TY - JOUR
AU - Jawannatoom, Chayapol
AB - Abstract Background Hemoglobin E (HbE) is common in Thailand and the Indochina subcontinent. Implementation of a simple assay to screen for HbE is crucial for use in reproductive counseling, as HbE/β-thalassemia is a condition with high morbidity and mortality. Methods This report developed the alcohol-based HbE screening test named the Alcohol HbE Test (AET). This test was composed of 19% isopropanol in 0.005% Triton-X-100 aliquoted as a 500-µL volume in a 1.5 mL transparent microcentrifuge tube. In performing the AET, 50 µL of EDTA blood was added into the tube, incubated at 37 °C for 30 min before watching for precipitation by eye, which was seen for HbE carriers, not in normal. The test was validated, and assessed for the effect of hematocrit, stability of blood samples, stability of the AET reagent, and interference of HbH. Results The AET was effective, simple, and less costly than the conventional dichlorophenol indophenol precipitation (DCIP) test. Hematocrit had no effect on the AET results. EDTA blood samples kept in cold conditions (4 °C) were stable for at least 30 days, but only for 2 days at room temperature (30 °C). The aliquoted AET reagent was stable for 30 days in both conditions, while the unaliquoted AET reagent was stable for 6 months. Finally, HbH showed false positive results for the AET. Conclusions The AET was successfully developed, and can be an alternative screening test for HbE carriers. Introduction Impact Statement The Alcohol HbE Test (AET) developed in this report is useful in countries where HbE is frequently found, e.g., Southeast Asian countries and other parts of the world due to migration. Thus, screening for HbE in the general population before sending samples for the more sophisticated and robust screening techniques is important, e.g., to reduce cost. The high effectiveness, clear distinction between positive and negative results, simplicity, and durability of the test should strengthen its potential in HbE screening. Finally, this AET test is truly novel as it has never been established by any other authors. HbE (HBB: c.79G>A) is an abnormal hemoglobin formed from assembly of a normal α-globin chain and abnormal β-globin chain in which glutamic acid at codon 26 is replaced by lysine. This abnormal hemoglobin is very common in Thailand and the Indochina region. It is also found in several western countries as a result of immigration (1, 2). This abnormal hemoglobin itself is not harmful to the patient. It causes only slight deviation of hematologic phenotypes. However, compound heterozygosity of HbE gene, βE, with β-thalassemia gene (βThal) leads to thalassemia syndrome called HbE/β-thalassemia (3, 4). This thalassemia syndrome may be life-threatening if management is not adequate and efficient. Therefore, screening for HbE carriers is essential and several countries implement prevention and control programs of this thalassemia syndrome in national plans. Screening for carriers of β-thalassemia and HbE is one of the important steps of prevention and control of HbE/β-thalassemia. Initial screening tests for these 2 carriers typically rely on a group of laboratory tests, including the one-tube osmotic fragility test (OFT), mean corpuscular volume (MCV), and dichlorophenol indophenol precipitation (DCIP) test (5–8). OFT and MCV have 100 and 92.9% sensitivity, respectively, in screening for β-thalassemia carriers (6, 8). Only some HbE carriers are detected by OFT and MCV. Combined OFT and DCIP tests have been shown to have 100% sensitivity, 97.1 specificity, 94.9% positive predictive value, and 100% negative predictive value for HbE carriers (6). Therefore, the DCIP test is now widely performed in Thailand to screen for HbE carriers. The major drawbacks of the DCIP test were its deep blue color that made result reading difficult, as well as its short shelf-life due to the natural oxidizing property. This problem has encouraged the invention of new HbE screening tests operated under various principles. These included the KKU-DCIP diagnostic kit, which still used DCIP as the main ingredient, but added a clearing reagent at the end of incubation time to fade the deep blue color of the DCIP (9, 10), the HbE tube test (11), and the microcolumn-based HbE screen test (12). The last 2 techniques were operated via anion exchange chromatography. Isopropanol is the alcohol that was used to establish the screening test for HbE in