Hematology 1: Bone Marrow Examination & Lymphoid Organs PDF

HEMATOLOGY 1 PRELIMS LEC 02 - BONE MARROW EXAMINATION AND LYMPHOID ORGANS BONE MARROW INDICATIONS FOR BONE MARROW EXAMINATION Soft tissues inside the bones...

HEMATOLOGY 1 PRELIMS LEC 02 - BONE MARROW EXAMINATION AND LYMPHOID ORGANS BONE MARROW INDICATIONS FOR BONE MARROW EXAMINATION Soft tissues inside the bones Indication Examples Contains hematopoietic cells, stromal cells, and blood vessels Neoplasia Acute leukemias ○ stromal cells - support differentiation of cells Diagnosis Myeloproliferative neoplasms such ○ anchor HSC to help it survive as chronic leukemias, myelofibrosis 3.4% to 5.9% of body weight Myelodysplastic neoplasms such as ○ 1600 - 3700 grams / 1.6 - 3.7 kg refractory anemia Lymphoproliferative disorders such ○ 30 - 50 mL per kg of body weight as acute lymphoblastic leukemia Main function: production and proliferation of blood cells Immunoglobulin disorders such as All blood-formed elements ultimately develop from this plasma cell myeloma, undifferentiated precursor macroglobulinemia ○ fats are needed in the bone marrow for bone Metastatic tumors integrity only pathologists and doctors are the ones taking the Neoplasia Hodgkin and non-Hodgkin specimen; MTs are the ones examining it Diagnosis lymphoma and Staging Marrow failure: Hypoplastic or aplastic anemia Cytopenias Pure red cell aplasia Idiosyncratic drug-induced marrow suppression Myelodysplastic syndromes such as refractory anemia Marrow necrosis secondary to tumor Marrow necrosis secondary to severe infection such as parvovirus B19 infection Immune versus amegakaryocytic thrombocytopenia Red BM - Site of blood cell formation Sickle cell crisis Differentiation of megaloblastic, iron deficiency, sideroblastic, RED BONE MARROW hemolytic, and blood loss anemia Composed of the hematopoietic cells and macrophages Estimation of storage iron to assess arranged in extravascular cords for iron deficiency Infiltrative processes or fibrosis ○ extravascular niche - where hematopoietic cells develop; outside the blood vessels Metabolic Gaucher disease mesoblastic phase occurs Disorders Mast cell disease extravascularly while medullary - intra Main Functions: Infections Granulomatous disease ○ Production of Blood Cells Miliary tuberculosis ○ Iron storage Fungal infections ○ B-cell development Hemophagocytic syndromes must be supplied with nutrients particularly oxygen ○ if not = tissue will die Monitoring of After chemotherapy or radiation once a mature cell is formed, it binds to endothelial cells Treatment therapy to assess minimal residual disease then goes to the peripheral blood After stem cell transplantation to both nutrient & peripheral arteries will supply to the assess engraftment osseous bones RE: BONE MARROW EXAMINATIONS BONE MARROW SPECIMEN COLLECTION SITES reduced now in requests due to alternatives (molecular tests) ○ blood tests can be alternative Posterior Superior Iliac Crest requested by doctors during blood abnormalities and ○ best site, far from risk of lethal injuries metabolism problems Anterior Superior Iliac Crest bleeding on the BM site must be avoided Sternum ○ risky since pericardium can be punctured coagulopathy = bleeding / clotting disorder ○ below angle of lewis; second intercostal space not allowed to collect bone marrow specimen Anterior Medial Surface of Tibia ○ best for 2 years old and below = aspiration common complication - infection and bleeding Spinous processes of vertebrae Garcia | Montano (2023) 1  BONE MARROW SPECIMENS 1. Aspirate To identify the types and proportions of hematologic cells and to look for morphologic variance ○ differential count in blood cells, changes in size etc primarily the liquid component, quite diluted with peripheral/sinusoidal blood; can be diluted with blood 14 - 18 gauge needle; needs anesthesia, scalpel (to make a 3 mm incision) simple hematologic examination - fats are seen, not evident 2. Core Biopsy Demonstrates bone marrow architecture Important for evaluating diseases that characteristically produce focal lesions puncture needle with obturator = guide as you puncture, Also allows for morphologic evaluation of bony spicules removed and replaced with aspiration needle directly the bone marrow is extracted, much deeper core biopsy needle with expulsion stylus - for biopsy estimate the celluliarity of the bone marrow collection ○ how much stromal