|Year : 2017 | Volume
| Issue : 4 | Page : 135-139
Utility of screening golf ball inclusions in hemoglobin H disease and its clinico-hematological profile
Dinesh Chandra1, Seema Tyagi2, Roopam Deka2, Richa Chauhan2, Jasdeep Singh2, Tulika Seth2, Hara Prasad Pati2, Renu Saxena2
1 Sanjay Gandhi Post Graduate Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 All India Institute of Medical Sciences, New Delhi, India
|Date of Web Publication||12-Jan-2018|
Dr. Seema Tyagi
Room No 207, Second Floor, New Private Ward, All India Institute of Medical Sciences, New Delhi
Source of Support: None, Conflict of Interest: None
BACKGROUND: In India, hemoglobinopathy screening programs focus on reducing the burden of beta-thalassemia by screening the antenatal females and young adolescents. Although alpha (α)-thalassemia is common in the Mediterranean countries, alpha (α)-thalassemia screening and diagnosis are performed in a few centers in India. HbH disease is an uncommon clinical phenotype of α-thalassemia with variable clinical presentation. The hallmark feature of HbH is the presence of golf ball inclusions in the red cells demonstrated with supravital staining.
PATIENTS AND METHODS: Here, we discuss an algorithmic approach to screen hemograms for thalassemic indices, followed by evaluation of reticulocyte preparation after 40 minutes (supravital stain preparation for golf ball inclusions). If golf ball inclusions were positive, Hb analysis on Hb-capillary zone electrophoresis (Hb-CZE)was done.
RESULTS: We received 28,000 samples for complete hemogram and blood film examination, over a period of 8 months. Reticulocyte count was required in 11,600 samples out of which 13 cases were positive for golf ball inclusions. Of these, nine were identified as HbH disease on Hb-CZE. These patients presented with pallor and generalized weakness. On examination, jaundice (3/9 patients) and hepatosplenomegaly (4/9 patients) were relatively uncommon. The majority of them (6/9) required infrequent blood transfusions. All patients had microcytic hypochromic red cell indices with median HbA of 89% (range: 68.1%–96.9%), HbA2 of 1.4% (range: 0.8%–1.7%), and fast-moving band in zone 15 (HbH) of 5.6% (range: 1.4%–30.3%) on Hb-CZE.
CONCLUSION: We emphasize a systematic screening of hemograms and peripheral smears along with a simple and cost-effective screening test for golf ball inclusions to diagnose HbH disease that often poses a clinical dilemma due to its variable clinical presentation.
Keywords: Alpha-thalassemia,Golf ball inclusions, Hemoglobin H disease, Hemoglobinopathy
|How to cite this article:|
Chandra D, Tyagi S, Deka R, Chauhan R, Singh J, Seth T, Pati HP, Saxena R. Utility of screening golf ball inclusions in hemoglobin H disease and its clinico-hematological profile. J Appl Hematol 2017;8:135-9
|How to cite this URL:|
Chandra D, Tyagi S, Deka R, Chauhan R, Singh J, Seth T, Pati HP, Saxena R. Utility of screening golf ball inclusions in hemoglobin H disease and its clinico-hematological profile. J Appl Hematol [serial online] 2017 [cited 2018 Mar 17];8:135-9. Available from: http://www.jahjournal.org/text.asp?2017/8/4/135/223178
| Introduction|| |
Patients with alpha (α)-thalassemia present with a variable clinical phenotype depending on the number of α-gene deletions or mutations present. Person with one or two α-gene deletions is clinically asymptomatic while deletion of three α-genes leads to hemoglobin H (HbH) disease with marked reduction in α-globin chains. The excess β-globin chains form aggregates as unstable HbH (β4-tetramers) which are toxic and leads to hemolysis. Supravital stains such as brilliant cresyl blue (BCB), gentian violet, and new methylene blue upon prolonged incubation stain these precipitated globin chains. They appear as numerous dot size inclusions inside the red cells and are known as “golf ball inclusions.”, The majority of patients with HbH disease have compensated hemolytic anemia with an average Hb level of more than 9 g/dL. Patients with HbH disease may require infrequent blood transfusion, splenectomy, or chelation therapy.