a classic test, the so-called isopropanol precipitation test invented in 1980 by Ali and coworkers (13). In our previous work, we modified its protocol and found that this alcohol-based test was still effective in screening for HbE carriers (T. Tatu, E. Potjanasit, and K. Anusak, unpublished observations). In this report, we further simplified the test so that whole-EDTA blood, instead of blood lysate, was used with the new test kit. We found that mixing the isopropanol alcohol in Triton-X 100 in the new test kit simplified it and so it could be performed as a routine test. Materials and Methods Blood Samples Blood samples anticoagulated in EDTA were used in this study. Three sets of these EDTA blood samples were analyzed in this study. The first set consisted of 3 blood samples of known cases of HbE carriers and 3 blood samples of normal individuals with no HbE. The second and third sets of blood samples comprised 114 and 110 unknown EDTA blood samples collected from Central Laboratory Department, Maharaj Nakorn Chiang Mai Hospital. The protocol of this study was reviewed and the ethics approved by the Research Ethics Committee of the Faculty of Associated Medical Sciences, Chiang Mai University (ethics approval no. 273/2020). Alcohol HbE Test (AET) The AET was a 500-µL reagent composed of 19% isopropanol (RCI Labscan) in 0.005% Triton-X 100 (Sigma-Aldrich) aliquoted in a 1.5-mL transparent microcentrifuge tube. The test was performed by mixing 50 µL of EDTA blood with the AET reagent in the microcentrifuge tube. The mixture was then incubated in a 37 °C water bath for 30 minutes, and monitored by eye for precipitation inside the tube. Cellulose Acetate Electrophoresis at Alkaline Condition (CAE) for Hemoglobin Separation The procedure of CAE was modified from that described earlier (14). The test started with preparation of hemolysate by mixing 200 µL of packed red cells in 600 µL of 0.05% Triton-X 100 in DW to be used immediately. Twelve µL of hemolysate was then applied onto a cellulose acetate plate (Titan III H, Helena Laboratories), presoaked in Tris-Borate-EDTA (0.1 M Tris, 0.025 M boric acid, 0.003 M EDTA) or TBE buffer pH 8.6, using a sample applicator (Helena Laboratories). Electrophoresis was performed for 10–15 minutes under a constant voltage of 270 V in TBE buffer pH 8.6. At the end, banding patterns of hemoglobin on the cellulose acetate plate were visualized without staining. A HbE carrier showed a Hb type of AE, while a normal individual showed A2A. Dichlorophenol Indophenol Precipitation (DCIP) Test The DCIP test is presently the conventional test for HbE screening in Thailand. We performed this test to compare the results with those generated by the newly established AET kit. This test was performed according to the instructions of the manufacturer (THALCONTM, Qualified Group). Briefly, 20 µL of whole-EDTA blood was mixed with 2 mL of DCIP reagent. The mixture was then incubated at 37 °C for 1 hour before adding decolorizing reagent and monitored for precipitation inside the tube. Precipitation was seen only in samples with HbE such as HbE carriers, but not in samples with no HbE. Validation of AET Validation of the AET was performed on 114 unknown blood samples. The results were validated against those obtained from CAE at alkaline condition (pH 8.6). Sensitivity and specificity of AET and DCIP were calculated against CAE as the standard technique. Determination of the Effect of Hematocrit (Hct) on the AET Test Results This experiment was aimed to determine the effect of Hct on results of the AET. The test was performed on 110 EDTA blood samples with different levels of hematocrit. These blood samples were grouped into 6 groups by hematocrit levels, including 13 samples with Hct 11–20%, 38 with Hct 21–30%, 33 with Hct 31–40%, 23 with Hct 41–50%, 1 with Hct 51–60%, and 2 with Hct 61–70%. The CAE was performed for all 111 blood samples to identify types of hemoglobin, i.e., whether HbE was present or not. The true positive, false positive, true negative, and false negative results of the AET of all these groups were compared. Determination of the Impact of Blood Storage on the Test Results This experiment aimed to determine optimal storage conditions for EDTA blood samples subjected to the AET. Two known EDTA blood samples were analyzed during this stage. These included EDTA blood from HbE carriers and from normal individuals having no HbE. Two sets of blood samples of each