The idea behind the constitution of Study Groups on the “Bence Jones proteins and Free Light Chains” subject arose on verifying

the clinical-diagnosis importance of determining
Bence Jones Proteinuria (BJP)

lacked adequate standardization

of methods, study protocols and reference samples, or even for reporting on results.

To this end, and for the purpose of advancing on said standardization, the “Forlí” Commission was set up in May 1993, to which other commissions were subsequently added.

Regarding determination of the Bence Jones Proteins, and more generally of the Free Light Chains in urine, the work of the Study Groups has aimed to:

• verify the different operational strategies existant, and
• evidence and document the analytical problems

then from here, respecting particular realities and demands at all times, verify the possibility of standardizing for the BJP:

• assessment
• interpretation of results
• reporting the results

As to standardizing the determination of the Bence Jones Proteins, the Study Groups established from the beginning that

the first step and non-negotiable objective is

for a sample to result,
“BJP-Positive” or “BJP-Negative”
in all laboratories

dispensing with the method or protocol (i.e. all the first level and in-depth methods) used.

The strategy agreed and adopted by the Study Groups is based on:

• comparing operational experiences of real day-to-day routine of laboratories with diversified dimensions, structures and organizations
• starting with the verification and definition of the “real characteristics” of usable techniques and methods in practice

The work methodology is defined in the following phases:

• experimental phase, comprising multicentric assessment of samples with agreed and known characteristics
• information of participants phase on all the results obtained and preparation of said results
• holding round table “co-ordination meetings” to:

° discuss together the results of the experimental work performed
° individualization, based on consensus, of conclusions and objectives reached, likewise eventual corrective actions to be taken both within the group and those considered convenient to propose outside
° define work to be performed with a view to the following meeting

The “starting point” has been verification and definition of the “real characteristics” of usable techniques and methods in practice.

Therefore, it has been deemed necessary prior to doing anything else, to:

assess the “sensitivity” and “accuracy”
of each method
both intra and inter-laboratory,

and thus, with sufficient judgement elements, be able to

set them
within the BJP
search and study strategy

For the first verification of methods sensitivity and accuracy the elemental, but linear, model has been taken comprising scaling dilutions of urine samples with important BJP and merely traces of other proteins.

This kind of model enables assessment and comparison of the method sensitivity and accuracy in evidencing “sample abnormality” ignoring considerations about the absolute concentration and the specific characteristics of the BJP.

Since the Study Groups started in 1993, they have carried out experiments and held meetings leading to a significant advance by the participants in the standardization of the BJP determination.

Furthermore, interesting results and conclussions have emerged, some really unpredictable and disconcerting because they were unexpected, regarding the real “sensitivity” and “accuracy” of commercial methods.

The Study Groups reflections on results and work methodology have:

• confirmed the good quality of the strategy and procedures followed,
• enabled formulation of operational proposals and recommendations,
• suggested continuing along the path started not only to improve and complete the proposal, but also to have the results obtained under control,
• induced expanding the experiment horizon on applying the same strategy to the study of proteinuria in general. The objective here is also to verify and improve standardization by comparing the separative traditional methods (Electrophoresis, SDS, etc.) with dosing some specific proteins (so-called “Proteinuria markers”) aided by software used to interpret results and prepare the report.

The "Forlì" Commission named after the town where the first meeting was held, groups some 30 laboratories from Emilia-Romagna and other surrounding Italian region. The commission’s scientific secretariat is run by Dr. Gualtiero Pallotti (Ospedale Pierantoni – Forlì).

The characteristics of this first meeting organized together with Dr. Palloti, on 28th May in 1993 in Forlì, in response to requests received from the different parties, were atypical; in its aim to freely express doubts, problems, opinions, sensations, results and any other question related to the topic proposed.

Since its spontaneous constitution, the commission has held over a dozen meetings on the initial object argument of the study groups: assessment of methods to determine Free Light Chains (FLC) and Bence Jones Proteins (BJP) in urine.