Here, we have analyzed the clinico-pathological profile of nine patients with HbH, who were diagnosed during evaluation for anemia based on their clinical profile.
| Patients and Methods|| |
We received 28,000 samples for complete hemogram and blood film examination, from September 2015 to April 2016. Reticulocyte count was required in 11,600 samples. Complete blood counts were carried out using an automated cell counter, Sysmex XT-1800i. Peripheral blood films were stained with Leishman-Giemsa for morphology. Golf ball inclusions were screened on 1% BCB stain with prolonged incubation period of 40 minutes in all patients. Thirteen patients were positive for golf ball inclusions on 1% BCB preparation [Figure 1]b and were further analyzed by Hb-capillary zone electrophoresis (Hb-CZE) using the automated Capillary Mini Flex Piercing analyzer, Sebia, France. Electrophoresis was done using alkaline buffer pH 9.4, provided by the manufacturer. Hbs were detected at 415 nm and electrophoretograms were recorded providing identification of Hbs in specific zones. In all these, thirteen patients detailed clinical history and general physical examination were conducted.
|Figure 1: (a) Peripheral smear showing microcytic hypochromic red cells with mild anisocytosis and target cells, (b) 1% brilliant cresyl blue preparation showing golf ball inclusions in red cells at 40 minutes of incubation (c) Hb-CZE showing a peak in zone Z15 (HbH)|
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| Results|| |
Of the 11,600 samples with complete hemograms, blood film, and reticulocyte count, golf ball inclusions were detected in thirteen patients. Nine out of these thirteen had microcytosis with erythrocytosis on hemogram [Figure 1]a and were diagnosed as HbH disease on Hb-CZE with peak in zone 15 [Figure 1]c. The remaining three out of four patients were diagnosed as Hb-E/Bart, Hb-D Punjab/Hb-Bart, and myelodysplastic syndrome. One patient remained undiagnosed even after extensive workup.
These nine patients were young with median age of 24 years (1–33 years). There was slight male predominance (M:F=5:3). Pallor and generalized weakness were the most common presentation (88.8% and 77.7%, respectively). Jaundice was noticed in three patients, and splenomegaly was found in four patients. Six out of nine patients required intermittent blood transfusions. Clinical characteristics are given in [Table 1] and [Table 3].
|Table 1: Clinical characteristics of 13 patients with golf ball inclusions on supravital stain|
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|Table 3: Clinical and laboratory characteristics of patients with HbH disease|
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Complete hemogram findings in the nine definite cases of HbH were as follows: median Hb was 8.8 g/dL, red cell count 4.43 × 106/μL, mean cell volume 68.7 fl (range: 56.7–87.3), mean cell Hb 18.1 pg (range: 17.2–24.4), mean cell Hb concentration 28.2 g/dL (range: 25.7–31.5), and red cell distribution width 21.1% (15.2–26.7) [Table 2].
|Table 2: Hematological profile and Hb-CZE findings of 13 patients with golf ball inclusions on supravital staining|
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Hemoglobin capillary zone electrophoresis findings
Median HbA in the 13 patients was 89% (range: 68.1%–96.9%), median HbA2 - 1.4% (range: 0.8–1.7%), and the fast-moving band in zone 15 was 5.6% (range: 1.4%–30.3%).
| Discussion|| |
In India, screening for hemoglobinopathies is skewed toward diagnosing beta-thalassemia trait in antenatal patients due to high burden of beta-thalassemia. The laboratories are also well equipped with state of the art instruments such as high-performance liquid chromatography (HPLC) (β-thalassemia short program) and mutation analysis for β-thalassemia. The same is not true for α-thalassemia. There is a tendency to underdiagnose α-thalassemias which can be easily misinterpreted as iron deficiency anemia (IDA), an extremely common condition in India. Also being asymptomatic, α-thalassemia with one or two gene deletions does not require intensive investigation. Hb Barts with 4-gene deletion is incompatible with life. HbH (3-gene deletion) has a clinical phenotype similar to thalassemia intermedia. Rarely, it may be missed on HPLC due to its elution before the start of chromatogram. The molecular tests are not widely available in most of the laboratories and also not cost effective.
BCB staining is a simple test used for detecting red blood cell inclusions in α-thalassemia and is very useful screening test for HbH disease in a resource-limited laboratory setup., BCB acts as an oxidant to denature HbH as intracellular inclusions. Lin et al. used prolonged incubation time from 30 min to 3 h for 1% BCB staining and found enhanced sensitivity of 91% for detecting α-thalassemic traits. They found no false-positive staining in other microcytic hypochromic anemia, for example, β-thalassemia and iron deficiency anemia. Fucharoen et al. enhanced the sensitivity and specificity (94% and 99%, respectively) of HbH inclusion test by enriching young red blood cells fraction using dextran sedimentation and then incubating with 2% BCB for 30–90 min.
