🦴 Bone Marrow Aspirate and Biopsy — Explained Simply

What Is Bone Marrow?

• Skull
• Sternum (breastbone)
• Scapulae (shoulder blades)
• Ribs
• Pelvic bones
• Upper ends of long bones (like the thigh bone)

Structure of Bone (Simplified)

1. Cortex — the hard outer shell (like the crust of a loaf of bread). Made of compact bone with tiny canals (called Haversian canals) running through it.
2. Medulla — the inner spongy part, made of a lattice of thin struts called trabeculae (like a sponge). The spaces between trabeculae hold the marrow.

• Osteoblasts — build new bone
• Osteocytes — maintain bone
• Osteoclasts — break down old bone

What's Inside the Marrow?

1. Parenchyma (the working cells)

• Haematopoietic stem cells — the "mother cells" that give rise to all blood cells
• Developing red blood cells (erythroid cells), white blood cells (myeloid cells), and platelet-producing cells (megakaryocytes)

2. Stroma (the supporting tissue)

• Fat cells, fibroblasts, histiocytes (scavenger cells), blood vessels, and the gel-like matrix that holds everything together

Why Is Bone Marrow Examined? (Purpose)

The Key Principles:

1. Bone marrow has an organised structure — cells are arranged in predictable patterns
2. In a healthy person, blood cells have specific numerical and spatial relationships with each other
3. Each cell type has a distinct appearance (cytological features)
4. Cell appearance reflects its lineage (what type) and maturity (how developed it is)
5. Any of these can be disrupted in disease

When Is Bone Marrow Examination Ordered? (Indications)

Anaemia (Low blood count):

• Microcytic anaemia (small red cells): to check iron stores and look for sideroblasts (iron-loaded cells)
• Macrocytic anaemia (large red cells): to confirm if it's megaloblastic (due to B12/folate deficiency)
• Normocytic anaemia (normal-sized red cells): when reticulocyte count is not increased, to look for production or maturation problems

Abnormal Blood Counts:

• Neutropenia (low neutrophils), thrombocytopenia (low platelets), pancytopenia (all cells low): to check if precursor cells are present and functioning normally, or if there's leukaemia

Cancer Diagnosis and Staging:

• Non-Hodgkin's lymphoma
• Hodgkin's lymphoma
• Metastatic carcinoma (cancer spread from another organ)
• Small round cell tumours (common in children)

Bone Marrow Structural Problems:

• Fibrosis (scar tissue replacing marrow)
• Necrosis (dead marrow tissue)
• Gelatinous marrow transformation (marrow replaced by gel-like substance)

Other Reasons:

• Unexplained leukoerythroblastic blood picture (immature cells appearing in blood)
• Suspected multiple myeloma (plasma cell cancer)
• Pyrexia of unknown origin (unexplained fever)
• Granuloma or metastatic focal lesions
• Amyloidosis (protein deposits in tissues)
• Metabolic bone diseases

Contraindications (When NOT to Do It)

• Biopsy in clotting disorders (coagulopathies): Aspiration can still be done, but clotting factor replacement and 24–48 hours of observation are needed first
• Sternal aspirate in osteoporosis or children: Risk of the needle going too deep and perforating underlying major blood vessels or the right atrium

Where Is It Done? (Sites)

The Procedure — Step by Step

1. Consent: A written informed consent is obtained from the patient
2. Relevant history is collected — clinical impression, lab results (iron, B12, folate), transfusion history, chemotherapy history
3. Lignocaine sensitivity test is done (lignocaine = local anaesthetic)
4. Local anaesthesia is applied to numb the area
5. A special needle is inserted through the skin and bone cortex into the marrow space
6. For aspiration: marrow fluid is sucked out using a syringe
7. For biopsy (trephine): a core of bone and marrow tissue is removed

Two Types of Bone Marrow Procedures

Handling the Sample

Aspirate:

• Smears should be made immediately at the bedside (cells deteriorate fast)
• Remaining material goes into an EDTA bottle (anticoagulant)
• If immunophenotyping or cytogenetics are needed, preservative-free heparin is used instead
• Some material can be fixed in fixative for histological (tissue) sections
• Films (smears) are fixed in absolute methanol
• Too much anticoagulant causes a pink-staining amorphous material and clumping of erythroblasts — this must be avoided

Centrifugation (spinning):

Direct Films:

• A drop of marrow is placed on a slide
• Spread into a film 3–5 cm long, not wider than 2 cm
• Marrow particles are dragged (not squashed) behind the spreader, leaving a trail of cells

Trephine Biopsy — Processing

What Makes a Good Biopsy?

• Length: 1.5–2.5 cm (optimal ~1.6 cm)
• Note: bone shrinks ~25% during processing
• Should contain 5–6 trabecular spaces
• Good quality staining throughout

Complications

• Haemorrhage (bleeding)
• Pain
• Infection
• Perforation of major vessels (especially with sternal approach)
• Risks of general anaesthesia or sedation if used

Summary in One Paragraph

Immunology: Detailed Outline

Sources: Robbins & Cotran Pathologic Basis of Disease (9th/10th ed.) + Guyton & Hall Textbook of Medical Physiology

PART I — OVERVIEW: TWO ARMS OF IMMUNITY

"The adaptive immune response typically takes 3 to 7 days to become fully active; innate immune mechanisms provide host defense during this critical early window." — Robbins & Kumar Basic Pathology, Ch. 5

PART II — INNATE IMMUNITY

A. Components (Robbins)

1. Epithelial barriers — skin, mucosal linings; physically block microbial entry
2. Phagocytic cells — neutrophils and macrophages engulf and destroy pathogens
3. Dendritic cells (DCs) — sentinel cells; capture antigens, bridge innate to adaptive
4. Natural Killer (NK) cells — kill virus-infected cells and tumour cells without prior sensitisation; express inhibitory receptors for MHC-I (spare normal healthy cells)
5. Innate lymphoid cells (ILCs)
6. Plasma proteins — complement system (~20 proteins)

B. How Innate Cells Recognise Pathogens — Pattern Recognition Receptors (PRRs)

• PAMPs (Pathogen-Associated Molecular Patterns): shared structures on microbes essential for their survival — LPS, flagellin, dsRNA, etc.
• DAMPs (Damage-Associated Molecular Patterns): molecules released from injured/necrotic host cells
• Innate immunity uses ~100 receptors to recognise ~1,000 patterns

Classes of PRRs:

TLR signalling → activates transcription factors → produces cytokines (TNF, IL-1, IL-12), interferons (IFNs), and costimulatory molecules (B7 proteins)

C. Guyton & Hall — Innate (Non-specific) Mechanisms

1. Lysozyme — mucolytic enzyme; lyses bacterial cell walls
2. Basic polypeptides — inactivate certain gram-positive bacteria
3. Complement system — activated to destroy bacteria directly
4. Natural killer lymphocytes — recognise and kill foreign/tumour/virus-infected cells
5. Species resistance — humans are naturally immune to many animal diseases (e.g., hog cholera, cattle plague, distemper); animals are immune to polio, mumps, cholera, measles

D. Key Functions of Innate Immunity

• Inflammation: phagocyte recruitment, cytokine release, vascular changes
• Antiviral defence: Type I IFNs (IFN-α, IFN-β) → activate enzymes that degrade viral RNA, inhibit replication
• Complement activation (see below)
• Danger signalling to activate adaptive immunity

E. Complement System (Guyton & Hall)

1. Opsonisation — C3b coats bacteria → enhanced phagocytosis
2. Chemotaxis — C5a recruits neutrophils and macrophages
3. Mast cell / basophil activation — C3a, C5a (anaphylatoxins) → histamine release
4. Direct lysis — Membrane Attack Complex (MAC = C5b–C9) punches holes in bacteria
5. Inflammatory augmentation — increased blood flow, capillary leakage, tissue fluid coagulation (traps invading organisms)
6. Viral neutralisation — lysis of enveloped viruses
7. Immune complex clearance

PART III — ADAPTIVE IMMUNITY

A. Overview (Both Sources)

B. Antigens — What Triggers Adaptive Immunity (Guyton & Hall)

• A foreign invader contains specific chemical compounds (proteins or large polysaccharides) not found in the host
• These are called antigens (antibody generators)
• Must have MW ≥ 8,000 daltons
• Must have recurring epitopes (stereochemical surface groups) that the immune system can recognise
• Different epitopes on one antigen activate different lymphocyte clones

C. Cells of Adaptive Immunity (Robbins & Cotran)

1. Lymphocytes

• Constantly circulate via blood and lymphatics — immune surveillance throughout body
• Naïve lymphocytes: have never encountered antigen; quiescent
• On activation: differentiate into:
  • Effector cells — carry out immune functions
  • Memory cells — long-lived; enable rapid response on re-exposure

Clonal Selection (fundamental principle):

Lymphocytes specific for many antigens exist before exposure. When an antigen appears, it selectively activates the antigen-specific clone → clonal expansion

2. T Lymphocytes (Thymus-derived)

• Kill cells displaying foreign peptides on MHC I
• Mechanism: release perforin (pore-forming) + granzymes (proteases → apoptosis)
• Also produce IFN-γ

3. B Lymphocytes (Bone marrow-derived)

• Recognise intact, unprocessed antigens (unlike T cells which need MHC presentation)
• On activation → proliferate → plasma cells (antibody factories)
• Each mature plasma cell secretes ~2,000 antibody molecules/second (Guyton & Hall)
• Some become memory B cells

4. Antigen-Presenting Cells (APCs)

• Dendritic cells — most potent; distributed throughout body; professional APCs
• Macrophages — present antigens + destroy phagocytosed microbes
• B lymphocytes — present antigen to T cells, receive T-cell help

PART IV — HUMORAL IMMUNITY IN DETAIL

A. Process (Guyton & Hall + Robbins)

1. Antigen enters → macrophages phagocytose and present to B lymphocytes
2. T-helper cells simultaneously activated → provide additional stimulation to B cells
3. B cells → transform to lymphoblasts → plasmablasts → plasma cells
   • Division ~every 10 hours for 9 divisions over 4 days → ~500 plasma cells per original plasmablast
4. Plasma cells secrete antibodies → carried via lymph → blood → systemic circulation

B. Memory and Secondary Response

• Some lymphoblasts become memory B cells → populate lymphoid tissue throughout body
• Primary response: 1-week lag; weak; short-lived
• Secondary response: rapid (hours); far more potent; lasts months
• This is why immunisation uses multiple spaced doses
• Long-lived plasma cells reside in bone marrow + gut-associated lymphoid tissue → lifelong antibody production (e.g., measles, smallpox immunity lasting decades)

C. Antibodies (Immunoglobulins)

D. Functions of Antibodies (Robbins & Cotran)

1. Neutralisation — block viral/toxin binding to host cells
2. Opsonisation — coat bacteria → enhanced phagocytosis (Fc receptor recognition)
3. Complement activation — classical pathway → lysis, opsonisation, inflammation
4. ADCC (Antibody-Dependent Cell-Mediated Cytotoxicity) — NK cells kill IgG-coated targets
5. Passive immunity to neonates — IgG crosses placenta; secretory IgA in breast milk

E. T-cell Help for B Cells (Robbins & Cotran)

• CD4+ Tfh cells express CD40L → engages CD40 on B cells (critical co-stimulatory signal)
• Signal 1: Antigen + BCR
• Signal 2: CD40L–CD40 + cytokines (IL-4, IL-21)
• → Germinal centre formation in lymph node follicles
• → Somatic hypermutation → antibody affinity maturation
• → Isotype class switching (IgM → IgG, IgA, IgE)

PART V — CELL-MEDIATED IMMUNITY IN DETAIL

A. Process (Guyton & Hall + Robbins)

1. Antigen captured by dendritic cells in epithelia/tissues
2. DCs mature → upregulate MHC molecules + B7 costimulators → migrate to lymph nodes
3. Naïve T cells in lymph node recognise MHC-peptide complex via T-cell receptor (TCR)
4. Two-signal activation:
   • Signal 1: TCR + MHC-antigen
   • Signal 2: CD28 (T cell) + B7/CD80/CD86 (APC) — without this signal, T cell becomes anergic (tolerant)
5. → T cell secretes IL-2 + upregulates IL-2 receptors → autocrine proliferation
6. → Clonal expansion → effector T cells + memory T cells

B. Effector Functions

CD4+ Helper T Cells:

• Th1 + IFN-γ + CD40L → classical macrophage activation → microbicidal machinery (reactive oxygen species, NO) → kills intracellular bacteria (TB, Listeria)
• Th2 + IL-5 → eosinophil activation → kills helminths; IL-4/IL-13 → alternative macrophage activation → tissue repair/fibrosis
• Th17 + IL-17 → neutrophil/monocyte recruitment → defence against fungi and extracellular bacteria; implicated in autoimmune diseases

CD8+ Cytotoxic T Cells:

• Kill any cell displaying foreign peptide on MHC I (virus-infected, tumour cells, allograft cells)
• Killing mechanisms:
  • Perforin-granzyme pathway: perforin creates pores; granzyme B enters and activates caspases → apoptosis
  • Fas–FasL pathway: T cell Fas ligand binds Fas on target → apoptosis
• Also produce IFN-γ (antiviral, activates macrophages)

C. T-Cell Memory (Guyton & Hall)

• Memory T cells spread to all lymphoid tissue in the body
• On re-exposure to the same antigen anywhere in the body → rapid, amplified response
• No lag period; immediate T-cell activation

D. MHC Restriction (Guyton & Hall + Robbins)

PART VI — LYMPHOID ORGANS & CELL TRAFFICKING (Robbins)

• Lymph nodes: B cells in follicles (cortex); T cells in parafollicular cortex; germinal centres form after antigen stimulation
• Spleen: T cells in periarteriolar lymphoid sheaths (PALS); B cells in follicles
• Lymphocytes continuously recirculate through blood, lymphatics, and tissues — enables immune surveillance

PART VII — CYTOKINES: MESSENGER MOLECULES (Robbins)

PART VIII — TOLERANCE AND SELF/NON-SELF DISCRIMINATION

Central Tolerance (Guyton & Hall — Thymus & Bone Marrow):

• Immature T cells in the thymus that react to self antigens → deleted (apoptosis) — called clonal deletion
• Immature B cells in bone marrow similarly purged
• Ensures the immune system does not attack the body's own tissues

Peripheral Tolerance (Robbins):

• Mature self-reactive lymphocytes that escape → rendered anergic (functionally inactive)
• Regulatory T cells (Tregs) suppress self-reactive lymphocytes
• Failure of tolerance → autoimmune disease

Signal 2 Requirement (Robbins):

• Simply presenting a self-antigen on MHC is not enough to activate T cells — costimulatory molecules (B7) must also be upregulated (which only happens during infection/injury)
• This prevents auto-immunity from routine self-antigen presentation

PART IX — SEX DIFFERENCES IN IMMUNITY (Guyton & Hall)

• Females generally mount stronger innate and adaptive immune responses than males
• Higher antibody production and more vigorous T-cell responses
• Explains the higher susceptibility of females to autoimmune diseases (lupus, rheumatoid arthritis, multiple sclerosis)
• Oestrogens enhance immune responses; androgens are relatively immunosuppressive

PART X — COMPARATIVE SUMMARY TABLE

KEY DISTINCTIONS TO REMEMBER

1. Innate vs. Adaptive = speed vs. specificity — innate is fast but broad; adaptive is slow but exquisitely specific
2. Humoral vs. Cell-mediated = extracellular vs. intracellular pathogens — antibodies can't follow viruses inside cells; CTLs can
3. CD4 vs. CD8 = helper/coordinator vs. killer — CD4 recognises MHC II (extracellular antigen); CD8 recognises MHC I (intracellular antigen)
4. B cells recognise intact antigen; T cells require processed peptide + MHC
5. Two signals needed for lymphocyte activation — antigen alone causes anergy (tolerance), not activation
6. Memory is the immunological basis for vaccines — secondary response is dramatically faster and more powerful