Packed Cell Volume (PCV) and Red Cell Osmotic Fragility Study Guide

Introduction to Packed Cell Volume (PCV)

• Definition: The Packed Cell Volume (PCV) is a measurement of the volume of whole blood that is occupied by red cells.
• Blood Sources: The blood sample used can be derived from capillary, venous, or arterial sources.
• PCV vs. Haematocrit (Hct):
  • PCV: A directly measured value of packed red cells obtained by centrifuging anticoagulated whole blood in a capillary tube.
  • Haematocrit (Hct): A derived, calculated value obtained by multiplying the Red Blood Cell (RBC) count with the Mean Cell Volume (MCV), often generated by automated analyzers.

Principles and Procedures of PCV Determination

• Principle of Centrifugation: PCV is determined by centrifuging a column of blood in a tube of uniform bore that is closed at one end.
• Centrifugation Parameters:
  • Performed in a special high-speed centrifuge.
  • Speed and Force: $11,000$ to $12,000\,G$ or $11,000\,\text{rpm}$.
  • Duration: $5\,\text{minutes}$.
  • Requirement: Centrifugation must continue until the packing of cells is as complete as possible, meaning further centrifugation under identical conditions leaves the cell column length unaltered.
• Measurement: The PCV is the length of the RBC column after centrifugation relative to the total length of the column.
• Evolution of Techniques:
  • Macro-haematocrit technique: First described in $1929$; it is no longer in use.
  • Micro-haematocrit technique: Developed by Maxwell Wintrobe in $1934$. It involves centrifuging whole blood in a micro-haematocrit tube at a constant speed to separate blood into layers.

Components and Separation of Centrifuged Blood

• Layer Formation: At the end of centrifugation, whole blood separates into five distinct layers:
  1. An uppermost layer of plasma.
  2. A very small cream-coloured layer of platelets.
  3. A greyish–red layer of leukocytes.
  4. A thin black band of deoxygenated RBCs.
  5. A larger layer of oxygenated RBCs.
• The Buffy Coat:
  • This is the yellowish-white layer of leukocytes and platelets forming on top of the red cell column.
  • Clinical Utility: The thickness provides an estimate of the White Blood Cell (WBC) count. In leukopenia, it is extremely thin (a "delicate scum"); in leukaemia, it may be very thick.
  • Diagnostic Use: It is useful for preparing thin films to search for leukaemic or malignant cells in leukopenic patients where such cells are scanty in peripheral films.

Practical Requirements and Methodology

• Necessary Items: Micro-haematocrit centrifuge, capillary tubes, haematocrit reader, and plasticine/cristaseal clay.
• Capillary Tube Specifications: Length of $75\,\text{mm}$ with a uniform bore of $1\,\text{mm}$.
• Anticoagulant Selection:
  • Options: $K_2EDTA$, $K_3EDTA$, or $Na_2EDTA$.
  • Preference: $K_2EDTA$ is preferred.
  • Limitations: $K_3EDTA$ is liquid and causes a $1$–$2\%$ dilution of blood; $Na_2EDTA$ is less soluble. Heparinized tubes are also acceptable.
• Sample Stability: PCV gradually increases if blood is stored at room temperature. Samples must be run within $6\,\text{hours}$ of collection, or within $24\,\text{hours}$ if refrigerated at $4^\circ\text{C}$.
• Step-by-Step Procedure:
  1. Perform a capillary puncture or use venous blood.
  2. Fill the tube by capillary action to the indicated mark (about $1\,\text{cm}$ from the end remains empty).
  3. Seal the filled end with plasticine clay.
  4. Place the tube in the radial groove of the centrifuge with the sealed end facing outward (to prevent spillage). A rubber gasket cushions the tube.
  5. Balance the load, secure the lid, and centrifuge at $11,000\,\text{rpm}$ for $5\,\text{minutes}$.
  6. Read the value using a haematocrit reader based on the principle of similar triangles.

