A total of 536 patients with suspected haemoglobinopathy were studied. The age group of the study population ranged from 0.2 to 73 years with a mean age of 26.43 years. There were more females in the study group 377 females (70.34%) and 159 males (29.66%) owing to a large number of antenatal cases that were screened. The most common subset was patients referred for antenatal checkups 224(41.79%). Clinically pallor had been documented in 84 (15.67%) patients and 112(20.90%) cases had been sent as family screening cases.17 cases out of 536 (3.17%) presented had jaundice on presentation while a history of transfusion was documented in 53 cases. Out of a total of 536 patients that were screened 141 patients were detected to have underlying thalassemia or haemoglobinopathy. The majority of these patients were BTT 97(68.79%) followed by compound heterozygous Hb E -beta-thalassemia 15(10.64%) and heterozygous E state 12(8.51%). There were 02 (1.42%) homozygous beta-thalassemia patients and 03 (2.13%) each with Homozygous HbS and Heterozygous sickle cell state. One each (0.71%) of compound heterozygous HbS beta-thalassemia, compound heterozygous delta beta-thalassemia, HbD Iran, Heterozygous HbO-Indonesia, Haemoglobin G Sriraj /O Arab were also identified. Three samples had a previous history of transfusion and 01 sample showed a carryover of HbD of 2.7%. A repeat HPLC/HbCZE was advised for these patients but they were lost to follow up. The distribution of subgroups is depicted in table 1. The erythrocyte indices of the commonest subgroups are shown in table 2.
Table 01:-Distribution of subgroups of study subjects
Subgroups
|
Frequency
|
Percentage
|
Homozygous beta thalassemia
|
2
|
1.42%
|
Heterozygous beta thalassemia
|
97
|
68.79%
|
Heterozygous E state
|
12
|
8.51%
|
Compound heterozygous Hb E -beta-thalassemia
|
15
|
10.64%
|
Homozygous HbS
|
3
|
2.13%
|
Heterozygous sickle cell state
|
3
|
2.13%
|
Compound heterozygous HbS beta-thalassemia
|
1
|
0.71%
|
Compound heterozygous delta beta /beta-thalassemia
|
1
|
0.71%
|
HbD Iran
|
1
|
0.71%
|
Heterozygous HbO-Indonesia
|
1
|
0.71%
|
Haemoglobin G Sriraj / O Arab
|
1
|
0.71%
|
Post transfusion sample /carry over samples
|
4
|
2.84%
|
Total
|
141
|
100.00%
|
Table 2. Erythrocyte indices of the commonest subgroups heterozygous beta-thalassemia, Compound heterozygous Hb E beta-thalassemia, Heterozygous E state
Erythrocyte indices
|
Heterozygous beta thalassemia(n=97)
|
Compound heterozygous Hb E beta-thalassemia(n=15)
|
Heterozygous E state(n=12)
|
Haemoglobin(g/dL)
|
11.3 ± 2.14
|
8.13 ± 3.46
|
11.61 ± 1.63
|
TRBC(x1012/l )
|
5.57 ± 1.11
|
4.52 ± 1.15
|
4.62 ± 1.1
|
HCT
|
36.62 ± 6.32
|
29.11 ± 11.02.