Therefore, we adopted a sequential approach of screening thalassemic indices on hemogram with reticulocyte count and then screen for golf ball inclusions using prolonged incubation with 1% BCB preparation. We further analyzed, these golf ball inclusion positive cases for Hb variants on Hb-CZE. On supravital staining of 11,600 cases, We detected golf ball inclusions in 13 patients. Iron deficiency was ruled out by measuring serum ferritin levels where cutoffs for iron replete were defined as serum ferritin more than 30 μg/L. Nine of 13 patients were confirmed to be HbH disease by eliciting a migration pattern of an abnormal Hb peak in zone 15 on Hb-CZE. Although the number of cases detected by demonstrating golf ball inclusions was minuscule, it helped to pick up HbH disease cases, especially asymptomatic (cases 3 and 6) that would otherwise remain undetected.
All patients had a common clinical presentation of pallor and weakness (88.8% and 77.7%). Median age was 24 years (age range: 1–33 years). Growth and development were normal in all of our patients.
Regular requirement of blood transfusions is not a feature of α-thalassemia and so was the situation with our patients. Two-third of the patients required transfusions at some point. This was slightly more than in the Chinese cohort of 114 patients (46%). Average number of transfusion requirement in our patients was 2.5.
Only four of nine patients presented with mild-to-moderate splenomegaly; however, none of them required splenectomy. Clinically, some cases in review of literature were initially suspected to have kala-azar or hypersplenism. Hepatosplenomegaly and jaundice were attributed to nondeletional mutations of α-gene due to tendency for hemolysis rather than ineffective hematopoiesis. This was also evident in the Thai population due to higher prevalence of nondeletional mutations. Only three patients in our cohort presented with jaundice.
Gallstones were found in only two of our patients (15.3%). Fucharoen and Viprakasit and Chui et al. found over 30% of HbH disease with gallstones.
Two cases were associated with β-chain variants, i.e., HbE and HbD Punjab. As stated in previous literature, coinheritance of α-thalassemia with HbE (Hb AE Bart's disease) leads to marked reduction in percentage of HbE to 13%–15% due to reduced affinity of α-globins for βE than βA resulting in thalassemic disease of intermediate severity. Another patient was diagnosed as heterozygous for HbD Punjab with Hb Bart's Hb.
One patient on further workup was diagnosed as a case of myelodysplastic syndrome. Acquired HbH disease is known to occur in various myeloproliferative disorders as well as myelodysplasia in elderly males. Some trans-acting genes are known to control the function of α-genes on chromosome 16. Thus, mutations of such trans-acting genes may lead to HbH without actually having dysfunctional α-alleles.
In our cohort, the percentage HbH ranged from 1.6% to 30.3%. The HbH level can be detected by Hb electrophoresis, isoelectric focusing, or HPLC. We did not find significant elevation of HbF levels. However, HbA2 levels were low as expected.
The molecular pathology of HbH disease is heterogeneous. Out of 35 patients were analyzed by Unal et al., the most common mutations was −α3.7 (62.8%). They found incidence of HbH to be 17.5% of all hemoglobinopathies in Turkey. In Taiwan (-SEA) type of α0-thalassemia mutation was detected in the majority of patients (>95%). The most common genotype was(-SEA/−α3.7). In India, Bhat et al. found an −α3.7 single-gene deletion in their patient using multiplex PCR reverse dot blot hybridization assay. The deletional mutations are the most prevalent α-thalassemia mutation in India, and −α3.7 homozygosity has been described commonly. In four of our patients, we extended our workup to screen for 4 α-thalassemia mutations namely del 3.7, del4.2, SE, and SEA. One of our patients, who also had a positive family history (patient 3), showed homozygous −α3.7 rightward deletion on molecular analysis by PCR. The results of other three were negative for these four common mutations studied.
| Conclusion|| |
In country like India, where the prevalence of HbH disease is low, the diagnosis is often complicated and missed due to variable presentation. HbH inclusion test, a simple screening test that is known for long but used infrequently, can serve as a screening test for unexplained moderate anemia, jaundice, or splenomegaly. Furthermore, here, we showed that routine workup and diagnosis of HbH does not have to hinge on only molecular studies where stepwise approach, diligent screening of peripheral smears, and a simple supravital stain preparation along with Hb-CZE or Hb-HPLC can be used for screening and diagnosis of HbH disease correctly and cost effectively.
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Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3]