phenotype were prepared, 1 for RT storage and 1 for 4 °C storage. The AET was performed for these blood samples on blood collection day (Day 0) and every subsequent day until the test results could not be interpreted, i.e., the same results were seen for both samples with and without HbE. Determination of Storage Effect on Stability of AET This experiment aimed to determine optimal storage conditions for the AET. Practically, 2 sets of AET were prepared, 1 kept at room temperature (about 30 °C) and 1 in a 4 °C refrigerator. The test was performed every day, using blood from the HbE carrier and a normal individual as positive and negative samples, respectively. In addition, the unaliquoted AET reagent kept in the stock container was also tested for its stability at both room temperature and 4 °C conditions. The test was stopped when the test results were the same for both positive and negative samples, i.e., no precipitation was observed and 2 observers agreed. AET Results of Blood Sample of HbH Disease HbH (β4) is an unstable hemoglobin produced in a considerable amount in patients with HbH disease. To determine whether HbH also gave a positive AET result, we performed the AET for 4 different blood samples of HbH disease. Results AET Results for HbE Containing Blood Samples The AET clearly showed positive and negative results. Precipitation was clearly seen in the blood sample of a known case of HbE carrier with Hbs A and E, which was reported as “positive.” In contrast, a “negative” result was reported in the blood sample of a normal individual who had no HbE, but Hbs A and A2 (Fig. 1). Fig. 1 Open in new tabDownload slide Results of the Alcohol HbE Test (AET) for HbE screening. The tube labeled “Positive” was the result seen in the case of HbE carrier who had Hbs A and E. The tube labeled “Negative” was the result from a normal individual whose Hb typing result was A2A. Precipitation is seen only in the “Positive” tube. Fig. 1 Open in new tabDownload slide Results of the Alcohol HbE Test (AET) for HbE screening. The tube labeled “Positive” was the result seen in the case of HbE carrier who had Hbs A and E. The tube labeled “Negative” was the result from a normal individual whose Hb typing result was A2A. Precipitation is seen only in the “Positive” tube. Validation of AET for Screening of HbE Four groups of blood samples were found after typing of the hemoglobin by the standard CAE at alkaline condition, including A2A, AE, AH, and EF (Supplemental Table 1). For the DCIP test, 7, 10, and 60 blood samples had true positive, false positive, and true negative results, respectively. No false negative results were observed. For AET, 10, 12, and 92 blood samples had true positive, false positive, and true negative results. None of these blood samples had false negative results. Based on this information, sensitivities of both AET and DCIP tests were 100%, and specificity of AET and DCIP tests in screening for HbE were 88.5 and 85.7%, respectively. Effect of Hematocrit on the AET Test Results False positive AET results, but no false negative ones, were observed in almost all ranges of hematocrit tested. A high proportion of blood samples of hematocrit range of 41–50% showed false positive AET results (Supplemental Table 2). Denaturation of hemoglobins during room temperature storage was thought to be the cause of this phenomenon. Stability of Hemoglobins with Storing Condition Blood samples were stable for 2 days storage at room temperature. Testing blood stored at room temperature on the third day after blood collection yielded substantial false positive results. However, the AET result was still readable and interpretable, even for blood samples that had been stored at 4 °C for at least 30 days (Fig. 2). Fig. 2 Open in new tabDownload slide Determination of stability of hemoglobin with storing conditions. The tube labeled AE was the AET result from a blood sample collected from a HbE carrier. The tube labeled A2A was the AET result from blood sample collected from a normal individual. Day 0 refers to the blood collection day, while Day 3 and Day 30 refer to the fourth and 31st days after blood collection. RT means room temperature. Fig. 2 Open in new tabDownload slide Determination of stability of hemoglobin with storing conditions. The tube labeled AE was the AET result from a blood sample collected from a HbE carrier. The tube labeled A2A was the AET result from blood sample collected from a normal