**4. Analysis of onboard techniques on electrophoresis systems**

The following theories are drawn mainly from the book *Appliances and Methods in Biochemistry and Molecular Biology*, whose pedagogical approach seems clearer.

#### **4.1 General principles of electrophoresis**

Electrophoresis has established itself over time as the method of choice for the qualification and quantification of different fractions in the management of hemoglobinopathy. It involves methods often embedded in laboratory materials. We review below the most common methods in electrophoresis of hemoglobin.

#### *4.1.1 Definitions*

Electrophoresis is a physical method of separating molecules based on their difference in mobility, under the effect of an electric field. Zone electrophoresis, carried out on a solid support, is used to essentially separate the ionizable biological macromolecules, that is to say proteins, nucleic acids, and certain polyosides and proteoglycans.

Liquid vein electrophoresis, currently capillary electrophoresis, is also applied to small molecules, organic or mineral, and not necessarily ionizable. In the most common case, the movement of the molecule depends on several intrinsic (due to the molecule itself) and extrinsic parameters, in particular linked to migration buffers which play the role of solvent [22].

#### *4.1.2 Zone electrophoresis*

This is the electrophoresis whose migration medium is stabilized by a real or sometimes virtual porous support as in the density gradient. In the case of a porous substrate, it is soaked with a buffer solution that both ensures conductivity and stabilizes the pH at the desired value. The molecules separate according to their different mobility in the system (they appear as migration zones) and will be visualized in a second time ("revelations"); we can even isolate them from the support for the preparatory purpose.

Zone electrophoresis is mainly applied to the separation of macromolecules.

#### *4.1.2.1 Characteristics*

These electrophoreses are often characterized by strong electroosmotic currents and sometimes intense Joule effect. The most common electroosmotic current is the electroendosmosis current, especially in polyosidic supports used at pH alkaline: the walls are negatively ionized as the macromolecules to be separated; positive buffer charges are attracted to the cathode and create a current that is in the opposite direction of electrophoretic migration.

Another electroosmosis phenomenon is related to the structure of the support, which can be assimilated to a capillary network; the friction forces are greater on the *Contribution of Biomedical Equipment Management to Better Management of Sickle Cell Disease… DOI: http://dx.doi.org/10.5772/intechopen.92546*

edges of the support, and the center moves faster, distorting the migration band. Finally, the Joule effect heats the substrate and therefore evaporates the solvent; this is gradually replaced by the liquid of the vessels which rises in the support by capillary action, opposite both ends of the support, and annulling in the middle [23].

## *4.1.2.2 Supports*

In the present case of the management of sickle cell disease and in order to make

The following theories are drawn mainly from the book *Appliances and Methods in Biochemistry and Molecular Biology*, whose pedagogical approach seems clearer.

Electrophoresis has established itself over time as the method of choice for the qualification and quantification of different fractions in the management of hemoglobinopathy. It involves methods often embedded in laboratory materials. We review below the most common methods in electrophoresis of hemoglobin.

Electrophoresis is a physical method of separating molecules based on their difference in mobility, under the effect of an electric field. Zone electrophoresis, carried out on a solid support, is used to essentially separate the ionizable biological macromolecules, that is to say proteins, nucleic acids, and certain polyosides and

Liquid vein electrophoresis, currently capillary electrophoresis, is also applied to

small molecules, organic or mineral, and not necessarily ionizable. In the most common case, the movement of the molecule depends on several intrinsic (due to the molecule itself) and extrinsic parameters, in particular linked to migration

This is the electrophoresis whose migration medium is stabilized by a real or sometimes virtual porous support as in the density gradient. In the case of a porous substrate, it is soaked with a buffer solution that both ensures conductivity and stabilizes the pH at the desired value. The molecules separate according to their different mobility in the system (they appear as migration zones) and will be visualized in a second time ("revelations"); we can even isolate them from the

Zone electrophoresis is mainly applied to the separation of macromolecules.