|
35.93 ± 4.5
|
Mean corpuscular haemoglobin (fl)
|
55.3 ±85.3
|
61.32 ±8.32
|
73.79 ±4.38
|
Mean corpuscular haemoglobin (pg)
|
20.67 ±3.21
|
17.83 ± 3.7
|
23.81 ± 1.97
|
Mean corpuscular haemoglobin concentration (g/dL)
|
32.67 ± 2
|
28.79 ± 2.6
|
31.48 ± 2.39
|
On HbCZE the haemoglobin A values in the study group ranged from 3.2-99% with a mean HbA of 93.74 ± 1.Haemoglobin A2 (HbA2) constitutes less than 3% of the total haemoglobin (Hb) in adults and has almost no physiological importance however the determination of Hb A2 levels is important to diagnose the beta thalassemia trait (BTT).The haemoglobin A2 values in the study group ranged from 3.2-99% with a mean HbA of 93.74 ± 12.66 %. For the sake of statistical analysis HbA2 values were divided into the following groups for ease of comparison <2%, 2.0-3.3%, 3.4-3.7%, 3.8-7.0% and >7% with HbA2 < 2% indicative of IDA, alpha thalassemia, HbH disease δβ thalassaemia (if haemoglobin F was also elevated), 2.0 - 3.3% was essentially the normal values for HbA2, HbA2 of 3.4-3.7% was indicative of a borderline elevated HbA2, or severe iron deficiency in β thalassaemia trait. β thalassaemia trait with additional δ chain variant may also have lower values of HbA2. In the presence of haemoglobin S, HbA2 values are raised on HPLC and may fall in this borderline zone. Haemoglobin A2 values of >7.0% are rare and a structural variant needs exclusion after repeating HbA2 estimation. The majority of the patients 340 (63.43%) had levels of HbA2 within the normal range i.e. 2.0-3.3 % while the second most commonest was HbA2 levels of 3.8-7.0% totalling to 149 pts (27.80%). Six pts had borderline values of between 3.4 and 3.7% only two pts (0.37%) had values over 7%.
On HPLC 352 (65.67%) pts had normal HbA2 values, and 136(25.37%) pts had HbA2 which fell into the range of 3.8-7.0%. 31 pts had values that were more than 7% and these were later sub-grouped based on the percentage of HbA2 present as Hb Lepore, HbE, and HbD Iran coeluate in this window. Values of HbA2 between 10-14% are indicated of Hb Lepore, 25-40% Hb E heterozygous, 44-48% HbD Iran and 70-90 % E homozygous. While the P2 window has the glycated Hb eluting in it, in the P3 window values up to 6% are acceptable 6-12% are indicative of sample deterioration and 15-25% are indicative of Hb J Meerut an alpha chain variant. In our study mean P2 values were 3.52 ± 1.05 while mean P3 values were 4.67 ± 1.43. No abnormal variant was detected in the P3 window.
Four rare variants were picked up in this study these included Compound heterozygous delta beta/ beta thalassemia, HbD Iran, heterozygous HbO Indonesia , Hb G Sriraj /O Arab.
- HbD Iran
The first rare variant picked up was Hb D Iran in a lady referred for antenatal screening. She had a Hb of 13.2gm/dl, TRBC-4.39 million/mm3 and normal RBC indices with MCV 89.1 fl, MCH 30 pg, MCHC 33.8gm/dl. On HPLC HbA2 was 41.1% with a retention time of 3.62 ,Hb A of 49.8%. On the HbCZE this abnormal haemoglobin was seen in the D window with HbA2 of 1.8%. Her sickling test was negative. Based on this she was diagnosed as a case of Hb D Iran. Reportedly in HPLC, nine abnormal Hbs elute in the Hb A2 window (3.27–3.83 as per the manufacturer's guidelines in the operating software): Hb Deer Lodge, Hb Lepore, Hb D Iran Hb E, Hb Hamadan, Hb Osu-Christiansborg, Hb Tianshu, Hb G Honolulu and Hb G Copenhagen. Among these, Hb Deer Lodge, Hb Lepore and Hb D Iran elute prior to the standard RT of Hb A2 (3.6 min) while others have higher RT to that of Hb A2. Interestingly, Hb Lepore has the lowest average quantity (7–15%) followed by Hb G Honolulu (about 15% of total hemoglobin quantity) and Hb E (about 30% of total hemoglobin in absence of α-thalassemias). All the other variants eluting in the Hb A2 window have variant hemoglobin quantities higher than 30% on average under heterozygous conditions, making it difficult to distinguish in HPLC. Amongst these, Hb D Iran has been reported to elute in this window at a RT of 3.49–3.58 min; almost in the middle of the window (3.27–3.83).A number of reasons contribute to the apparent low prevalence of Hb D Iran, more so because Hb D Iran when in heterozygous and homozygous form is clinically asymptomatic except for cases of compound heterozygosity with β/Hb D Iran where there is mild to moderate anemia and easy fatiguability. Majority of homozygous and heterozygous cases are detected retrospectively as a part of extended family screening. Hence most of the Hb D Iran cases are missed because of its asymptomatic phenotype.(7)
