Bone Marrow & Stains.2022.st.pdf

BONE MARROWS & SPECIAL STAINS MDLS 4226/5226 Hematology II OBJECTIVES: • In accordance with MLS student guidelines, at the completion of this lecture the student is expected to use the information gained to correctly: 1. State the 3 main indications for a bone marrow evaluation. 2. Evaluate the pr...

BONE MARROWS & SPECIAL STAINS MDLS 4226/5226 Hematology II OBJECTIVES: • In accordance with MLS student guidelines, at the completion of this lecture the student is expected to use the information gained to correctly: 1. State the 3 main indications for a bone marrow evaluation. 2. Evaluate the process of collecting a bone marrow sample. 3. Differentiate between a bone marrow aspirate and a bone marrow core biopsy. 4. Analyze how verification of bone marrow aspirate is obtained. 5. List the preferred locations for a bone marrow tap, in order of preference. 6. Distinguish the 5 cell types found in normal bone marrow and their predominance. 7. Assess the 6 aspects of a routine bone marrow evaluation. 8. State the 3 main indications for a bone marrow evaluation. OBJECTIVES: 9. Evaluate how one can verify that bone marrow aspirate has been obtained. 10. Examine the following key terms as they relate to the assessment of bone marrow: differential cell count, M:E ratio, & aplastic / hypoplastic / hyperplastic marrow. 11. Validate the procedure for performing a bone marrow differential. 12. Design a study aid to diagram all of the various cytochemical & immunocytochemical staining techniques used in the diagnosis of leukemias. 13. Given the appropriate information, calculate an LAP score on a bone marrow, & evaluate the criteria for such an evaluation. Hematopoietic Sites (courtesy Peripheral Blood Tutor CD) Bone Marrow • Fat cell conversion (yellow marrow) • 4 years of age • 18 years of age sternum Proximal end of large bones vertebrae Iliac Crest skull pattern • Spoke-like of venous sinuses and hematopoietic tissue (red marrow) cords of • Contains all the developing blood cell lines Retrogression – the process of replacing the active marrow by fat tissue during development; results in restrictive active marrow sites Bone Marrow Functions • Minor function in antigen processing of cellular and humoral immunity • Major function is the production and proliferation of blood cells Bone Marrow Procedures • Three main reasons for performing a bone marrow evaluation: 1. In pts. with solid malignant tumors (Ex., lymphomas, carcinomas & sarcomas, with possible “mets” to bone marrow) 2. As part of initial workup of unexplained ↑ or ↓ in RBCs, WBCs, &/or plts. 3. 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) 1. 2. 3. 4. Posterior iliac crest (adults & children Sternum (adults) Vertebrae (in adults) Tibia (children < 1 yr. only) Posterior superior iliac crest “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. Jamshidi bone marrow needle Westerman-Jensen needle “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 as gray particles floating in among blood & fat droplets. https://www.youtube.com/watch?v=EYd7OnCt7ug “Bone Marrow Tap” Procedure Cont. 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), etc. Many special stains also are available. https://www.youtube.com/watch?v=0ZTGoPmCV1w Bone Marrow Aspirate Smear Bone marrow components Five Types of NORMAL Bone Marrow Cells: 1. Developing hematopoietic cells (blasts of all types, normally at overall 5% cellularity). 2. Macrophages or Histiocytes - large cells, with abundant cytoplasm & debris-filled vacuoles, & irregular, “spreading” shape. - Will be ↑ in disorders with rapid cell turnover (such as leukemias & leukemoid reactions.) Bone Marrow Macrophage Five Types of NORMAL Bone Marrow Cells: More rarely: 3. Megakaryocytes - involved in platelet formation through endomitosis. 4. Osteoblasts - part of bone marrow stroma; specialized bone matrixsynthesizing cells. Oval, elongated cells with eccentric nuclei & cometary-appearing cytoplasm. Rare in normal adult bone marrow! 