individual. Day 0 refers to the blood collection day, while Day 3 and Day 30 refer to the fourth and 31st days after blood collection. RT means room temperature. Stability of the AET Reagent The results of AET stored at room temperature (30 °C) and 4 °C for up to 30 days were still readable. However, false negative AET results were seen when the AET tubes were stored under both conditions for 6 months. However, we found that the unaliquoted AET reagent stored at 4 °C in a stock container with the lid tightly closed was stable for at least 6 months (Fig. 3). Fig. 3 Open in new tabDownload slide Determination of the stability of aliquoted AET stored at room temperature (around 30 °C) (RT) and 4 °C for 30 days (30th day) [A] and of unaliquoted AET reagent stored at 4 °C in a large container for 6 months [B]. “+” and “−” indicate positive and negative AET, respectively. Fig. 3 Open in new tabDownload slide Determination of the stability of aliquoted AET stored at room temperature (around 30 °C) (RT) and 4 °C for 30 days (30th day) [A] and of unaliquoted AET reagent stored at 4 °C in a large container for 6 months [B]. “+” and “−” indicate positive and negative AET, respectively. AET Results for Blood Samples of HbH Disease The 3 blood samples that were tested for HbH disease had Hb typing results by capillary zone electrophoresis (CZE) of A2AH with HbH levels of 1.1, 10.1, and 11.9%, whereas 1 sample had Hb typing of CsA2ABart’s H with an HbH level of 11.1% (Fig. 4). Positive AET results were observed for all of these 4 samples with the pattern of precipitation identical to that of HbE carriers as shown in Fig. 4. Fig. 4 Open in new tabDownload slide CZE and AET results of blood samples collected from 4 patients with HbH disease. HbH peaks in CZE chromatograms are marked with arrows. The AET result of each sample is embedded inside the relevant CZE chromatogram. “N” and “HD” stand for normal and HbH disease, respectively. Fig. 4 Open in new tabDownload slide CZE and AET results of blood samples collected from 4 patients with HbH disease. HbH peaks in CZE chromatograms are marked with arrows. The AET result of each sample is embedded inside the relevant CZE chromatogram. “N” and “HD” stand for normal and HbH disease, respectively. Discussion HbE is still one of the health burdens in several Southeast Asian countries and, now, in other parts of the world (1, 3). HbE carriers seeking to become pregnant need to know their status as carriers, i.e., to prevent birth of a child with HbE/β-thalassemia, a condition with significant morbidity and even mortality if management is not adequate. Thus, an initial screen for HbE carriers in the general population must be conducted to provide proper counseling to those at risk of having HbE/β-thalassemia offspring. In theory, all screening tests must be simple, cheap, and durable with high sensitivity and moderate specificity. Here, we invented a new in-tube screening test for HbE and arbitrarily named it the Alcohol HbE Test, abbreviated to AET. The main ingredient of this test kit was isopropanol (C3H8O), a polar alcohol generally used for preparing disinfectant and DNA extraction (15–17). Although the first attempt to employ this alcohol in HbE screening was performed by Ali et al. in 1980 (13), this test has not substantially been in routine use, particularly in Thailand where HbE is very common. This might be due to the invention of the dichlorophenolindophenol precipitation (DCIP) test, which was also simple to use for HbE screening (18). The DCIP test has been in conventional use for HbE screening in the general population of Thailand since then. Several authors have evaluated and modified the protocols of the DCIP test (5, 6, 9, 10, 12, 19, 20). However, one thing that no-one could modify in the DCIP test was dichlopheholindophenol (C12H7Cl2NO2), which is the main ingredient of the DCIP test. This chemical is an oxidizing agent with a deep blue color. Therefore, it is easily reduced, leading to loss of its oxidizing property. Moreover, visualizing the precipitation in the tube was difficult due to its color. This therefore has been realized as the weak point of the DCIP test. AET was a test that we modified from the original test by Ali et al. We previously modified the protocol of this test and named it the “improved protocol of isopropanol precipitation test for hemoglobin E screen” (IIPT). We found that the improved protocol was effective in screening for