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Hemoglobin Measurement – Pre-analytical Factors and Comparison Between Analytical Methods

By Editor on 3/16/2020

Manisha Shah, IQMH Chemistry Scientific Committee; Danijela Konforte, IQMH Chemistry Scientific Committee; Miranda Wozniak, IQMH Hematology Scientific Committee

Hemoglobin (Hb) is a blood protein that carries oxygen to the body’s organs and tissues. Its measurement is part of a complete blood count – a commonly ordered blood test that aids in the screening and diagnosis of many diseases.

A person’s hemoglobin level or concentration (measured in g/L) is the most commonly used indicator of anemia. Anemia is defined by a Hb concentration below an age, sex and life stage-specific reference interval, keeping in mind that reference intervals may vary based on methods and guidelines.1 Given its utility in screening and evaluating the impact of clinical intervention2, accurate Hb measurement is essential for optimal patient care.

This article provides an overview of the pre-analytical factors that affect Hb measurement, common analytical methods used in measurement of Hb, as well as the interchangeability of results between methods.

Pre-analytical factors

There are a number of physiological and sample collection factors that can affect Hb measurement.  

Biologic variation: Hb measurement can vary over time, even in stable patients. Systematic review and meta-analysis of within-subject (CVi) and between-subject (CVg) biological variation estimated CVi of 2.85% and CVg of 6.8% for Hb.3 The CVi and CVg estimates are based on 21 different publications where sampling intervals vary from hourly to monthly.

Exercise: Sustained exercise (i.e. endurance exercise or training) produces an increase in plasma volume which may lead to a decrease in Hb.

Positioning during phlebotomy: Hb results can be as much as 10 g/L lower if a sample is obtained from a patient in upright versus supine position due to changes in plasma volume.

Blood source: Arterial Hb measurements can be on average 7–10 g/L lower than venous measurements mostly due to decreased plasma volumes in arterial blood.4 Capillary blood samples also show broad variation in hemoglobin values. For example, capillary blood Hb results obtained from different fingers on the same patient can have a deviation of ±7 g/L.5 This could be due to the fact that capillary sample collection sometimes involves squeezing (“milking”) of a finger or heel; this dilutes the sample with interstitial fluid leading to a falsely low Hb result.

IV line contamination: Contamination with infusion fluids when a specimen is collected close to an infusion site can cause spuriously low Hb due to dilution.

Prolonged tourniquet use: Venous stasis of more than one minute during venipuncture can increase Hb by as much as 4 g/L due to hemo-concentration.6

Inadequate sample mixing: Variability of sample manual mixing techniques can lead to falsely increased Hb results in cases where samples are not homogenously mixed before analysis.

Analytical methods used in Hb testing

Most hemoglobin values used for clinical purposes are obtained from invasive methods. However, Hb can be measured by invasive and noninvasive analytical methods.

Noninvasive methods: these methods include pulse oximetry and occlusion spectroscopy which have the ability to estimate Hb concentration through the skin of a finger using a spectrophotometric sensor or by assessing the colour of the conjunctiva of the eye (Photo biosensor). These methods are used predominantly for Hb screening in blood donors or monitoring hemoglobin concentration during surgery in hospitalized patients.7

Invasive methods: these methods involve obtaining a blood sample. Examples include automated hematology analyzers, CO-oximeters, or point-of-care (POC) devices.

Invasive analytical methods for obtaining a hemoglobin value can be divided into two general categories – measured and calculated.

Measured Hb Methods

Hematology analyzers

The hemoglobincyanide method (HiCN) has traditionally been the standard quantitative measurement of Hb. Potassium ferricyanide oxidizes Hb in the whole blood to form methemoglobin (metHb). Potassium cyanide then combines with metHb to form hemoglobincyanide (HiCN) which is a stable pigment read photometrically at a wavelength of 540 nm.

Due to concerns about the disposal of potassium cyanide, most automated hematology analyzers use a photometric method based on sodium lauryl sulfate (SLS) which is similar to the HiCN method. SLS is a surfactant which lyses erythrocytes and forms a complex with the released Hb called SLS-MetHb. This complex is stable for a few hours and has characteristic spectrum with maximum absorbance at 539 nm.8

CO-oximeters are specialized spectrophotometers

A CO-oximeter measures concentrations of different hemoglobin derivatives — oxygenated hemoglobin, deoxygenated hemoglobin, carboxyhemoglobin and methemoglobin. Blood gas analyzers can have an incorporated CO-oximeter. Each hemoglobin derivative absorbs light at a specific wavelength and has characteristic absorbance spectra. Absorbance measurements of a lysed blood sample at multiple wavelengths (520–620 nm) are used by the CO-oximeter software to calculate the concentration of each hemoglobin derivative, the sum of which is equal to total Hb concentration.10

Sample turbidity caused by lipemia, high plasma protein, and cellular matter (e.g. high white blood cell counts) can sometimes interfere with photometric Hb methods.

