{"id":1633,"date":"2021-09-06T14:07:50","date_gmt":"2021-09-06T14:07:50","guid":{"rendered":"https:\/\/haema-journal.gr\/?p=1633"},"modified":"2021-09-11T09:59:22","modified_gmt":"2021-09-11T09:59:22","slug":"sep6","status":"publish","type":"post","link":"https:\/\/haema-journal.gr\/?p=1633","title":{"rendered":"Haemoglobin electrophoresis and HPLC"},"content":{"rendered":"

Haema 2021; 12(1): 39-41
\n<\/em><\/p>\n

Konstantinos Liapis<\/p>\n

Consultant Haematologist, Georgios Gennimatas Hospital<\/p>\n

Full PDF<\/a> | \"\"<\/a><\/p>\n

<\/p>\n

    \n
  1. Haemoglobin electrophoresis in cellulose acetate at alkaline pH <\/strong>(cellogel; pH=8.3 or pH=8.6). At alkaline pH, haemoglobins are negatively charged proteins so they move toward the anode (+), as shown in Figure S1.<\/li>\n<\/ol>\n

    \"\"<\/p>\n

    Figure S1.<\/strong> Alkaline haemoglobin electrophoresis.<\/p>\n

     <\/p>\n

      \n
    1. Haemoglobin electrophoresis in agarose citrate at acid p<\/strong>\u0397 <\/strong>(agarose gel, pH=6.0 or pH=6.2 or pH=6.5). At acid pH, haemoglobins are positively charged proteins so they migrate toward the cathode (-), as shown in Figure S2.<\/li>\n<\/ol>\n

      \"\"<\/p>\n

      Figure S2.<\/strong> Acid haemoglobin electrophoresis.<\/p>\n

      At alkaline p<\/strong>\u0397<\/strong>, haemoglobins S, D, G migrate together at the same position. Hb Lepore<\/strong> (\u03b4-\u03b2 fusion hybrids) migrates very close to S\/G\/D. Their distinction is possible with electrophoresis at acid pH, HPLC and sickling test. Three Lepore haemoglobins have been identified on the basis of \u03b4-\u03b2 crossover: Lepore-Boston (also called Lepore-Washington or Hb Pylos), Lepore-Hollandia, and Lepore-Baltimore. Hb Lepore results in a \u03b2-thalassaemia-like condition: heterozygous Hb Lepore resembles thalassaemia minor and the homozygous state results in a thalassaemia major-like condition. Hb D<\/strong> has a limited distribution (Punjab region at India-Pakistan border, where its incidence is 3%) and is clinically mild. Hb D heterozygotes are completely asymptomatic; Hb D homozygotes have mild anaemia with many target cells in the blood film or they are asymptomatic. Hb G<\/strong> is a rare \u03b1 chain variant seen in Ghana and in African-Americans (\u0397b GPhiladelphia<\/sup>). Hb G is stable and is not associated with haematological abnormalities.<\/p>\n

      There are 6 haemoglobins associated with the sickling phenomenon<\/strong> except Hb S (they all have the mutation \u03b26: Glut\u00e0Val plus one additional point mutation): \u0397b CHarlem<\/sup>, Hb CGeorgetown<\/sup>, Hb SAntilles<\/sup>, Hb SOman<\/sup>, Hb STravis<\/sup>, and Hb SProvidence<\/sup>. They are associated with a (+) sickling test and (+) solubility test, but migrate at a different position on alkaline Hb electrophoresis and HPLC. Clinically, these haemoglobins behave as Hb S.<\/p>\n

      Hb I<\/strong> (an \u03b1 chain variant, stable, no symptoms) and a large quantity of Hb Barts (\u03b34) may give a (+) solubility test. The clinical importance of Hb I is that it migrates at the same position as Hb \u0397 in alkaline electrophoresis (fast Hb variant). Hb I is not associated with Hb H inclusions or golf-ball cells. Hb I is found in the Mediterranean littoral and in Africa.<\/p>\n

