Point-of-care hemoglobin testing: Methods and relevance to combat anemia

Anemia is a condition that causes a high degree of personal disability but, historically, has lacked adequate resourcing in many public health systems. This situation is even less understandable when you consider that the main diagnostic, hemoglobin testing, is one of the most commonly used  point-of-care (POC) tests, and one of the easiest to perform.

POC hemoglobin testing is often needed in settings where the use of a benchtop laboratory hematology analyzer is not practical. It is ideal for use in settings where resources are poor, or there is a need for mobility and simplicity in field use, or where turnaround time (TAT) for the test result needs to be short, as in acute clinical situations.

It is vital for healthcare professionals working in these settings to be able to rely on the result given by the POC hemoglobin analyzer and not have to order a confirmatory test from the laboratory. Accuracy and reliability are therefore the crucial characteristics for POC hemoglobin analyzers. Reliability includes traceability to established reference methods, regular quality assurance checks and continuous operator education.

POC hemoglobin testing: where and why?

Anemia is the most common blood disorder, affecting around 25 percent of the global population,¹ and it can be caused by poor nutrition or various diseases. It is a condition in which the number of red blood cells or the availability of hemoglobin falls below the body’s physiological needs. The different causes of anemia can be divided into three groups:

• Blood loss caused by trauma, heavy menstrual bleeding, gastrointestinal bleeding, or other bleedings.
• Decreased or faulty red blood cell production, which is often due to deficiency of iron, vitamin B12 and folate, cancer, HIV/AIDS, or chronic inflammatory or bone marrow diseases.
• Destruction of red blood cells (hemolytic anemias), which can be inherited, (e.g., sickle cell anemia or thalassemias), acquired in the course of autoimmune diseases, or induced by drug exposure or mechanical stress.

The most common cause of anemia is iron deficiency (IDA= iron deficiency anemia), as iron is the central, oxygen-binding molecule of the heme group in each of the four subunits of the hemoglobin protein. The most prominent symptom of anemia is fatigue, often accompanied by shortness of breath, dizziness and headaches, cold hands and feet, pale skin, and chest pain.

Since anemia reduces an individual’s wellbeing, physical productivity, and work performance, timely treatment can restore personal health and raise national productivity levels by as much as 20 percent in developing countries,² where the prevalence of anemia is the highest.

Notably, anemia also contributes to 20 percent of all maternal deaths.² Many countries conduct interventions during pregnancy to reduce anemia and its adverse effects.

Pre-donation hemoglobin testing is an integral part of blood donor assessment in many countries. International and national guidelines commonly recommend minimum hemoglobin levels of 12.5 g/dl for females and 13.5 g/dl for males³ to prevent the risk of inducing anemia by the donation.

Methods of testing hemoglobin

The measurement of hemoglobin (Hb) is the most used parameter in POC hematology,⁴ and it can be accomplished by a variety of methodologies.

Cyanmethemoglobin method (HiCN)

The cyanmethemoglobin method works on the principle of conversion of hemoglobin to cyanmethemoglobin by the addition of potassium cyanide and ferricyanide, whose absorbance is measured at 540 nm in a photometer against a standard solution.⁵

Based on the first initiatives to standardize this method by Drabkin and Austin, the “International Committee for Standardization in Haematology“ (ICSH) was founded in 1964 and published recommendations for the measurement of hemoglobin in 1967 (updated in 1995).⁶ The CLSI (at that time NCCLS) converted these recommendations into a formal standard named NCCLS H15-A3.⁷

Today the HiCN method is still in routine use in laboratories in rural countries. But because it is time-consuming and dependent on cyanide (toxic) agents, it is predominantly used as a reference method for the calibration of modern POC hemoglobin devices and lab analyzers. Standardized, stable reference material is used for the calibration and ensures traceability of POC results back to the HiCN reference method.

Alkaline hematin detergent (AHD575) method

In the alkaline hematin detergent (AHD575) method, red blood cells are lysed with Triton X-100 and sodium hydroxide (NaOH) at pH 13, which converts hemoglobin to hematin, forming a stable complex with Triton X-100. The optical density of hematin is measured at 575 nm to 578 nm. Measurement can also be carried out at 540 nm and 546 nm.⁸

Despite several methodological advantages, the AHD575 has not replaced the more established HiCN method as a reference method for POC hemoglobin analyzers. A direct use of the AHD method in POC testing is possible on a small, portable photometer but still involves several manual steps like sample pipetting or blank measurements of liquid reagents.

Vanzetti´s azide methemoglobin method

The first generation of portable POC hemoglobin devices with single-use, dry reagent cuvettes operates based on a modification of Vanzetti´s azide methemoglobin method.⁹ The blood is drawn into the cuvette by capillary action, and the walls of the red blood are hemolyzed by the reagent. The free hemoglobin is oxidized to methemoglobin, which is then converted into azide methemoglobin. This stable colored complex is measured photometrically at 570 nm. A second measurement is taken at 880 nm for compensation of turbidity. A high sensitivity and specificity has been described for this method,⁵ and today it is the most common and established POC method for measuring hemoglobin in clinical as well as in blood collection settings.

