|Year : 2022 | Volume
| Issue : 4 | Page : 208-212
Frequency of sickle cell hemoglobin in high-performance liquid chromatography received in a centralized laboratory
Hareem Alam, Natasha Ali
Department of Pathology and Laboratory Medicine, Aga Khan University, Karachi, Pakistan
|Date of Submission||03-Jul-2022|
|Date of Decision||07-Aug-2022|
|Date of Acceptance||16-Aug-2022|
|Date of Web Publication||18-Oct-2022|
Dr. Hareem Alam
P O Box 3500, Stadium Road 74800, Aga Khan University, Karachi
Source of Support: None, Conflict of Interest: None
BACKGROUND: Sickle cell disease and its variants result from an abnormal hemoglobin, hemoglobin S (HbS), caused by a single point mutation in the Beta-Globin gene. Hypoxia causes polymerization and distortion of Hb S containing red blood cells resulting in sickle crisis and hemolytic anemia. Common diagnostic methods include high-performance liquid chromatography (HPLC) and hemoglobin electrophoresis. The purpose of this research is to determine the frequency of sickle cell hemoglobin in HPLC samples and its geographical distribution in Pakistan.
MATERIALS AND METHODS: Data was collected from the samples received from 1st February 2020 to 31st January 2021. Proforma included demography, complete blood count parameters, and variants of sickle cell disease which were extracted from hospital records. Analysis was done using the SPSS (version 26).
RESULTS: Out of 14,740 samples, 295 (2%) revealed HbS. These patients had a mean age of 14.2 years. The male-to-female ratio was 1.5:1. The samples were received from Baluchistan (43%), followed by Sindh (32.1%), Khyber Pakhtunkhwa (16%), and Punjab (8.9%). Mean hemoglobin was 8.6 ± 2.6 g/dl, lowest and highest were 2.4 g/dl and 16.9 g/dl, respectively. Sickle cell trait was found in 21.3% of the patients, homozygous HbSS in 27.7%, sickle beta thalassemia in 30.8%, 4.4% were compound heterozygotes for Hb S and Hb D, whereas 15.5% were posttransfusion samples.
CONCLUSION: Our analysis showed that the highest frequency was of sickle beta thalassemia and other variants being low. This study also proved it to be more prevalent in Baluchistan with relatively high male preponderance.
Keywords: Complete blood count parameters, high-performance liquid chromatography, sickle cell disease[/TAG:2]
|How to cite this article:|
Alam H, Ali N. Frequency of sickle cell hemoglobin in high-performance liquid chromatography received in a centralized laboratory. J Appl Hematol 2022;13:208-12
|How to cite this URL:|
Alam H, Ali N. Frequency of sickle cell hemoglobin in high-performance liquid chromatography received in a centralized laboratory. J Appl Hematol [serial online] 2022 [cited 2022 Dec 3];13:208-12. Available from: https://www.jahjournal.org/text.asp?2022/13/4/208/358713
| Introduction|| |
Sickle cell disease and its variants are the consequence of genetic variation of hemoglobin, that is, mutated hemoglobin hemoglobin S (HbS). The molecular defect is a single nucleotide change in the beta-globin gene resulting from substitution of valine for glutamic acid at position 6. Under the conditions of low oxygen concentration, HbS polymerizes and produces fibrous precipitates, distorting the structure of the red blood cell (RBC) and rendering it vulnerable to destruction.
Sickle cell disease inherits in an autosomal recessive pattern. Apart from homozygous HbSS and heterozygous sickle cell trait, several other sickle cell syndromes result from the sickle cell gene inherited in compound heterozygosity with other mutant beta globin genes, including HbSC disease, sickle beta thalassemia, compound heterozygote for HbS and HbD, and others.
Clinically, significant aspects include hemolytic anemia and vaso-occlusion, resulting in acute and chronic discomfort, tissue ischemia, including splenic infarction. The major diagnostic methods used are high-performance liquid chromatography (HPLC) and hemoglobin electrophoresis. HPLC is the method of choice for diagnosing this condition in adults because of its accuracy in identifying and quantifying hemoglobin.