2.Hb G-Sriraj /O Agenogi
The suspected Hb G-Sriraj /O Agenogi where the HPLC showed a peak in the C window of 87.7% with a retention time of 4.98 seconds an HbA2 of 7.7 % while on HbCZE HbA2 was 3.3% and a peak in the E window was found of 91.6%.( Figure B) On the HPLC O Arab, O Indonesia, Constant Spring, Agenogi, Sriraj may be found in the C window with a retention time ranging from 4.90 to 5.30. While on the HbCZE Hb E, Hb Köln (Ube-1), Hb Buenos Aires (minor peak), Hb Agenogi, Hb G-Sriraj, Hb Santa Ana, Hb A2-Babinga, Hb M-Saskatoon (minor peak), “M-Iwate” Hb A2 variant, denatured Hb are found.(8) He had a Hb of 7.5gm/dl, TRBC-4.01million/mm3, HCT 23, MCV of 57.6%, MCH 19pg, MCHC -32.5. bilirubin was raised at 1.40mg/dl but no splenomegaly was present. Based on this a provisional diagnosis of Hb G-Sriraj /O Arab was rendered. Due to rarity the exact incidence is not known. The carriers in the heterozygous state are clinically asymptomatic. Nonetheless, its complex interaction with other β-thalassaemia could give rise to different clinical phenotypes, ranging from mild thalassaemia intermedia to thalassaemia major.(7) The establishment of the diagnosis is challenging and laborious and the only confirmatory test is by molecular analysis.
3.Hb O Indonesia
The third rare case a case of Hb O Indonesia had microcytic hypochromic indices with Hb of 9.7g/dl, MCV of 77.6, MCH of 24pg, MCHC of 31.1g/dl. This again was an ANC case referred for screening. The HBCZE showed HbA of 86.2% and HbS of 12% HbA2-2.3. The HPLC on the other hand showed an abnormal peak of 11.1% in the C window with a RT of 4.86 and HbA of 78.5 ,HbA2 of 2.3. Sicking test was negative. Hemoglobin O (HbO) is a rare type of hemoglobin in which there is a substitution of glutamic acid by lysine but at different positions. In Hb O Indonesia this replacement is at the position 116 of the beta globin chain.Hemoglobin O Indonesia is a was first reported in the Bugis population of Sulawesi Island in Indonesia, and hence designated originally as hemoglobin Buginese-X thereafter phenotypically similar mutations were found in Iran and Italy. HbO Indonesia is asymptomatic however in heterozygous states especially with Hb S there may be mild sickle cell trait.(7,8)
4. Compound heterozygous delta beta/ beta-thalassemia
The compound heterozygous delta beta/ beta-thalassemia was picked up, on HbCZE and had values of 94.7% HbF, 1.6% HbA2 and acetylated Hb of 3.7% in the Z8 window. On HPLC there was HbF of 88.1%, HbA2 of 2.3%, and HbA of 5.5%. Two unknown peaks were found at retention times of 0.68 and 0.98 respectively with the percentage area of 0.2 and 0.4. Her Hb was 5.3% gm/dl ,TRBC was 2.33 million/mm3 and her indices were microcytic hypochromic with MCV of 77.7 fl, MCH of 23pg, and MCHC of 29.3g/dl. RDW was 26.5, based on the family screening studies she was diagnosed as compound heterozygous delta beta / beta-thalassemia with one parent showing raised HbF and the other parents being a beta thalassemia trait with raised HbA. Delta beta thalassemia results from the deletion of both delta and beta genes, homozygotes for δβ-thalassemia have 100% HbF and, because of the increased HbF have a thalassemia intermedia picture rather than thalassemia major. It is necessary to distinguish it from hereditary persistence of fetal hemoglobin (HPFH) which also has 100% HbF but is phenotypically different as HPFH is asymptomatic in contrast with delta beta thalassemia. The phenotype of delta beta thalassemia heterozygotes is similar to BTT but the HbA2 percentage is often normal. And HbF is raised varying from 5% to 20%.The incidence is unknown as only a handful of case reports are available.