cells/fats are present obturator - for biopsy, replaced with cutting blade sees the overall structure of the bone marrow ○ 3 mm incision ○ good for lesions (Hodgkin’s Lymphoma) spatial relationship of hematopoietic & stromal cells !!! both are used for checking cells core biopsy is done first before doing the aspirate 1st thing to do for both procedure - check for CBC result before collection, “time-out procedure” is done where the doctor confirms all patient details MATERIALS NEEDED Surgical gloves Disposable Jamshidi biopsy needle Westerman-Jensen needle Snarecoil biopsy needle In this example, the patient is placed in the prone position, the posterior, superior iliac crest is palpated (A) and then local anesthetic Shaving equipment University of Illinois aspiration is injected (B). The Jamshidi needle is inserted and a rotating motion needle is used (C and D) to core through the bone. The core biopsy specimen is removed and touch preps of the specimen are made on glass slides Antiseptic solution and Microscope slides or coverslips (E and F). Marrow aspiration (liquid) specimens are acquired (G and alcohol pads washed in 70% ethanol H) through the same biopsy site using an aspiration needle in a second quick accession. Drape material Petri dishes or shallow circular watch glasses CORE BIOPSY PROCEDURE Local anesthesia Wintrobe hematocrit tubes 1. After the incision, the physician inserts a Jamshidi outer ○ 1% Lidocaine - don’t cannula with the obturator in place through the skin and exceed 20 mL cortex of the bone. 2. Reciprocating rotation No. 11 scalpel blade Anticoagulantl liquid 3. Removal of obturator for skin incision tripotassium 4. Insertion of the biopsy needle through the canula EDTA rotate the biopsy needle w/o applying pressure 5. The physician changes the needle angle slightly Vials or test tubes with Zenker fixative closures ○ glacial acetic acid, 6. The biopsy needle and cannula are withdrawn from the bone mercuric chloride, 7. The biopsy needle is placed over an ethanol-cleaned slide sodium sulfate 8. The stylus is pushed through to dislodge the core cylinder also used for histologic B5 fixative onto slide examinations ○ has mercuric chloride 9. Touch preparation on slide sodium sulfate touch the glass slide before placing biopsy in slide 10% neutral formalin 10. The core cylinder is then transferred to the chosen fixative Gauze dressings ? - how to know if area punctured is correct? no resistance; soft area meaning it is the BM space Garcia | Montano (2023) 2 ASPIRATION PROCEDURE Direct transfer of freshly collected marrow specimen onto 6 Using the same insertion point, but in a separate location or more ethano-lwashed slides. from the biopsy site Preparation of wedge-smears same procedure with blood smear 1. Physician inserts a 14- to 18-gauge aspiration needle with avoid crushing the bony spicules obturator 2. The obturator is removed, and a 10- to 20-mL syringe is B. Anticoagulated Aspirate Smears attached Use of EDTA - preparation of HC to prevent distortion of cells obtain 1 - 1.5 mL marrow specimen; if more = before smearing, put first on an EDTA tube diluted with peripheral blood and destroyed 3. The physician withdraws the plunger C. Crush Smears of Bone Marrow Aspirate 4. The physician detaches the syringe and passes it A portion of the aspirate is expelled to a petri dish or watch immediately to the medical laboratory professional glass covered with a few mL of EDTA solution and the 5. Preparation of smears aspirate is spread over the surface with a sterile applicator 6. The physician may attach a second syringe to aspirate an For each slide, another glass slide is placed directly over the additional specimen specimen at a right angle and presses gently to crush the 7. The needle is then withdrawn, and pressure is applied to the spicules wound similar to direct asperate smear but crush bony spicules ** Addition of 22% albumin DRY TAP ○ prevent basket cells (degenerate cells of patient is punctured, yet no specimen is obtained lymphocytes), reduce smudge cells happens when bone marrow is fibrotic or filled with leukemic cells or aplastic D. Concentrate (Buffy Coat) Smears what to do: change angle; use different syringe; puncture Useful when there are sparse nucleated cells in the direct another one - if still none, dry tap positive marrow aspirate smear or when the number of nucleated ○ to confirm = check biopsy cells is