Hemoglobinopathy Screening by Osmotic Fragility Test Based on Flow Cytometer or Naked Eye

Transcription

1 Cytometry Part B (Clinical Cytometry) 90B: (2016) Original Article Hemoglobinopathy Screening by Osmotic Fragility Test Based on Flow Cytometer or Naked Eye Rinkle Mohapatra, Prashant Warang, Kanjaksha Ghosh, and Roshan Colah* Department of Haematogenetic, National Institute of Immunohaematology, Indian Council of Medical Research, KEM Hospital Campus, Parel, Mumbai, India Background: Diagnosis of hemoglobin (Hb) disorders is based mostly on abnormal red blood cell (RBC) indices, elevated levels of HbA2, HbF, or any other Hb on the Variant high performance liquid chromatography (HPLC) system, and confirmation by molecular methods. However, large scale population screening is of prime importance and requires a simple, accurate, and cost effective technique. We have tried to compare the sensitivity of the widely used Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) and the osmotic fragility described as % residual RBCs through flow cytometry for population screening. Methods: The count of residual red cells was measured sequentially in real-time using flow cytometry. NESTROFT was performed using a 0.36% buffered saline. HbA2 and HbF levels along with other abnormal Hbs were determined on the Variant HPLC System. Molecular studies were done to confirm the diagnosis. Results: The normal group showed a significantly lower percentage of residual RBCs ( ) as compared to cases (b thalassemia trait , a thalassemia trait , and HbS trait ). The sensitivity and specificity of NESTROFT was high for both b thalassemia traits (98.33 and 96.72%, respectively) and a thalassemia traits (100 and 96.72%, respectively) but very low sensitivity for HbS traits (54.84%). Conclusion: Flow cytometric osmotic fragility was a more sensitive method to discriminate normal from the group of hemoglobinopathy carriers as compared to NESTROFT which missed majority of HbS carriers. However, in view of feasibility and cost effectiveness, NESTROFT could still be used for population screening of thalassemia. VC 2014 International Clinical Cytometry Society Key terms: flow cytometric osmotic fragility; Naked Eye Single Tube Red Cell Osmotic Fragility Test; hemoglobinopathies How to cite this article: Mohapatra R, Warang P, Ghosh K, and Colah R. Hemoglobinopathy Screening by Osmotic Fragility Test Based on Flow Cytometer or Naked Eye. Cytometry Part B 2016; 90B: Defects in the human globin genes give rise to a heterogeneous group of autosomal recessive disorders, the hemoglobinopathies. These disorders include, the thalassemias characterized by reduced synthesis of one or more globin chains and hemoglobin (Hb) variants characterized by the synthesis of structurally abnormal Hbs (1). Hemoglobinopathies are one of the commonest monogenic disorders globally and pose a major public health problem particularly in tropical countries like India, where they occur at a high frequency (2). Thus, screening of communities known to have a high prevalence of thalassemias and Hb variants is an important component for community control of these disorders. Diagnosis of hemoglobinopathies is based on measurement of red blood cell (RBC) indices and the estimation of HbA 2 levels, HbF levels, and the percentage of any other abnormal Hb if present which most frequently is *Correspondence to: Roshan Colah, National Institute of Immunohaematology, 13th Floor, Multistoreyed Building, KEM Hospital Campus, Parel, Mumbai , India. colahrb@gmail.com Grant sponsor: Indian Council of Medical Research, New Delhi, India. Received 2 September 2014; Revised 17 November 2014; Accepted 20 November 2014 Published online 17 December 2014 in Wiley Online Library (wileyonlinelibrary.com). DOI: /cyto.b VC 2014 International Clinical Cytometry Society