A review on different modalities including HPLC, isoelectric focusing, and Capillary Electrophoresis (CE) which was carried out on neonates as a screening tool proved to be highly sensitive and specific in detecting sickle cell disease. A study carried out to detect sickle cell disease in a low-income setup showed that point of care testing was 87% sensitive as compared to HPLC, which proved to be the gold standard in the detection of sickle cell variants. One of the studies estimated the percentage of hemoglobin A2 (Hb A2) in the samples by HPLC. The results showed that the level of Hb A2 was increased in patients with Hb S and Hb C.
Early identification of sickle cell disease aids in sickle crisis prevention and treatment. The sickle cell variants are caused by a concomitant hereditary mutation in another globin gene, and their clinical severity is different from the homozygous sickle cell disease. In Pakistan, around 0.5%–1% of the population is reported to have HbS or E; however, the actual prevalence has not been identified. In our country, the hemoglobin HPLC is usually requested in case of symptomatic individuals, or cascade testing or antenatal screening. Even though pre-marital screening programme has not yet been implemented in our country, findings of our study could form the basis of this being offered as a cost effective method for the prevention and early detection of sickle cell disease or variants in new borns.
Based on this background, our study's purpose was to ascertain the prevalence of sickle cell disease in the samples received for hemoglobin variant analysis by HPLC and also determined the geographical distribution of this disease in our country.
| Materials and Methods|| |
After getting approval from the Ethics Review Committee (AKUH-ERC 2020-1975-10475) of the Aga Khan University Hospital, data were collected from the samples received between February 2020 to January 2021. The samples were analyzed for hemoglobin variants by HPLC on Bio-Rad Variant II.
High-performance liquid chromatography
HPLC is an ideal and efficient method for detecting thalassemias and abnormal hemoglobins by evaluating hemoglobin fractions qualitatively and quantitatively simultaneously. It is an automated technique that offers superior results and is less time-consuming than many other methods. In the previous two decades, the utilization of this technology has expanded 12-fold. HPLC is fundamentally associated with the retention time (RT), the amount of time needed for gradient elution of the various hemoglobin fractions. Each hemoglobin has a unique retention period that the manufacturer specifies for that hemoglobin fraction. HbS elutes with a retention duration of 4.1–4.7 minutes. The graphical representation is plotted on a paper showing peaks with RT. The determination of known and unknown hemoglobin peaks is compared to known hemoglobin retention durations. Variants moving in the same range might be eluted out within the same window, their peaks may be comparable to HbS. Thus, a sickling test in conjunction with HPLC may provide a conclusive diagnosis.
Our center utilizes Bio-Rad Variant II Sampling Station for HPLC. Two dual-piston pumps and a gradient regulate the flow of elution buffer through the analytical cartridge. The changes in absorbance as detected by two different cartridges are tracked and shown as an absorbance versus time on chromatogram. We are also the only center in Pakistan to report peripheral blood film findings along with complete blood count (CBC) parameters and HPLC results.
Data for the study were collected according to a proforma, which included patient's age, gender, locality, CBC parameters, and variants of sickle cell disease along with percentages of hemoglobin variants. These details were extracted from the hospital records using Integrated Laboratory Management System and Patient Care Inquiry. A separate grid was maintained to compile medical record numbers with the serial number of the forms to avoid misuse of patient's information.
IBM SPSS version 26 (IBM® SPSS Statistics 26.0, Armonk, NY, USA) was used to analyze the data. Descriptive statistics including mean and standard deviation (SD) were calculated for all continuous data.
| Results|| |
Out of total 14,740 samples received for hemoglobin HPLC, 295 (2%) revealed HbS. The mean age of patients that were included in this study was 14.2 years ranging from 6 months to 59 years. The research sampled 119 females and 176 males. The majority of the samples received were from the province of Baluchistan (43%), followed by Sindh (32.1%), Khyber Pakhtunkhwa (16%), and Punjab (8.9%), as shown in [Figure 1].
|Figure 1: Geographical distribution of sickle cell hemoglobin in Pakistan|
Click here to view
The mean hemoglobin of patients in the study was 8.6 ± 2.6 gms/dl with the lowest recorded value of 2.4gms/dl and the highest being 16.9 gms/dl. The rest of the CBC parameters are described in [Table 1].
