Page 3 of 9
PA13.1-3 | Hematopoiesis & Blood Specimen Basics — Part 2
Extramedullary Haematopoiesis: When the Marrow Fails to Cope
In the foetus, haematopoiesis does not begin in the bone marrow — it migrates there over gestation. The sequence is:
- Yolk sac (weeks 3–8 of gestation) — primitive haematopoiesis, produces embryonic haemoglobin
- Liver and spleen (weeks 6 to 6 months of gestation) — definitive haematopoiesis takes over
- Bone marrow (from 5 months of gestation, sole site by birth)
The liver and spleen retain haematopoietic potential throughout life. When the bone marrow is overwhelmed, damaged, or infiltrated, this potential is reactivated — a process called extramedullary haematopoiesis (EMH) (extra- 'outside' + medulla 'marrow').
When does EMH occur?
• Chronic severe haemolytic anaemias — beta-thalassaemia major (the classic teaching case), sickle cell disease
• Myelofibrosis — marrow replaced by fibrous tissue, blood formation shifts outward
• Marrow infiltration — leukaemia, lymphoma, metastatic carcinoma pushing out normal cells
What do you find clinically?
• Hepatosplenomegaly — the organs enlarge because they are actively making blood
• Paravertebral masses — paraspinal soft tissue haematopoiesis can compress the spinal cord (rare but clinically critical)
• Skull X-ray: 'hair-on-end' appearance — in beta-thalassaemia major, the diploe (inner spongy layer of skull) is massively expanded by erythroid hyperplasia. On X-ray the vertical trabecular striations look like hairs standing on end. This is a spot diagnosis question in exams AND in real radiology rounds.
Peripheral blood smear clue: When EMH is active, leukoerythroblastic picture — nucleated red cells + immature granulocytes (myelocytes, metamyelocytes) + teardrop cells (dacrocytes) spill into the peripheral blood together. Seeing this triad demands a bone marrow biopsy.
Beta-Thalassaemia Major: Radiographic and Haematologic Findings
CLINICAL PEARL
Pearl: Beta-thalassaemia major and the 'hair-on-end' skull.
In beta-thalassaemia major, defective beta-globin chains mean the body cannot make normal HbA. The marrow responds by driving erythropoiesis to maximum — the red marrow expands so aggressively it erodes and widens the diploe of the skull. On a lateral skull X-ray you see the classic 'hair-on-end' or 'crew-cut' appearance: perpendicular trabecular striations at the outer table.
Same principle explains the 'rodent facies' (frontal bossing, maxillary overgrowth, prominent malar eminences) in older, untransfused patients — marrow expansion remodels facial bones. Once you have seen it, you will never forget it.
SELF-CHECK
A 3-year-old child from a consanguineous family presents with pallor, massive splenomegaly, and frontal bossing. Peripheral blood smear shows nucleated red cells, target cells, and a leukoerythroblastic picture. Skull X-ray demonstrates the 'hair-on-end' appearance. What is the most likely diagnosis?
A. A. Iron-deficiency anaemia
B. B. Aplastic anaemia
C. C. Beta-thalassaemia major
D. D. Autoimmune haemolytic anaemia
Reveal Answer
Answer: C. C. Beta-thalassaemia major
Correct — Beta-thalassaemia major. The constellation of early childhood presentation, consanguinity, massive splenomegaly (EMH), frontal bossing (marrow expansion of facial bones), target cells (HbF/HbA2 dominance with excess alpha chains), leukoerythroblastic picture (EMH-driven release of precursors), and 'hair-on-end' skull (diploe expansion from erythroid hyperplasia) is pathognomonic. Iron-deficiency anaemia does not cause splenomegaly or EMH at this severity. Aplastic anaemia shows a hypocellular marrow with pancytopenia, not erythroid hyperplasia. AIHA can cause splenomegaly but not this degree of bony change.
