Mednotes

L01 Blood Introduction to the Blood Screen

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tagsblood, lecture
aliasesL01 Blood Introduction to the Blood Screen

14 min read

Overview

This lecture introduces the blood count (full blood screen/CBC) as the most commonly requested blood test, covering what it measures, how automated analysers generate the numbers, the biology of haematopoiesis that produces the cells being counted, and how the red cell indices are used together to classify anaemia. Real patient blood count tables run throughout to illustrate how the numbers are interpreted in practice.

The blood count

  • The blood count (complete blood count, CBC; full blood screen, FBS) has two parts:
    • Quantitative: haemoglobin concentration; red cell count, size and content; leucocyte count; platelet count.
    • Qualitative: blood film examination to check cell morphology.
  • It is the most commonly requested blood test; a clinician will request hundreds every month.
  • Key points when reading a blood count table: not every reported value is equally useful diagnostically. In one worked case (87-year-old female), the “numbers that matter” were highlighted as Haemoglobin, MCV, MCH, Platelets and Neutrophils, while RBC, Hct and WBC were de-emphasised as redundant/less informative.

Normal blood film morphology

  • Stained with a Romanowsky stain (e.g. Wright-Giemsa).
  • Red cells: relatively uniform in size; central pale zone occupies up to about 1/3 of the red cell diameter; about 1% of red cells may normally be oval or an odd shape.
  • Platelets: pale purple cytoplasm with central granules.
  • Leucocytes: morphology is evaluated on the film.
  • Leucocyte types identifiable on a film:
    • Neutrophil: multilobed (segmented) purple nucleus.
    • Band form: curved, unsegmented nucleus (immature neutrophil).
    • Eosinophil: bilobed nucleus, orange-pink granular cytoplasm.
    • Basophil: nucleus obscured by dense dark-purple granules.
    • Monocyte: large cell with a folded/kidney-shaped nucleus and abundant pale cytoplasm.
    • Lymphocyte: small cell, dense round nucleus, scant cytoplasm; a granular lymphocyte shows visible azurophilic granules in the cytoplasm.

Haematopoiesis

  • All blood cells derive from a pluripotent (haematopoietic) stem cell, which splits early into:
    • Lymphoid stem cell → T lymphocytes (via the thymus), NK lymphocytes, and B lymphocytes → plasma cells. Lymphocyte development is very distinct from the other lineages.
    • Myeloid stem cell → erythrocytes (via an erythroid progenitor), megakaryocytes (→ platelets, blood clotting) via a megakaryocyte progenitor, and granulocytes (neutrophils, eosinophils, basophils) and monocytes (→ macrophages) via a shared granulocyte/monocyte progenitor.
  • Site of production changes with development:
    • Fetus: mainly liver and spleen.
    • Child: bone marrow, including the limb bones.
    • Adult: bone marrow of the axial skeleton mainly (skull, vertebrae, ribs, sternum, pelvis); long-bone shafts/limbs contain yellow (fatty, non-haematopoietic) marrow in the adult.
  • A normal bone marrow biopsy shows marrow cells occupying the spaces within bone trabeculae (the trabecular/cancellous bone framework).
  • Principles of regulation:
    • Self-renewal of pluripotent stem cells continues through life.
    • Stem cells differentiate into specific cell lineages.
    • Cytokine signals induce growth and differentiation, e.g. erythropoietin (EPO), secreted by the kidney, and thrombopoietin (TPO), secreted by the liver.
    • Haematopoiesis is supported by marrow stromal cells, which produce factors and receptors supporting stem cell quiescence, proliferation and migration.
  • Regulatory signals act at different stages:
    • Haematopoietic stem cells: SCF (stem cell factor).
    • Multipotential progenitor cells: IL-3, G-CSF, GM-CSF, IL-6, TPO.
    • Committed progenitor cells (used clinically): G-CSF → neutrophils; EPO → red cells; TPO → platelets; IL-5 → eosinophils.
  • Clinical illustration: a patient given G-CSF therapy after chemotherapy showed pancytopenia (low Hb, platelets, WBC/neutrophils) recovering over days; as neutrophil counts rose, immature granulocyte forms (blast cells, myelocytes, metamyelocytes) appeared in the blood, reflecting stimulated granulopoiesis.

Automated haematology analysers

  • Provide an automated count of cells based on cell size, cytoplasmic/nuclear complexity, and nucleic acid fluorescence.
  • Technology:
    • Single cells are assessed by changes in electrical impedance and by laser beam light absorption and scattering.
    • Red cells and platelets are measured by changes in electrical impedance as they flow single-file through a narrow aperture.
    • White cells are measured using a laser (flow cytometry-style light scatter and fluorescence).
    • Automated white cell identification uses light scatter (SSC) and fluorescence (SFL): each leucocyte type (immature granulocytes, monocytes, lymphocytes, neutrophils, eosinophils) occupies a distinct cluster on an SFL vs SSC scatter plot (e.g. lymphocytes lowest, neutrophils/basophils and eosinophils at high SSC).
    • Cell counts of the order of 10,000 cells give high precision.
    • Haemoglobin concentration is measured by colour absorbance.