2 280 MOHAPATRA ET AL. Table 1 The Mean Hematological Parameters in Different Groups Groups Sex (no.)age (Years) Hb (g/dl) RBC (310 6 /ml) MCV (fl) MCH (pg) RDW(%) Normal (59 cases) Males (31)/ Females (28)/ b Thalassemia Males (33)/ traits (59 cases) Females (26)/ a Thalassemia Males (03)/ traits (8 cases) Females (05)/ Males (11)/ Females (06)/ HbS traits (17 cases) determined by automated high performance liquid chromatography (HPLC). However, the most simple and cost effective preliminary method used for population screening for b thalassemia is the Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT), (3), a test with high sensitivity but low specificity. We have evaluated the usefulness of the recently described flow-cytometric osmotic-fragility test (FCM- OF) which is useful for detecting red cell membrane disorders (4), for screening of different hemoglobinopathies in comparison to NESTROFT. MATERIALS AND METHODS Subjects A total of 143 individuals were included in this study. They included 59 b-thalassemia traits, eight a- thalassemia traits, 17 sickle cell traits, and 59 healthy normal controls. The study was approved by the Institutional Ethics Committee of National Institute of Immunohaematology, Mumbai. Laboratory Investigations The samples were collected in ethylenediaminetetraacetic acid vials after an informed consent and were analyzed within 2 h of sample collection. Hematological parameters were measured on an Automated Cell Counter (Sysmex K-1000; Sysmex Corporation, Kobe, Japan). HbA 2, HbS, and HbF were quantitated using cation exchange HPLC on the VARIANT Hemoglobin Testing System (Biorad Laboratories, Hercules, CA). DNA was extracted from the leukocytes using the standard phenol chloroform method. The b thalassemia/sickle mutations were characterized by Covalent Reverse Dot Blot hybridization (5) or by Amplification Refractory Mutation System (6). The a thalassemia mutations were characterized by multiplex polymerase chain reaction (PCR) (7). NESTROFT NESTROFT is a single tube qualitative osmotic fragility test based on the limit of hypotonicity which the red cells can withstand. Two milliliters of 0.36% buffered saline was taken in a corning glass tube ( mm 2 ). Twenty microliters of whole blood was added to the tube and after proper mixing was allowed to stand at room temperature for 30 min. The results were recorded as positive (1), negative (2), or doubtful (1/2) by observing the tubes against a white paper with a sharp black thin line (8). If the line was not visible, the sample was considered positive (1), if clearly visible, it was graded as negative (2) and if not clearly visible it was graded as doubtful (1/2). Preparation of Red Cell Suspension for Flow Cytometry A two-step dilution method with normal saline (4,9) was used to prepare the red cell suspension. The first dilution involved dilution of a certain volume of blood depending on the red cell count of each individual per 1 ml of drawn blood (to maintain uniformity in every

3 SCREENING OF HEMOGLOBINOPATHIES 281 FIG. 1. The Time/FSC plot. (A) % residual RBCs in normal 42.96%, (B) % residual RBCs in b thalassemia trait 76.24%, (C) % residual RBCs in a thalassemia trait 65.83%, and (D) % residual RBCs in HbS trait 87.40%. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

4 282 MOHAPATRA ET AL. Table 2 Comparison of NESTROFT and FCM-OF With a % Residual RBC Cut Off of 60 (Mean of Normal 6 1 SD) Normal b Thal. trait a Thal. trait HbS trait All hemoglobinopathies NESTROFT 1/6 1/6 1/6 1/6 1/6 % Residual RBCs < % Residual RBCs > Total no tube) in 1.1 ml normal saline. The amount of blood required was calculated using the formula: Blood volume (ml) 5 130/(Number of red cells per ml)/10 6 The second dilution step involved transfer of 10 ml of the first dilution to a fluorescence-activated cell sorting (FACS) tube that already had 1.1 ml of normal saline. This FACS tube was finally ready for acquisition. The second dilution step was performed just before acquisition. Flow-Cytometric Osmotic Fragility Test Forward Scatter Characteristics (FSC) and Side Scatter Characteristics