Reading and Reporting Results

• Reading Method: Align the zero mark ($0\,\%$) with the bottom of the RBC column and the $100$ mark with the top of the plasma column. Adjust the reader to the upper end of the deoxygenated RBC column (the auxiliary line through the border of the Erythrocyte and Leukocyte layers).
• Expression of Results: PCV should be reported as a decimal fraction (e.g., $0.45\,l/l$) rather than a percentage, per recognized haematology bodies.
• Reference Ranges ($20$–$50\,\text{years}$ at sea level):
  • Women: $0.37$–$0.47\,l/l$
  • Men: $0.40$–$0.52\,l/l$
• Variation Factors: Diurnal variations, exercise, and posture affect blood distribution and PCV values.

Variables and Sources of Error in Manual PCV

• Systemic Variables:
  • Trapped Plasma: A small amount of plasma ($\approx 2\%$) remains between cells even at maximum sedimentation. This increases to up to $20\%$ in conditions like iron deficiency anaemia, thalassaemia, spherocytosis, and sickle cell disease (marked poikilocytosis and anisocytosis).
  • Dehydration of RBCs: Caused by high temperatures, $K_3EDTA$, or direct glass toxicity (erythrocyte shrinkage). This counteracts the trapped plasma bias.
  • Tube Material: Soda-lime glass reduces PCV twice as much as borosilicate glass.
  • Tube Diameter: Smaller diameters result in lower PCV values.
• Procedural Errors:
  • Inadequate mixing of blood.
  • Sample dilution (e.g., from interstitial fluid during capillary puncture).
  • Haemolysis (e.g., ejecting blood forcefully through a needle).
  • Presence of clots.
  • Excess anticoagulant causing RBC shrinkage.
  • Inadequate centrifugation.
  • Including the buffy coat in the final reading.

Clinical Significance of PCV Levels

• Causes of Elevated PCV:
  • Dengue Haemorrhagic Fever: High haematocrit is a danger sign for dengue shock syndrome.
  • Polycythemia Vera (PV): A myeloproliferative disorder with excessive RBC production.
  • Hypoxia-related Conditions: Chronic Obstructive Pulmonary Disease (COPD) or pulmonary issues trigger erythropoietin production by the kidneys.
  • Anabolic Androgenic Steroid (AAS) Use: Compounds like boldenone and oxymethelone increase RBC counts.
  • Dehydration: Causes hemoconcentration.
• Causes of Low PCV (Anaemia):
  • Infants with inadequate iron intake.
  • Children during rapid growth spurts.
  • Women of child-bearing age (menstrual blood loss).
  • Pregnancy (fetal iron demand).
  • Chronic Kidney Disease (CKD) due to insufficient erythropoietin secretion.

Red Blood Cell (RBC) Indices

Indices describe the size and haemoglobin content of RBCs, essential for the morphological classification of anaemia.

• Mean Corpuscular Volume (MCV):
  • Definition: The average volume (size) of a single RBC.
  • Formula: $\text{MCV} = \frac{\text{PCV}}{\text{RBC count}} \times 10$
  • Units: femtolitres ($fL$)
  • Reference Range: $80$–$100\,fL$
  • Interpretation: $< 80\,fL$ (Microcytic); $80$–$100\,fL$ (Normocytic); $> 100\,fL$ (Macrocytic).
• Mean Corpuscular Haemoglobin (MCH):
  • Definition: The average amount of haemoglobin in a single RBC.
  • Formula: $\text{MCH} = \frac{\text{Hb}}{\text{RBC count}} \times 10$
  • Units: picograms ($pg$)
  • Reference Range: $27$–$33\,pg$
• Mean Corpuscular Haemoglobin Concentration (MCHC):
  • Definition: The average concentration of haemoglobin within the packed red cell volume.
  • Formula: $\text{MCHC} = \frac{\text{Hb}}{\text{PCV}} \times 100$
  • Units: $g/dL$
  • Reference Range: $32$–$36\,g/dL$
  • Interpretation: $< 32\,g/dL$ (Hypochromic); $32$–$36\,g/dL$ (Normochromic); $> 36\,g/dL$ (Hyperchromic - rare).
• Red Cell Distribution Width (RDW-CV):
  • Definition: Statistical measure of variation in RBC size (anisocytosis).
  • Units: Percentage ($\%$)
  • Reference Range: $11$–$15\%$

Clinical Case Studies

Case Study 1

• Patient: $28$-year-old female.
• Symptoms: Fatigue, dizziness, palpitations for $4\,\text{months}$, heavy menstrual bleeding for $1\,\text{year}$.
• Parameters:
  • Hb: $8.2\,g/dL$
  • PCV: $28\%$
  • RBC Count: $3.8 \times 10^{12}/L$
  • WBC Count: $6.5 \times 10^9/L$
  • Platelet Count: $480 \times 10^9/L$
  • RDW: $19.5\%$
• Tasks: Calculate MCV, MCH, and MCHC, and classify the anaemia morphologically.