Red cell indices

  • Measured directly by the automated analyser: haemoglobin (Hb, g/L, the primary measure of oxygen-carrying capacity), mean cell volume (MCV, fL), and red blood cell count (RBC, x10^12/L, which doesn’t provide much additional information on its own).
  • Calculated from the measured values:
    • MCH (mean cell haemoglobin, pg) = Hb / RBC.
    • Hct (haematocrit, L/L) = RBC x MCV.
    • MCHC (mean cell haemoglobin concentration, g/L) = MCH / MCV; seldom diagnostically useful.
  • MCH and MCV are parallel measures of cell size/content, so volume (MCV) tends to be used preferentially. Hct parallels Hb and adds little extra information. RBC and MCHC are typically not reported since they add little to the diagnostic process.
  • Practical approach to reading red cell values: always assess Hb and MCV first, as they carry the essential information.

Reference intervals

  • A reference interval is the central 95% of values obtained from healthy individuals of a specified population for a test (2.5% excluded at each tail of the distribution).
  • “Reference interval” is the most correct term; “normal range” and “reference range” are also used.
  • Factors affecting reference intervals for blood cells: developmental age (infant, childhood, adult), sex, physiology (e.g. pregnancy, altitude), and ethnic (genetic) differences.
  • NZ reference intervals vary substantially by age/sex, e.g.:
    • Hb (g/L): female adult 115-155, male adult 130-175, newborn cord blood 124-192, 2-3 months 97-130, 4-7 years 113-145.
    • MCV (fL): adult (combined) 80-99, newborn 99-119, 2-3 months 72-91, 4-7 years 74-87.
    • MCH (pg): adult (combined) 27-33, newborn 32-39, 2-3 months 24-32, 4-7 years 24-29.
    • Hct (L/L): female adult 0.35-0.46, male adult 0.40-0.52, newborn 0.37-0.56, 2-3 months 0.29-0.38, 4-7 years 0.33-0.42.

Anaemia: definition and classification

  • Defined as a haemoglobin below normal for age, sex and pregnancy status; a low Hb indicates reduced ability to transport oxygen in the blood.
  • WHO definition: female Hb <120 g/L; male Hb <130 g/L.
  • Morphological classification is by cell size (adult MCV), since cell size parallels the amount of haemoglobin in a cell:
    • Microcytic: MCV <80 fL (small cells).
    • Normocytic: MCV 80-99 fL (normal-sized cells).
    • Macrocytic: MCV >99 fL (large cells).
  • Corresponding blood film appearances: normocytic anaemia shows normal-sized red cells but fewer of them; microcytic anaemia shows small, under-filled red cells containing less Hb; macrocytic anaemia shows large red cells.

Iron deficiency (microcytic anaemia)

A common cause of microcytic anaemia. Microcytic is identified from the MCV on the automated analyser; hypochromic (pale) is identified from the MCH and confirmed under the microscope. With early treatment response, some well-filled (normally haemoglobinised) red cells appear alongside the small pale ones. Illustrative case: 87-year-old female with progressively falling Hb (93→75→54 g/L), MCV (83→76→72 fL) and MCH (25→22→22 pg), with rising platelets and neutrophils over the same period — a microcytic, progressively worsening anaemia with a reactive leucocyte/platelet picture.

  • Normocytic anaemias: a large, common group. Important causes include:

    • Haemodilution after acute blood loss.
    • Renal failure (low EPO).
    • Bone marrow failure or infiltration.
    • Anaemia of inflammation (anaemia of chronic disease): occurs with chronic inflammation, infection or malignancy; mechanism is reduced red cell production due to reduced iron availability; red cells tend to be smaller than usual, becoming microcytic when severe.
    • Many other causes.
    • Illustrative case: 70-year-old woman with a major acute GI bleed showed a normocytic anaemia (MCV stayed 90-94 fL while Hb fell from 104→97→52 g/L) with a markedly raised reticulocyte count (303, ref 10-100) reflecting an appropriate marrow response to acute blood loss. By contrast, a 10-year-old girl with a normocytic-range anaemia (Hb fell to 69 g/L) had a low reticulocyte count (5, ref 10-100), indicating an inadequate marrow response [slide does not elaborate on the underlying cause in this case].
    • Contrasting case (not anaemia): a 27-year-old male with cannabis-induced hyperemesis and marked dehydration showed haemoconcentration — raised Hb (up to 200 g/L) and Hct (up to 0.56), i.e. an apparent rise in red cell measures due to reduced plasma volume rather than true polycythaemia.

