A different ontogenesis for chronic lymphocytic leukemia cases carrying stereotyped antigen receptors: molecular and computational evidence.

Chronic lymphocytic leukemia (CLL) is uniquely characterized by the existence of subsets of cases with quasi-identical, 'stereotyped' B-cell receptors (BCRs). Herein we investigate this stereotypy in 2662 patients with CLL, the largest series yet, using purpose-built bioinformatics methods based on sequence pattern discovery. Besides improving the identification of 'stereotyped' cases, we demonstrate that CLL actually consists of two different categories, based on the BCR repertoire, with important biological and ontogenetic differences. The first ( approximately 30% of cases) shows a very restricted repertoire and is characterized by BCR stereotypy (clustered cases), whereas the second includes cases with heterogeneous BCRs (nonclustered cases). Eleven major CLL clusters were identified with antigen-binding sites defined by just a few critically positioned residues, regardless of the actual immunoglobulin (IG) variable gene used. This situation is closely reminiscent of the receptors expressed by cells participating in innate immune responses. On these grounds, we argue that whereas CLL cases with heterogeneous BCRs likely derive from the conventional B-cell pool, cases with stereotyped BCRs could derive from progenitor cells evolutionarily adapted to particular antigenic challenges, perhaps intermediate between a true innate immune system and the conventional adaptive B-cell immune system, functionally similar to what has been suggested previously for mouse B1 cells.

Molecular evidence for EBV and CMV persistence in a subset of patients with chronic lymphocytic leukemia expressing stereotyped IGHV4-34 B-cell receptors.

The chronic lymphocytic leukemia (CLL) immunoglobulin repertoire is uniquely characterized by the presence of stereotyped B-cell receptors (BCRs). A major BCR stereotype in CLL is shared by immunoglobulin G-switched cases utilizing the immunoglobulin heavy-chain variable 4-34 (IGHV4-34) gene. Increased titers of IGHV4-34 antibodies are detected in selective clinical conditions, including infection by B-cell lymphotropic viruses, particularly Epstein-Barr virus (EBV) and cytomegalovirus (CMV). In this context, we sought evidence for persistent activation by EBV and CMV in CLL cases expressing the IGHV4-34 gene. The study group included 93 CLL cases with an intentional bias for the IGHV4-34 gene. On the basis of real-time PCR results for CMV/EBV DNA, cases were assigned to three groups: (1) double-negative (59/93); (2) single-positive (CMV- or EBV-positive; 25/93); (3) double-positive (9/93). The double-negative group was characterized by heterogeneous IGHV gene repertoire. In contrast, a bias for the IGHV4-34 gene was observed in the single-positive group (9/25 cases; 36%). Remarkably, all nine double-positive cases utilized the IGHV4-34 gene; seven of nine cases expressed the major BCR stereotype as described above. In conclusion, our findings indicate that the interactions of CLL progenitor cells expressing distinctive IGHV4-34 BCRs with viral antigens/superantigens might facilitate clonal expansion and, eventually, leukemic transformation. The exact type, timing and location of these interactions remain to be determined.

The effect of red cell shape on the measurement of red cell volume. A proposed method for the comparative assessment of this effect among various haematology analysers.

Shape changes of abnormally deformed red cells in aperture impedance haematology analysers are known to affect MCV, MCHC and haematocrit estimation. However, different counters vary in the manifestation of this effect. We performed a comparative study among five analysers. Three of them are based on impedance without hydrodynamic focusing (Coulter STKR, Cell-Dyn3000 Abbott and K-1000 Sysmex). The other two use hydrodynamic focusing, either with impedance (NE-8000 Sysmex) or two angle laser scatter (H*1 Bayer). A novel method of analysis was applied. Two hundred and three specimens with abnormal red cells and 50 normal specimens (according to ICSH criteria) were assayed. In all samples the PCV was estimated by the reference method without correction for trapped plasma. A true MCHC value was estimated from the mean haemoglobin value and the PCV. The shape effect was assessed by three linear regressions: 1) haematocrit deviations from PCV (corrected for any calibration bias) versus true MCHC; 2) analyser MCHC vs. true MCHC; 3) MCV vs. MCH. The regressions for the analysers with hydrodynamic focusing indicated no significant shape effect. Aperture impedance analysers without focusing varied in their behaviour. The Coulter STKR and the Cell-Dyn3000 both showed strong correlation of haematocrit deviations with true MCHC, poor MCHC correlations and linear MCV-MCH regressions. The K-1000 showed minor indications of such an effect. We conclude that comparative studies are needed to quantitate red cell shape effect errors among various impedance analysers.

The anemia of childhood revisited.

The question of the mechanism of the physiological anemia of childhood was reexamined; we took into consideration the fact that microcytosis is a feature of this anemia as well as the evidence suggesting that the 2,3 DPG/hemoglobin ratio may depend in part on red cell size. In a group of normal, not-iron deficient children a high inverse correlation was obtained between the MCV and the 2,3 DPG/Hb ratio; this latter ratio did not correlate with serum phosphorus levels, previously incriminated in the indirect causation of the anemia of childhood.