These electrophoreses are often characterized by strong electroosmotic currents and sometimes intense Joule effect. The most common electroosmotic current is the electroendosmosis current, especially in polyosidic supports used at pH alkaline: the walls are negatively ionized as the macromolecules to be separated; positive buffer charges are attracted to the cathode and create a current that is in the opposite

Another electroosmosis phenomenon is related to the structure of the support, which can be assimilated to a capillary network; the friction forces are greater on the

his contribution relevant and effective, the biomedical engineer must make an inventory of the existing situation in the field, evaluate the technologies in the state of the art, and propose material solutions that present a better compromise between technical and technological contributions and optimization of the financial aspect.

**4. Analysis of onboard techniques on electrophoresis systems**

**4.1 General principles of electrophoresis**

*Human Blood Group Systems and Haemoglobinopathies*

buffers which play the role of solvent [22].

support for the preparatory purpose.

direction of electrophoretic migration.

*4.1.1 Definitions*

proteoglycans.

*4.1.2 Zone electrophoresis*

*4.1.2.1 Characteristics*

**184**

The supports must be chemically inert (low adsorbent) and homogeneous (regular microporous structure), have good mechanical resistance (handling), and possibly allow densitometric reading [24].

## *4.1.2.2.1 Paper*

Paper is a natural cellulose; it is no longer used much because it is not homogeneous. Paper electrophoresis provides a strong electroendosmosis current and is a source of parasitic adsorptions (added chromatography), resulting in poor resolution; the Joule effect is important with heating, evaporation, and even electrolysis of the buffer. At high pressure (1000–3000 V), paper electrophoresis is mainly used to separate peptides and amino acids.

#### *4.1.2.2.2 Cellulose acetate*

Cellulose acetate is much more homogeneous than paper; this support allows densitometric reading, but the electroendosmosis current remains high. The applications of cellulose acetate are mainly found in medical biology, allowing a quantitative densitometric reading of the protein fractions rather roughly separated (plasma and urinary proteins, lipoproteins, and hemoglobins), or finer (isoenzymes), applying the potential gradients of the order of 30 V cm<sup>1</sup> . Resolution is poor, and reproducibility is average.

However, at alkaline pH (typically pH 8.6), Hb A2, Hb C, Hb E, and Hb O migrate to the same area, and Hb S, Hb D, and Hb G migrate at the same rate. In the case of suspicions of such hemoglobin abnormalities, an additional technique should therefore be considered [25].

#### *4.1.2.2.3 Starch gel (cross-linked starch)*

Starch gel is a polyoside; electrophoresis on this gel allows the separation of complex or heterogeneous oligomeric protein associations. Starch gel is little used because it is opaque, fragile, and not very reproducible.

#### *4.1.2.2.4 Agarose gel*

Agarose is desulfonated agar (purified agar); removal of sulfonates greatly limits the flow of electroendosmosis; agarose gels between 0.5 and 2% are not very viscous. They make it possible to carry out native electrophoresis as with the previous supports, that is, without denaturation of the macromolecules. Potential gradients up to 50 V cm<sup>1</sup> are usable for protein separation; agarose gel is gradually replacing cellulose acetate in most biomedical applications because agarose improves resolution and remains colorless, allowing a good densitometric reading. The agarose gel is also very homogeneous, thus ensuring good reproducibility, and is well adapted to zymographic reading [23].

The distinction between the different variants Hb A2, Hb C, Hb E, and Hb O, as well as Hb S, Hb D, and Hb G, is most often made by electrophoresis on agarose gel at acidic pH (pH 6.0), which allows to separate Hb C, from Hb E and Hb O, as well as Hb S, from Hb D and Hb G. On the other hand, Hb E and Hb O, as well as Hb D and G, still cannot be differentiated by combining these two electrophoretic methods (cellulose acetate, agarose gel). In addition, these techniques have the disadvantage of consuming time and labor.