5. Osteoclasts – 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. Rare in normal bone marrow! Megakaryocyte in bone marrow Osteoblasts in bone marrow Seven Aspects of Routine Bone Marrow Evaluation: 1. Cellularity - 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). (FYI: Infant bone marrow has little to no fat!) 2. Differential cell count (cellular distribution) – evaluated on 100X oil immersion. Requires counting 500-1000 cells! Results highly variable. After count, M:E ratio is calculated (normally M:E ratio ranges from 2:1 - 4:1, & is slightly higher in infants.) 3. Type & concentration of abnormal aggregates – especially estimation of storage Fe (essential in severe anemia diagnosis). Requires Prussian Blue stain. (FYI: Fe stores reported as absent, ↓, adequate, mod. ↑ or mkd. ↑.) Seven Aspects of Routine Bone Marrow Evaluation (cont.): 4. Number & morphology of megakaryocytes (largest cells in normal bone marrow!) 5. Presence & degree of fibrosis (if any). 6. Presence of abnormal intra- or extra-cellular material (if any). 7. Presence of abnormal changes in bony ultrastructure (if any) due to severe “space-occupying lesions.” NOTE: This evaluation is usually done in the Pathology department, but can be done in the Hematology department of Oncology centers. Bone Marrow Aspirate Microscopic Examination Guidelines for Normal Adult Bone Marrow Diffs in Concentrated Smears (1000-Cell Counts) Guidelines for Normal Adult Bone Marrow Diffs in Concentrated Smears (1000-Cell Counts) • According to the preceding table, which single cell line is most abundant in adult bone marrow? Neutrophils (various stages) • Which single cell line is the 2nd most abundant in adult bone marrow? RBCs & their precursors • How does this correlate with what you already knew about bone marrow cellularity & distribution in adults & children? Adult M:E ratio is range of 2:1 – 4:1 (Infant M:E ratio ranges from 5:1 – 6:1) Special Cytochemical Stains: 1. Myeloperoxidase (MPO or MPX) M1 – M4 • Pos. in which AMLs? _______ • Pos. cells show gray-black or red-brown cytoplasmic granules. • Stain reacts w/ lysosomal enzyme in 1o granules of myeloid & (to lesser extent) of monocytic cells. • 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 & ALL!! MPO Stain MPO Stain Special Cytochemical Stains: 2. Sudan Black B (SBB) M1 – M4 • Pos. in which AMLs?________. • SBB stains lipoproteins & phospholipids. • Found in 1o (azurophilic) & 2o granules of mature & immature neutrophils (& in eos, & slightly in monos & monoblasts.) • Neg. for lymphs, megakaryocytes & erythroid precursors. • Results parallel those for MPO! • So this stain is also best used for differentiating AML from ALL. FYI: MPO faster, but SBB more stable & can be run on older specimens SBB Stain MPO vs. SBB Special Cytochemical Stains: 3. Specific Esterase (SE, or Napthol AS-D Chloroacetate Esterase, NASD) M1 – M4 • Pos. in AMLs? _______ • Pos. cells show reddish staining of cytoplasmic granules. • Useful in separating monocyte precursors from granulocyte precursors • SE present in 1o granules of neutrophils (& mast cells), but shows a negative or weak positive reaction in monocytes or lymphocytes. • Auer rods of AML +! Why? bc. they’re fusedmyelocytes 1o granulesstrongly & contain SE! ____________________________________ SE (NASD or AS-D) Stain Specific Esterase Stain Special Cytochemical Stains: 4. Nonspecific Esterase (Alpha-Napthyl Butyrate Esterase, NBE) • Pos. cells show reddish appearance. • NBE + in monocytic cells, and T-cells • NBE – in myeloid cells and megakaryocytes • The NSEs primarily are used to differentiate myeloid leukemias from monocytic ones! • NAE & NBE frequently done together: just called “Nonspecific Esterases [NSEs]”. (They can be done along with the Specific Esterase stain, as well.) Special Cytochemical Stains: 4. Nonspecific Esterase (Alpha-Napthyl Butyrate Esterase, NBE) • the “fluoride inhibition step” makes the nonspecific esterase, more specific • NaFl inhibits the enzymatic activity in monocytes….other cells are uneffected NBE Stain Special Cytochemical Stains: 5. Nonspecific Esterase (Alpha-Napthyl Acetate Esterase, NAE) • More sensitive than NBE. • Pos. cells show brownish appearance. • NAE strongly + in monocytes, T-cells, & megakaryocytes FYI: If you add NaFl, this reaction goes away in monocytes: called “NaFl inhibition”. NAE Stain NAE Stain The bone marrow aspirate specimen (a–d). A May-Grunwald-Giemsa-stained bone marrow aspirate smear showed numerous hypergranular blasts with intense azurophilic granulation and Auer rods (faggot cells) (a). On cytochemical staining, the blasts were strongly positive on myeloperoxidase (MPO) staining (b), and strongly dual-positive on double-staining by naphthyl butyrate (nonspecific) esterase (NSE) and naphthol-ASD-chloroacetate esterase (CAE) (c). Sodium fluoride (NaF) inhibited NSE staining (d) Special Cytochemical Stains: 6. Periodic Acid Schiff (PAS) • PAS stains glycogen. • Many cell types stain positive… it’s the pattern that’s unique. (Ex., plts. can be 4+, monos can be 1+.) • Pos. cells show red color. • Staining intensity & pattern varies with cell type/maturity. • Most common reaction patterns are: diffuse, granu-lar, & mixed. • Remember: • Normal RBC precursors are NOT PAS + ! This is important in identifying which of the AMLs? Special Cytochemical Stains: 6. PAS (cont.) 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!) PAS Reactions PAS Reactivity Special Cytochemical Stains: 7. Acid Phosphatase (ACP) • Used to identify T-cell subsets of ALL & lymphomas. • Granules stain red • ACP + in monocytes, neutrophils, and T-cells • Pos. in which AMLs? _______ AMegL 7a. Tartrate Resistant Acid Phosphatase (TRAP) Principle: Add LTartaric 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 +”. • Used to diagnose Hairy Cell Leukemia (HCL) bc. only these cells strongly + with this modified stain. Acid Phosphatase Stain TRAP stain reactivity Special Cytochemical Stains: 8. Leukocyte Alkaline Phosphatase (LAP) • FYI: Pos. cells show red ppt. with fast red violet stain; show black ppt. with fast blue violet stain. • Only neutrophils contain this enzyme (in varying amounts) in their 2o granules. • Used to help differentiate early CML from other conditions like leukemoid rxn. or PV (at a screening level only.) LAP scoring: • Count only segs & bands! • Count 100 cells. • Normal Score ~ 20-100 (varies by institution) • 0 = no granules • 1+ = very few granules (< 50% of cytoplasm) • 2+ = mod. granules scattered throughout (50 – 80% of cytoplasm) • 3+ = numerous granules starting to coalesce (80 – 100% of cytoplasm) • 4+ = cytoplasm packed with granules (only nucleus remains visible) LAP scoring: • To calculate 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; our N.R. = ? 20-100 LAP reactivity LAP Scores in Various Diseases: Increased (> 110) Normal Decreased (< 15) Polycythemia vera Late CML Early CML Leukemoid reaction CML in remission PNH Bacterial infections Secondary erythrocytosis Sideroblastic anemia 3rd trimester pregnancy Marked eosinophilia Steroid therapy Sickle cell anemia Chronic Granulocytic Leukemias Improper technique Blast Crises Myelodysplastic disorders Chronic Neutrophilic Leukemias PNH CML w/ infections Viral infections Myelofibrosis Special Cytochemical Stains: 9. (Perl's) Prussian Blue Iron Stain • Principle: Fe3+ (ferric) + potassium cyanide --> ferricyanide (blue-green ppt.) • Used to evaluate RBC Fe stores; reported semiquantitatively. • 3 conditions with increased bone marrow [Fe] are: ACD Hemochromatosis Sideroblastic anemias. AML Stain Positivity Memory Aid Mo M1 TdT M2 M3 APL AMML M4 MPO SBB SE Auers (M1-M4) ---------------------------------- M5 AMoL AEL M6 M7 AMegL PAS ----(M5-M7)---- NSEs ----(M4-M6)--- ACP NBE – NAE + Summary Table of Cytochemical Reactions: Stain Name / Substrate Cell Specificity Myeloperoxidase (MPO) Granulocytes, monocytes Naphthol AS-D chloroacetate esterase (NASD, aka. Specific Esterase, or SE) Neutrophils Alpha-naphthyl acetate Monocytes, esterase (NAE, a megakaryocytes, nonspecific esterase) plasma cells Alpha-naphthyl butyrate esterase (NBE, a nonspecific esterase) Monocytes Summary Table of Cytochemical Reactions (cont.) Stain Name/Substrate Cell Specificity Acid phosphatase T lymphoblasts & megakaryocytic precursors Tartrate-Resistant Acid Phosphatase (TRAP) Hairy cells Leukocyte Alkaline Phosphatase (LAP) Mature neutrophils Periodic Acid-Schiff (PAS) Abnormal blast cells Sudan Black B (SBB) Granulocytes; slt. in monos