These rare variants and their corresponding findings on HPLC and HbCZE are depicted in table 3 below
Table 3. Clinical and laboratory findings in the rare variants picked in this study.
CLINICAL FINDINGS
|
HPLC
|
HbCZE
|
DIAGNOSIS
|
Antenatal screening
Normocytic Normochromic indices
|
HbA2- 41.1%(RT 3.62)
Hb A - 49.8%
|
D window -43.4%
HbA2 of 1.8%
HbA 54.8%
|
Hb D Iran
|
Microcytic hypochromic anemia ,
h/o gall stones
|
C peak 87.7 (RT 4.98)
HbA2- 7.7%
HbA -3.1%
|
E window-91.6%
HbA2 -3.3%
HbA- 3.2%
|
HbG-Sriraj /O Agenogi
|
Microcytic hypochromic anaemia
|
HbF of 88.1%
HbA2 of 2.3%,
HbA of 5.5%.
|
Hb F- 94.7%
HbA2- 1.6%
Acetylated Hb - 3.7%
|
Compound heterozygous delta beta/beta-thalassemia
|
Microcytic hypochromic anaemia
|
abnormal peak of 11.1% (RT4.86)
HbA-78.5
HbA2-2.3
|
HbA -86.2%
HbS of 12%
HbA2-2.3
|
Hb O Indonesia
|
On comparing the HbA2 values between HbCZE and HPLC, HPLC showed higher values for HbA2 with a median value of 2.9% as compared to HbCZE where the value was 2.65%. The mean HbA2 for HbCZE was 3.21 ± 1.35%. In the case of cumulative HbA2 for HPLC, it was higher because the HbE, HbD Iran, and glycated HbS also fell in the HbA2 window thereby raising the value. When HbA2 values were compared by HbCZE in patients with HbS and without HbS the mean HbA2 was higher in patients with HbS being 3.44 ± 1.25% in patients with HbS while in patients without HbS it was 3.2 ± 1.35%. These were not statistically significant. When the same values were compared on the HPLC machine the mean for patients with HbS was 4.07 ± 1.22% while for those without HbS (also excluding HbE/HbC/HbD-Iran) it was3.38 ± 1.18% and this difference was statistically significant. We know that glycated adducts have a retention time similar to HbA2 thereby falsely elevating the percentage of HbA2 in the case of HbS on the HPLC machine and this was the reason for these elevated values. On correlating HbA2 between HPLC and HbCZE values in patients with HbA2 <3.5% the correlation coefficient was 0.867 with a significant p-value of < 0.001 while in patients with HbA2 > 3.5 %the correlation coefficient was 0.369 with a p-value of <0.001 thereby implying the poor correlation between HbA2 values between HbCZE and HPLC at values over 3.5%.This is depicted in Figure 1. which compares HbA2 using HPLC and HbCZE with values of HbA2 <3.5% and >3.5%
As Hb E, HbD Iran also fell into the HbA2 window in the HPLC this may lead to elevated values as already discussed. The bias for low HbA2 < 3.5% was -0.2882 while for higher values >3.5% it was - 8.0012. The HbF agreement within both the methods was good as evidenced by Figure 2.
Figure 1. Comparison of HbA2 using HPLC and HbCZE with values of HbA2 <3.5% and >3.5%
Fig 2. Comparison of HbF using HPLC and HbCZE showing a good agreement with both methods.