Vilma Valceckiene
email: vilma.valceckiene@santa.lt

Rima Kontenyte

Arturas Jakubauskas

Laimonas Griskevicius

Selection of reference genes for quantitative PCR studies in purified B cells from B cell chronic lymphocytic leukaemia patients

Keywords: B-CLL, B cells, qPCR, reference genes, normalisation

Clinical heterogeneity of B-cell chronic lymphocytic leukaemia (B-CLL) makes it necessary to identify potent prognostic indicators to predict individual clinical course and select risk-adapted therapy. During the last years numerous gene expression models have been suggested as prognostic factors of B-CLL. Today quantitative polymerase chain reaction (qPCR) is a preferred method for rapid quantification of gene expression and validation of microarray data. Reliability of qPCR data is highly dependent on the use of appropriate reference genes for normalisation. To date, no validated reference genes have been reported for normalisation of gene expression in B-CLL. Therefore, the present study was conducted to identify suitable reference genes for gene expression studies in CD19+ B cells isolated from B-CLL patients' peripheral blood. The stability of ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, MRPL19, TBC and UBC genes was determined by three different descriptive statistics, geNorm, NormFinder and BestKeeper-1, which produced highly comparable results. Based on our results, B2M, HPRT1, and GUSB were found to be the most suitable reference genes for qPCR studies in B-CLL patients' peripheral blood B cells.

Introduction

B-CLL is the most common form of adult leukaemia in the Western world.

It follows a highly heterogeneous clinical course; some patients survive for many years without any treatment, whereas others progress very quickly and die within a few years [START_REF] Zenz | From pathogenesis to treatment of chronic lymphocytic leukaemia[END_REF]. To predict individual clinical course and select optimal therapy, powerful prognostic factors are needed. The clinical heterogeneity of B-CLL was shown to correlate with the pattern of genetic changes [START_REF] Stilgenbauer | Genetics of chronic lymphocytic leukemia: genomic aberrations and V(H) gene mutation status in pathogenesis and clinical course[END_REF]. Gene expression profiling of B-CLL samples using microarray platforms identified a number of genes that appeared to be under-or overexpressed in B-CLL. (Falt, et al 2005[START_REF] Jelinek | Identification of a global gene expression signature of Bchronic lymphocytic leukemia[END_REF][START_REF] Klein | Gene expression profiling of B cell chronic lymphocytic leukemia reveals a homogeneous phenotype related to memory B cells[END_REF], Rosenwald, et al 2001). Based on these findings, the role of gene expression levels in B-CLL prognosis assessment was intensively studied [START_REF] Joshi | ATM, CTLA4, MNDA, and HEM1 in high versus low CD38 expressing B-cell chronic lymphocytic leukemia[END_REF], Nuckel, et al 2009, Oppezzo, et al 2005[START_REF] Stamatopoulos | Quantification of ZAP70 mRNA in B cells by real-time PCR is a powerful prognostic factor in chronic lymphocytic leukemia[END_REF][START_REF] Van Bockstaele | Lipoprotein lipase mRNA expression in whole blood is a prognostic marker in B cell chronic lymphocytic leukemia[END_REF]. The high expression of ZAP70 (coding for tyrosineprotein kinase ZAP-70) [START_REF] Catherwood | ZAP-70 mRNA quantification in B-cell chronic lymphocytic leukaemia[END_REF][START_REF] Stamatopoulos | Quantification of ZAP70 mRNA in B cells by real-time PCR is a powerful prognostic factor in chronic lymphocytic leukemia[END_REF] and AICDA (coding for activation-induced cytidine deaminase) (Palacios, et al 2010) was found to be associated with unfavourable prognosis of B-CLL. Similar observations were made for lipoproteinlipase (encoded by LPL) and disintegrin and metalloproteinase domain-containing protein 29 (encoded by ADAM29) (Oppezzo, et al 2005[START_REF] Van Bockstaele | Lipoprotein lipase mRNA expression in whole blood is a prognostic marker in B cell chronic lymphocytic leukemia[END_REF]. All these data indicate a significant role of gene expression parameters in evaluation of biological mechanisms of B-CLL and in identification of potential markers for prognostic assessment.

Today qPCR has become a method of choice for gene expression studies.

In addition, it is also used to validate microarray data. Most frequently, gene expression quantification involves the analysis of target gene expression relative to the reference gene. This allows control for possible non biological variations, since the reference genes are exposed to the same preparation steps as the gene of interest. An ideal reference gene has to be expressed constitutively and uniformly in all test samples. Significant fluctuations or changes in the expression of chosen reference genes between samples can lead to the loss of detection of small differences between expression of genes of interest or to other erroneous results. Therefore, it is extremely important to find appropriate reference genes with minimal variability between the test samples.

However, this task is often omitted and many studies have used previously published reference genes without appropriate analysis of their expressional stability in the test tissue.

The accurate selection of the reference control for gene expression studies in B-CLL is crucial as sometimes even small changes in the gene expression level may differentiate patient groups with different prognosis [START_REF] Catherwood | ZAP-70 mRNA quantification in B-cell chronic lymphocytic leukaemia[END_REF][START_REF] Stamatopoulos | Quantification of ZAP70 mRNA in B cells by real-time PCR is a powerful prognostic factor in chronic lymphocytic leukemia[END_REF][START_REF] Van Bockstaele | Lipoprotein lipase mRNA expression in whole blood is a prognostic marker in B cell chronic lymphocytic leukemia[END_REF]. To the best of our knowledge, there have been no reports on the suitability of reference genes for qPCR studies in B-CLL samples.

The aim of this research was to select the reference genes best suited for gene expression studies in B cells purified from B-CLL patients' blood. The expression stability of nine potential reference genes was compared using BestKeeper-1, NormFinder and geNorm applications.

Materials and Methods

Samples

Peripheral blood samples were obtained from 30 B-CLL patients. The diagnosis of B-CLL required a persistent lymphocytosis of more than 5.0×10 9 /L and a typical CD5+, CD19+, CD20+ (low), CD23+, sIg low, CD79b+ (low) immunophenotype as revealed by flow cytometry of peripheral blood cells. CD19+ cells from blood samples were isolated by positive immunomagnetic selection with Dynal ® magnetic beads against CD19 (Invitrogen, Carlsbad, CA, USA) according to vendor's recommendations. CD19 selection typically resulted in >97 % purity as assessed by flow cytometry. The study was approved by the Lithuanian Bioethics Committee and all patients gave informed consent to use their blood samples.

Primers

Primer pairs for B2M, HPRT1, GUSB, MRPL19, HMBS and UBC were designed using Primer3Plus software (Untergasser, et al 2007) taking into account primer dimer, self-priming formation, and primer melting temperature. Primers for ACTB, GAPDH and TBP were published previously (Lossos, et al 2003[START_REF] Zhang | Development of a real-time polymerase chain reaction-based method for the measurement of relative allelic expression and identification of CYP2A13 alleles with decreased expression in human lung[END_REF]. All primer pairs were chosen to span an exon-intron boundary to exclude amplification of genomic DNA. The non-specific amplification was tested on genomic DNA (gDNA) samples. Primers for ACTB, GAPDH, GUSB and TBP were synthesized Germany). Primer sequences and GenBank accession numbers of selected reference genes are listed in Table I.

qPCR analysis

Total RNA was extracted from purified CD19+ cells using a QIAmp RNA Blood Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The concentration and purity of each RNA sample was evaluated spectrophotometrically by Smart-Spec Plus (Bio-Rad Laboratories, Hercules, CA, USA). First strand cDNA synthesis was carried out in triplicates using 1 µg of RNA, random hexamers and First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) according to the manufacturer's recommendations. After reaction cDNA samples were diluted up to 60 µl with nuclease-free water, triplicate samples were pooled, aliquoted and stored at -20 ˚C. To minimize experimental variation all reference genes were quantified on the same batch of cDNA. For each reference gene all cDNA samples were amplified in the same qPCR run. All samples were amplified in duplicates, as well as non-template control (NTC) samples without cDNA to check for reagent contamination. qPCR amplification efficiencies for all primer pairs were evaluated using the serial 10-fold dilutions of the same cDNA sample (a pool of cDNAs prepared from CD19+ cells purified from 5 B-CLL patients' blood samples). Amplification curves were plotted using RotorGene 6000 software and amplification efficiencies were calculated automatically from raw fluorescence data taken from the RotorGene 6000 detection system.