were acquired as a linear amplification on a FACS Caliber flow cytometer (Becton Dickinson, San Jose, CA). As mentioned by Won and Suh (4), a Time/FSC acquisition plot was demarcated with eight identical regions. The final suspension in the FACS tube was then installed at the sample injection port and the acquisition started. After the first region in the plot had elapsed (22 s), the tube was removed from the port without halting the acquisition and 0.9 ml deionized distilled water was spiked to the tube. The acquisition FIG. 2. ROC curve for determination of the sensitivity and specificity of the FCM-OF test for screening of hemoglobinopathies. The area under the ROC curve is and the best cut off value of % residual red cells for both high assay sensitivity and high specificity is 58.2%. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.] was then resumed and continued until the acquisition of events had passed through region 8 of the time versus FSC plot. The degree of osmotic lysis was expressed as % Residual RBCs and was calculated by the formula % Residual RBCs Mean event count of last two regions 5 Event count in first region 3 1:1=2: ð% Þ A correction factor, 1.1/2.0 is also introduced in the formula to counter the dilution in Region 1 as compared to dilution after spiking with distilled water in other regions. Statistical Tests The data is presented as mean 6SD. The sensitivity, specificity, positive and negative predictive values, and receiver operating characteristic (ROC) curve were determined using MedCal Software bvba, (Ostend, Belgium) and McNemar s test was done using the Graph Pad Software, Prism 6, (CA). RESULTS Based on the complete blood count (CBC), HPLC, and molecular analysis, the individuals screened were divided into four groups, normal, b thalassemia trait, a thalassemia trait, and sickle cell (HbS) trait. The mean hematological parameters in each group are shown in Table 1. The 59 b thalassemia traits were heterozygous for the following mutations: IVS 1 5 (G!C)230, Codon 8/ 9(1G)24, Codon 15(G!A)27, IVS 1-1 (G!T)24, Codons 41/42(-CTTT)21, Codon 30 (G!C)21, 619 base pair deletion-4, Capsite11 (A!C)26, and 288 (C!T)22. The eight a thalassemia traits had the following mutations: 2a 3.7 /aa 2 4, 2a 4.2 /aa 2 2, and 2a 3.7 / 2a The 17 sickle cell traits were heterozygous for the b-codon 6 (GAG! GTG) mutation. The % residual RBCs in hemoglobinopathies as determined by FCM-OF is shown in Figure 1. The mean % residual RBCs show a variation among the cases with different hemoglobinopathies and normal. b Thalassemia traits had a mean % residual RBC count of (6SD 12.20), a thalassemia traits had a mean of (6SD 8.34), HbS traits showed a mean of (6SD 4.05) while the normal group had a mean of (6SD 11.87). The b thalassemia traits and HbS traits showed comparatively higher % residual RBCs compared to the normal.

5 Table 3 Sensitivity and Specificity of NESTROFT and FCM-OF for Screening of Carriers of b Thalassemia, a Thalassemia, and HbS Cases Sensitivity (%) Specificity (%) SCREENING OF HEMOGLOBINOPATHIES 283 Positive predictive value (%) Negative predictive value (%) Sensitivity and specificity of NESTROFT b Thalassemia trait a Thalassemia trait HbS trait Sensitivity and specificity of FCM-OF b Thalassemia trait a Thalassemia trait HbS trait The a thalassemia traits showed comparatively lower % residual RBCs, however, this value was also significantly high as compared to normal. The 10 normal individuals showing % residual RBCs of >60 could be due to latent iron deficiency or IDA which was not ruled out. The NESTROFT results have been summarized in Table 2 in relation to the % residual RBCs seen on flow cytometry taking a cut off of 60. The positive and doubtful cases in NESTROFT were grouped together for analysis as both these groups would need further investigations. Out of 59 normal, two false-positive results were seen by NESTROFT. Among the hemoglobinopathy cases, 1 b thalassemia trait and 14 sickle