HbE, the results of which were comparable to the conventional DCIP test. However, the IIPT could not be used for mass screening of HbE as it required preparation of hemolysate prior to testing. AET was then further established with the ultimate aim to simplify it for routine use in mass screening of HbE. This test still used isopropanol as the main working ingredient, but Triton-X-100 (TX100) at a final concentration of 0.005% (v/v) was used as the diluent. Triton-X 100 (C14H22O(C2H4O)n) is the nonionic detergent routinely used in several laboratories to destroy cells or to permeabilize the cell membrane. Therefore, mixing TX100 with isopropanol should directly lyse red blood cells, and preparing of hemolysate is not required. The results of this report proved this concept clearly. This protocol thus made AET much simpler than the previously established IIPT. We also tested several concentrations of TX100 and found false positive results at high concentrations of TX100. This was explained by the fact that, at high TX100 concentration, RBC died and aggregated inside the solution, thus making it turbid (21). However, we found that the 0.005% (v/v) concentration of TX100 was optimal in this protocol since positive results were clearly differentiated from the negative ones. Efficiency of the new AET is important. Thus, we evaluated its validity in unknown samples and the results indicated high efficiency of this test as shown by high sensitivity and specificity. Although AET did not appear superior to the conventional DCIP test in terms of sensitivity and specificity, its ease of preparation and operation together with its low cost per test (approximately $1/test for the DCIP test, <$0.5 for AET) would encourage its routine use in under-developed and developing nations (Supplemental Table 3). It has been shown that when the total Hb level decreased, the HbE level also reduced (22). This point was also of concern in the DCIP test. Fucharoen et al. found no effect of anemia in iron deficiency anemia on DCIP results (23). However, Sangkitporn et al. observed a false negative DCIP result in a sample of AEBart’s disease (24). AEBart’s disease is a complex form of thalassemia disease caused by combination of HbE heterozygote and HbH disease (βA/βE and –/–α). Therefore, HbE level was the main factor determining the DCIP results. Our AET results performed at several hematocrit levels did not show false negative results. However, our samples were not severely anemic. Therefore, we could not conclude that anemia had no impact on this AET. Studies on samples with Hb less than 11 g/dL such as AEBart’s disease and EF Bart’s disease were planned. We expected that these 3 groups of samples would also have the same positive results after being tested with this AET. Moreover, as blood samples used in this evaluation were 1–3 days old, this should explain false positive results in all groups studied. HbE is mildly unstable and has increased sensitivity to oxidants (3, 22). Therefore, it may denature easily over storing time and under some conditions. Thus, using aged blood in this AET might lead to false negative results. Therefore, we tested the stability of blood samples subjected for the AET. As expected, blood kept at a room temperature of around 30 °C lost stability rapidly and blood kept in colder conditions of about 4 °C stabilized better. Practically, the shelf-life of any test kit must be as long as possible. Thus, we tested for the shelf-life of the aliquoted AET kit kept at both room temperature in Thailand (30 °C) and in the cold (4 °C). The 500-µL aliquoted AET remained stable for at least 1 month in both conditions. The reagent of the AET was kept in a 1.5-mL microcentrifuge tube with a firmly closed lid. Closing the lid tightly should prevent evaporation of alcohol. However, keeping the aliquoted AET for quite a long time might lead to evaporation of isopropanol. This was the reason why false negative results were observed for the 6-month stored AET. Interestingly, we found that keeping the unaliquoted AET reagent in a stock container with the lid tightly closed in a cold place such as in a refrigerator could maintain its shelf-life for up to 6 months. Therefore, if long-term storage is required, the AET reagent should be kept cold in a sealed stock container. HbH disease is the severe nonfatal form of α-thalassemia syndrome caused by deletions or mutations of α-globin genes in chromosome 16p13.3 