This technique, capable of separating hemoglobin variants with isoelectric points different from 0.02 pH units, has excellent resolution and is very useful for detecting abnormal hemoglobin in the newborn. In fact, it allows a good separation of hemoglobins F, A, and S. Moreover, the electrical isofocusing is perfectly adapted to the analysis of large series. On the other hand, the main limitations of this method are a long and complex implementation. Therefore, its use is almost

*Contribution of Biomedical Equipment Management to Better Management of Sickle Cell Disease…*

Typically, capillary electrophoresis is performed in a fused silica capillary coated with a polyamide layer of 20–200 μm of internal diameter and 20–200 cm of length. The capillary, placed in a thermostatization system, is filled with a buffer solution and plunges into two tanks containing the same solution. Each tank is connected to an electrode connected to a current generator. A large potential difference (several

thousand volts) is applied to the terminals of each capillary to separate the

The use of a capillary has a double advantage: increases the sensitivity of the detection since a reading window in the capillary allows an absorbance reading with a very small optical path and increases the resolution by applying the potential difference of more than 10,000 V since it is easy to regulate the capillary in

In this method, the buffer solution in contact with the two tanks of the system constitutes the support. Since liquid has no specific form, the buffer uses the capillary

There are several methods used in capillary electrophoresis including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and micellar electrokinetic chromatography (MECC) [32]. In this study, we will limit ourselves to capillary zone electrophoresis which is the most exploited for hemoglobinopathy. This method can be performed on a single-fused silica capillary in which an electroosmotic flux develops. It causes the negative molecules to the cathode where

The electroosmotic flux depends on the temperature, the ionic force, and the

We will limit ourselves here to the most common routine techniques for the analysis of hemoglobinopathies in the Democratic Republic of the Congo in particular and in sub-Saharan Africa in general. They are often affected by accessibility during acquisition, ease of commissioning, operation, maintenance and

On one side, our analysis is based globally on equipment meeting international standards such as ISO, FDA, and CE certification. Field experience shows that such

as a solid support, contributing also to electroendosmosis current production.

molecules on the basis of their charge/mass ratio [30].

*4.1.3.3 Liquid vein electrophoresis methods*

concentration of organic solvent.

the detection is carried out, the injection being anodic.

reserved for neonatal screening of hemoglobinopathies [28, 29].

*4.1.3 Liquid vein electrophoresis*

*DOI: http://dx.doi.org/10.5772/intechopen.92546*

*4.1.3.1 Characteristics*

temperature [31].

*4.1.3.2 Support*

**4.2 Facilities**

supply, and cost.

**187**

In addition, they lack precision for the quantification of hemoglobin in low concentrations, such as Hb A2, and for the detection of fast-migrating variants, such as Hb H or Hb Bart's. It is even now accepted that the quantification of variants by densitometry lacks precision and that these two electrophoresis techniques must be used for qualitative purposes. They are therefore most often used today in combination with another method, mainly high-performance liquid chromatography, which has a much higher accuracy.

A 1999 study by the College of American Pathologists showed a coefficient of variation (CV) of 33.6% for the quantification of Hb A2 at a concentration of 2.41% by densitometry from electrophoretic gels. By HPLC, the CV was 4.3% for Hb A2 at a concentration of 3.47%. Thus, the combination of these electrophoresis techniques with HPLC allows the identification and quantification of hemoglobin, the latter being performed by HPLC only [26].

#### *4.1.2.2.5 Polyacrylamide*

It is a polymer of acrylamide and N,N<sup>0</sup> -methylene-bisacrylamide (Bis), the acrylamide gel polymerization being obtained in the presence of a catalyst (ammonium persulfate) and a cross-linking agent (N,N,N<sup>0</sup> ,N<sup>0</sup> -tetramethyl-ethylenediamine [TEMED]). The porosity of gels can be very precise; it depends on the relative concentrations of acrylamide and Bis.

The polymer obtained is very hydrophilic although insoluble in water and easy to mold even under small thicknesses (<1 mm); it is thermostable, not fragile, transparent, and inert chemically. There is almost no electroendosmosis flow and no macromolecules are absorbed. The resolutive power is generally superior to that of polyosidic gels using gradients of similar potential. The main disadvantage is that the acrylamide in solution is neurotoxic but also that the resulting porosities are very poorly adapted to very large molecules [27].