|Table 1: Complete blood count parameters of patient with sickle cell disease|
Click here to view
Out of 295 specimens positive for HbS, sickle cell trait was found in 21.3% of the patients, homozygous HbSS in 27.7%, sickle beta thalassemia in 30.8%, compound heterozygote for Hb S and Hb D in 4.4%, while 15.5% were posttransfusion samples and therefore were unclassifiable [Figure 2]. Out of all 14,740 samples received for HPLC, the perecentage of sickle variants was sickle cell trait (0.42%), homozygous hbSS (0.55%), sickle beta thalassemia (0.61%), and compound heterozygote for Hb S and Hb D (0.08%).
The mean hemoglobin in sickle cell trait was 10.7 gm/dl, in homozygous HbSS was 8.30gm/dl and sickle beta thalassemia it was 8.07 gm/dl, respectively. The difference among each variant in terms of hematocrit, mean corpuscular volume (MCV), RBC, and MCHS is compared in [Table 2].
|Table 2: Complete blood count parameters in patients with sickle cell disease and its variants|
Click here to view
The mean percentage of hemoglobin F (HbF) was 22.94% in patients with homozygous HbSS and 1.54% in patients with sickle cell trait. The rest of the distribution of HbF is given in [Table 3]. We performed sickling test in all n = 293 samples and the results were positive. The mean percentage of HbA2 in sickle beta thalassemia was 5.28% and in sickle cell trait was 2.8%. A comparison of mean percentage of HbA2 is shown in [Table 3].
|Table 3: Percentage of hemoglobin fetal and hemoglobin A2 in patients with sickle cell disease and its variants|
Click here to view
| Discussion|| |
Sickle cell disease is more frequent among people from sub-Saharan Africa, Saudi Arabia, India, and the Mediterranean nations. Africa has the greatest frequency of sickle cell disease. According to one research, around 0.5%–1% of the Pakistani population has HbS or Hemoglobin E, although the precise prevalence is unknown. There is little data available on its prevalence in Pakistan.
In 1990, Kazmi and Rab reported eight cases of sickle cell anemia in a two-year period indicating that this hemoglobinopathy was not totally non-existent in Pakistan.
Hashmi et al. described chromatographic analysis to diagnose different sickle cell diseases in Pakistan population during years 2005 and 2006, in which percentage of HbS was 1.92% in an analysis of 15,699 samples. The different genotypes were identified such as Sβ0 (46.7%), SS (19.2%), SA (11.6%), Sβ+ (8.6%), and SD (2.3%). The highest prevalence was reported from Balochistan in their study. The percentage of HbS in our population was also close to this study, i.e., 2%. Our study also demonstrated the highest frequency in Balochistan followed by Sindh.
Waheed et al. used hemoglobin electrophoresis to determine the incidence of hemoglobinopathies in 504 consecutive patients in single-center research in 2011. Approximately, 25% of subjects had hemoglobinopathies, out of which 1.4% demonstrated HbS or HbD. Although the sample size was small, this data also corresponded to the already reported prevalence.
According to Sameen et al., sickle cell anemia affects 14% of the study population belonging to the Sheedi/Makrani caste living in Lyari, Karachi. This study specifically targeted one ethnic population. The highest frequency found in their data must be attributable to the fact that Sheedi/Makrani are descendants of East Africans. Another screening study performed in the Karachi by Ghani et al. showed 5.1% cases of sickle cell disease. Both these studies focused on one ethnic region only, hence provided a higher frequency. Our study has included subjects from all over Pakistan as we are placed as a reference laboratory with collection centers across the country; hence, it can be assumed that the geographical data is representative of the occurrence of sickle cell and its variants across the country.
In Southern Iran, the prevalence of sickle cell trait and homozygous hbSS is estimated to be around 1.43% and 0.1%, respectively. In Central Iran (South-east Isfahan), sickle cell trait was detected at a frequency of 8.33% The Baluchistan province is situated on the Iranian plateau's southeasternmost rim. Around 50% of the ethnic Baloch population lives in Pakistan's Baluchistan region, while 40% dwell in Sindh. Because of the similar ethnic origin of the Baloch population and Iranians, a high frequency of sickle cell anemia in Baluchistan and Sindh provinces was reported. Consanguinity also contributed to this high frequency.
The mean hemoglobin in sickle cell trait was 10.7 ± 2.8 gm/dl while it was lower in homozygous hbSS and sickle beta thalassemia, i.e., 8.30 ± 2.44 gm/dl and 8.07 ± 2.09 gm/dl, respectively. The RBC indices including MCV and MCH were normal in homozygous hbSS and lower in sickle beta thalassemia, as expected. However, patients with sickle cell trait exhibited borderline microcytosis and hypochromia which could be due to underlying iron deficiency or concomitant alpha trait. Further workup including alpha gene squencing by Multiplex Ligation-dependent Probe Amplification and iron profile were not a part of this study.
HbF is the most potent modifier of sickle cell anemia's clinical and hematologic characteristics. Increased HbF levels are related to a decreased incidence of acute painful episodes, fewer leg ulcers, less osteonecrosis, fewer acute chest symptoms, and a decreased severity of illness. In our study, the mean percentage of HbF found in homozygous hbSS was 22.9% while it was 21% in sickle beta thalassemia which is higher than the values reported in the literature. Subsequently, this higher percentage of HbF leads to a protective effect and decreased episodes of pain crisis and other complications associated with this disease.
Newborn babies with sickle cell disease are usually healthy at birth but show symptoms in later stage. Therefore, an early detection helps in establishing the diagnosis and preventing neonates from developing infections, pain crisis, frequent hospitalizations which results in management of the disease as early as possible. As of 2008, all 50 states of the United States mandate newborn screening for sickle cell disease but such expensive methods are not practical in developing countries such as Pakistan. That is the reason why most of the cases stay undiagnosed until they develop crisis or require blood transfusion.
Currently, Deoxyribonucleic Acid (DNA) testing is utilized to determine hemoglobin abnormalities. These protocols may ultimately replace HPLC. DNA testing may be necessary when the HbF concentration is elevated, raising the possibility of hereditary persistenc of fetal hemoglobin; however, this technique requires good financial architecture and therefore makes it a less viable option for the third world countries.
As recent data for sickle cell disease are sparse in Pakistan, it is essential to determine the frequency of sickle cell disease. Data were collected from HPLC samples that were brought from all over the country; therefore, an interventional study would have required more resources. This prospective study helped us identify the frequency of each variant which can form the basis of HPLC as the first line diagnosis for sickle cell disease and can also be utilized at the National level for policy development of guidelines for hemoglobinopathy screening.
| Conclusion|| |
Analysis of 14,740 samples received for hemoglobin HPLC showed HbS frequency as 2%. The highest frequency was of sickle beta thalassemia followed by homozygous hbSS. This study once again showed that sickle cell disorders are more prevalent in Baluchistan with relatively high male preponderance. Large scale multicenter study is recommended for better estimation of the disease prevalence and its demographic distribution within Pakistan so that population-oriented programs can be initiated for newborn/family screenings and premarital testing.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
| References|| |
Elsayid M, Al-Shehri MJ, Alkulaibi YA, Alanazi A, Qureshi S. Frequency distribution of sickle cell anemia, sickle cell trait and sickle/beta-thalassemia among anemic patients in Saudi Arabia. J Nat Sci Biol Med 2015;6:S85-8.
Wastnedge E, Waters D, Patel S, Morrison K, Goh MY, Adeloye D, et al.
The global burden of sickle cell disease in children under five years of age: A systematic review and meta-analysis. J Glob Health 2018;8:021103.
Lervolino LG, Baldin PE, Picado SM, Calil KB, Viel AA, Campos LA. Prevalence of sickle cell disease and sickle cell trait in national neonatal screening studies. Rev Bras Hematol Hemoter 2011;33:49-54.
Frömmel C. Newborn screening for sickle cell disease and other hemoglobinopathies: A short review on classical laboratory methods – Isoelectric focusing, HPLC, and capillary electrophoresis. Int J Neonatal Screen 2018;4:2-10.
