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Abstract
Background:
We analyzed T cell receptor β chain (TCRB) gene to investigate the presence of putative T cell clones and its clinicopathologic implications in Korean patients with aplastic anemia (AA).
Methods:
Twenty-nine bone marrow specimens were collected from 20 AA patients, 19 specimens from initial diagnosis and 10 from follow-up. T cell clonality assay was performed using IdentiClone™ TCRB Gene Clonality Assay kit (InVivoScirbe Technology, USA) and automatic genetic analyzer. Patients' clinical information and laboratory parameters were also analyzed.
Results:
Five patients had definitive underlying factors related with aplastic anemia, such as hepatitis B virus (4 cases) and benzene exposure (1 case). Putative T cell clones were detected in bone marrow specimens of 11 (58%) out of 19 patients at diagnosis. The location of putative T cell clones of TCRB gene (diversity region, Dβ; joining region, Jβ; variable region, Vβ) was distributed in Dβ2+ Jβ2 (6 cases), Dβ1+Jβ1 (3 cases), Vβ+Jβ1 (2 cases), and Dβ1+Jβ2 (2 cases). Interestingly, among seven patients who underwent stem cell transplantation, five patients with no T cell clones detected at diagnosis developed new T cell clones during the follow-up.
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Fig. 1.
Schematic diagram of strategy for T cell clonality assay. A representative rearranged T cell receptor beta gene on chromosome 7 (7q35) shows the approximate placement of the upstream and downstream DNA primers. The numbers of primers and their specificity are listed for master mix tubes A, B and C.
Fig. 2.
Capillary electrophoretogram of amplified T-cell receptor beta chain (TCRB) gene. Positive peaks (presence of clonal cell population) are observed in IVS-0004 positive control DNA at 295 base pairs (bp) (A), and patient No. 13 at 21 days after bone marrow transplantation in TCRB variable and joining region (B). Typical polyclonal peaks are observed in Patient No. 20 at 240-285 bp (C). The definition of positive peaks was at least three times the amplitude of the third largest peak in the polyclonal background within the valid size range.
Table 1.
Patient characteristics and laboratory data
| Patient No. | Age | Sex | Underlying factor | BM cellularity (%) | WBC (ANC) (×103/μL) | Hemoglobin (g/dL) | Platelet (×103/μL) | Corrected reticulocyte count (%) | Severity | Treatment |
|---|---|---|---|---|---|---|---|---|---|---|
| 1∗ | 35 | M | HBV, benzene | 5 | 1,900 (600) | 7.4 | 10 | 1.5 | IST | |
| 2 | 38 | M | - | 10 | 940 (400) | 4.4 | 28 | 0.1 | Severe | IST |
| 3 | 36 | M | - | 10 | 2,440 (100) | 10.0 | 2 | 0.4 | Very severe | BMT |
| 4 | 21 | M | HBV | 10 | 400 (70) | 7.8 | 36 | 0.1 | Very severe | PBSCT |
| 5 | 25 | F | - | 10 | 600 (80) | 7.0 | 52 | 0.1 | Very severe | IST |
| 6 | 33 | M | HBV | 10 | 1,700 (360) | 7.3 | 24 | 0.4 | Severe | |
| 7 | 50 | F | - | 10 | 2,000 (70) | 8.7 | 7 | 0.4 | Very severe | BMT |
| 8 | 69 | M | - | 10 | 1,700 (160) | 6.0 | 33 | 0.1 | Very severe | |
| 9 | 28 | F | - | 10 | 2,400 (620) | 6.3 | 10 | 0.4 | Severe | BMT |
| 10 | 27 | F | - | 10 | 2,000 (390) | 5.7 | 9 | 0.1 | Severe | BMT |
| 11 | 73 | F | - | 10 | 3,100 (500) | 5.5 | 5 | 0.2 | Severe | IST |
| 12 | 26 | F | - | 10 | 4,700 (1780) | 8.4 | 14 | 0.9 | Severe | |
| 13 | 36 | F | - | 10 | 800 (550) | 6.9 | 9 | 0.2 | Severe | BMT |
| 14 | 57 | F | - | 15 | 3,700 (1510) | 11.3 | 67 | 0.8 | ||
| 15 | 23 | F | - | 15 | 1400 (20) | 5.7 | 4 | 0.1 | Very severe | PBSCT |
| 16 | 25 | M | - | 20 | 900 (290) | 5.0 | 11 | 0.3 | Severe | IST |
| 17 | 69 | F | HBV | 10 | 3,800 (1700) | 10.0 | 73 | 0.9 | ||
| 18 | 33 | F | - | 10 | 3,200 (1120) | 9.9 | 27 | 0.8 | ||
| 19 | 26 | M | - | 5 | 2,600 (1200) | 15.1 | 86 | 0.8 | ||
| 20 | 18 | F | - | 10 | 2,740 (750) | 5.7 | 23 | 0.8 |
Abbreviations: -, not detected; HBV, hepatitis B virus; a, degree of severity of aplastic anemia (‘severe’ was defined as two of following criteria: neutrophils ≤500×103/μL, platelet ≤20×103/μL, corrected reticulocyte count ≤1% and ‘very severe’ defined as the criteria of ‘severe’ plus neutrophils ≤200×103/μL); IST, immunosuppressive therapy; BMT, bone marrow transplantation; PBSCT, peripheral blood stem cell transplantation.
Table 2.
T cell clonality detected in bone marrow specimens of aplastic anemia patients
Table 3.
Changes of T cell clonality in bone marrow specimens after stem cell transplantation
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