Data analysis

Individual quantification cycle (C q ) values were obtained by setting a threshold manually and obtained mean C q values were used for further analysis. Data from RotorGene 6000 were imported into Microsoft Excel and transformed to relative quantities Q using Q=E (minCq-sampleCq) equation, where amplification efficiency (E) was specific for each reference gene. The data obtained were converted into correct input files, according to the requirements of the particular software, and analyzed using three different Visual Basic for Applications (VBA) applets, geNorm (version 3.5) [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]), NormFinder (Pfaffl, et al 2004) and BestKeeper-1 [START_REF] Andersen | Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[END_REF].

Results

Expression profile of reference genes

In order to identify the best reference genes for gene expression studies in B cells from B-CLL patients, we designed a qPCR assay based on SYBR Green I detection for the expression analysis of nine selected genes (ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, MRPL19, TBC and UBC). The amplification specificity for each primer pair was confirmed by a single band of expected size in agarose gel electrophoresis and by a single-peak melting curve of PCR products (data not shown).

No signals were detected in NTC samples. Amplification efficiency ranged from 88.9 % for ACTB to 102 % for B2M (Table I).

The RNA transcription levels were analysed by directly comparing Cq values. The C q is defined as the number of cycles needed for the fluorescence to reach a specific threshold level of detection and is inversely correlated with the amount of template nucleic acid present in the reaction [START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF]. The lowest C q value recorded was 13.63 cycles for UBC and the highest was 31.83 for TBP. The lowest geometric mean of C q values was 16.40 for B2M, and the highest was 25.74 for MRPL19 (Table II andFig 1). MRPL19, B2M, HPRT1 and GAPDH exhibited the smallest gene expression variation (less than 4 cycles) among the studied reference genes, while UBC and TBP had much higher expression variation (more than 9 cycles).

geNorm analysis

The geNorm calculates the gene expression stability measure (M) for the reference genes. The M value is defined as an average pair-wise variation (V) of a particular gene with all other tested reference genes, whereas the variation of this gene to another is determined as the standard deviation of the log2-transformed expression level ratios [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. The gene with the lowest M value was considered to have the most stable expression, while the gene with the highest M value had the least stable expression. Table II indicates To evaluate the optimal number of genes required for accurate normalization, geNorm calculates whether the stepwise addition of less stable gene to the normalization factor (NF n ) affects the variance (V n/n+1 ) compared to (NF n+1 ). A large pair-wise variation implies that the added gene has a significant effect on normalization and should be included in calculation of a reliable normalization factor [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. To meet the default cut-off V value of 0.15 (the point at which it is unnecessary to include additional genes in a normalization strategy) the geNorm indicated the use of four from the five most stable genes (Fig 2B).

BestKeeper-1 analysis

BestKeeper-1 estimates inter-gene relations of possible reference gene pairs by performing numerous pair-wise correlation analysis using raw C q values of each gene (Pfaffl, et al 2004). Unstable genes show the standard deviation (SD Cq ) of > 1.0, indicating variation in the starting template by the factor 2. However, the qPCR reaction efficiency in biological samples is rarely 100 %, and default setting of BestKeeper-1 might be too strict. Therefore it was suggested to adjust the SD threshold (ln2/lnE) for each gene to its specific efficiency [START_REF] Axtner | Validation of internal reference genes for quantitative real-time PCR in a non-model organism, the yellow-necked mouse, Apodemus flavicollis[END_REF]. As indicated in Table II, ACTB, HMBS, TBP and UBC had SD Cq values of > 1.0 and were ranked by BestKeeper-1 as unstable, whereas GAPDH, HPRT1, B2M, GUSB and MRPL19 showed SD Cq values of less than 1.0 and were considered to be stably expressed. The calculated SD threshold did not change the tested gene stability: GAPDH, HPRT1, B2M, GUSB and MRPL19 were considered as stable reference genes as the SD Cq was lower than their individual SD threshold value whereas the remaining genes were ranked as unstable (Table II). The range of expression stability calculated by BestKeeper-1 was (from the most stable to the least stable): MRPL19, GUSB, HPRT, B2M, GAPDH, HMBS, UBC, ACTB and TBP (Table III).

NormFinder analysis

NormFinder calculates gene expression stability value (not comparable to the M value of genNorm) for each single gene independently and ranks the set of potential reference genes according to their expression stability [START_REF] Andersen | Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[END_REF].

The more stable gene expression is indicated by a lower expression stability value. The NormFinder calculated stability values for tested reference genes and their ranking II andIII, accordingly. The expression of B2M gene was defined to be the most stable, whereas TBP gene was indicated as the most unstable.

Evaluation of reference gene stability

In order to select the most stable reference gene for accurate normalization of gene expression in purified B cells from B-CLL patients' blood, we compared data obtained by three different statistical approaches: geNorm, NormFinder and BestKeeper-1. All three applications gave comparable results (Table III). The five stable reference genes identified by BestKeeper-1 (GAPDH, HPRT1, B2M, GUSB and MRPL19) did not differ from those identified by geNorm application, though they were arranged in a different order. NormFinder ranking order differed from geNorm and BestKeeper-1 ranking, but B2M, HPRT1 and GUSB, the most stable genes in NormFinder analysis, were among the top four most stable genes identified by geNorm and BestKeeper-1. TBP and UBC were the most unstable genes in all three applications. The calculated mean ranking indicated B2M, GUSB and HPRT1 as the most stable and these genes could be considered as suitable reference genes for gene expression studies in B-CLL patients' B cells (Table III).

Discussion

Gene expression studies by qPCR play an important role in understanding the pathogenesis of B-CLL, and in identification of novel prognostic markers as well as potential targets for therapy. In order to get reliable qPCR results, appropriate normalization of relative qPCR data is required to control for non-biological variation [START_REF] Bustin | Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays[END_REF][START_REF] Bustin | The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments[END_REF], Nolan, et al 2006).

Nine potential reference genes were analyzed for their expression stability in the purified B cells obtained from B-CLL patients' blood samples. These genes were ACTB, B2M, GAPDH, GUSB, HMBS, HPRT1, MRPL19, TBC and UBC. To select the most stably expressed genes, three publicly available programs: BestKeeper-1 (Pfaffl, et al 2004), geNorm [START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF] and NormFinder [START_REF] Andersen | Normalization of real-time quantitative reverse transcription-PCR data: a model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets[END_REF] were used.

Although geNorm, BestKeeper-1 and NormFinder are based on different algorithms and analytical programs, all three programs showed consistent results with only slight differences in the ranking order except for MRPL19 gene which was the most stable gene in BestKeeper-1 analysis but became the seventh in the NormFinder gene stability ranking. In general, analysis indicated B2M, HPRT1 and GUSB as the most stable reference genes, as these three genes were always classified among the four best performing reference genes. On the other hand, UBC and TBP were ranked poorly based on all three software programs, indicating that these two genes should not be used as reference controls for gene expression studies in B-CLL patients. Based on the summarized data (Table III), the use of ACTB, GAPDH and HMBS as reference controls could not be recommended as well. Several studies have scrutinized the stability of commonly known reference genes GAPDH and ACTB and have demonstrated that these genes should be used with caution as their expression varied considerably and they were consequently unsuitable as reference genes in some cases [START_REF] Deindl | Differential expression of GAPDH and beta3-actin in growing collateral arteries[END_REF][START_REF] Glare | beta-Actin and GAPDH housekeeping gene expression in asthmatic airways is variable and not suitable for normalising mRNA levels[END_REF][START_REF] Tong | Selection of reliable reference genes for gene expression studies in peach using real-time PCR[END_REF]. Present study identified ACTB and GAPDH among the unstable reference genes as well. However, literature search revealed that GAPDH and ACTB were among the most often used reference genes in B-CLL gene expression studies [START_REF] Jelinek | Identification of a global gene expression signature of Bchronic lymphocytic leukemia[END_REF][START_REF] Joshi | ATM, CTLA4, MNDA, and HEM1 in high versus low CD38 expressing B-cell chronic lymphocytic leukemia[END_REF][START_REF] Kienle | Gene expression factors as predictors of genetic risk and survival in chronic lymphocytic leukemia[END_REF], Nuckel, et al 2009). This underscores the importance of careful selection of reference genes.

The geNorm model makes an assumption that the candidate reference genes are not co-regulated as this may lead to the erroneous choice of the best gene pair.

Whereas geNorm is susceptible to identification of co-regulated genes as an optimal reference gene pair, NormFinder and BestKeeper-1 do not have this problem. As all three softwares gave consistent results, one can assume that the studied reference genes are not co-regulated in the analysed samples.

