Introduction

The term hematocrit is derived from the English prefix hemato- and the Greek word krites. Hematocrit measures the volume of packed red blood cells (RBCs) relative to whole blood cells (WBCs). Hence, it is also known and reported as a packed cell volume. Hematocrit is a simple test used to identify conditions such as anemia or polycythemia and to monitor the response to treatments. A glass tube and a centrifuge machine are sufficient to measure hematocrit. After centrifugation, the blood component separates into 3 distinct parts—the bottom layer of RBCs, a middle layer of WBCs and platelets, and a top layer of plasma. This method of determining hematocrit using a Wintrobe hematocrit tube is known as the macrohematocrit method (see Image. Wintrobe Hematocrit Tube Containing Blood Components After Centrifugation).[1] 

The Wintrobe tube is a narrow glass tube measuring 110 mm long, with graduation from 0 to 100 mm in ascending and descending order. This method has been replaced by the microhematocrit method, which uses a small capillary tube instead of a Wintrobe hematocrit tube. The microhematocrit method requires a smaller blood sample and less time for the testing procedure, making it beneficial for patients where blood collection is difficult, such as pediatric patients or those with hypovolemia. However, the principle of the test remains the same as the macrohematocrit method. Hematocrit calculation is performed by dividing the lengths of the packed RBC layer by the length of total cells and plasma. As it is a ratio, it does not have any unit. Multiplying the ratio by 100 gives the accurate value, which is the accepted reporting style for hematocrit. For healthy adults, normal hematocrit ranges from 40% to 54% in males and 36% to 48% in females.[2] Although these 2 methods are still used in some primary care settings and medical teachings, they are widely replaced in most settings by an automated analyzer, where hematocrit reports are generated along with the complete blood count.

Specimen Requirements and Procedure

Blood specimen handling should be performed using proper aseptic precautions, and testing should be performed immediately after collection. Prolonged storage at room temperature can alter the shape of RBCs due to metabolism. After approximately 6 hours, the risk of hemolysis increases, leading to inaccurate results.[3][4]

For the macrohematocrit method, venous blood is collected as a random sample, requiring no special precautions other than aseptic technique. The blood is typically collected in a vacutainer containing EDTA or in a vial or test tube with EDTA if vacutainers are unavailable.[5] Proper care must be taken when filling the Wintrobe hematocrit tube. If the tube is reused, it should be thoroughly cleaned, as any foreign particles could interfere with the RBC or plasma column, leading to errors.

For the microhematocrit method, a smaller blood sample is required, and a single finger-prick sample is sufficient. Blood is collected in a heparin-filled capillary tube; if anticoagulated blood is available from other hematologic tests, a capillary tube without heparin can be used. The sealing of the capillary tube must be secure to prevent leakage.[6]

For hematocrit measurement using an automated hematologic cell counter, blood collected with an anticoagulant, such as that used for a complete blood count, is required.[7]

During centrifugation, whether for macrohematocrit or microhematocrit methods, the centrifuge machine must not be opened; interrupting the centrifugation process increases the likelihood of inaccurate results. The operator should wait until the centrifuge has completely stopped before opening the lid.[8][9]

Testing Procedures

The macrohematocrit method uses a Wintrobe hematocrit tube, a centrifuge machine, and a Pasteur pipette. Blood is placed into a Wintrobe hematocrit tube up to the 100-mm mark using a Pasteur pipette. Care is taken not to leave any bubbles in the blood column. To avoid bubbles, the pipette tip should remain below the surface of the blood column during filling. Once filled, the tube is placed in the centrifuge. When testing a single specimen, another blood-filled Wintrobe hematocrit tube is kept on the opposite holder to counterbalance. The centrifuge is set to 3000 rpm for 30 min. After completion of the centrifugation, the tube is removed, and the height of the RBC column is reported as hematocrit. During the reporting, special precaution is necessary to omit the buffy coat, a combination of WBC and platelets. This layer should not be included in the hematocrit, which may lead to false positive results.

For the microhematocrit method, blood is drawn into a capillary tube, typically 75 mm long and 1 mm in diameter. Both ends of the tube are sealed with clay sealant or heat. The tube is centrifuged at 11,000 to 12,000 rpm for 4 to 5 minutes. The hematocrit is read using a scale on the tube holder or a microhematocrit card reader.

The automated analyzer measures the average RBC size and number using the Coulter principle.[10] In this method, the size and number of the RBCs are measurable by detecting impedance while the blood passes through a space between 2 electrodes.

Interfering Factors

The hematocrit may deviate from its normal range due to several physiological and pathological conditions. Newborns have a high hematocrit, which gradually decreases during the neonatal period.[11] Adult males have a higher hematocrit compared to adult females.[12] Pregnant patients may have a lower hematocrit due to hemodilution. The number of RBCs is elevated in patients living at high altitudes due to persistent hypoxia; the inhabitants of high altitudes have a higher hematocrit.