  • Macrocytic anaemias: common in liver disease. Folic acid or vitamin B12 deficiency causes megaloblastic anaemia:

    • Folate and B12 are needed for nucleic acid synthesis.
    • Deficiency causes delayed and abnormal maturation in the marrow.
    • Dividing cells die from lack of nucleotides and inability to synthesise DNA.
    • This affects all tissues, but blood abnormalities are usually how it is diagnosed.
    • Illustrative case: 23-year-old with severe diarrhoea, macrocytic anaemia (MCV 110-111 fL, MCH 38-39 pg) due to vitamin B12 deficiency. B12 is absorbed in the terminal ileum (terminal end of the small intestine); Crohn disease particularly affects this part of the gut, raising it as a differential requiring further investigation.
    • Illustrative case: cumulative data from 2018 to 2023 showed emergence of a macrocytic anaemia (MCV rising from 90 to 110 fL, MCH from 28 to 35 pg) together with falling Hb, platelets and neutrophils — i.e. pancytopenia (reduction in all three main lineages: red cells, platelets, and neutrophils) alongside macrocytosis.

Erythropoiesis

  • Driven by EPO (erythropoietin).
  • Marrow stages (about 8-9 days of development): proerythroblast → erythroblasts (nucleated red cell precursors), which divide, take up iron, and synthesise haemoglobin, before extruding their nucleus.
  • Blood stages: reticulocyte, an immature red cell that completes clearance of RNA, mitochondria and Golgi apparatus while migrating into the blood over 1-2 days; it then becomes a mature red blood cell, which survives approximately 100-120 days.
  • Terminology: “erythroblast” = nucleated cell; “erythrocyte” = the cell once its nucleus has been discarded.
  • Erythroblasts, in order:
    1. Proliferate only in early stages.
    2. Take up large amounts of iron.
    3. Produce haemoglobin.
    4. Finally remove mitochondria, endoplasmic reticulum, ribosomes and Golgi apparatus, and extrude the nucleus.
  • Erythroblasts mature in “erythroblastic islands”, clusters of erythroblasts at different maturation stages surrounding a central macrophage.
  • Expansion: an average adult produces roughly 200-300 x 10^9 red cells per day. Each division from haematopoietic stem cell through proerythroblast, basophilic erythroblast and polychromatic erythroblast stages roughly doubles cell numbers, before the nucleus is lost and cells migrate into blood as reticulocytes then mature erythrocytes.

Self-test

  1. Define the blood count (CBC/FBS) and list what it quantitatively measures.
  2. Which measures were highlighted as “the numbers that matter” in a routine blood count, and which are considered largely redundant?
  3. Describe the normal appearance of red cells, platelets and leucocytes on a Romanowsky-stained blood film.
  4. List the two major lineages that arise from the pluripotent (haematopoietic) stem cell, and name the mature cell types each gives rise to.
  5. Where is haematopoiesis located in the fetus, the child, and the adult?
  6. Name the cytokines that act on haematopoietic stem cells, multipotential progenitor cells, and each of the committed progenitor cells (neutrophils, red cells, platelets, eosinophils), and state the source organ of EPO and TPO.
  7. Explain the two physical principles automated analysers use to count and classify blood cells.
  8. State the three red cell measures obtained directly from the analyser, and give the formulas for the three calculated values derived from them.
  9. Define a reference interval, and list two factors that can alter it for a given test.
  10. State the WHO haemoglobin thresholds that define anaemia in women and in men.
  11. Distinguish microcytic, normocytic and macrocytic anaemia by their MCV cut-offs.
  12. Describe the mechanism of anaemia of inflammation and its effect on red cell size.
  13. List at least three causes of normocytic anaemia besides anaemia of inflammation.
  14. Explain how iron deficiency anaemia is identified from the blood count and blood film, and what an early treatment response looks like on the film.
  15. Explain the mechanism by which folate or vitamin B12 deficiency causes megaloblastic (macrocytic) anaemia.
  16. A patient with severe diarrhoea has a macrocytic anaemia. Which vitamin deficiency is most likely, where is it absorbed, and what underlying gut disease should be considered?
  17. Describe the stages of erythropoiesis from proerythroblast to mature red blood cell, including where the nucleus is lost and the approximate lifespan of a mature red cell.
  18. A patient has a normocytic anaemia after acute GI blood loss and a very high reticulocyte count, while another patient has a similar degree of anaemia but a low reticulocyte count. What does the reticulocyte count tell you about the marrow’s response in each case?

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