Reagent-less methods

A constant drawback of the azide methemoglobin method is the susceptibility of the reagent to humidity, especially in challenging climate conditions. Cuvettes need to be stored in a carefully closed canister with desiccant and removed directly before usage. The shelf-life after opening of the canister is limited.

POC hemoglobin analyzers using reagent-less cuvettes were developed to overcome these limitations. They either measure the absorbance of whole blood photometrically at the isosbestic point—the wavelength in which the absorbance the two main hemoglobin derivatives, oxy-hemoglobin (HbO2) and deoxy-hemoglobin (Hb), is the same—or, more recently, use broad spectrum photometry with multiple wavelength to get an overall picture of the absorbance spectrum, while scattered light is prevented from arriving at the sensor. This is essential in order to obtain accurate results when measuring non-hemolyzed blood.

Non-invasive methods

Recently, non-invasive methods have become commercially available using near-infrared spectroscopy to identify the spectral pattern of Hb in an underlying blood vessel and derive a measurement of Hb concentration. Other non-invasive devices use white light and capture the reflected transmission data in order to measure Hb levels in tissue capillaries or multiple wavelength light absorption to calculate the Hb concentration. A finger-clip or ring is used to apply the sensor.

Given the advantage of obsolescence of finger-sticks and the option for more frequent measurements in clinical settings, the literature remains unclear about the precision and accuracy of the current non-invasive hemoglobin monitors.^10,11 In practical use, patient movement, nail polish, skin color, or ambient light have been shown to influence the measurement.¹²

Sahli´s method

Sahli’s manual hemoglobinometer operates based on the conversion of hemoglobin to acid hematin by the action of HCL. The acid hematin solution is diluted until its color matches exactly with that of the permanent standard of the comparator block. The hemoglobin concentration is read directly from the calibration tube. In developing countries like India, Sahli’s method, invented in 1902, is still the most common method used for hemoglobin estimation. The method is simple and cheap but rather inaccurate.

The color developed is unstable and must be read after 10 minutes standing. There is inter-observer variability and the use of manual pipetting makes it prone to errors, and there is no international standard.⁵

Hemoglobin color scale (HbCS) method

The hemoglobin color scale (HbCS) method relies on comparing the color of a drop of blood absorbed on a test strip of special chromatography paper with standard colors on a laminated card displayed in increments of 2g/dL (20 g/L). The cost per test is very low, and apart from materials for taking the blood sample, no technical equipment is needed. However, the visual comparison is susceptible to inter-observer variability, and a low sensitivity has been found in some studies.⁵

Copper sulphate (CuSO4) method

The copper sulphate (CuSO4) method is mostly used to ensure a certain Hb level for blood donation. A blood droplet is allowed to fall into copper sulphate solution of a specific gravity, equivalent to that of blood with the cut-off hemoglobin level, e.g. 12.5 g/dL (125 g/L). If the drop of blood floats or takes too long to sink, the donor is deferred.

The CuSO4 method is hampered by a lack of quality control, problems with disposal of the biohazardous solutions, and erroneous results in individuals with a very high serum protein concentration. Furthermore, there are concerns that the CuSO4 method might give falsely high deferral (“false-fail”) rates for donors, particularly in women.⁴ A common practice in many settings is to re-test donors who failed in the CuSO4 test with a quantitative measurement.

Hematology analyzer/CBC method

Automated hematology analyzers can provide high precision, enable high sample throughputs, and analyze a number of different types of red and white blood cells (3-part, 5-part or 7-part differential), blood platelets, hemoglobin, and hematocrit levels from the same blood sample.

However, the investment costs for the analyzer are high and it may not be suitable outside a laboratory environment. Regular maintenance, control of calibration, and stable climate conditions are needed to operate hematology analyzers, and operator lab experience is required.

Blood gas analyzer (BGA) method

Blood gas analyzers are used to measure combinations of pH, blood gases (i.e., pCO2 and pO2), hemoglobin, electrolytes, and metabolites from whole blood samples, mainly from arterial blood. They are commonly used in critical care units, operating theatres, delivery wards, and emergency rooms. Technical improvements such as ready-to-use sensor cassettes and solution packs have made the usage of BGA much more convenient, but maintenance is still required.

Recently, hand-held devices with single-use cartridges have become available for POC use, but the costs per test are high compared to a POC hemoglobin analyzer—if Hb is the primary interest. Also, some cartridges require cool storage and pre-warming before the test can be done.