Methodological variation may provide a minor deviation of hematocrit tested for the same sample. In the macrohematocrit method, there is an increased amount of trapped plasma (approximately 2%) in the packed RBC, potentially resulting in a higher hematocrit reading. This factor is minimized with the microhematocrit method, where the amount of trapped plasma is less as the diameter of the capillary tube is less than that of the Wintrobe hematocrit tube. Blood collected from different sources may also show variation. Venous blood demonstrates a higher hematocrit compared to arterial blood. However, there is no difference in hematocrit between venous and finger-prick blood samples.[13]

Results, Reporting, and Critical Findings

The Wintrobe hematocrit tube is graduated from 0 to 100 from bottom to top, with the highest level indicating the packed RBC as a percentage of hematocrit. For the microhematocrit method, the reading is from the hematocrit card or scale. However, for these 2 methods, even without any graduation or scale, the hematocrit value can be calculated with a simple scale by comparing the length of the RBC column with the total length of the fluid column. The final report is a percentage. Hematocrit, along with RBC count and hemoglobin (Hb) concentration, is used to report other blood indices manually as follows:

The mean corpuscular volume (MCV) calculation uses hematocrit and RBC count.[14]

• MCV (fL) = hematocrit (%) × 10 / RBC count (10¹²/L)

Mean corpuscular hemoglobin concentration (MCHC) is calculated using Hb concentration and hematocrit.

• MCHC (%) = Hb concentration (g/dL) / hematocrit (%) × 100

Clinical Significance

In primary healthcare settings, especially in resource-limited settings, macrohematocrit and microhematocrit methods are 2 low-cost, simple tests for determining RBC levels in the blood. Clinically, hematocrit is used to identify anemia and polycythemia along with other parameters, such as RBC count and Hb concentration. In anemia, where there are fewer RBCs in the circulating blood relative to the total blood volume, the hematocrit decreases.[15] In polycythemia, an increased number of RBCs leads to a higher hematocrit.[16] Smokers and chronic obstructive pulmonary disease patients also have high hematocrit due to chronic hypoxia.[17] The increase in hematocrit increases the viscosity of the blood, and so does the peripheral resistance. Hence, patients with higher hematocrit may have higher blood pressure.[18]

Quality Control and Lab Safety

Internal quality control (QC) for hematocrit tests is essential to ensure the accuracy and reliability of test results in clinical laboratories.[19] Internal QC assesses a measurement procedure by periodically testing control samples, such as those with known hematocrit values. If the result for a QC sample is within acceptable limits, the measurement process is confirmed to function correctly, allowing patient results to be confidently reported for clinical use. However, if a QC result falls outside acceptable limits, it indicates a problem with the measurement procedure, and there is a high probability that patient sample results may not be reliable. In this case, corrective action must be taken, and patient samples may need to be retested once the system is stabilized.[20] A corrected report must be issued if incorrect results are reported before the issue is identified.[21]

A Levey-Jennings chart is a commonly used tool in laboratory QC, particularly for tracking the performance of assays such as hematocrit tests. This chart offers a visual representation of QC data over time, enabling easy assessment of measurement stability and accuracy. Regular plotting of QC results allows laboratories to quickly identify trends or shifts that may affect test accuracy.[22][23]

External quality assessment (EQA) is a critical component of quality assurance in clinical laboratories.[24] This assessment evaluates and improves the quality of laboratory testing through independent assessment and comparison of results among participating laboratories. EQA providers send test samples with unknown values to participating laboratories. Each laboratory measures the external quality assessment/proficiency testing (EQA/PT) samples as patient samples and reports the results to the EQA/PT provider for evaluation. The EQA/PT provider sets target values for the EQA/PT samples and determines whether the results for an individual laboratory align closely enough with the target value to demonstrate acceptable measurement procedure performance.[25] EQA/PT programs that use commutable samples are preferred whenever available.[26]

Lab safety is essential for ensuring a secure working environment and preventing accidents or exposure to hazardous materials.[27] Regardless of size, every clinical laboratory must implement a comprehensive safety program. A designated safety officer should oversee the program, with overall responsibility starting from laboratory leadership, including directors and managers, and delegated through them. The safety officer or committee guides leadership in maintaining a safe workplace for all employees.[28]

Key elements of the safety program include educating and motivating staff on safety protocols. New employees should receive a copy of the laboratory safety manual during orientation, and ongoing education should include regular safety discussions.[29]

The laboratory environment must meet accepted safety standards, including proper labeling of chemicals, functional fire extinguishers, and hoods; adequate grounding of electrical equipment; ergonomic considerations; and safe handling and disposal of biohazardous materials. The Occupational Safety and Health Administration mandates that employees are provided with appropriate personal protective equipment, such as lab coats, gloves, and eye protection, in necessary areas.[30]

Safety equipment, such as eye washers, face washers, and safety showers, should be strategically located and regularly tested to ensure they meet safety requirements. A good laboratory practice is to form a safety inspection team composed of laboratory staff. This team is responsible for conducting regular, scheduled safety inspections of the laboratory.[31][32]

Enhancing Healthcare Team Outcomes

With the advent of automated hematology cell counters, the macrohematocrit and microhematocrit methods have become less commonly used in many clinical settings. However, these techniques remain vital in resource-poor environments where access to advanced technology may be limited. In these contexts, macrohematocrit and microhematocrit methods are essential for diagnosing conditions such as anemia and polycythemia and monitoring treatment responses. Notably, in rural healthcare facilities, the microhematocrit method is often more reliable for identifying anemia compared to total RBC counts, which are prone to significant errors in manual assessment.

The microhematocrit method, in particular, offers advantages that make it suitable for mass surveys and screening programs. This method requires a smaller blood sample, which benefits patients, especially when blood volume is a concern, such as in pediatric populations or individuals with difficult venous access. In addition, the testing time for microhematocrit is significantly shorter compared to that for the macrohematocrit method, allowing for a quicker turnaround in results. This efficiency enhances the treatment of patients and facilitates timely interventions in public health initiatives, making it a valuable tool in settings where rapid diagnosis is crucial.