Although most studies used only a single reference gene for normalization, it had been suggested that the use of two or more reference genes for qPCR studies might generate more reliable results [START_REF] Tricarico | Quantitative real-time reverse transcription polymerase chain reaction: normalization to rRNA or single housekeeping genes is inappropriate for human tissue biopsies[END_REF][START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF]. geNorm analysis indicated that four genes would be required for accurate normalization of qPCR data in B-CLL patients' blood cells (Fig 2B). However, the number of reference genes needs to be balanced between accuracy and practical consideration thus the cut off V value of 0.15 suggested by geNorm should not be taken too strictly as it is suggested by the geNorm manual itself. The calculated V 3/4 value was 0.153 which is only slightly higher than the geNorm cut off value and we would suggest using three reference genes from the four most stable ones. The optimal set of reference genes should be defined for each study since it may depend on the gene of interest. For example, B2M is a highly expressed gene in most tissues [START_REF] Rulcova | The effect of total-ABL, GUS and B2M control genes on BCR-ABL monitoring by real-time RT-PCR[END_REF][START_REF] Vandesompele | Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes[END_REF] and in blood B cells from B-CLL patients as well (Fig 1).

Highly expressed reference genes are not recommended for normalization of target genes with low expression levels because the copy number of reference and target gene should fall in the same linear measurement scale.

In conclusion, B2M, HPRT1, and GUSB genes were identified as the most stably expressed genes in CD19+ B cells purified form B-CLL patients' blood and they are recommended for the normalization of gene expression results in these cells. Calculated by Rotor-Gene 6000 * , BestKeeper-1 ** , NormFinder ♦ and geNorm φ softwares. C q : quantification cycle, SD Cq : C q standard deviation, CV: C q coefficient of variation. SD threshold = ln2/lnE. Every bar represents change in normalization accuracy when more reference genes are added stepwise according to the ranking. geNorm proposed cut-off value below which the inclusion of an additional reference gene is not required is 0.15.

F o r P e e r R e v i e w

  by Integrated DNA Technologies (Coralville, IA, USA) whereas primer pairs for B2M, HMBS, HPRT1, MRPL19 and UBC were synthesized by Metabion (Martinsried,

  qPCR reactions were performed in a 20 µl reaction volume containing Maxima™ SYBR Green I qPCR Master Mix (Fermentas), 0.2 µM final concentration of each primer, and 5 µl cDNA solution. Uracil-DNA glycosylase (Fermentas) was added into each reaction mix (0.4 units) to prevent qPCR cross-contamination. qPCR was performed on RotorGene 6000 (Corbett Life Science, Australia) using the following cycling conditions: 2 minutes at 50 °C followed by 5 minutes at 95 °C and 45 amplification cycles at 95 °C for 20 seconds, 60 °C for 15 seconds and 72 °C for 20 seconds. The melting curve analysis involved a rise of °C for 5 seconds, cooling to 65 °C for 90 seconds, and then heating back to 95 °C while continuously monitoring fluorescence.

  the M values for all tested reference genes. GUSB, GAPDH, HPRT1, B2M and MRPL19 had M values less than geNorm default threshold of 1.5, while ACTB, HMBS, TBP and UBC M values exceeded the threshold. In a stepwise progression, geNorm excludes the least stable gene, and recalculates M values for the remaining genes, which results in the characterization of the stability of each gene on a ranked scale and the identification of the two most stably expressed genes. The average expression M values of the nine tested reference genes were plotted in Fig 2A.TBP and UBC were respectively the first and the second genes excluded from the analysis on the basis of instability whereas HPRT1 and B2M were identified as the most stable reference gene pair in purified CD19+ cells(Fig 2A).

  Figure legends.

Fig 2

 2 Fig 2 geNorm analysis of reference genes. Results are presented as an output file of

  .S., Czerwinski, D.K., Wechser, M.A. & Levy, R. (2003) Optimization of quantitative real-time RT-PCR parameters for the study of lymphoid malignancies. Leukemia, 17, 789-795. Nolan, T., Hands, R.E. & Bustin, S.A. (2006) Quantification of mRNA using real-time RT-PCR. Nature Protocols, 1, 1559-1582. Nuckel, H., Collins, C.H., Frey, U.H., Sellmann, L., Durig, J., Siffert, W. & Duhrsen, U. (2009) FCRL2 mRNA expression is inversely associated with clinical progression in chronic lymphocytic leukemia. European Journal of Haematology, 83, 541-549. Oppezzo, P., Vasconcelos, Y., Settegrana, C., Jeannel, D., Vuillier, F., Legarff-Tavernier, M., Kimura, E.Y., Bechet, S., Dumas, G., Brissard, M., Merle-Beral, H., Yamamoto, M., Dighiero, G. & Davi, F. (2005) The LPL/ADAM29 expression ratio is a novel prognosis indicator in chronic lymphocytic leukemia. Blood, 106, 650-657. Palacios, F., Moreno, P., Morande, P., Abreu, C., Correa, A., Porro, V., Landoni, A.I., Gabus, R., Giordano, M., Dighiero, G., Pritsch, O. & Oppezzo, P. (2010) High expression of AID and active class switch recombination might accounts for a more aggressive disease in unmutated CLL patients: link with an activated microenvironment in CLL disease. Blood. Pfaffl, M.W., Tichopad, A., Prgomet, C. & Neuvians, T.P. (2004) Determination of stable housekeeping genes, differentially regulated target genes and sample integrity: BestKeeper--Excel-based tool using pair-wise correlations. Biotechnology Letters, 26, 509-515.

  Cq values for reference genes. Expression data displayed as Cq values for each reference genes in all B cell samples. The median is depicted as a line across the box. The box indicates the 25th and 75th percentiles. Whiskers represent the maximum and minimum values.

  reference genes. Results are presented as an output file of the geNorm program. (A) Average expression stability (M) of reference genes during stepwise exclusion of the least stable gene. (B) Determination of the optimal number of reference genes for normalization.

Table II .

 II Descriptive statistics of the tested reference genes

		GUSB	ACTB	GAPDH	HMBS	TBP	HPRT1	B2M	UBC	MRPL19
	amplification efficiency (E) *	1.996	1.889	1.996	1.997	1.982	2.000	2.022	2.044	1.999
	geometric mean [C q ] **	25.29	19.71	18.60	23.18	24.71	23.75	16.40	19.40	25.74
	arithmetic mean [C q ] **	25.31	19.81	18.62	23.23	24.97	23.77	16.42	19.50	25.75
	Minimum C q **	23.42	16.29	17.08	19.49	21.15	22.00	14.13	13.63	23.96
	Maximum C q **	27.54	25.02	20.92	26.95	31.83	25.06	17.78	22.64	27.43
	M value φ	1.303	1.812	1.276	1.454	3.643	1.274	1.304	1.947	1.353
	stability value ♦	0.113	0.150	0.144	0.151	0.233	0.111	0.099	0.172	0.159
	SD Cq **	0.686	1.547	0.800	1.086	3.206	0.715	0.723	1.532	0.678
	SD threshold	1.003	1.090	1.003	1.002	1.013	1.000	0.984	0.969	1.001
	CV [%C q ] **	2.71	7.81	4.30	4.67	12.84	3.01	4.40	7.86	2.63

Table III .

 III Ranking order of the reference genes calculated by NormFinder, BestKeeper-1 and geNorm algorithms.

		B2M	HPRT1	GUSB	MRPL19	GAPDH	HMBS	ACTB	UBC	TBP
	NormFinder	1	2	3	7	4	6	5	8	9
	BestKeeper-1	4	3	2	1	5	6	8	7	9
	geNorm	1.5	1.5	3	4	5	6	7	8	9
	Mean rank	2.17	2.17	2.67	4.00	4.67	6.00	6.67	7.67	9.00

British Journal of Haematology

Acknowledgements

This study was supported by the European Economic Area (Iceland, Liechtenstein and Norway) and Norwegian Financial Mechanism grant No.

2004-LT0040-IP-1EEE.