#### *4.1.2.3 Zone electrophoresis methods*

#### *4.1.2.3.1 Native electrophoresis (without denaturation)*

It is made on paper, starch, cellulose acetate, agarose gel, and sometimes polyacrylamide whenever we do not want to touch the tertiary and quaternary structures of macromolecules, thus their biological activities. This method without denaturation is a priori applicable to all types of macromolecules, both in vertical and horizontal tanks [27].

#### *4.1.2.3.2 Isoelectrofocusing*

Isoelectrofocusing, carried out on agarose gel or polyacrylamide gel, separates hemoglobin in a pH gradient according to their isoelectric point. To do this, ampholytes are introduced into the gel in order to create a continuous pH gradient under the effect of an electric field. The different hemoglobins contained in the sample to be analyzed will migrate to the region where the pH is equal to their isoelectric pH. At this position, the net load is zero, and the hemoglobin ceases to migrate and focuses into a narrow band.

#### *Contribution of Biomedical Equipment Management to Better Management of Sickle Cell Disease… DOI: http://dx.doi.org/10.5772/intechopen.92546*

This technique, capable of separating hemoglobin variants with isoelectric points different from 0.02 pH units, has excellent resolution and is very useful for detecting abnormal hemoglobin in the newborn. In fact, it allows a good separation of hemoglobins F, A, and S. Moreover, the electrical isofocusing is perfectly adapted to the analysis of large series. On the other hand, the main limitations of this method are a long and complex implementation. Therefore, its use is almost reserved for neonatal screening of hemoglobinopathies [28, 29].

## *4.1.3 Liquid vein electrophoresis*

#### *4.1.3.1 Characteristics*

at acidic pH (pH 6.0), which allows to separate Hb C, from Hb E and Hb O, as well as Hb S, from Hb D and Hb G. On the other hand, Hb E and Hb O, as well as Hb D and G, still cannot be differentiated by combining these two electrophoretic methods (cellulose acetate, agarose gel). In addition, these techniques have the

In addition, they lack precision for the quantification of hemoglobin in low concentrations, such as Hb A2, and for the detection of fast-migrating variants, such as Hb H or Hb Bart's. It is even now accepted that the quantification of variants by densitometry lacks precision and that these two electrophoresis techniques must be used for qualitative purposes. They are therefore most often used today in combination with another method, mainly high-performance liquid chromatogra-

A 1999 study by the College of American Pathologists showed a coefficient of variation (CV) of 33.6% for the quantification of Hb A2 at a concentration of 2.41% by densitometry from electrophoretic gels. By HPLC, the CV was 4.3% for Hb A2 at a concentration of 3.47%. Thus, the combination of these electrophoresis techniques with HPLC allows the identification and quantification of hemoglobin, the latter

acrylamide gel polymerization being obtained in the presence of a catalyst (ammo-

The polymer obtained is very hydrophilic although insoluble in water and easy to mold even under small thicknesses (<1 mm); it is thermostable, not fragile, transparent, and inert chemically. There is almost no electroendosmosis flow and no macromolecules are absorbed. The resolutive power is generally superior to that of polyosidic gels using gradients of similar potential. The main disadvantage is that the acrylamide in solution is neurotoxic but also that the resulting porosities are

It is made on paper, starch, cellulose acetate, agarose gel, and sometimes polyacrylamide whenever we do not want to touch the tertiary and quaternary structures of macromolecules, thus their biological activities. This method without denaturation is a priori applicable to all types of macromolecules, both in vertical

Isoelectrofocusing, carried out on agarose gel or polyacrylamide gel, separates

hemoglobin in a pH gradient according to their isoelectric point. To do this, ampholytes are introduced into the gel in order to create a continuous pH gradient under the effect of an electric field. The different hemoglobins contained in the sample to be analyzed will migrate to the region where the pH is equal to their isoelectric pH. At this position, the net load is zero, and the hemoglobin ceases to

diamine [TEMED]). The porosity of gels can be very precise; it depends on the



,N<sup>0</sup>

disadvantage of consuming time and labor.

