pink.
H&E (Hematoxylin and Eosin Stain) in a cell
In a Cell:
○Nuclei in blue/purple
○ Basophils in purplish red
○ Cytoplasm in red
○ Erythrocytes in cherry red
Prussian Blue
○ stains Iron Blue
○ Prussian blue (HCl + K ferrocyanide)
○ To differentiate iron deficiency anemia from iron overload (i.e. Thalassemia, sickle cell disease)
○ Ringed sideroblasts are (+)
Molecular Analysis
PCR analysis of specific genome mutations
○ BCR-ABL1 gene
○ JAK2 (MPNs) - ET, PV, MF, CML
Immunophenotyping
○ technique used to study protein expression on cells (CD markers)
○ Done on tissue, cell suspensions, blood, bone marrow
○ Labels WBC with antibody against surface proteins on the cell membrane
○ Processed with a flow cytometer (laser based)
CD markers
cluster of differentiation
CD Markers to know (important!)
● HSC (Stem Cell) CD34 +
● All leukocytes show CD45+
● B-cells: CD19+ many are CD20+
● T-Cells: CD3+ CD4+ CD8+
● Monocytes: CD14+
● Monocytes and Myeloid Cells: CD13+ CD33+
CD Markers to know cont'd (important!)
● Plasma Cell Neoplasms: CD138+ CD38+
● Sezary Syndrome: CD4+
● Hairy Cell Leukemia: CD103+
FACTS TO KNOW 1
●Bone marrow cellularity is the volume of hematopoietic cells/cells + fat
●Bone marrow cellularity is highest in infants and children and decreases with age
●The M:E (myeloid:NRBC) ratio is 3:1 or 4:1
●LAP is increased in a leukemoid reaction
●LAP is decreased in CML
●Hairy cells are TRAP positive
FACTS TO KNOW 2
● The PAS reaction stains glycogen pink
● Lymphoblasts and erythroblasts are PAS positive
● Monoblasts stain positive with non-specific esterase stains
● Sudan Black B stains phospholipids in primary and secondary granules and Auer rods
● Myeloblasts are SBB and myeloperoxidase positive; Lymphoblasts are SBB and peroxidase negative
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