cell traits were NESTROFT negative, however, the % residual RBCs were >60. The diagnostic sensitivity of FCM-OF and NES- TROFT in the total group of all hemoglobinopathies was significantly different (98.8 and 82.1%, respectively, P , McNemar s test). Using the ROC curve, the group of hemoglobinopathies was picked up by the FCM-OF test with a sensitivity of % and specificity of 83.05%. The area under the ROC curve was (Fig. 2). As summarized in Table 3, the sensitivity of NESTROFT varied considerably among the various conditions. HbS traits showed a high number of false negatives. However, the b thalassemia traits and a thalassemia traits showed a higher sensitivity (98.33 and 100%, respectively). FCM-OF was a more sensitive method (100%) mainly for screening for HbS as compared to NESTROFT which missed majority of HbS carriers. DISCUSSION Screening for carriers of different hemoglobinopathies is an important component of a community control programme. Many studies have been undertaken using red cell indices with cut off values for mean corpuscular volume (MCV) (<80 fl) and/or mean corpuscular hemoglobin (MCH) (<27 pg) or the single tube osmotic fragility test (NESTROFT) mainly for preliminary screening of b thalassemia carriers in population surveys (8,10 16). Both these approaches are associated with variable percentages of false-negative or false-positive results. Although false positives could be due to the presence of iron deficiency anemia, prevalent in countries like India and would mean further analysis by HPLC or other electrophoretic techniques for estimation of HbA 2, HbF, and other Hb variants in larger number of samples, false negatives would mean missing carriers of hemoglobinopathies who would remain undiagnosed. It has also been shown that combining the use of red cell indices with NESTROFT would reduce the error rate but increase the cost of screening. Old et al. (17), argue about the limited success of NESTROFT in large population screening programmes due to the associated false-negative errors associated with this test. The FCM-OF has conventionally been used for screening of red cell membrane disorders where a decrease in the percentage of residual red cells after spiking with deionized water is used for discrimination (4,9). The FCM-OF test has greater test efficiency than the conventional OF test and the FCM-OF test results quantitatively reflect the clinical severity of hereditary spherocytosis (HS) (18). We evaluated this same approach to see whether we could discriminate individuals with hemoglobinopathies like a thalassemia carriers, b thalassemia carriers, and sickle cell carriers from normal individuals based on an increase in the percentage of residual red cells. At the same time, we also compared this FCM-OF with the visual NESTROFT for discriminating these cases. The % residual RBCs as screened by flow cytometry was able to discriminate the hemoglobinopathies significantly from the normal. The % residual RBCs in normal and b thalassemia traits were in concordance with the findings of Warang et al. (9) on a few samples. The type of the b thalassemia mutations did not have any effect on the percentage of residual RBCs. There was a significant difference between the mean % residual RBCs in normal and hemoglobinopathy samples. However, to differentiate among the different hemoglobinopathies, this method proved to be ineffective. Using both these approaches one case of b thalassemia trait gave a false-negative result but the a thalassemia trait cases were correctly picked up although the number of cases in this group was small. A large number of HbS traits were missed by NESTROFT, the sensitivity being only 54.84%, however, they were all picked up correctly by FCM-OF. If access to a flow cytometer is available as is often the case in many Hematology laboratories, the FCM-OF could be a first line screen for hemoglobinopathies before proceeding to HPLC as it is simple and requires only