Alvarez OA, Hustace T, Voltaire M, Mantero A, Liberus U, Saint Fleur R. Newborn screening for sickle cell disease using point-of-care testing in low-income setting. Pediatrics 2019;144:e20184105.
da Fonseca SF, Amorim T, Purificação A, Gonçalves M, Boa-Sorte N. Hemoglobin A2 values in sickle cell disease patients quantified by high performance liquid chromatography and the influence of alpha thalassemia. Rev Bras Hematol Hemoter 2015;37:296-301.
Davies EG, Riddington C, Lottenberg R, Dower N. Pneumococcal vaccines for sickle cell disease. Cochrane Database Syst Rev 2004; (1):CD003885.
Smith EW, Conley CL. Clinical features of the genetic variants of sickle cell disease. Bull Johns Hopkins Hosp 1954;94:289-318.
Ahmed S, Saleem M, Modell B, Petrou M. Screening extended families for genetic hemoglobin disorders in Pakistan. N Engl J Med 2002;347:1162-8.
Hemoglobin and Clinical Microscopy Reference Committee. Hemoglobinopathies Survey Proficiency Reports 1994-2003. Chicago, IL: College of American Pathologists; 2009.
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;50:1736-47.
Keren DF, Hedstrom D, Gulbranson R, Ou CN, Bak R. Comparison of sebia capillarys capillary electrophoresis with the primus high-pressure liquid chromatography in the evaluation of hemoglobinopathies. Am J Clin Pathol 2008;130:824-31.
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;31:110-5.
Ranney HM, Jacobs AS, Nagel RL. Haemoglobin New York. Nature 1967;213:876-8.
Bunn HF. Pathogenesis and treatment of sickle cell disease. N Engl J Med 1997;337:762-9.
Adewoyin AS. Management of sickle cell disease: A review for physician education in Nigeria (sub-saharan Africa). Anemia 2015;2015:791498.
Janjua TK, Haider SA, Raza N. Multiple complications in sickle cell anaemia. J Pak Med Assoc 2018;68:154-6.
Kazmi KA, Rab SM. Sickle cell anaemia in Pakistan. Br J Clin Pract 1990;44:503-5.
Hashmi NK, Moiz B, Nusrat M, Hashmi MR. Chromatographic analysis of Hb S for the diagnosis of various sickle cell disorders in Pakistan. Ann Hematol 2008;87:639-45.
Waheed U, Satti HS, Farooq N, Zaheer HA. Frequency of haemoglobinopathies: A single-centre, cross-sectional study from Islamabad, Pakistan. East Mediterr Health J 2012;18:1257-9.
Sameen D, Parveen S, Danish F, Salam H, Agha A, Sharafat S. Sickle cell anemia in Sheedi population of Lyari: Hemoglobinopathy seen in a neglected population. Int J Pathol 2018;16:119-22.
Ghani R, Manji MA, Ahmed N. Hemoglobinopathies among five major ethnic groups in Karachi, Pakistan. Southeast Asian J Trop Med Public Health 2002;33:855-61.
Nasiri A, Rahimi Z, Vaisi-Raygani A. Hemoglobinopathies in Iran: An updated review. Int J Hematol Oncol Stem Cell Res 2020;14:140-50.
Steinberg MH, Chui DH, Dover GJ, Sebastiani P, Alsultan A. Fetal hemoglobin in sickle cell anemia: A glass half full? Blood 2014;123:481-5.
Shokrani M, Terrell F, Turner EA, Aguinaga MD. Chromatographic measurements of hemoglobin A2 in blood samples that contain sickle hemoglobin. Ann Clin Lab Sci 2000;30:191-4.
US Preventive Services Task Force. Screening for sickle cell disease in newborns: Recommendation statement. Am Fam Physician 2008;77:1300-2.
Michlitsch J, Azimi M, Hoppe C, Walters MC, Lubin B, Lorey F, et al.
Newborn screening for hemoglobinopathies in California. Pediatr Blood Cancer 2009;52:486-90.
[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3]