Giorgos ; Chouliaras
email: georgehouliaras@msn.com

Giorgos L Chouliaras

Antonis Kattamis

Vasilis Berdoukas

Efstathios D Gotsis

Sophie Mavrogeni

Vassilis Ladis

MD Giorgos Chouliaras

CARDIAC MAGNETIC RESONANCE IN TRANSFUSION DEPENDENT THALASSAEMIA: ASSESSMENT OF IRON LOAD AND RELATION TO LEFT VENTRICULAR EJECTION FRACTION

Keywords: Thalassaemia, Cardiac Magnetic Resonance Imaging (CMR), Left ventricular ejection fraction (LVEF), Transfusion Iron Overload

Cardiac Magnetic Resonance (CMR) has replaced all other surrogate measurements in the determination of transfusional cardiac iron overload in patients with thalassaemia major. We aimed to determine the diagnostic value of CMR T2* with respect to cardiac dysfunction (CD) as determined by CMR-derived left ventricular ejection fraction (LVEF). Cardiac T2* values and LVEF measured by CMR were recorded in 303 patients with thalassaemia major, at the time of their first CMR. T2* was correlated with LVEF (regression coefficient: 0.57, p<0.001).

The prevalence of CD was 32.9% in patients with T2*≤ 8ms, 12.5% in patients with T2*> 8ms and ≤14 ms and reduced to 9.1% in patients with T2* between 14-20 ms. As the probability of CD is progressively, and not suddenly, reduced with increasing values of T2*, CMR has a limited diagnostic value for cardiac dysfunction (ROC Analysis, AUC = 0.68). Patients with cardiac T2* ≤ 8 ms require careful and intensive management. This risk decreases with increasing values of T2* but even in mildly loaded patients the probability of impaired LVEF is not negligible.

Introduction

Over the last ten years, Magnetic Resonance Imaging (MRI) has been increasingly used for the assessment of transfusional iron overload [START_REF] Anderson | Cardiovascular T2-star (T2*) magnetic resonance for the early diagnosis of myocardial iron overload[END_REF][START_REF] Berdoukas | The efficacy of iron chelator regimes in reducing cardiac and hepatic iron in patients with thalassaemia major: a clinical observational study[END_REF].

Cardiac T2* and left ventricular ejection fraction (LVEF), derived by Cardiac Magnetic Resonance (CMR), are the indices mainly used to evaluate cardiac iron load and function.

Nevertheless, the relation between these two parameters is, as yet, not completely understood. As the major cause of premature death in patients with thalassaemia remains iron related cardiac disease, timely intervention is of vital importance [START_REF] Borgna-Pignatti | Survival and complications in patients with thalassemia major treated with transfusion and deferoxamine[END_REF][START_REF] Ladis | Longitudinal study of survival and causes of death in patients with thalassemia major in Greece[END_REF]. To date there is only one large prospective study on the role of cardiac T2*, as a prognostic factor of cardiac failure [START_REF] Kirk | Cardiac T2* magnetic resonance for prediction of cardiac complications in thalassemia major[END_REF]. However, studies showing the distribution of CMR-derived LVEF in thalassaemia major patients are sparse.

As there is increasing access to CMR-derived LVEF, such information comparing it to cardiac T2* may be indispensable in implementing the best therapeutic iron chelation approach. In this single institution study we evaluated the distribution of CMR-derived LVEF in thalassaemia major patients as well as the relation of cardiac T2* to CD.

Patients and Methods

In this cross sectional study, we have retrospectively recorded the results of the first-ever CMR (T2* values and LVEF) performed in patients with thalassaemia major, who have been followed in our unit. The clinical management of these patients includes regular blood transfusions at 14-21 day intervals and chelation therapy. Pre-transfusion haemoglobin (Hb) Patients under the age of 18 were excluded from the analysis. There were no patients with congenital heart defects. The Ethics Committee of the hospital approved permission for medical review, waiver of informed consent and anonymous publication of data according to the Declaration of Helsinki.

Magnetic resonance studies were performed using a cardiac-dedicated 1.5 Tesla magnet (Signa CVI, GE, Milwaukee, USA) with 40 mT/m gradients and appropriate cardiac software.

For cardiac T2* determination a single breath multi-echo fast gradient-echo sequence was used with a fixed TR of 25.6 ms, with 10 echoes acquired in the range of 2.2-22.6 ms and an inter-echo spacing of about 2.2 ms. Additional imaging parameters included 1 excitation, matrix of 160x256, bandwidth 62.5 KHz, slice thickness of 8 mm, ECG-gating and total acquisition time ranging from 15-20 msec. The calculation of T2* was made off line by an exponential least-squares fit of the multi-echo signal intensity data (SI) versus echo time (TE), with the use of a PC and standard fitting programs (GRAFIT or SigmaPlot 2001).

Ejection fractions were calculated at a separate workstation (Windows Advantage 4.3, GE, Milwaukee, USA) and GE proprietary software (MASS 6). A FIESTA multi-phase sequence was used in the 2-chamber and 4-chamber long axis views (mid slice) and in the short axis view (10-12 multi-phase slices). No statistically significant differences were found for the LVEF calculated in the short (one multi-phase mid slice) and long axis (multi slice, multi-phase) views. In order to establish LVEF reference limits for thalassaemia major patients without cardiac iron overload, we assessed the distribution of LVEF in patients with T2*>20 ms, which is currently considered, arbitrarily, the lower limit of the normal values [START_REF] Anderson | Cardiovascular T2-star (T2*) magnetic resonance for the early diagnosis of myocardial iron overload[END_REF].

Cardiac dysfunction (CD) was defined as LVEF below -1.96xSD from the mean of the population without cardiac iron overload. Subsequently, the relation between LVEF and T2*, in those patients with T2*≤ 20 ms, was evaluated. In addition, we assessed the percentage of patients with cardiac dysfunction, in each group of cardiac iron load according to their T2*, namely T2* ≤ 8 (severe iron load), >8 and ≤14 (moderate iron load), >14 and ≤20 (mild iron load). Finally, the ability of T2* to discriminate between patients with and without cardiac dysfunction (among those with cardiac iron overload) was also evaluated.

Statistical analysis

Continuous variables are presented as mean ± standard deviation (SD), while categorical variables are described by absolute (n) and relative frequencies (%). The reference range of LVEF in the cardiac iron-free patients (T2*>20 ms) was determined by the mean-1.96x(SD) which corresponds to the lower 2.5% of subjects in that population. Means were compared by Student t-test or analysis of variance (ANOVA, applying the Bonferroni correction for multiple comparisons) in cases of more than two groups, while percentages were compared by Fisher's exact test. Correlations between continuous variables were assessed by Results are presented as odds ratios (OR) and p-values. The probabilities of having CD for every distinct value of T2* were also estimated by logistic regression analysis. Finally, receiver operating characteristic analysis (ROC) was used to evaluate T2*'s ability to discriminate between CD and CD-free patients. The ROC analysis allows the evaluation of T2* in distinguishing between CD and non-CD patients characterized according to the LVEF.

The measure of accurate classification by T2* is the area under the ROC curve (AUC). The results for AUC lie between 1 (perfect classification) and 0.5 (of information, which is equivalent to classifying individuals by chance). Confidence intervals (95% c.i) are provided along with the estimation of AUC. Data were analyzed using Stata 11.2 statistical software (Stata Corporation, College Station, Texas, USA).

Results

A total of 303 patients (48.2% males) with a mean age of 29.4 ± 5.6 years were included in the analysis. Figure 1 shows the relationship of LVEF to T2*.

According to the >20 ms threshold, 128 patients (46.9% males) were cardiac iron-free at the time of their first CMR. The mean value of LVEF in that group was 67.8±5.3%. Cardiac ironfree males had lower LVEF compared to females (66.7±5.1 vs 68.8±5.3, respectively, p=0.026). The lower limit for LVEF in cardiac iron-free patients was 56.7% for males and 58.4% for females, thus we set different lower LVEF limits according to gender. This finding of small gender-related differences is in accord with a previous report, which has also shown that women with thalassaemia major exhibit higher levels of LVEF [START_REF] Westwood | Normalized left ventricular volumes and function in thalassemia major patients with normal myocardial iron[END_REF]. In Pearson's r (r=0.08, p=0.3 and r=-0.06, p=0.6, respectively).

In the patients with cardiac iron overload (T2* ≤ 20 ms), LVEF was significantly related to T2* values. Linear regression revealed that an increase of T2* by 1 ms increases the LVEF by 0.57% (β=0.57, p<0.001), while in the same regression model age had also a borderline positive effect on LVEF (β=0.21, p=0.060). Gender was not statistically significant.

The mean values of LVEF and the ratio of patients with CD to those without CD differed significantly between the four groups of cardiac iron load as shown in table 1. Logistic regression analysis showed that patients with severe cardiac iron load had 4.9 and 3.7 times higher probability of exhibiting CD compared to mild and moderate cardiac iron load, respectively (OR=4.8, p=0.008 and OR=3.7, p=0.005, respectively, results adjusted for age).

The odds of having CD between the mild and moderate iron load group did not differ significantly.

Compared to the non-loaded population (T2*>20 ms) patients with T2*≤ 8ms were 13.0 times more likely to have impaired LVEF (OR=13.0, p<0.001). The respective relative risks for those with moderate (8-14 ms) and mild (14-20 ms) iron overload were 4.3 (OR=4.2, p=0.014) and 3 (OR=3, p=0.1).

With respect to the capability of T2* to discriminate between CD and CD-free patients among those with cardiac iron overload, the ROC analysis estimated an area under the curve equal to 0.68 (figure 2, 95% c.i. 0.58-0.77). At T2* cut-off values of 14, 10 and 8 ms, the percentages for sensitivity/specificity were 88.6/27.9, 77.1/50.

Discussion

This analysis confirms that the risk of cardiac dysfunction steadily increases with the greater deposition of iron as determined by CMR. It concurs with the recently published, prospective study, from London, in which patients with excessive cardiac iron had a marked increase in the risk of developing cardiac failure [START_REF] Kirk | Cardiac T2* magnetic resonance for prediction of cardiac complications in thalassemia major[END_REF]. Previous, smaller studies have also shown a strong correlation between cardiac T2* and CMR-derived LVEF in thalassaemic patients [START_REF] Anderson | Cardiovascular T2-star (T2*) magnetic resonance for the early diagnosis of myocardial iron overload[END_REF][START_REF] Patton | Observational Study Of Iron Overload As Assessed By Magnetic Resonance Imaging (MRI) In An Adult Population Of Transfusion Dependent Patients With beta Thalassaemia: Significant Association Between Low Cardiac T2* < 10 ms And The Occurrence Of Cardiac Events[END_REF].

Our evaluation was based on a single time point in a patient's life and indicates that undoubtedly there is a relationship between the degree of cardiac iron load and the cardiac status at a particular point in time. For those patients with a T2* <8ms the probability of having cardiac dysfunction is 32.9% and is 13.0 times more likely than in those with T2* > 20ms. This is also demonstrated by the probabilities estimated by the regression analysis.

From 20 ms to 8ms there is an increase in risk from 5.7% to 21.0%. It is apparent that the risk of CD rises in a steep fashion, 5.7% to 11.3% as the T2* decreases from 20 to 14 ms and continues to rise, from 9.4% to 21.0%, as the T2* decreases further (from 14ms to 8ms). Below 8ms the risk reaches its maximum of approximately 35%. This gradual increase of the risk throughout the range of abnormal T2* values is in contrast with the results of the prospective study by Kirk et al [START_REF] Kirk | Cardiac T2* magnetic resonance for prediction of cardiac complications in thalassemia major[END_REF] who identified 10 ms as the threshold above which the risk of heart failure drops rapidly and that is practically minimized. Our results indicate that a non-negligible risk exists even for values proximal to the lower normal level of 20 ms. Another observational study [START_REF] Patton | Observational Study Of Iron Overload As Assessed By Magnetic Resonance Imaging (MRI) In An Adult Population Of Transfusion Dependent Patients With beta Thalassaemia: Significant Association Between Low Cardiac T2* < 10 ms And The Occurrence Of Cardiac Events[END_REF], also, identified that 50% of patients with T2*> 10ms had LVEF < 58%. This difference could be attributed to the different outcome assessed by Kirk et al. An abnormally low LVEF may precede clinically evident heart failure for long periods of time.

F

Therefore, the possibility that a proportion of the patients with T2*>10 ms may have impaired LVEF in the absence of heart failure cannot be excluded. In addition, moderate cardiac iron overload may require substantially more time in order to cause the damage necessary for symptoms of heart failure to appear. The relatively short period of follow up (1 year) in the above mentioned study may have not permitted this effect to become apparent.

These reasons could, also, explain the high percentage (approximately 70%) of patients with T2* ≤8 ms who were CD-free. It may take a period of time before the excessive iron overload can result in clinically obvious cellular dysfunction or there may be a protective effect of ongoing chelation in the clearance of toxic free iron [START_REF] Esposito | Labile plasma iron in iron overload: redox activity and susceptibility to chelation[END_REF][START_REF] Maggio | Improving survival with deferiprone treatment in patients with thalassemia major: A prospective multicenter randomised clinical trial Under the auspices of the Italian Society for Thalassemia and Hemoglobinopathies[END_REF].

In addition, genetic factors and susceptibility to excess iron are likely to play a role [START_REF] Economou-Petersen | Apolipoprotein E epsilon4 allele as a genetic risk factor for left ventricular failure in homozygous beta-thalassemia[END_REF][START_REF] Ferrara | Role of apolipoprotein E (APOE) polymorphism on left cardiac failure in homozygous beta thalassaemic patients[END_REF]. However, as it is still possible and even likely that these patients could progress to cardiac dysfunction or even sudden cardiac death, it is essential that they are prescribed intensive chelation in order to clear cardiac iron.

It is clear that the amount of cardiac iron load is related to the prevalence of cardiac dysfunction at a particular point in time. However, the exact level of iron load at which the risk of cardiac dysfunction is minimized cannot be identified by this analysis and as shown, this minimum level may vary according to the definition of cardiac dysfunction. In terms of impaired LVEF, our results indicate that even mildly loaded patients could benefit from a further reduction of their cardiac iron load. As this is a cross sectional analysis it does not provide prospective risk for the development of cardiac dysfunction. As the probability of CD is progressively, and not rapidly, reduced with increasing values of T2* the ROC analysis showed that T2* has limited ability in predicting the risk of abnormal LVEF. In order to obtain a sensitivity > 90%, it would require a cut off value of T2* = 15ms. Such a cutoff would reduce specificity to a non-acceptable level (23%), which would lead a significant number of non-loaded patients being subjected to unnecessary intensive chelation.

As with any cross sectional study, the main drawback is that results are drawn from a static picture of our population and correlations are based on characteristics that could be present for an undetermined period of time. In addition, the analysis was not performed according to chelation regime which may be related to the prevalence of cardiac dysfunction.

Another issue with our study was related to the determination of the LVEF. To date there has not been standardization of the methodology for this measurement. The method used in our centre is different to that previously described, which resulted in higher normal values than ours [START_REF] Westwood | Normalized left ventricular volumes and function in thalassemia major patients with normal myocardial iron[END_REF]. Our method may not be ideal, as it is based on the assumption that CD is solely due to cardiac iron overload. However, as we used the same analysis in assessing LVEF in "normal" individuals (patients with T2*>20ms) the determination of CD or non CD would be expected to be appropriate for the purposes of this analysis.

CMR, providing both T2* values and LVEF, is an essential tool for evaluating patients with transfusion dependent thalassaemia. This study provides strong evidence that even low levels of cardiac iron may be related to cardiac dysfunction and efforts should always be The probabilities were estimated by logistic regression analysis. 56x33mm (600 x 600 DPI)

  above 95g/l. Chelation regimes include deferoxamine, deferiprone, combination of deferoxamine and deferiprone and deferasirox.

  Pearson's coefficient (r). Linear regression analysis, reporting β coefficients and p-values, was used to evaluate the effect of different factors on LVEF. The relative risk of having CD groups defined by T2* was estimated by logistic regression analysis.

  iron free patients, age and T2* values did not affect LVEF as assessed by

  figure 3.

  levels of T2* > 20ms. Patients with T2* ≤ 8ms are at great risk of having cardiac dysfunction and require reversal of such iron load without delay.Authorship and DisclosuresAll authors contributed in study design and writing of the manuscript. GC and VB were involved in data entry. GC performed the statistical analysis. GC, SM and EG have no interests to declare. AK has participated in study monitoring committees for Novartis and has also participated at Exjade speaker Bureau for Novartis. He has attended sponsored meetings from Novartis and ApoPharma VB is a remunerated consultant with ApoPharma Inc and a consultant with Novartis Inc with respect to the development of deferasirox. He has received honoraria from Demo AE (Greece) and served on the Advisory Board of ApoPharma and has attended Novartis sponsored meetings on iron chelation. VL has been remunerated for clinical trials from both ApoPharma Inc and Novartis Inc and has attended sponsored meetings from both. VL has also served on an ApoPharma advisory board.

Figure 1 .

 1 Figure 1. Scatter plot and fitted regression lines, of left ventricular ejection fraction according

Figure 3 .

 3 Figure 3. Showing the estimated probability of having cardiac dysfunction according to T2*

Figure 1 .Figure 2 .Figure 3 .

 123 Figure 1. Scatter plot and fitted regression lines, of left ventricular ejection fraction according to cardiac T2* for patients with (T2 *≤ 20 ms, black dots) and without (T2*> ms, grey squares) cardiac iron overload 56x33mm (600 x 600 DPI)

Table 1 .

 1 Proportion † of patients with and without cardiac dysfunction and left ventricular ejection fraction (LVEF) according to cardiac iron load at the time of their first CMR , p<0.001. Significant comparisons (considering p=0.009 as the level of significance according to the Bonferroni correction): severe vs moderate p<0.001, severe vs mild p<0.001, severe vs normal p<0.001.

				Cardiac T2* values
			≤ 8 ms	>8 & ≤14 ms	>14 & ≤20ms	> 20 ms
	Cardiac dysfunction free, n (%)	46 (66.7)	55 (87.3)	39 (90.7)	123 (96.1)
	Cardiac dysfunction, n (%)	23 (33.3)	8 (12.7)	4 (9.3)	5 (3.9)
	Total, n (%)	69 (100)	63 (100)	43 (100)	128 (100)
	LVEF*, mean (SD)	60.8 (10.6)	65.6 (6.3)	67.5 (5.9)	67.8 (5.3)
	†Comparison of ratio CD to CD-free between T2* groups: Fisher's exact, p-value<0.001
	* ANOVA F-test

†

British Journal of Haematology


Thierry Passerat De Silans

Isabelle Maurin

Athanasios Laliotis

Pedro Chaves De Souza Segundo

Daniel Bloch
email: daniel.bloch@univ-paris13.fr

Pedro Chaves

EXTRA SUB-DOPPLER LINES IN THE VICINITY OF THE THIRD RESONANCE 6S-8P OF ATOMIC Cs ATTRIBUTED TO OPTICALLY INDUCED Cs DIMERS

d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

I. Introduction

The spectroscopy of alkali metal vapors is very well-known, and saturated absorption (SA) spectroscopy of alkali vapor provides among the most valuable and easily identified narrow spectral references. It is hence a surprise, when extra-resonances appear on such reference spectra. However, when the optical irradiation is strong, and/or when atomic density is relatively high, several phenomena, related to resonant non linear optical propagation, are susceptible to induce a spontaneous symmetry break [START_REF] Chaves | Honda Hexagonal pattern formation due to counterpropagation in KNbO 3[END_REF]. It is also known that in the atomic vapor of an alkali metal, formation of molecules (alkali dimers) may occur in a reversible manner, and that in dimer spectroscopy, richer than atomic spectroscopy, some resonances nearly coincide with those of atomic spectroscopy for the same species.

We report here on observations performed in the vicinity of the third resonance line of Cs (6S→8P), at 388 nm (6S 1/2 -8P 3/2 ) and 389 nm (6S 1/2 -8P 1/2 ). The experiment was initially implemented in view of getting atomic reference lines in the course of a selective reflection experiment [START_REF] Chaves De Souza Segundo | Selective Reflection Spectroscopy on the UV Third Resonance Line of Cs : Simultaneous Probing of a van der Waals Atom-Surface Interaction Sensitive to Far IR Couplings and of Interatomic Collisions[END_REF]. The third resonance line has an oscillator strength of about 10 -3 (at 388 nm), the 389 nm line being much weaker (3.10 -4 [START_REF] Cataliotti | Doppler-free excitation of the weak 6S 1/2 -8P 1/2 cesium transition at 389 nm[END_REF]) due to the well-known Fermi anomaly of Cs [START_REF] Fülöp | Emission and absorption measurements of the anomalous cesium principal-series doublet oscillator-strength ratios[END_REF]. The lifetime of Cs(8P) (~265 ns) [START_REF] Lingard | Transition probability for the isoelectronic sequences: Li I, Na I, K I, Rb I, Cs I[END_REF] is governed by a variety of decay channels, making the natural width of the transition as large as ~ 0.6 MHz (NB: a smaller value is mentioned in [START_REF] Liu | Measurement of the caesium 6S 1-2 -8P 1/2 transition frequency[END_REF]).

A modest heating-up of the Cs vaporto increase atomic density -, as well as focusing the beams, are required to obtain a saturated absorption spectrum owing to the weak oscillator strength of the considered line. Our experiments show that when the pump and probe are relatively intense, there are some additional (unreported) narrow lines inside the Doppler width, aside from the narrow saturated absorption components from which the structure (fig. 1) of the hyperfine manifolds of Cs (8P 1/2 ) is resolved (A =42.97  0.1 MHz, F=3-F=4 : 172 MHz) and of Cs (8P 3/2 ) (F=2-F=3 : 23 MHz, F=3-F=4 : 30 MHz, F=4-F=5 : 37 MHz) [START_REF] Arimondo | Experimental determinations of the hyperfine structure in the alkali atoms[END_REF][START_REF] Nesmeyanov | Vapor Pressures of Chemical Elements[END_REF]. Complementary experiments, performed with two independent lasers, lead to analogous observations in various experimental schemes, notably including co-propagating geometries. Similar extra-lines appear in non degenerate three-level schemes, when the pump and probe beams are resonant for different transitions, starting from the two hyperfine levels of the ground state (separation 9.192 GHz), or even for different fine-structure components [6S 1/2 -8P 3/2 and 6S 1/2 -8P 1/2 ]. These observations help us to rule out the possibility to explain these observations by resonant nonlinear optics, and establish that these extra lines are of a spectroscopic origin, attributed to a selective population of molecular caesium induced by the strong light excitation, as the atomic density is unusually low to enable a notable dimer population at thermal equilibrium.

II. Experimental set-up

A-Lasers

The experiments were conducted both with: (i) a low-power (~1 mW) diode laser, operating with a specific chip (for 388 -389 nm) mounted in an extended cavity (DL 100 from Toptica, see [START_REF] Chaves De Souza Segundo | Selective Reflection Spectroscopy on the UV Third Resonance Line of Cs : Simultaneous Probing of a van der Waals Atom-Surface Interaction Sensitive to Far IR Couplings and of Interatomic Collisions[END_REF]), and (ii) the high-power (~100mW) second harmonic generation of the amplified emission of an extended cavity diode laser emitting at ~776-778 nm (SHG 110 from Toptica). For both sources, the overall linewidth was  4 MHz. Some imperfections are present in the initial spatial mode structure (due to the intrinsic modal asymmetry of "blue" laser chips, and to the nonlinear frequency doubling and amplification process as well), but the implementation of an optical filtering system (pinhole) did not modify any of our observations. For a better monitoring of the frequency scan, applied through piezo-elements in the laser cavity, an auxiliary part of the laser beam is sent to a long and stable Fabry-Perot marker ( FSR : 83 MHz). In spite of this, relatively large fluctuations (~10 MHz), that cannot be corrected, appear on long scans owing to acoustical noise and piezo-elements nonlinearities.

B-Cs vapor cells

Our experiments were performed on three different sealed Cs vapor cells. Two of them were glass cells, whereas the third one was made of sapphire windows and a copper body.

These cells are heated-up in an oven, allowing a control of the Cs density (for the density / temperature dependence, see e.g. ref [START_REF] Nesmeyanov | Vapor Pressures of Chemical Elements[END_REF]). The observable differences between the various cells are essentially connected to differences in the absorption length (1-5cm) of these cells, and this rules out effects associated with a cell contamination by impurities. In addition, the narrow linewidths obtained for the main lines of atomic saturated absorption confirm the absence of notable impurities.

C-Experimental conditions

In a typical experiment, the pump beam is amplitude modulated with a mechanical chopper (occasionally with an acousto-optic modulator that enables a high modulation speed, but induces laser power losses), and the induced modulation on the counter-propagating probe beam is detected through a phase-sensitive amplifier. The pump and probe beams are either issued from the same laserwith a possible frequency shift between them, when an acoustooptic frequency shifter (typically at 80 MHz or 200 MHz) is introduced -, or they originate from two independent sources, of which, only one is frequency-scanned. Feedback is suppressed by optical isolators, and in most cases the beams bear a linear and orthogonal polarization.

Both beams are focused in the cell (typical waist ~ 50-100 µm) to provide a relatively high intensity. In spite of the saturation, the absorption is non negligible (typically up to 50 %) due to the relatively high Cs density; the temperature is typically ~ 80 °C (atom density ~ 10 13 cm -3 ). The choice of Cs density is not critical, as observations could be performed even at 45 °C (atom density ~ 5.10 11 cm -3 ) in a 5cm-long cell. A too strong absorption precludes an effective overlap of the counter-propagating beams. Also, if the probe intensity is weak, or the Cs density low, only the usual (Doppler-free) components of saturated absorption are observed.

Complementary experiments with two independent lasers were also performed in a nearly co-propagating geometry. The relevant spectroscopic parameter is thus the frequency difference between the two beams, while one beam is kept at a constant frequency and monitored in an auxiliary SA set-up. In this geometry, there is often a remaining overlap of the two nearly co-propagating beams, so that the probe beam is contaminated with a residual modulation of the pump beam. An additional frequency modulation (FM) on the probe beam, allows the detection of the frequency-derived spectra, and enhances the narrow (sub-Doppler) components. A cascade demodulation (FM on the probe and AM on the pump) enables us to observe only the joint effect of the pump and of the probe

III Experimental observations

A) Experiments with a single laser or around a single atomic resonance Extra sub-Doppler resonances, such as the ones shown in figure 2, are observed in the conditions of a standard saturated absorption experiment with the pump and probe beams issued from a single source, and require a relatively high intensity of the probe beam (typically  5-10 mW/mm² for the transition to the 8P 3/2 level), and high Cs density (typically  10 12 cm -3 for the transition to the 8P 3/2 level). The intensity of the pump beam is also at least as large as 5-10 mW/mm².

These resonances are characterized by the following features:

-Their typical width is 30-50 MHz : although broader than the individual atomic saturated absorption hyperfine components, whose observed width is ~ 4-6 MHz (mostly due to the laser linewidth), they remain clearly below the Doppler width (governed by ku ther = (2) 500 MHz, with k = 2/, and u ther the most probable velocity).

-They remain clearly narrower that the overall hyperfine structure of the Cs(8P) (see fig. 1).

-Their amplitude compares with the main SA resonances (see fig. 3). The influence of the beam polarization on the amplitude of these resonances is marginal.

-These sub-Doppler resonances most often correspond to an enhanced absorption (when the pump is on), while a standard SA atomic resonance corresponds to a reduced absorption (although the sign of the SA atomic resonances can be reversed in 4-level processes, notably in the presence of optical pumping at a relatively high irradiation intensity).

-More extra-resonances can appear under strong irradiation/density conditions (see fig. 4).

They can be located either between the first appearing set of extra-resonances and the main

SA resonances, or more remotely, but they remain in all cases inside the Doppler-broadened absorption.

-The position of these extra-resonances, provided in table 1, is remarkably insensitive to the experimental conditions, although their amplitude is not. The shifts, relatively to the main SA atomic lines, differ according to the considered ground state [6S 1/2 (F=3) or 6S 1/2 (F=4)], and do not exhibit a significant red-blue symmetry.

Quantitatively, at the onset of their appearance, the amplitude of these extraresonances exhibits a strong non linear dependence on probe intensity (fig. 3), and Cs density (fig. 5).

In a SA experiment, an extra degree of flexibility is provided when introducing a frequency shift between the pump and the counter-propagating probe. This gives us the possibility to address various velocity groups, and is particularly susceptible to modify the relative amplitudes of cross-over resonances, which are governed by the population of the probed velocity group. Here, such flexibility was provided either by an acousto-optic shifter or, once a second laser has become available, by keeping one laser at a fixed frequency and scanning the other laser across the same resonance. In such experiments, equivalent to the experiment with a single frequency laser in a moving frame, we have observed that it is the

B) Experiments around nondegenerate three-level saturated absorption

Using the set-up with two independent lasers, we have also performed non-degenerate velocity-selective three-level saturation spectroscopy, with the pump and probe frequencies shifted by ~9 GHz, resonant to transitions starting from different hyperfine components of the ground state. In this case, "extra" resonances are also observed aside from the main threelevel (-type) saturation signal, in experimental conditions similar to those mentioned above (saturating intensities of both beams, relatively high atomic density). Analogous observations can even be performed in a V-type non degenerate three-level scheme based upon the fine structure of the Cs(8P) level, e.g. with the pump resonant on the 6S 1/2 → 8P 1/2 line (389 nm) and the probe resonant on the 6S 1/2 → 8P 3/2 line (388 nm).

-The set-up with two independent tuneable lasers offers the versatility to study the copropagating geometry. Typically, one laser is kept at a constant frequency inside the Doppler-broadened absorption, and the other one is frequency scanned (across the same transition for a 2-level SA signal, across a different one for a 3-level scheme): such a set-up yields a spectrum revealing the hyperfine structure, independently of the velocity selected by the "pump" laser (assumed to be the laser at a fixed frequency, the detected probe laser being the frequencyscanned laser). The influence of the pump laser is limited to governing the height of the signal, according to the population of the selected velocity group, i.e. according to the position within the Doppler width. The same experimental conditions (i.e. vapor density, and beam intensity) allowing for "extra" resonances in the counter-propagating geometry apply in the co-propagating geometry (fig. 6). These resonances exactly appear as an extra spectroscopic structure, i.e. like a remote addition to the hyperfine structure, whatever is the actual frequency chosen to be kept constant. Most often, their position appears consistent with those mentioned in table 1 (once the required factor of 2 is introduced in the frequency scale, depending on whether a single or both beams are frequency-scanned). In some other cases, notably for a probing resonant to a transition starting from the 6S 1/2 (F=3) level -and the pump resonant to a transition starting from the 6S 1/2 (F=4) level -, the position of the extraresonances differs from those observed in the counter-propagating geometry.

C) Effect of a modulation

In all cases (counter-or co-propagating geometry, 2-level or 3-level spectroscopic scheme), increasing the speed of the modulation applied to the pump beam leads to attenuated signals.

This most probably indicates a process in which, at some step, the optical excitation induces optical pumping inside a long-lived level, as it is the case when a hyperfine transfer F=3→ F=4 occurs in the 6S 1/2 ground state.

IV Attempts of interpretation

It has been known for a long time [START_REF] Chaves | Honda Hexagonal pattern formation due to counterpropagation in KNbO 3[END_REF] resonances relatively to the experimental conditions. This is an essential point ruling out a possible interpretation of the observed extra-resonances as the signature of a resonant non linear emission process. In the experiments with co-propagating beams, a frequency shift within the Doppler width, applied to both beams, provides a simple way to modify the nonlinear propagation, but not the overlap between the two beams: it is hence particularly significant to note that the typical features of the spectrum (position of the extra-resonances) do not appear affected by such a frequency-shift.

B Spectroscopy of dimers

The stability of the spectral structures that we observe (i.e. the distance between the extra-resonances and the identified atomic transitions), whatever is the frequency of the pump beam within the Doppler width, is a good indication that the observed structures on the spectrum are truly of a spectroscopic origin. It is hence natural to look for spectral features associated to Cs dimers. Various spectral bands of Cs 2 were identified, and it has been noted for a very long time that there exists dimers transitions in the vicinity of the Cs 8S→8P line (388 nm and 389 nm) [START_REF] Loomis | The Band Spectrum of caesium[END_REF]. However, while an identification of dimers around the second resonance line of Cs was performed recently [START_REF] Pichler | Observation of blue satellite bands and photo-association at ultracold temperatures[END_REF], the molecular energy levels in this region of the third resonance line [START_REF] Vadla | Line-broadening studies of excited diatomic homoatomic and heteroatomic Rydberg molecules formed by potassium, rubidium, and cesium atoms[END_REF] have remained largely unexplored, most probably due to the lack of convenient sources, rendering very difficult the spectroscopic identification of a given line. In addition, the considered levels are too energetic to be reached efficiently by energypooling processes when optical excitation is limited to the first resonance line.

B1 Main features of dimer generation

Caesium dimers (Cs 2 ) can be produced by collisions as well as photoassociation. In the conditions of thermal equilibrium, the caesium vapor is a mix of atomic and molecular vapor, but it is only when the temperature exceeds ~200 °C that a notable fraction (typically exceeding 1%) of the vapor consists in dimers (from ground state Cs atoms, three-body collisions are needed to sustain the generation of Cs 2 ). It has also been noted that the efficiency of the dimer generation in a hot vapor can be modified through light irradiation, notably through an optical pumping mechanism inducing hyperfine redistribution in the ground state [START_REF] Allegrini | Molecule formation in an optically oriented vapor[END_REF]. Alternately, via photoassociation of laser-cooled atoms, the relative number of Cs 2 dimers can be quite large in spite of the relatively low atomic density of cold Cs atoms [START_REF] Jones | Ultracold photo-association spectroscopy: Long-range molecules and atomic scattering[END_REF]. Indeed, for cold atoms, the interaction between two atoms is much more efficient owing to an interaction time no longer limited by a fast relative (thermal) motion.

B2. Interpreting extra-resonances as connected to dimer spectrum

Although it is not unexpected that dimer lines are observed in close vicinity to atomic lines, the narrow extra-resonances that we observe cannot be simply attributed to saturated absorption lines of the Cs 2 that already exists in the vapor at thermal equilibrium. Indeed, the temperature is so low that the Cs 2 density is for sure a very small fraction of the atomic density: this is confirmed by the fact that with an extra diode laser operating in the infrared around 780 nm, it was only for a temperature higher than ~200 °C that we could observe (on the same set-up) the relatively strong (i.e requiring a relatively modest optical power) saturated absorption line of Cs 2 . Here, a quite low Cs density allows us to observe a signal height comparable to the atomic SA signal itself. Another major argument confirming that the observed extra-resonances are not simply the saturated absorption signature of pre-existing dimers, is that all attempts to observe some of these lines outside the Doppler resonance (our laser sources have a large tunability around 388 nm) have remained unsuccessful.

Dimer population is expected to depend nonlinearly upon the relevant atomic density, at least quadratically for a two-body process (NB: the "relevant" density is not necessarily the total atomic density, and could include a kind of selection, in atomic excitation or velocity).

Figure 5 shows a fast growth (nearly cubic for the first three points) of the amplitude of the extra-resonances with atomic density. However, the absorption coming along with propagation in the Cs cell is non negligible, so that this density parameter could not be modified without affecting other experimental parameters.

A remarkable point, already mentioned, is that the beam on which the detection is operated ("probe" beam) must be intense. This is unusual in spectroscopy, as it is ordinarily expected that the "probe" beam measures the linear absorptionor gainof the medium, or its pump-induced modifications. This indicates that the extra-resonances do not even originate in a saturated absorption process, in which the intense pump beam would both favour the production of dimers, and mark ( i.e. velocity select) this population of dimers. The variety of possible processes explaining these extra-resonances can include highly nonlinear spectroscopic process, such as a process relying on a Raman excitation performed by the two intense beams, followed by a linear spectroscopic probing. In particular, the co-propagating geometry, with extra-resonances observed for fixed frequency differences between the irradiating beams, resembles a Raman process, yielding Doppler-free resonances (conversely, for a counter-propagating geometry, a standard Raman process is sensitive to the Dopplerbroadening). Extended information on the spectroscopic structure of molecular levels would then require a third laser, if the probed level is here populated only as the result of a Ramaninduced dimer population. Such a scheme has been already considered and provided novel spectroscopic data [START_REF] Lyrra | all-optical triple resonance spectroscopy[END_REF], but was never implemented with the near UV wavelengths required for the considered dimer band.

B3 Sub-Doppler width and velocity selection

As long as it appears that the extra-resonances cannot be simply interpreted as a saturated absorption signal, it is worth discussing the narrow (sub-Doppler) width of the observed resonance. A remarkable feature of these extra lines is that they are not only sub-Doppler, but narrower (~30MHz) than the hyperfine structure manifold of the atomic Cs (8P) level: this is compatible with the fact that they are not of atomic origin. In the context of cold atoms, photoassociation leading to dimer formation is favoured because the two atoms can interact for a very long time, owing to the cooling of the thermal motion. In contrast, for atoms in a thermal vapor, the usual velocity selection processes solely apply to an axial selectivity, while the duration of interaction between two atoms is limited by the thermal and unselected motion perpendicular to the light propagation axis. This makes it uneasy to ascribe the sub-Doppler features of these extra-resonances to a velocity-selective process specifically required to induce the formation of dimers. Rather, among the multiple steps requiring a high intensity for both the pump and the probe, some of these processes, including optical pumping (see subsection III-C), are able to induce a (molecular) velocity selection. It may be also worth wondering if the atomic densityhigher than for photoassociation of cold atoms-is not sufficient to allow a process that would be limited to relatively slow atoms in the plane perpendicular to the beam axes ("slow" atoms corresponding to a Doppler width ~ 30 MHz, i.e. the width of the extra-resonances)

V. Conclusion

We report on novel spectroscopic lines, observed in a very elementary spectroscopic set-up, and this has motivated us to initiate a relatively systematic study. Because the investigation of a weak atomic line requires a relatively high light intensity, our experimental conditions favor nonlinear processes that could lead to the formation of molecular dimers, or even higher order aggregates such as clusters. Moreover, the high level of excitation of Cs (8P) allows complex step transfers through levels at an intermediate energy. The relatively high density of highly excited atoms allows the observation of molecular Cs at remarkably low overall atomic densities, without laser cooling technology. One of the intriguing results of these experiments is the sub-Doppler nature of the observed resonances, which is a possible indication of a high-order nonlinear process, combining selective formation of molecules and a standard nonlinear spectroscopy sub-Doppler process. A more systematic spectroscopic study would require a third independent laser beam, in order to allow a genuine spectroscopic probing (i.e. with a weak beam) of the molecular states produced in a complex nonlinear process.

Table caption

Frequency shifts of the extra-resonances, relatively to the cycling transitions F=3F'=2, or F=4 F'=5 of the third resonance component 6S

  whole spectrum that becomes frequency shifted, i.e. the extra-resonances are shifted by the same amount as expected for the main SA resonances. However, the relative amplitude of the extra-resonances is affected, and for large frequency shifts between the pump and the probe, more remote extra-components can appear, located on the side for which the probe beam remains inside the Doppler width. Such extra-resonances have been observed up to 1 GHz from the main SA atomic lines. All efforts to obtain extra-resonances away from the domain of (Doppler-broadened) probe absorption yielded negative results.

  that in the simple geometry of a SA experiment, intense counter-propagating beams are susceptible to induce strong nonlinear effects, leading to a spontaneous symmetry breaking. Such strong and resonant non linear effects can appear as narrow extra-resonances inside the Doppler-broadened resonant width, corresponding to a combination of strong local nonlinearities -nonlinear dispersive effects, possibility of gain at some frequencies-, with propagation in an optically thick medium [possibility of a strong depletion through an absorption mechanism, and of an oscillation in directions specifically favourable to gain mechanism, ...]. Hence, modifying the propagation conditions, notably through a change of the atomic density -i.e. vapor temperature-, or through the number of velocity-selected interacting atoms -controlled through the laser detuning-should severely affect the frequency position of the non linear Doppler resonant processes. In contrast, a major feature of our observations is the independence of the extra narrow (sub-Doppler)

Figure 1 :Figure 2 :

 12 Figure 1 : Scheme of the relevant energy levels of Cs, and additional indication of the two first resonant levels

Figure 3 (Figure 4 (Figure 5 (

 345 Figure 3 (Color online) : Height of the signals observed in a saturated absorption experiment (counter-propagating geometry) as a function of the probe intensity. The regular SA peak (full circles ) increases sub-linearly with the probe intensity, owing to saturation effects, and in spite of a more efficient transmission. The amplitude of the extra-resonances (empty circles ○ for the blue-shifted peak, triangles Δ for the red-shifted peak) increase nonlinearly with the probe intensity, with a fitting provided in the insert. In these experiments, the pump power is ~ 50 mW/mm²

Figure 6 : 2 (

 62 Figure 6 : FM transmission spectrum around the transition 6S 1/2 (F=4)-8P 3/2 (F'=3,4,5), when a

Figure 1 6

 1 Figure 1

Figure 5 Figure 6 -

 56 Figure 5

  1/2 -8P 3/2 , as measured in a single laser experiment. The typical accuracy is ± 10 MHz. The most peripheral resonances are observed only for strong intensities, and adequate densities.

	Table 1
	Hyperfine ground state		Positions of the extra-resonances
	F=3	-425 MHz	-180 MHz	210 MHz	300 MHz	500 MHz
	F=4	-650 MHz	-355 MHz	310 MHz	-	-

Acknowledgements. P. Todorov and H. Failache (ECOS-Sud U08E01) have also participated to some steps of this work. We thank G. Pichler for stimulating discussions. We thank Thierry Billeton for the design of the Fabry-Perot marker. This work was partly supported by INTAS South-Caucasus Project (grant 06-1000017-9001).