The specific first results were published in Biochimica Clinica in 1995, vol. 19, n. 5, entitled "L'angolo dell'Elettroforesi" of F. Aguzzi with the title "Valutazione Multicentrica di metodi e protocolli per le Catene Leggere Libere e Proteine di Bence Jones in urine - Primi risultati". Subsequent results, obtained in joint experiments with other subsequently constituted commissions, were presented at different scientific society Meetings and Congresses; since the 1999 edition the commissions have been invited to present their results and conclusions at the CEFAR Courses “Proteins: from the Laboratory to the Clinic”.

Different meetings subsequent to the experiments have enabled the commission to formulate a draft on “Operational Recommendations” , which proposes some base characteristics the methods and protocols used for qualitative and quantitative BJP determination should comply with. Other conclusions drawn from the experiments and detail of the results obtained appear in the paragraph on results and conclusions.

Lately, at their recent meetings, the commission has been expanding the horizons of its experiments on applying the same methodology to proteinuria study in general. The objective, as initially for FCL and BJP, is to advance in the standardization of proteinuria study; by comparing traditional separative methods (EF, SDS, etc.) with dosing specific marker proteins in urine.

In 1997 the Ligure Sections of the SIBioC – AIPAC – SIMEL decided to commence in conjunction an analogous initiative to that of the "Forlì" Commission, also co-ordinated organizationally by NSC; and constituted the "Liguria" Commission, whose scientific secretariat is run by Dr. Liliana Burlando (già Ospedale Galliera – Genova) and Dr. Giovanna Zaninetta (Ospedale S. Martino - Genova), grouping some 14 laboratories in the area.

The "Liguria" commission has kept close contact with the other commissions, and particularly with the "Forlì" commission, leading them to present their results and conclusions jointly at different scientific society Meetings and Congresses, in particular those of SIBioC and the CEFAR courses.

The result of these discussion meetings after the experiments, has led the commission to formulate its own “Operational Recommendations”, whose base lines coincide with the "Forlì" commission document, complemented with two proposals:

• considerations and mask for the report
• quantitative determination assessment proposal

Both the “Forlì” and "Liguria" commissions are expanding their works to assessment of methods for the general study of proteinuria, to advance in their standardization.

Two new commissions were added to those of “Forlì” and “Liguria” in 2001, grouping between them some 30 laboratories from Lazio, Toscana and Umbria .

The “Lazio” Commission co-ordinated by Dr. Maria Teresa Muratore (Ospedale Civile – Viterbo), held their first meeting on 10th April 2001 in Vitorchiano, while the “Toscana-Umbría” Commission, whose scientific secretariat is run by Dr. Enzida Piazza (Ospedale Careggi – Firenze), was constituted on 24th May 2001.

The results and conclusions of their experiments were recently presented in conjunction with those of the “Forlì” and “Liguria” commissions, in the 2001 edition of the CEFAR Course (Castrocaro).

At the end of 2002, NSC launched in Spain the start of a multicentric experiment, analogous to those previously performed by the Italian commissions centered on an experimental level in two differentiated geographical areas:

• The “Castilla-Madrid” area, grouping laboratories from the Madrid area and both Castilas
• The “Catalunya” area, grouping laboratories throughout Catalonia

Some laboratories from other communities particularly interested in this initiative joined the laboratories of these two areas.

Taking advantage of the experience acquired in Italy, which was considered a good starting point; it was proposed to the participating laboratories to study the same samples analysed by the Italian groups.

The degree of participation was very satisfactory, 25 laboratories between both groups, which achieved the objective proposed, i.e. arouse the participants’ interest in discussing and exchanging experiences on the “Free Light Chains and Bence Jones Proteins determination” argument.

Although the experiment was performed jointly, for operational reasons the meetings were held separately. The “Castilla-Madrid” Group met in Madrid on 26th March 2003, and the “Catalunya” Group, met in Barcelona on 2nd April 2003, which was also attended by laboratories from other areas.

At the meetings it turned out clearly the need to advance in standardization of the methods used in the study of Bence Jones Proteins (BJP), and the groups agreed to perform a new experiment to this effect at the end of 2003.

The second experiment involved over 40 laboratories, since it represents the constitution of two new study groups who have joined the initiative.

In the said experiment, samples of the same origin were analysed but with greater BJP concentration, trying to obtain results which were more coherent with the different methods (and laboratories) than those obtained in the initial experiment.

The results were presented at the meetings meetings held by the different groups during 2004.