      Hb OArab<\/sup><\/strong> is rare in the tropics. Hb O is a \u03b2 haemoglobin variant: Glut \u00e0 Lys (\u03b2121). Hb O is characterised by the formation of denser and more spherical erythrocytes, leading to elevated MCHC in combination with a slight decrease in MCV. The clinical importance of this haemoglobin is that it migrates at the same position as Hb C in alkaline Hb electrophoresis, but they are separated on acid Hb electrophoresis. Haemoglobin O-Arab heterozygotes show no clinical manifestations; homozygotes present with mild haemolysis and splenomegaly of minimal clinical significance, but may develop haemolytic anaemia during infection or severe illness.\u00a0 Importantly, the anaemia caused by combinations of Hb O-Arab with \u03b2 thalassaemia trait (\u03b2+ or \u03b20<\/sup>) varies from benign to transfusion-dependent, and sickling is enhanced when Hb S and Hb OArab<\/sup> coexist. Although Hb OArab<\/sup> is widely distributed, it is mostly detected in Eastern Mediterranean and Middle East populations. The Greek Pomaks, a Muslim population of the mountainous area of Thrace, demonstrate Hb OArab<\/sup> in impressively high percentage (5.076%), which reaches 27.4% in selected villages (Hb OThrace<\/sup>).<\/p>\n

        \n
      1. Cation-exchange High Performance Liquid Chromatography (HPLC)<\/strong><\/li>\n<\/ol>\n

        The normal HPLC pattern is shown in Figure S3.<\/p>\n

        \"\"
        \nFigure S3.<\/strong> Normal HPLC pattern.<\/p>\n

        Normal values: Hb<\/strong>\u0391<\/strong>2 <\/strong>= 1.9-3.3% and HbF<\/strong> = 0-2%<\/p>\n

        Example 1:<\/strong> A 13-year-old girl of Filipino descent, with hypochromia, microcytosis, and many target cells. No history of transfusion and her parents are healthy. Figure S4 shows her HPLC. Diagnosis: Hb \u0395 heterozygote (Hb A\u0395).<\/p>\n

        Example 2:<\/strong> A 33-year-old man from \u039digeria with anaemia (Hb 10.0 g\/dl, MCV 82 fl), splenomegaly and recurrent leg pain. No history of transfusion. His family history is unknown. Figure S5 shows his HPLC. Diagnosis: Hb SC disease.<\/p>\n

        \"\"<\/p>\n

        Figure S4<\/strong>. HPLC consistent with heterozygous HbE (HbAE).<\/p>\n

        B. S. Haldane first suggested that that the geographical co-incidence of malaria and \u03b2-thalassaemia major (Cooley\u2019s anaemia) could be due to the heterozygotes (\u03b2-thalassaemia minor) being at genetic advantage through a partial protection against P. falciparum<\/em>. A relative resistance to malaria was confirmed in Liberian children with thalassaemia minor (\u03b2\/\u03b2+<\/sup>). Another classic example of what Haldane called balanced polymorphism<\/strong> (i.e. heterozygotes are protected against malaria while the harmful genetic effects are restricted to homozygotes) is Hb S. African children who are heterozygous for Hb S are 10 times less likely to develop life-threatening complications of P. falciparum<\/em> infection than those who lack this allele.<\/p>\n

        \"\"<\/p>\n

        Figure S5.<\/strong> HPLC consistent with HbSC disease.<\/p>\n

        Tips:<\/strong><\/p>\n

          \n
        1. Always obtain a family history when haemoglobinopathy or thalassaemia is suspected!<\/strong><\/em><\/li>\n
        2. The diagnosis of heterozygous \u03b2-thalassaemia (\u03b2-thalas\u00adsaemia minor) depends upon finding an increased Hb A2 >3.5%, usually 4-6% (a higher value may be seen in some cases but values of Hb A2 >7% are rare). – -Hb F is slightly increased in 40-50% of individuals with heterozygous \u03b2-thalassaemia (usually up to 3%; in \u03b2\/\u03b20<\/sup> trait up to 5%). In cases of:<\/strong><\/em>
          \n-HbF >5% \u2192 consider \u03b4\u03b2-thalassaemia carrier (Hb A2 <3%) or HPFH heterozygote (Hb F 5-16%).<\/strong><\/em>
          \n-low Hb \u03912 (<1.9%) \u2192 consider co-inheritance of \u03b4-tha\u00adlas\u00adsaemia<\/strong><\/em>
          \n-Hb A2 \u226519% \u2192 consider Hb \u0395 (Hb \u0395 migrates at the same position as Hb\u03912 on alkaline and acid Hb electrophoresis and HPLC).<\/strong><\/em><\/li>\n
        3. In carriers of sickle cell anaemia (Hb AS), the percentage of \u0397b S is usually 35-45% (because the rate of \u0397b S synthesis is slower than Hb \u0391). If:<\/strong><\/em>
          \n-Hb S is <33% \u2192 consider S-\u03b1 thalassaemia co-inheritance.<\/strong><\/em>
          \n-Hb S is \u226550% \u2192 consider S-\u03b2 thalassaemia (also has an increased Hb A2 3.5-5% and HbF 5-10% or more) or sickle cell anaemia and recent blood transfusion.<\/strong><\/em><\/li>\n<\/ol>\n

