Comprehensive Guide to Hematocrit and Hemoglobin Determination

Definition and General Principles of Hematocrit

• Definition: Hematocrit is the ratio of the volume of packed red blood cells (RBCs) to the total volume of whole blood in a specimen.

• Alternative Terminology: It is also frequently referred to as Packed Cell Volume (PCV), where the "packed cells" specifically refer to the red blood cells.

• Manual Determination:

  • Blood is transferred to a plastic tube with a uniform bore.

  • The tube is centrifuged to pack the cells.

  • The column of RBCs is measured and then divided by the total length of the column (RBCs plus plasma).

• Reporting Units: Hematocrit is reported as a percentage ($\%$) or in liters per liter ($L/L$).

• The Buffy Coat:

  • A light-colored layer often visible between the packed RBCs and the plasma.

  • Contains white blood cells (WBCs) and platelets.

  • This layer is strictly excluded from the calculation during hematocrit determination.

Normal Hematocrit Reference Values

• At Birth: $45 \%$ to $60 \%$

• Adult Women: $36 \%$ to $48 \%$

• Adult Men: $40 \%$ to $55 \%$

Methods of Hematocrit Determination

• Manual Methods: Classified into the Macromethod and the Micromethod.

• Automated Methods:

  • Hematocrit is a computed parameter in automated cell counters.

  • The calculation used is: $\text{Hct} = \text{MCV} \times \text{RBC}$.

• Common Methodologies:

  • Microhematocrit centrifuge.

  • Conductometric methods.

  • Calculations via automated cell counters.

Conductivity Method for Hematocrit

• Principles of Measurement:

  • Plasma is a conductor of electrical current.

  • White blood cells (WBCs) act as insulators.

  • The conductivity of a whole blood specimen depends on the concentration of electrolytes in the plasma portion.

• Specific Devices:

  • i-STAT (Abbott Laboratories): Portable instrument used for measuring hematocrit and other blood parameters.

  • Epoc (Siemens Healthineers): Another device utilizing conductivity for determination.

• System Corrections (i-STAT): Before converting conductance to a hematocrit value, corrections are applied for:

  1. Temperature of the specimen.

  2. Size of the fluid segment being measured (determined from the calibrant conductance).

  3. Relative conductivity of the plasma component (determined from the measured concentrations of sodium and potassium in the specimen).

• Sources of Error in Conductivity Methods:

  • Low Total Protein: Falsely decreases the hematocrit.

  • Lipids: Presence of lipids can interfere with the measurement.

  • Increased WBC Count: Falsely increases the hematocrit as conductivity does not distinguish RBCs from other non-conductive elements.

  • Cold Agglutinins: Falsely decrease the hematocrit.

Detailed Microhematocrit Manual Procedure

• Tube Selection:

  • Fill two plain capillary tubes approximately three-quarters full.

  • Specimens should be anticoagulated with EDTA or heparin.

  • Mylar-wrapped tubes are recommended by the National Institute for Occupational Safety and Health (NIOSH) to mitigate the risk of injury from broken glass.

  • Alternatively, blood may be collected directly into heparinized capillary tubes via skin puncture.

• Sealing the Tube:

  • Wipe excess blood from the exterior of the tube.

  • Seal the end with the colored ring using non-absorbent clay.

  • Hold the tube horizontally and press the dry end into the sealing compound at a $90$-degree angle. Rotate slightly to remove.

  • The clay plug must be at least $4\,mm$ long.

• Centrifugation:

  • Balance tubes in the microhematocrit centrifuge with the clay-filled ends facing outward, touching the rubber gasket.

  • Tighten the head cover and close the centrifuge top.

  • Centrifuge at $10,000\,g$ to $15,000\,g$ for the time required to achieve maximum packing of RBCs.

  • Do not use the brake to stop the centrifuge.

• Reading Results:

  • Use a microhematocrit reading device immediately to determine the level of RBC packing.

  • Exclude the buffy coat from the measurement.

  • Precision Requirement: Duplicate results should agree within $1\%$ ($0.01\,L/L$).

Specialized Centrifuge Equipment

• READACRIT Centrifuge (Becton, Dickinson): Uses precalibrated capillary tubes and features built-in hematocrit scales, removing the need for external reading devices.

• SUREPREP Capillary Tubes (Becton, Dickinson): Features a factory-inserted plug that seals automatically upon contact with blood, eliminating the need for clay sealants.

Sources of Error in Centrifuge-Based Methods

• False Decreases:

  • Improper Sealing: Causes blood leakage during centrifugation. Because RBCs pack at the bottom, more cells are lost than plasma, lowering the reading.

  • Short Draw: An increased concentration of anticoagulant (e.g., too little blood in an EDTA tube) causes RBC shrinkage.

  • Specimen Collection Errors: Introduction of interstitial fluid during skin puncture or improper flushing of an intravenous catheter.

  • Acute Blood Loss: Immediately after loss, plasma is replaced faster than RBCs, leading to a temporary low reading.

• False Increases:

  • Insufficient Centrifugation: The RBCs are not fully packed.

  • Delayed Reading: Reading long after centrifugation has stopped.

  • Inclusion of Buffy Coat: Artificially elevates the RBC column height.

  • Dehydration: Fluid loss decreases plasma volume, falsely elevating the hematocrit.

• Other Errors:

  • Improper Mixing: Can lead to either increased or decreased results depending on the portion of the sample drawn.

  • Trapped Plasma: Specific disorders prevent perfect packing, causing plasma to be trapped in the RBC layer. This results in hematocrit readings $1\%$ to $3\%$ ($0.01$ to $0.03\,L/L$) higher than automated methods. Disorders include:

    • Sickle cell anemia.

    • Macrocytic anemias.

    • Hypochromic anemias.

    • Spherocytosis.

    • Thalassemia.

Hemoglobin (Hgb) Concentration Determination

• Estimation Techniques: Hemoglobin is estimated using light intensity or color-matching techniques, primarily via a spectrophotometer.

• Primary Chemical Methods:

  • Hemoglobin Cyanide Method (HiCN): The international reference method and the standard recommended for measurement.

  • Oxyhemoglobin Method.

• Other Determination Methods:

  • Colorimetric: Direct (Acid hematin with $0.1\,N\,HCl$; Alkali hematin with $0.1\,NaOH$) or Indirect (Cyanmethemoglobin).

  • Gasometric: Van Slyke Oxygen Capacity method.

  • Specific Gravity: Copper sulfate ($CuSO_4$) method.

  • Physical Method: Rule of Three ($\text{Hct} \approx 3 \times \text{Hgb}$).

  • Chemical Method: Measurement of iron content.

Hemoglobin Cyanide (HiCN) / Cyanmethemoglobin Method

• Principle: Blood is diluted in Drabkin solution, which contains potassium cyanide ($KCN$) and potassium ferricyanide ($K_3Fe(CN)_6$).

• Chemical Process:

  1. Potassium Ferricyanide: Converts hemoglobin iron from the ferrous state ($Fe^{2+}$) to the ferric state ($Fe^{3+}$), forming methemoglobin (Hi).

  2. Potassium Cyanide: Converts methemoglobin to cyanmethemoglobin or hemiglobincyanide (HiCN).

• Reagent Components:

  • Nonionic Detergent: Enhances red cell lysis and reduces turbidity caused by abnormal proteins such as lipoproteins.

  • Dihydrogen Potassium Phosphate: A substitute for the original Drabkin's sodium bicarbonate ($NaHCO_3$), allowing the test to be read after $3$ minutes instead of $15$.

• Procedure Details:

  • Dilution is typically $1:201$ ($20\,\mu L$ of blood in $4\,mL$ of diluent).

  • The solution stands for $5$ to $10$ minutes to ensure complete conversion.

  • Absorbance is measured at $540\,nm$ against a reagent blank.

  • Optical density (OD) is directly proportional to hemoglobin concentration (Beer's Law).

• Calculations:

  • $\text{Hgb concentration} = \frac{\text{Absorbance of test sample} \times \text{Concentration of standard} \times \text{Dilution factor}}{\text{Absorbance of Standard} \times 1000}$

• Turbidity Interferences (False Highs):

  • Lipemia: Correct by using a patient blank (plasma + reagent).

  • High WBC (> $30 \times 10^9/L$): Correct by centrifuging the reaction tube and reading the supernatant.

  • Hb S or Hb C: Cells are resistant to lysis. Dilute the mixture $1:1$ with distilled water, read, and multiply the result by $2$.

• Method Notes:

  • Measures all forms of hemoglobin except sulfhemoglobin.

  • Results are not affected by over-anticoagulation.

Normal Hemoglobin Reference Values

• At Birth: $15$ to $20\,g/dL$

• Adult Women: $12$ to $16\,g/dL$

• Adult Men: $13$ to $18\,g/dL$

Additional Hemoglobin Estimation Methods

• Gasometric Method (Van Slyke Oxygen Capacity):

  • Regarded as having the greatest accuracy.

  • Estimates the oxygen-combining power of blood.

  • Calculation: $1\text{ gram Hgb} = 1.34\,mL \text{ of } O_2$.

  • Uses the Van Slyke-Neill Apparatus or the Natelson Microgasometer.

• Specific Gravity (Copper Sulfate Method):

  • A drop of blood is dropped into a $CuSO_4$ solution with a specific gravity of $1.05$.

  • Used as a screening tool for minimum hemoglobin levels:

    • Women: Specific gravity of $1.053$ corresponds to roughly $12.5\,g/dL$.

    • Men: Specific gravity of $1.055$ corresponds to roughly $13.5\,g/dL$.

  • Interferences include myeloma proteins, abnormal globulins, and radiographic contrast media.

• Chemical Method for Iron Content:

  • Kennedy's / Wong's methods: Based on the calculation that $1\text{ gram Hgb} = 3.47\,mg \text{ of Iron}$.

  • Alba's method: Uses the calculation that $1\text{ gram Hgb} = 3.35\,mg \text{ of Iron}$.