*Human Blood Group Systems and Haemoglobinopathies*

phy, which has a much higher accuracy.

being performed by HPLC only [26].

It is a polymer of acrylamide and N,N<sup>0</sup>

relative concentrations of acrylamide and Bis.

very poorly adapted to very large molecules [27].

*4.1.2.3.1 Native electrophoresis (without denaturation)*

*4.1.2.3 Zone electrophoresis methods*

and horizontal tanks [27].

*4.1.2.3.2 Isoelectrofocusing*

**186**

migrate and focuses into a narrow band.

nium persulfate) and a cross-linking agent (N,N,N<sup>0</sup>

*4.1.2.2.5 Polyacrylamide*

Typically, capillary electrophoresis is performed in a fused silica capillary coated with a polyamide layer of 20–200 μm of internal diameter and 20–200 cm of length. The capillary, placed in a thermostatization system, is filled with a buffer solution and plunges into two tanks containing the same solution. Each tank is connected to an electrode connected to a current generator. A large potential difference (several thousand volts) is applied to the terminals of each capillary to separate the molecules on the basis of their charge/mass ratio [30].

The use of a capillary has a double advantage: increases the sensitivity of the detection since a reading window in the capillary allows an absorbance reading with a very small optical path and increases the resolution by applying the potential difference of more than 10,000 V since it is easy to regulate the capillary in temperature [31].

#### *4.1.3.2 Support*

In this method, the buffer solution in contact with the two tanks of the system constitutes the support. Since liquid has no specific form, the buffer uses the capillary as a solid support, contributing also to electroendosmosis current production.

#### *4.1.3.3 Liquid vein electrophoresis methods*

There are several methods used in capillary electrophoresis including capillary zone electrophoresis (CZE), capillary gel electrophoresis (CGE), and micellar electrokinetic chromatography (MECC) [32]. In this study, we will limit ourselves to capillary zone electrophoresis which is the most exploited for hemoglobinopathy.

This method can be performed on a single-fused silica capillary in which an electroosmotic flux develops. It causes the negative molecules to the cathode where the detection is carried out, the injection being anodic.

The electroosmotic flux depends on the temperature, the ionic force, and the concentration of organic solvent.

#### **4.2 Facilities**

We will limit ourselves here to the most common routine techniques for the analysis of hemoglobinopathies in the Democratic Republic of the Congo in particular and in sub-Saharan Africa in general. They are often affected by accessibility during acquisition, ease of commissioning, operation, maintenance and supply, and cost.

On one side, our analysis is based globally on equipment meeting international standards such as ISO, FDA, and CE certification. Field experience shows that such equipment can operate for about 7 years, if the manufacturer's operating recommendations are followed.

*4.2.2 Semiautomatic systems*

*DOI: http://dx.doi.org/10.5772/intechopen.92546*

modules, which considerably reduce user handling.

module, and the application programs for the two modules.

Semiautomatic systems for native electrophoresis on cellulose acetate or agarose gel offer the possibility of processing large series of samples. Although the cost of the system is still high for a large number of health facilities, the manual routine is clearly improved. Semiautomatic systems (on cellulose acetate or agarose gel) are embedded on compact systems generally comprising a migration and coloring

*Contribution of Biomedical Equipment Management to Better Management of Sickle Cell Disease…*

The reading system, often equipped with advanced post-processing software, can be incorporated or remote. Nevertheless, the results obtained show the same limits as in the case of manual methods because the operating principles of the migration and

The semiautomatic system is generally composed of a compact unit comprising a thermoregulated migration module connected to a current generator and a fluidic module for coloring migrated gels. Technologically, an intelligent electronic unit manages the high voltage of the migration module, the fluidics of the coloring

Generally, devices typically contain conventional electronic parts and boards. The process control is often ensured by position and temperature sensors. As their complexity is not great, maintenance can be easily carried out by a duly trained