anticipated to be small ○ if positive for dry tap - improper collection Compensates for hypocellular marrow and allows for ○ if biopsy positive = no second aspirate, as is examination of a large number of nucleated cells without ○ if biopsy denied = another aspirate interference from fat or RBC if cellularity is wanted; cells are distorted = don’t use BC 2500 gf for 10 minutes ADVANTAGES & DISADVANTAGES layering: ○ topmost = fats Aspirate Smear Advantages: ○ 2nd = plasma Fast ○ 3rd = 5-8 % myeloid to erythroid layer - where No need for decalcification of precursors can be obtained the specimen aspirated and placed on slide; smeared Quantification of cell type ○ 4th = RBCs differential count Material for ancillary studies (flow, molecular) E. Core Biopsy Imprints (Touch Preparations) Valuable when the specimen has clotted or there is a dry tap Disadvantages: May not represent all cells F. Histologic Sections (Cell Block) of the Core Biopsy ○ stromal not included For histologic examinations; has fixative Dry tap in cases of fibrosis or hypocellularity Does not represent architecture G. Marrow Core Biopsy and Aspirate Smear Stains Inability to analyze the stroma Wright or Wright-Giemsa Stains - commonly used in aspirates; HnE Stains - biopsies Core Biopsy Advantages: Prussian Blue Ability to analyze both cells and ○ to see iron components stroma ○ look for cells sideroblasts - immature cells Represents all cells containing iron deposits Explains dry taps ○ iron must always be looked when doing BM testing Disadvantages: ○ iron components of dead RBCs are recycled in Slow processing the BM to synthesize hemoglobin Decalcification precludes if immature cells are not synthesizing certain ancillary studies Hgb = high iron storage ○ calcium removal for iron studies, use aspirate Inability to perform quantitative differential count ○ non-quantitative CYTOCHEMICAL STAINS (identifies Leukemia) USED PREPARATION OF THE BONE MARROW SPECIMENS Cytochemical Stain Application Myeloperoxidase Detects myeloid cells by staining A. Direct Aspirate Smears Garcia | Montano (2023) 3  (MPO) cytoplasmic granular contents Iron stores Categorization of findings as increased, uses fresh specimen; stained normal, within 24 hrs or decreased iron stores Sudan black B (SBB) Detects myeloid cells by staining cytoplasmic granular contents BONE MARROW ASPIRATE MICROSCOPIC SMEAR used if not deteriorated stored specimen staining - fat EXAMINATION components of the cells Low Power 10x Objective Lens (100x total Periodic acid-Schiff Detects lymphocytic cells and certain magnification) (PAS) abnormal erythroid cells by staining of Assess peripheral blood cytoplasmic glycogen dilution abnormalities in RBC Find bony spicules and areas precursors of clear cell morphology Observe fat-to-marrow ratio, Esterases Distinguish myeloid from monocytic estimate cellularity maturation stages (several esterase Search for tumor cells in substrates) clusters differentiate specific & Examine and estimate nonspecific granules megakaryocytes Tartrate-resistant acid Detects tartrate-resistant acid High Power 50x and 100x Objective Lenses phosphatase (TRAP) phosphatase granules in hairy cell (500x and 1000x total leukemia magnification) ideal for recovering hairy cells Observe granulocytic and - b cells with leukemia erythroid maturation Distinguish abnormal distribution of cells or cell maturation stages EXAMINING BONE MARROW ASPIRATE OR IMPRINT Perform differential count on 300–1000 cells Compute myeloid-to-erythroid COMPONENTS OF A BONE MARROW EXAMINATION ratio Component Description BONE MARROW ASPIRATE SMEARS Cellularity Hypocellular, normocellular, o hypercellular classification based on ratio of hematopoietic cells to adipocytes Megakaryocytes Estimate using 10x objective lens (100x magnification), compare with reference interval and comment on morphology Maturation Narrative characterizing the maturation of the myeloid and erythroid (normoblastic, rubricytic) series Additional hematologic Narrative describing numbers and Myeloid stages include a myeloblast (MyBl), promyelocyte (ProMy), cells morphology of eosinophils, basophils, and myelocyte (Myel). The lymphocyte (Lymph) diameter illustrates its mast cells, lymphocytes, plasma cells, size relative to the myeloid stages. The source of the lymphocyte is monocytes, and histiocytes if appropriate, sinus blood. (Wright-Giemsa stain, x1000.) with reference intervals Stromal cells Narrative describing numbers and morphology of osteoblasts, osteoclasts, bony trabeculae, fibroblasts, adipocytes, and endothelial cells; appearance of sinuses; presence of amyloid, granulomas, fibrosis, necrosis Differential count Numbers of all cells and cell stages observed after performing a differential count on 300–1000 cells and comparing results with reference intervals Myeloid stages include a myeloblast (MyBl), promyelocytes (ProMy), Myeloid-toerythroid Computed from nucleated hematologic myelocytes (Myel), and a metamyelocyte (Meta). One orthochromic ratio cells excluding lymphocytes, plasma cells, monocytes, and histiocytes normoblast (OrthoN) and one lymphocyte (Lymph) are present. (Wright-Giemsa stain, x1000.) Garcia | Montano (2023) 4  DISTRIBUTION OF CELLS AND CELL MATURATION STAGES IN ASPIRATES OR INPRINTS Cell or Cell Normal Cell or Cell Normal Maturation Distribution Maturation Distribution Stage Stage Myeloblasts 0%–3% Pronormoblasts/ 0%–1% rubriblasts Myeloid stages include myelocytes (Myel), a metamyelocyte (Meta), Promyelocytes 1%–5% Basophilic 1%–4% and neutrophilic bands. (Wright-Giemsa stain, x1000.) normoblasts/ prorubricytes Myelocytes 6%–14% Polychromatophili 10%–20% c normoblasts/ rubricytes Metamyelocytes 3%–20% Orthochromic 6%–10% normoblasts/ metarubricytes Neutrophilic 9%–32% Lymphocytes 5%–18% bands Note the neutrophilic bands and segmented neutrophils (SEGs). (Wright-Giemsa stain, x1000.) Segmented 7%–30% Plasma cells 0%–1% neutrophils Eosinophils and 0%–3% Monocytes 0%–1% eosinophilic precursors Basophils and 0%–1% Histiocytes 0%–1% mast cells Megakaryocytes 2–10 visible Myeloid-to-erythro 1.5:1–3.3:1 per low-power id ratio field Note the island of erythroid precursors with polychromatophilic and orthochromic normoblasts. (Wright-Giemsa stain, x1000.) !!! megakaryocytes - largest; 2-10 megakaryocytes per field neutrophils and their precursors - most abundant RBCs - fried-egg appearance, 2nd most abundant PRUSSIAN BLUE IRON STAIN EXAMINATION Useful for highlighting the presence of ring sideroblasts color blue = iron deposits Note the cluster of osteoblasts that superficially resemble plasma cells. iron is a component of heme; without heme = no hemoglobin Osteoblasts have round to oval eccentric nuclei and mottled blue cytoplasm that is devoid of secretory granules. They may have a clear area within the cytoplasm but lack the well-defined central Golgi complex of the plasma cell. (A, B, C, Wright-Giemsa stain, x1000.) The large multinucleated cell near the endosteal surface is an osteoclast (arrow), a cell that reabsorbs bone. The spindle-shaped cells are fibroblasts. (Hematoxylin and eosin stain, x500.) Garcia | Montano (2023) 5  EXAMINING BONE MARROW CORE BIOPSY SPECIMEN ○ can iron stain be used in biopsies? yes but be careful Bone marrow core biopsy specimen and imprint examinations are essential when the aspiration procedure yields a dry tap ○ prepare touch preparation first Preservation of bone marrow architecture To assess cellularity disadvantage: can’t clearly see morphologically since obscured by other tissues Immature erythroid (RBC) progenitor cells Normal cellularity, approximately 50% fat and 50% hematopoietic ○ two or three comma-shaped nucleoli, one cells. (Hematoxylin and eosin stain, x50.) nucleolus touching the nuclear envelope ○ tend to cluster with more mature normoblasts and often surround histiocytes ○ have centrally placed, round nuclei that stain intensely (Poly and Ortho) ○ “fried egg” appearance cytoplasm - not total stained but plasma membrane is clearly observed ○ they are more easily recognized using the 10X objective lens Lymphocytes are difficult to recognize Plasma cells are difficult to distinguish from myelocytes in Hypocellularity with only fat and connective tissue cells from a patient H&E stained sections with aplastic anemia. (Hematoxylin and eosin stain, x100.) ○ ”clock face” nucleus STAINS USED IN EXAMINATION OF BONE MARROW CORE BIOPSY SPECIMENS Stain Application Hematoxylin and Evaluate cellularity and hematopoietic eosin (H&E) cell distribution; locate abnormal cell clusters most common Hypercellularity from a patient with chronic myelogenous leukemia with Prussian blue (acidic Evaluate iron stores for deficiency or virtually 100% cellularity and no fat visible. (Hematoxylin and eosin potassium excess iron; decalcification may remove stain, 