leading to decreased production of α-globin chain (25). There are 2 broad types of HbH disease; deletional and nondeletion HbH diseases. Deletional HbH disease results from compound heterozygosity of αO-thalassemia gene and α+-thalassemia gene (–/–α). Mutated α-globin gene of Hb Constant spring (Cs) or αCs is common in Thailand. Therefore, compound heterozygosity of the αO thalassemia gene and αCs gene results in HbH-Cs disease (–/αCsα); a nondeletional HbH disease commonly found in Thailand. Hbs A2AH or A2ABart’sH are the typical hemoglobins found in the deletional HbH disease. In contrast, Hbs CsA2AH or Hbs CsA2ABart’sH are typical for HbH-Cs disease (26). A two α-globin chain assembles with a 2 β-globin chain to form adult HbA (α2β2). A reduced amount of the α-globin chain leads to an excess amount of the β-globin chain, which subsequently forms a β4 homotertramer called HbH (26). HbH has been known to be unstable and is easily denatured at high temperature and under oxidative stress. A large amount of HbH is formed in HbH disease and might give a positive result by this AET. We tested this hypothesis and found that HbH disease also showed positive AET results. False positive results of HbH disease are also seen for the DCIP test and in our previous IIPT test (20, 27). More importantly, evaluation of the original isopropanol precipitation test showed false positive results with other unstable hemoglobins (28, 29). HbH is an unstable hemoglobin frequently encountered in Thailand. Therefore, we performed the AET for HbH disease that had a considerable quantity of HbH, and false positive results were obtained as stated previously. HbF (α2γ2) is one of the human hemoglobins found predominately during the prenatal period. However, a minute amount (<0.6%) of HbF is synthesized in adults (30). Modestly high HbF levels (1–5%) may be found in adults in a condition termed the Swiss-type hereditary persistence of fetal hemoglobins (HPFH). The Swiss-type HPFH has been shown to be common in the Thai population, attaining a frequency of 32% (31). A previous study showed that the original isopropanol precipitation test (31) showed false positive results in blood samples with an HbF level >4% (32). Therefore, the false positive results shown with this AET test might also be due to this phenomenon. A further test was planned to determine this interference. Other unstable hemoglobins such as HbS and HbC, may also give false positive results. Unfortunately, we could not test these 2 hemoglobins as their prevalences are very low in Thailand. Further investigation on this aspect is still required. In conclusion, the new AET was successfully established and proven to be effective in screening for HbE, but less costly than the conventional DCIP test. Thus it can be an alternative test for HbE screening and help successful prevention and control of HbE/β-thalassemia in poor countries. Further investigations on the lowest HbE levels that still give positive AET results and whether it is possible to use heel-prick blood samples from neonates should strengthen the clinical application of this test. Supplemental Material Supplemental material is available at The Journal of Applied Laboratory Medicine online. Author Contributions: All authors confirmed they have contributed to the intellectual content of this paper and have met the following 4 requirements: (a) significant contributions to the conception and design, acquisition of data, or analysis and interpretation of data; (b) drafting or revising the article for intellectual content; (c) final approval of the published article; and (d) agreement to be accountable for all aspects of the article thus ensuring that questions related to the accuracy or integrity of any part of the article are appropriately investigated and resolved. Authors' Disclosures or Potential Conflicts of Interest: No authors declared any potential conflicts of interest. Role of Sponsor: No sponsor was declared. Acknowledgments: The authors thank the volunteers for donating the blood samples used in this study. 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TI - Screening of HbE by the New Screening Test: Alcohol HbE Test (AET)
JF - The Journal of Applied Laboratory Medicine
DO - 10.1093/jalm/jfaa229
DA - 2021-07-07
UR - https://www.deepdyve.com/lp/oxford-university-press/screening-of-hbe-by-the-new-screening-test-alcohol-hbe-test-aet-k0OXpNnzYp
SP - 881
EP - 891
VL - 6
IS - 4
DP - DeepDyve
ER -