Case Study 2

• Patient: $67$-year-old male.
• Symptoms: Weakness, numbness/tingling in feet, weight loss, jaundice, and glossitis.
• Parameters:
  • Hb: $7.5\,g/dL$
  • PCV: $24\%$
  • RBC Count: $1.9 \times 10^{12}/L$
  • WBC Count: $3.2 \times 10^9/L$
  • Platelet Count: $95 \times 10^9/L$
  • RDW: $22.8\%$
• Tasks: Calculate MCV, MCH, and MCHC, and classify the anaemia morphologically.

Red Cell Osmotic Fragility

• Definition of Osmosis: Movement of water molecules from a region of lower solute concentration to a region of higher solute concentration through a semi-permeable membrane.
• Definition of Osmotic Fragility: The susceptibility of RBCs to haemolyse (burst) when exposed to hypotonic saline solutions.
• Cell Membrane Structure: A bi-lipid layer composed of lipids, proteins, and carbohydrates. Integrity is maintained by specific proteins: Actin, spectrin, ankyrin, band 3, and protein $4.1$.
• Tonicity Effects:
  • Isotonic (Physiological): $0.85\%\,NaCl$. Cells remain stable.
  • Hypotonic: $< 0.85\%\,NaCl$. Cells swell and burst.
  • Hypertonic: $> 0.85\%\,NaCl$. Cells shrink (crenate).

Clinical Application of Osmotic Fragility Tests

• Indications: Evaluation of suspected hereditary spherocytosis (HS), thalassaemia, or immune haemolytic states.
• Hereditary Spherocytosis (HS):
  • Caused by a genetic defect in spectrin.
  • Results in membrane instability and decreased RBC surface-area-to-volume ratio.
  • Spherocytes lack central pallor and are trapped/haemolysed in the spleen sinusoids.
  • Fragility: Increased osmotic fragility (cells burst in higher concentrations of saline than normal).
• Thalassaemia:
  • Inherited reduced synthesis of normal globin chains.
  • Associated with target cells which are flatter than normal.
  • Fragility: Generally decreased osmotic fragility (cells are more resistant to bursting).
• Fragility Summary:
  • Decreased Fragility: Thalassaemia, Iron deficiency anaemia, Sickle cell anaemia.
  • Increased Fragility: Hereditary spherocytosis, Acquired spherocytosis, Malaria infection.

Methodology: Osmotic Fragility Test

• Purpose: To aid diagnosis of HS and Thalassemia and detect morphologic RBC abnormalities.
• Materials:
  • Specimen: $5\,ml$ whole blood.
  • Medium: EDTA, Na citrate, Na Heparin, or Lithium Heparin tubes.
  • Rejection: Haemolyzed specimens.
• Principle: Osmotic fragility is influenced by structural integrity and cell volume. Liberated haemoglobin concentration in the medium serves as an index of osmotic haemolysis.
• Procedure:
  1. Prepare 7 tubes with $10\,ml$ of $NaCl$ at concentrations: $0.85\%$, $0.7\%$, $0.6\%$, $0.5\%$, $0.4\%$, $0.3\%$, and $0.0\%$ (distilled water).
  2. Add $100\,\mu l$ of blood to each; mix and stand for $20\,\text{minutes}$.
  3. Centrifuge at $2500\,\text{RPM}$ for $10\,\text{minutes}$.
  4. Transfer supernatant to cuvettes and read absorbance at $540\,nm$ (Zero set with distilled water).
• Calculations:
  • $\% \text{ of haemolysis} = \frac{\text{Absorbance of tube}}{\text{Absorbance of tube with 100\% haemolysis}} \times 100\%$
  • The $100\%$ haemolysis point is defined by the tube containing distilled water ($0\%\,NaCl$).