6 284 MOHAPATRA ET AL. deionized water. However, a larger number of samples with different Hemoglobinopathies need to be investigated particularly cases of a thalassemia which often remain unidentified even after HPLC analysis. Recently, using flow cytometry, the ratio of intact RBCs in hypotonic solution has also been used as an indicator of osmotic fragility in hemolytic anemia (19). Nevertheless, in remote regions with economic constraints, it would not be possible to bring the samples collected for population surveys for analysis to the laboratory within 2 h and the simple and inexpensive NESTROFT could be used in spite of its limitations, but it should be combined with an additional sickling or solubility test to avoid missing HbS carriers for the provision of public health services. LITERATURE CITED 1. Weatherall DJ, Clegg JB. The Thalassemia Syndromes. 4th ed. Oxford, England: Blackwell Science; Mohanty D, Colah RB, Gorakshakar AC, Patel RZ, Master DC, Mahanta J, Sharma SK, Chaudhari U, Ghosh M, Das S, Britt RP, Singh S, Ross C, Jagannathan L, Kaul R, Shukla DK, Muthuswamy V. Prevalence of b-thalassemia and other hemoglobinopathies in six cities in India: A multicentre study. J Community Genet 2013;4: Kattamis C, Efremov G, Pootrakul S. Effectiveness of one tube osmotic fragility screening in detecting beta thalassemia trait. J Med Genet 1981;18: Won DI, Suh JS. Flow cytometric detection of erythrocyte osmotic fragility. Cytometry Part B 2009;76B: Colah RB, Gorakshakar AC, Lu CY, Nadkarni AH, Desai SN, Pawar NR, Lulla CP, Krishnamoorthy R, Mohanty D. Application of covalent reverse dot blot hybridization for rapid prenatal diagnosis of the common Indian thalassemia syndromes. Indian J Haematol Blood Transf 1997;15: Vaz FE, Thakur CB, Banarjee MK, Gangal SG. Distribution of beta thalassemia mutations in the Indian population referred to a diagnostic center. Hemoglobin 2000;3: Tan A, Quah T, Low P, Chong SS. A rapid and reliable 7 deletion multiplex polymerase chain reaction assay for a thalassemia. Blood 2001;98: Gorakshankar AC, Colah R, Nadkarni A, Desai S. Evaluation of the single tube osmotic fragility test in detection of beta thalassemia trait. Natl Med J India 1990;3: Warang P, Gupta M, Kedar P, Ghosh K, Colah R. Flow cytometric osmotic fragility An effective screening approach for red cell membranopathies. Cytometry Part B Clin Cytom 2011;80B: Clarke GM, Higgins TN. Laboratory investigation of hemoglobinopathies and thalassemias: Review and update. Clin Chem 2000;46: Mehta BC, Gandhi S, Mehta JB. Naked eye single tube red cell osmotic fragility test for beta thalassemia population survey. Indian J Haematol 1988;6: Raghavan K, Lokeshwar MR, Birewar N, Nigam V, Manglani MV, Raju N. Evaluation of naked eye single tube red cell osmotic fragility test in detecting beta thalassemia trait. Indian Paediatr 1991;28: Manglani M, Lokeshwar MR, Vani VG, Bhatia N, Mhaskar V. Nestroft. An effective screening test for beta thalassemia trait. Indian Paediatr 1997;34: Mamtani M, Jawahirani A, Das K, Rughwani V, Kulkarni H. Is NES- TROFT sufficient for mass screening for beta thalassemia trait? J Med Screen 2007;14: Chakrabarti I, Sinha SK, Ghosh N, Goswami BK. Beta thalassemia carrier detection by NESTROFT: An answer in rural scenario. Ir J Pathol 2012;7: Piplani S, Manan R, Lalit M, Manjari M, Bhasin T, Bawa J. NES- TROFT A valuable, cost effective screening test for beta thalassemia trait in north Indian Punjabi population. J Clin Diagn Res 2013; 7: Old JM, Olivieri NF, Thein SL. The laboratory diagnosis of thalassemias. In: Weatherall DJ, Clegg JB, editors. The Thalassemia Syndromes. Oxford: Blackwell Sciences; p Shim YJ, Won DI. Flow cytometric osmotic fragility testing does reflect the clinical severity of hereditary spherocytosis. Cytometry Part B Clin Cytom 2014;86B: Yamamoto A, Saito N, Yamauchi Y, Takeda M, Ueki S, Itoga M, Kojima K, Kayaba H. Flow cytometric analysis of red blood cell osmotic fragility. J Lab Autom 2014;19: