Basic Investigations in Haematology Lecture 2 - Packed Cell Volumes (PCV) and Determination of Red Cells Indices Notes

Packed Cell Volume (PCV)

PCV represents the proportion of whole blood that is actually occupied by erythrocytes.

PCV is usually written either as a percentage (e.g. 45 %) or as a ratio/decimal (e.g. 0.45 L/L).

ICS (International Council for Standardization in Haematology) terminology:

• “PCV” should be used when the blood is centrifuged in a micro-haematocrit (capillary) tube.

• “Haematocrit (HCT)” is preferred when an automated analyser mathematically derives the value (generally from MCV \times RBC).

• Because plasma becomes trapped between red cells, manual PCV values are typically 1–3 % higher than automated HCT results.

Relationship with haemoglobin: in rough screening, PCV \approx 3 \times Hb (where Hb is in g/dL and PCV in %).

Clinical/Diagnostic Reasons for Performing PCV

• Screening for anaemia when Hb cannot be measured.

• Monitoring or investigating:

  • Dehydration

  • Severe burns (loss of plasma)

  • Dengue haemorrhagic fever (haemoconcentration)

  • Polycythaemia (↑ RBC mass & Hb)

• Post-centrifugation plasma inspection for:

  • Trypanosomes in endemic African trypanosomiasis (parasites may swim at the plasma–buffy-coat interface).

  • Colour/clarity changes that give clues to pathology (see later list).

Visual Clues from Centrifuged Plasma

• Normal plasma → pale straw-yellow.

• Severe iron deficiency → colourless plasma.

• Haemolytic anaemia (↑ bilirubin) → deep yellow plasma.

• In-vitro haemolysis → pink-red plasma.

• Marked lipaemia → cloudy white plasma.

• Leukocytosis → widened buffy coat; should prompt a manual/automated WBC count.

Specimen & Equipment

Specimen choices:

• EDTA-anticoagulated venous blood (well-mixed).

• Capillary blood directly collected into a heparinised micro-capillary.

Consumables & apparatus:

• Glass or plastic micro-haematocrit capillary tubes, 75 mm long, OD ≈ 1 mm, wall 0.20–0.25 mm.

  • Red band = sodium heparin-coated (for direct capillary sticks).

  • Blue/no band = plain (for use with already anticoagulated venous blood).

• Plastic sealant or clay to close one end.

• Micro-haematocrit centrifuge capable of 12{,}000–15{,}000\;g (≈ rpm shown by manufacturer) for 3–5 min.

• Micro-haematocrit reader – a calibrated card or plastic device; alternatively a ruler.

Principle of the Test

Anticoagulated blood in a standardised capillary is spun at high RCF so that red cells form a tightly packed column. The packed height is divided by the total height (RBC + plasma + buffy coat) to obtain PCV.

Mathematically:
PCV = \frac{\text{Height of RBC column}}{\text{Total column height}}

Step-by-Step Procedure

1. Mix sample gently.

2. Fill capillary three-quarters by capillary action.

3. Seal one end (heat-sealing discouraged because of glass weakening).

   • If finger-stick: wipe first drop, fill a heparinised tube.

4. Load centrifuge with sealed ends outermost, balance opposite slots, note slot # on request form.

5. Spin 3–5 min at 12{,}000–15{,}000\;rpm; avoid brake (prevents cell disruption).

6. Retrieve tubes; immediately read with reader:

   • Align 0 with bottom of RBC pack.

   • Align 100 % with top of plasma meniscus.

   • Buffy coat (WBC + platelets) is excluded from RBC length.

If no reader is available:
PCV = \frac{\text{mm of RBCs}}{\text{mm total}} × 100 %.

When PCV > 50 %, re-spin an additional 3 min to ensure tight packing.

Quality Control & Error Sources

Duplicate analysis: two capillaries per patient; results should match within ±5 % (absolute value).

A. False ↑ PCV

• Low centrifugal force or inadequate spin time.

• Delay in reading (evaporation ↓ plasma height).

• Excess trapped plasma because of morphological change (spherocytosis, microcytosis, macrocytosis; up to +6 %).

• Sickle cell disease: rigid, misshapen cells cause up to +20 % error.

B. False ↓ PCV

• Over-anticoagulation (excess EDTA) → cell shrinkage.

• Clot formation due to poor mixing.

• Long storage (>6 h at room temp) can cause cell swelling; at 4\,^{\circ}C, stable ≤24 h.

C. Miscellaneous pitfalls

• Using non-standard tubes.

• Poorly maintained centrifuge.

Normal / Reference PCV Ranges (Sea-level)

• Newborn: 44–54 %

• Infants / children: 35–45 %

• Adult males: 40–54 %

• Adult females: 36–46 %

Physiological variations: higher at high altitude; lower in pregnancy (plasma expansion).

Pathological alterations: decreased in anaemia; increased in plasma loss (burns, dehydration) and polycythaemia vera or secondary polycythaemia.

Red Cell Indices

Introduced by Wintrobe (1929) to provide objective, numerical description of RBC size & Hb content.

Indices include:

1. Mean Cell Volume (MCV)

2. Mean Cell Haemoglobin (MCH)

3. Mean Cell Haemoglobin Concentration (MCHC)

4. Red Cell Distribution Width (RDW)

They are routinely generated by automated analysers and aid in anaemia classification and QC checks (comparing to Hct, Hb, RBC count consistency).

Source Parameters for Calculations

Need three directly measured values:

• Hb in g/L (or g/dL)

• Hct / PCV in L/L (or %)

• RBC count in \times 10^{12}/L

Metric Prefix Refresher

milli (10^{-3}), micro (10^{-6}), nano (10^{-9}), etc. (Full scale shown in transcript).

Mean Cell Volume (MCV)

Represents average red-cell volume (femtolitres, fL).

Formula:
MCV = \frac{PCV \; (L/L) \times 1000}{RBC \;(10^{12}/L)}
or using % PCV:
MCV = \frac{PCV\;(\%) \times 10}{RBC \;(10^{12}/L)}

Interpretation:

• Normocytic: 80–100 fL

• Microcytic: < 80 fL (iron deficiency, thalassaemia)

• Macrocytic: > 100 fL (B12 / folate deficiency, alcoholism, reticulocytosis)

Note: MCV is volume; smear cell diameter correlates but is not identical (flattened disc vs volume).

Example using male baseline (Hb 15 g/dL, PCV 45 %, RBC 5 \times 10^{12}/L):
MCV = \frac{45 \times 10}{5} = 90\;fL (normal).

Mean Cell Haemoglobin (MCH)

Average mass of Hb per red cell (picograms pg).

Formula:
MCH = \frac{Hb\;(g/dL) \times 10}{RBC\;(10^{12}/L)}

Typical reference: 28–34 pg.

With same example:
MCH = \frac{15 \times 10}{5} = 30\;pg

Conditions causing ↑ MCH (macrocytosis): B12/folate deficiency, reticulocytosis, alcoholism.

Causes of ↓ MCH: iron deficiency, thalassaemia, anaemia of chronic disease.

Mean Cell Haemoglobin Concentration (MCHC)

Average concentration of Hb within packed RBCs – essentially colour intensity, expressed as g/dL or g/L.

Formula:
MCHC = \frac{Hb\;(g/dL) \times 100}{PCV\;(\%) }
or using decimal PCV:
MCHC = \frac{Hb\;(g/dL)}{Hct\;(L/L)}

Reference: 32–36 g/dL.

Example: MCHC = \frac{15}{0.45} = 33.3 \; g/dL.

Interpretation:

• Normochromic: 32–36 g/dL.

• Hypochromic: < 32 g/dL (iron deficiency).

• True hyperchromia (>36 g/dL) only in spherocytosis (reduced surface area). Apparent high MCHC may reflect sample artefacts (in-vitro haemolysis, lipaemia, many Heinz bodies).

Red Cell Distribution Width (RDW)

Coefficient of variation of RBC volume distribution; highlights anisocytosis that MCV can mask.

Automated analysers compute:
RDW\,(\%) = \frac{\text{SD of MCV}}{\text{Mean MCV}} \times 100

Reference: 11.5–14.5 %.

Clinical points:

• ↑ RDW + low MCV is a classic pointer to iron-deficiency anaemia.

• Thalassaemia minor usually: low MCV but normal RDW (uniformly microcytic cells).

Combined Indices Patterns in Common Anaemias

(Arrow legend: ↓ = lower than reference; ↑ = higher; N = within reference; → = unchanged.)

Practical & Philosophical Connections

• PCV still critical in resource-limited settings where automated Hb or CBC instruments are unavailable or unreliable.

• Quick bedside estimation of PCV \div 3 for Hb enables emergency decision-making (transfusion, triage).

• Indices embed simple algebra yet reveal complex erythropoietic physiology; they exemplify how numerical reasoning underpins clinical judgement.

• Ethically, knowing limitations & error sources prevents mis-diagnosis that could lead to inappropriate transfusion or iron therapy.

Key Equations (LaTeX Form)

1. PCV = \frac{\text{RBC height}}{\text{Total column height}}

2. MCV = \frac{PCV\;(\%) \times 10}{RBC\;(10^{12}/L)}

3. MCH = \frac{Hb\;(g/dL) \times 10}{RBC\;(10^{12}/L)}

4. MCHC = \frac{Hb\;(g/dL) \times 100}{PCV\;(\%)}

5. RDW\,(\%) = \frac{SD\;of\;MCV}{MCV} \times 100

Adult Reference Summary

• MCV: 80–100\;fL

• MCH: 28–34\;pg

• MCHC: 32–36\;g/dL

• RDW: 11.5–14.5\;\%

• PCV: 40–54\;\% (males); 36–46\;\% (females)

These notes consolidate every major and minor point from the transcript, integrate all calculations, provide worked examples, link analytic reasoning to clinical practice, and list all normative data and error considerations necessary for examination or real-world laboratory application.