Factors influencing the measurement

Variability in reported hemoglobin values can be caused by a number of physiological factors and pre-analytical errors. It is of great importance to establish a standardized procedure when measuring hemoglobin on POC testing devices. The physiological factors include:

• Gender: The reference ranges for hemoglobin concentration in adults are 14.0-17.⁵ (mean 15.7) g/dL for men and 12.3-15.3 (mean 13.8) g/dL for women.¹³ For a given finger-stick result, the expected venous Hb value is 0.5 to 0.8 g/dL lower for women compared to men.¹⁴
• Sample type: Capillary blood has higher Hb than venous blood, especially in women and in men with severe iron depletion (median +0.67 g/dL or +6.7 g/L for iron-depleted women to -0.1 g/dL or -1 g/L for iron-repleted men).¹⁴ Venous blood has a slightly higher Hb than arterial blood.¹⁵
• Sampling site: Finger-stick sampling has been shown to more closely approximate venous Hb values, while ear-stick sampling tends to give higher results.¹⁷
• Tourniquet use: Tourniquet use longer than 30 seconds increases venous hemoglobin value.¹⁴
• Body position: Hb is higher in blood samples from standing subjects than in samples from sitting or supine subjects.¹⁴
• Diurnal variation: Hb tends to be higher in the morning and decreases throughout the day.¹⁴
• Dehydration: Loss of plasma volume, for example due to transpiration or insufficient fluid uptake, causes increased hematocrit and hemoglobin values.
• Altitude: The normal hemoglobin concentration increases at high altitudes (>1,500 m) to compensate for the lower concentration of oxygen in the air.¹⁶
• Smoking: Smoking is known to increase hemoglobin concentrations, an effect which is proportional to the amount of tobacco smoked.¹⁶
• Age: The highest hemoglobin concentrations are found in neonates on day one to day three after birth (mean 18.5 g/dL , -2SD: 14.5 g/dL). Hemoglobin levels constantly decline reaching the lowest point at three to six months (mean 11.5 g/dL , -2SD: 9.5 g/dL), when they are starting to increase gradually to adult levels.¹⁸
• Pregnancy: Hemoglobin concentrations decline during the first trimester in pregnancy, reaching their lowest point in the second trimester, diminishing by approximately 0.5 g/dL (5 g/L). Hemoglobin levels begin to rise again in the third trimester.¹⁶

Reference ranges may vary depending on the individual laboratory, instruments, and methods. When carrying out comparative Hb testing for studies or evaluations, the samples should be taken and analyzed under identical conditions.

As for pre-analytical errors, the most common sampling technique for POC hemoglobin tests is taking capillary blood from finger sticks. A free capillary flow is essential to obtain
correct hemoglobin results. Potential sources of error are:

• Choice of lancet: A penetration depth of 1.85 to 2.25 mm, depending on the thickness of the skin, is generally recommended to ensure an adequate flow of blood.
  Selection of puncture site: The middle or ring finger should be used, ideally of the non-dominant hand, as they are generally less calloused and less sensitive to pain. The puncture should be made slightly off center from the central, fleshy portion near the side of the fingertip. The hand must be warm and relaxed. The patient must not wear a ring on the finger as this may obstruct the blood circulation.
• Cleaning and disinfection: After cleaning and disinfection, the puncture site must be dried completely. Remnants of alcohol solution will dilute the blood and cause false low readings.
• Puncture: The finger should be supported by the operator´s hand. It can be massaged gently before and after the puncture to stimulate blood circulation. Maintaining a light pressure in the moment of the puncture helps to achieve a good penetration.
• Capillary flow: The first two or three drops of blood should be wiped away, using a clean lint-free gauze pad. Thinning and clotting of the blood must be avoided, as it causes incorrect results. A good capillary flow is normally found within 30 to 45 seconds after the puncture. The third or fourth drop of blood should be used for the hemoglobin measurement. The drop must be of sufficient size to gain the volume specified for the test. The finger must not be squeezed hard or “milked” to increase the size of the drop, as this will dilute the sample with interstitial fluid.

Conclusion

Point-of-care hemoglobin testing can deliver accurate results comparable to laboratory techniques when traceability to established reference methods is granted and quality control schemes and standardized procedures are implemented. The measurement of hemoglobin can be influenced by a number of physiological factors which need to be considered in order to apply correct reference ranges, and potential pre-analytical errors can be eliminated by continuous operator training. With this in place, hemoglobin testing can be undertaken in a wide range of POC settings.

Happily, there is a growing awareness of the prevalence and consequences of anemia in many emerging countries as they develop their public health services. Alternative test sites are starting to evolve, with pharmacies offering general health checks and mobile care services, taking POC testing into the patient´s home. The volume of hemoglobin tests is therefore expected to grow and will be a key contributor to reducing the global burden of anemia worldwide.

REFERENCES

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Katja Lemburg, a biomedical engineer, serves as Product Manager, Hematology, at EKF Diagnostics, which specializes in the development, production, and distribution of POC analyzers for use in the detection and management of diabetes, anemia, lactate, and kidney-related diseases.