          I have found the following references of considerable value in preparing this manuscript. Many further references will be found in each of these works.<\/p>\n

          References<\/strong><\/p>\n

            \n
          1. Lewis SM, Bain B, Bates I. Dacie, Lewis\u2019s Practical Haematology, 9th ed. Edinburgh: Churchill Livingstone; c2001.<\/li>\n
          2. Bain BJ. Haemoglobinopathy diagnosis. 2nd ed. London: Blackwell Publishing; c2006.<\/li>\n
          3. Steinberg MH, Forget BG, Higgs DR, Weatherall DG. Disorders of Hemoglobin. 2nd ed. Cambridge: Cambridge University Press; c2009.<\/li>\n
          4. Joutovsky A, Hadzi-Nesic J, Nardi MA. HPLC retention time as a diagnostic tool for hemoglobin variants and hemoglobinopathies: a study of 60000 samples in a clinical diagnostic laboratory. Clin Chem. 2004 Oct;50(10):1736-47.<\/li>\n
          5. Khera R, Singh T, Khuana N, Gupta N, Dubey AP. HPLC in characterization of hemoglobin profile in thalassemia syndromes and hemoglobinopathies: a clinicohematological correlation. Indian J Hematol Blood Transfus. 2015 Mar;31(1):110-5.<\/li>\n
          6. Luzzatto L. Haemoglobinopathies including thalassaemia. Part 3. Sickle cell anaemia in tropical Africa. Clin Haematol. 1981 Oct;10(3):757-84.<\/li>\n
          7. Colombo B, Mart\u00ednez G. Haemoglobinopathies including thalassaemia. Part 2. Tropical America. Clin Haematol. 1981 Oct;10(3):730-56.<\/li>\n
          8. Wasi P. Haemoglobinopathies including thalassaemia. Part 1: Tropical Asia. Clin Haematol. 1981 Oct;10(3):707-29.<\/li>\n
          9. Voskaridou E, Konstantopoulos K, Kollia P, Papadakis M, Loukopoulos D. Hb Lepore (Pylos)\/Hb S compounds heterozygosity in two Greek families. Am J Hematol. 1995 Jun; 49: 131-4.<\/li>\n
          10. Papadopoulos V, Dermitzakis E, Konstantinidou D, et al. The origin of Greek Pomaks based on HbO-Arab mutation history. Haema. 2006 Oct; 9(3):380-394.<\/li>\n
          11. Papadopoulos V, Vassiliadou D, Xanthopoulidis G, Petridis D, Agorasti A, Loukopoulos D. The implications of haemoglobin O-Arab mutation. Haema. 2003; 6(4): 296-303.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

            Haema 2021; 12(1): 39-41 Konstantinos Liapis Consultant Haematologist, Georgios Gennimatas Hospital Full PDF |<\/p>\n","protected":false},"author":1,"featured_media":0,"comment_status":"closed","ping_status":"open","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[101],"tags":[],"class_list":["post-1633","post","type-post","status-publish","format-standard","hentry","category-haema-2021-1"],"_links":{"self":[{"href":"https:\/\/haema-journal.gr\/index.php?rest_route=\/wp\/v2\/posts\/1633","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/haema-journal.gr\/index.php?rest_route=\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/haema-journal.gr\/index.php?rest_route=\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/haema-journal.gr\/index.php?rest_route=\/wp\/v2\/users\/1"}],"replies":[{"embeddable":true,"href":"https:\/\/haema-journal.gr\/index.php?rest_route=%2Fwp%2Fv2%2Fcomments&post=1633"}],"version-history":[{"count":6,"href":"https:\/\/haema-journal.gr\/index.php?rest_route=\/wp\/v2\/posts\/1633\/revisions"}],"predecessor-version":[{"id":1733,"href":"https:\/\/haema-journal.gr\/index.php?rest_route=\/wp\/v2\/posts\/1633\/revisions\/1733"}],"wp:attachment":[{"href":"https:\/\/haema-journal.gr\/index.php?rest_route=%2Fwp%2Fv2%2Fmedia&parent=1633"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/haema-journal.gr\/index.php?rest_route=%2Fwp%2Fv2%2Fcategories&post=1633"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/haema-journal.gr\/index.php?rest_route=%2Fwp%2Fv2%2Ftags&post=1633"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}