Blood Specimen Collection: Tubes, Anticoagulants, and Why They Matter
You have now built the conceptual map of haematopoiesis. Before the pathologist can examine that blood, someone has to collect it correctly. Competency PA13.3 (Shows How) makes this your procedural responsibility.
Venepuncture basics:
• Use a 21–23 gauge needle; larger gauge causes haemolysis, smaller is too slow
• Tourniquet on for <1 minute — prolonged stasis causes haemoconcentration (falsely elevated cell counts and haematocrit)
• Release tourniquet before withdrawing the needle
• Invert tubes gently 3–8 times after filling — vigorous shaking causes haemolysis
• Fill tubes in the correct order of draw (see below) to prevent cross-contamination of additives
The colour-coded tube system — international standard, used in SBV OPDs:
| Tube colour | Additive | Mechanism | Tests ordered |
|---|---|---|---|
| Purple / Lavender | EDTA (ethylenediaminetetraacetic acid) | Chelates calcium → prevents coagulation | CBC, PBF, reticulocyte count, HbA1c, blood grouping |
| Blue | Sodium citrate (3.2%) | Chelates calcium reversibly | Coagulation screen (PT, aPTT, INR, fibrinogen) |
| Green | Lithium heparin | Activates antithrombin → inhibits thrombin + Xa | Electrolytes, LFTs, RFTs, drug levels |
| Red / Gold | No additive (red) OR gel separator (gold SST) | Allows clotting → serum collected after centrifuge | Serology, biochemistry, tumour markers |
| Grey | Sodium fluoride + potassium oxalate | Fluoride inhibits glycolysis (enolase), oxalate chelates calcium | Blood glucose, lactate |
| Black | Sodium citrate (3.8%, higher concentration) | Same as blue | ESR (Westergren method) |
Order of draw (to prevent additive carryover into the next tube):Blood cultures → Blue → Red/Gold → Green → Purple → Grey
Mnemonic: Boy Before Reading Great Pathology Gets better (BC → Blue → Red → Green → Purple → Grey)
Blood Collection Tubes: Order of Draw Reference Card
Wrong Tube = Wrong Result: The Clinical Pathology Trap
The tube system is not arbitrary — each additive fundamentally alters the sample. Using the wrong tube is one of the most common pre-analytical errors in clinical pathology, and understanding why it matters is what separates a thinking clinician from a checkbox technician.
Scenario 1: Coagulation tests in an EDTA tube (purple) instead of citrate (blue)
• EDTA chelates calcium irreversibly and completely at 1.8 mg/mL concentration — all coagulation factors that need calcium are permanently inactivated
• The PT and aPTT cannot be measured from this sample
• Result: lab reports 'unsuitable sample — repeat in citrate tube'
• Delay → clinical decision delayed → risk if patient is actively bleeding or pre-op
Scenario 2: EDTA tube used for electrolytes
• EDTA is a calcium chelator — plasma calcium will read falsely LOW
• EDTA contains potassium salts (dipotassium or tripotassium) → plasma potassium reads falsely HIGH
• A 'result' of Ca 1.2 mmol/L and K 6.8 mmol/L could trigger emergency intervention for a tube artefact
Scenario 3: Heparin tube (green) sent for coagulation screen
• Heparin itself extends PT and aPTT — the result is falsely prolonged
• The lab cannot distinguish therapeutic heparin from the tube heparin
• Result: patient may be incorrectly labelled as coagulopathic
Scenario 4: Fluoride-oxalate (grey) tube used for CBC
• Oxalate osmotically distorts red cell shape → falsely abnormal MCV and MCHC
• White cell count is also suppressed by the additive
Scenario 5: Wrong citrate fill volume
• Citrate tubes MUST be filled to the indicated line — the citrate:blood ratio is 1:9
• Under-filled tube → excess citrate → falsely prolonged PT/aPTT
• Over-filled tube → insufficient citrate anticoagulation → micro-clots → falsely low platelet count
Blood Collection Tube Selection Decision Tree