100x.) ferrocyanide) iron stain iron from fixed specimens; thus EDTA chelation or the aspirate smear is preferred for iron store estimation Reticulin and Examine for marrow fibrosis trichrome stains Acid-fast stains Examine for acid-fast bacilli, fungi, or bacteria in granulomatous disease Gram stain Examine for acid-fast bacilli, fungi, or Note the many large lobulated megakaryocytes and increased blasts. bacteria in granulomatous disease (Wright- Giemsa stain, x400.) Immunohistochemical Establish the identity of malignant cells stains with staintagged monoclonal antibodies specific for tumo surface markers Wright or Wright- Observe hematopoietic cell structure; cell Giemsa stain identification is less accurate in a biopsy specimen than in an aspirate smear !!! aspirate? =yes even always iron stains biopsies - 50:50 decalcification - decrease iron = false negative Garcia | Montano (2023) 6 Note the abundant cytoplasm and irregular nuclei with open chromatin of the blasts. (Hematoxylin and eosin stain, x600.) Note the myelocytes, metamyelocytes, bands, segmented neutrophils, and bright red-orange eosinophils. (Wright-Giemsa stain, x400.) DEFINITIVE STUDIES PERFORMED ON SELECTED BONE MARROW SPECIMENS Bone Marrow Application Specimen Study Iron stain Identification of iron Fresh marrow deficiency, iron overload aspirate Cytochemical Diagnosis of leukemias and Fresh marrow studies lymphomas aspirate Cytogenetic Diagnosis of acute 1 mL marrow studies leukemias via deletions, in heparin translocations, and polysomy; remission studies LYMPHOID ORGANS Molecular studies Polymerase chain reactio fo 1 mL marrow diagnostic point mutations; in EDTA minimal residual disease PRIMARY LYMPHOID ORGANS studies A. Bone Marrow Fluorescence in Staining for diagnostic Fresh marrow Can be considered the largest tissue of the body situ hybridization mutations; minimal residual aspirate Functions as the center for antigen-independent disease studies lymphopoiesis ○ produce B and T cells without stimulation Flow cytometry Immunophenotyping, usually 1 mL marrow of malignant hematopoietic in heparin, B. Thymus cells, clonality; minimal EDTA, or ACD Small, flat, bilobed organ found in the thorax, or chest cavity, residual disease studies right below the thyroid gland and overlying the heart In humans, it weighs an average of 30 g at birth, reaches about 35 g at puberty, and then gradually atrophies ○ even it diminishes at 50 years old - still okay since we have enough T cells (immunity) ○ on thymus cortex = thymocytes ○ on thymus medulla = mature T cells SECONDARY LYMPHOID ORGANS BONE MARROW EXAMINATION REPORTS A. Spleen Largest secondary lymphoid organ Large discriminating filter graveyard of the body; removal of old and damaged cells Two main types of Splenic Tissue: ○ Red Pulp - destroys old red blood cells Garcia | Montano (2023) 7  ○ White pulp - contains the lymphoid tissue, which is arranged around arterioles in a periarteriolar lymphoid sheath (PALS) culling - totally remove the RBCs together with organelles pitting - spleen removes unwanted materials within RBCs ○ inclusion bodies are abnormal and may create scars engulfed by phagocytes slow transit pathway - slow passage of RBCs in the spleen rapif transit pathway - fast passage of RBCs causing the spleen to enlarge, happens during leukemia B. Lymph Nodes Located along lymphatic ducts and serve as central collecting points for lymph fluid from adjacent tissues. Filtration is a main function of these organs. primary lymph node = no germinal center; follicle only secondary lymph node = has germinal center adenitis = inflammation of lymph nodes C. Liver Serves as the major site of blood cell production during the second trimester of fetal development major metabolic organ of the body problems within liver can affect clotting factors Contains Kupffer cells ○ processing of drugs = during overdose, first organ to be damaged Extramedullary hematopoiesis Other Secondary Organs: MALT ○ GI tract, respiratory, urologic area BALT ○ Lymphoid tissue in the lower respiratory tract and hilar lymph nodes. ○ Associated with IgA production in response to inhaled antigens. GALT ○ Includes lymphoid tissue in the intestines (Peyer’s patches) and the liver. ○ Important for the development of tolerance to ingested antigens. CALT ○ cutaneous, skin Tonsils ○ prevent foreign bodues from coming into respiratory system Garcia | Montano (2023) 8