Point of Care Devices


These portable devices (e.g. HemoCue) also use photometry similar to automated analyzers but use different reagents and measure Hb at a wavelength of 506 nm. These devices are factory calibrated against the HiCN method.7

Calculated Hb Methods

Conductivity methods commonly used by certain POC devices measure electrical conductance of the blood sample, which then is converted to hematocrit (Hct). Hb concentration is then calculated based on the assumption that Hb is approximately one-third of the hematocrit (hematocrit × 0.34 = Hb).10

However, conductivity methods tend to underestimate the hematocrit and therefore the calculated Hb level. For example, Hct derived by conductivity has been shown to be inaccurate at a hematocrit below 0.30, or Hb levels of 100 g/L or less, making this methodology less accurate in detecting anemia.

Furthermore, the accuracy of hematocrit/Hb measurement by conductivity-based methods is affected by several factors such as sodium levels, protein concentrations in the blood, the use of plasma volume expanders, lipemia, and the presence of elevated white cell counts.9

Standardization of Hb Measurement

A reference Hb cyanide (HiCN) solution is used for standardization and calibration of whole blood Hb measurement on automated analyzers and hemoglobinometers. Its hemoglobin concentration is determined based on criteria assigned and reviewed periodically by the International Council for Standardization in Hematology (ICSH).12 Despite availability of reference HiCN solution there are still gaps in standardization between different Hb methods as described in the studies below.

Comparison Between Hb Methods

Automated hematology instruments are considered the standard laboratory method for hemoglobin measurement and all common platforms compare well to each other with regard to hemoglobin. In a 2015 publication, inter-instrument comparison of five automated hematology analyzers, Abbott CELL-DYN Sapphire, Beckman Coulter DxH 800,Siemens Advia 2120i, Sysmex XE-5000 and XN-2000 show good agreement for Hb using patient samples.13

However, studies comparing hemoglobin results from CO-oximetry and hemoglobinometers to automated methods have not shown such good agreement.

In two separate studies it was shown that CO-oximeters and POC devices generally overestimate hemoglobin concentration compared to standard laboratory methods. IL-GEM, Abbott i-STAT and Siemens RapidPoint all showed systematic overestimation of Hb values when compared to the automated hematology analyzers.11,14GEM 4000 consistently overestimated across the analytical range and i-STAT showed a negative bias at lower Hb values.

Additionally, in a study performed by UK NEQAS a significant variation was noted in the results from blood gas analyzers compared to standard laboratory methods. Using partially fixed whole blood samples with a mean Hb value of 93.8 g/L, the blood gas analyzers produced a hemoglobin result in the range of 83–111 g/L compared to hemoglobinometer (HemoCue) in the range of 93–103 g/L, and automated hematology analyzers in the range of 92–100 g/L.15

With regard to hemoglobinometers, the HemoCue has shown greater bias and higher variability in Hb measurement compared with automated hematology instruments. This discrepancy may be a result of different blood sampling procedures, biological differences in capillary versus venous blood, hydration status, or other factors.16

The IQMH proficiency testing (PT) performance expectations for both automated hematology analyzers in addition to CO-oximeters are also worth noting in the section.

The IQMH Hematology PT survey assesses Hb performance of automated lab analyzers based on the instrument mean using the following Allowable Performance Limits (APLs): <100 g/L ±4 g/L; ≥100 g/L ±5%. The IQMH survey material is instrument specific and therefore does not allow for comparison between different instrument groups.

The IQMH CHEM-OX survey assesses performance of CO-oximeters that includes total Hb. The survey is assessed based on all-methods’ mean (AMM) with the following APLs: <100 g/L ±15 g/L; if ≥100 g/L ±15%.

Desirable performance specifications based on biological variation are: Imprecision: 1.43%, Bias: 1.84%, Total Error: 4.19%.17


These studies collectively show it is important to consider the possible pitfalls associated with each analytical method when making a clinical decision regarding treatment or transfusion. Additionally, reference intervals should be adjusted to account for analytical biases to avoid misinterpretation of results.

The laboratory hematology analyzer is usually considered the reference platform within an organization. Regular sample comparisons and establishment of acceptable performance criteria are necessary in any quality assurance program. Observed differences when comparing Hb values from hematology instruments and POC devices that impact patient care must be shared with stakeholders to ensure quality clinical decision making.


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  2. Ontario Transfusion Quality Improvement Plan Committee, Ontario Regional Blood Coordinating Network, Ontario Transfusion Coordinators and Choosing Wisely Canada. (2017) Why give two when one will do? A toolkit for reducing unnecessary red blood cell transfusions in hospitals. Version 1.2 (Choosing Wisely Canada, Toronto) Available from:  .
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