These systems can equip medium-sized laboratories by planning either periodic operation or continuous operation, depending on the flow of samples. They are fairly widespread in the private laboratories which can obtain them and some public hospital laboratories often on behalf of specific programs. User maintenance monitoring must be ensured to guarantee proper functioning, and periodic annual maintenance must be carried out, insured in accordance with the manufacturer's

Automatic techniques (only capillary zone electrophoresis) are the best offer, both in terms of flexibility of use and technical performance. Prices are still very high for many customers in Africa. Nevertheless, a good expression of needs and an adequate exploitation planning can allow a return on investment in an acceptable

These techniques are carried out on compact systems, generally comprising capillaries at the ends plunging into reservoirs of buffer solution, themselves connected to the current generator. The apparatus also includes a detection system, most often a UV–visible spectrophotometer, linked to the wavelength of specific

coloring units taken separately are the same as those of the manual system.

*4.2.2.1 Offer*

*4.2.2.2 Facilities*

biomedical technician.

recommendations.

*4.2.3.1 Offer*

*4.2.3.2 Facilities*

absorption of hemoglobin at 415 nm.

time.

**189**

*4.2.3 Automatic techniques*

*4.2.2.3 Use*

On the other hand, high-performance liquid chromatography has been developed to allow both the detection and confirmation of hemoglobinopathy in newborns with high sensitivity and specificity. In fact, its good sensitivity to the major variants involved in pathology and its speed of completion (about 3 min per sample), allowing the analysis of a large number of samples, have made HPLC a particularly suitable method for screening for hemoglobin abnormalities [33].

However, we will not discuss this technique in this study because, since its performance is comparable to that of the HPLC method, capillary electrophoresis quickly became the method of choice, just like HPLC, for the study of hemoglobinopathy. In addition, it is of economic interest: although the material cost is comparable to that of the HPLC, the expenditures on reagents are much lower. Indeed, the price of a capillary is much lower than that of a chromatography column, and the volumes of buffer used are much lower, about 1000 times less [34].

#### *4.2.1 Manual systems*

#### *4.2.1.1 Offer*

Manual systems for native electrophoresis (on cellulose acetate and agarose gel) or isoelectrofocusing offer the best acquisition possibility both in terms of cost and operational constraints. Their limits both in the separation and in the identification of hemoglobin variants will be used for the routine forms to be specified by the customer (identification of the electrophoretic profile, identification of specific variants).

The coupling of these methods to the reading system (densitometers) makes it possible to quantify the separate variants. And from this point of view, agarose gel electrophoresis offers better performance than cellulose acetate. On the other hand, the reagents are in the form of combs which often require a minimum of seven samples. Such a constraint requires, for economic reasons, to launch the samples in series of seven, which requires a consequent sizing and proper holding wire.

#### *4.2.1.2 Facilities*

For native electrophoresis the system is usually composed of a current generator and a migration tank. For isoelectrofocusing, the system consists of a stabilized supply, an isoelectrofocusing, unit, and a circulating cryostat.

The devices typically contain conventional electronic parts and boards, used in the manufacture of power generators. These components are often not complex, and do not require advanced technical repair. In addition, it has been found that when equipment actually meets ISO, FDA, or CE marking standards, it works well and lasts for a long time.

#### *4.2.1.3 Use*

These systems can be used in very small laboratories, without large volume flow of samples, for the screening of hemoglobinopathies, by planning a periodic operation. Indeed, the pre-analytical phase requires a lot of sample preparation time and immobilizes the staff for quite a long time. They can also be used in medium laboratories as a backup system.

The isoelectrofocusing system will be more targeted for newborn screening because it allows for a good separation of the Hb F, Hb A, and Hb S fractions, which assumes that the system is usually installed near a maternity ward.

*Contribution of Biomedical Equipment Management to Better Management of Sickle Cell Disease… DOI: http://dx.doi.org/10.5772/intechopen.92546*

#### *4.2.2 Semiautomatic systems*
