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Acute lymphoblastic leukemia (ALL)

Region: IKZF1 7p12.2; PAX5 9p13.2; ETV6 12p13.2; RB1 13q14.2; BTG1 12q21.33; EBF1 5q33.3; CDKN2A-CDKN2B 9p21.3; Xp22.33

Intended purpose
The SALSA MLPA Probemix P335 ALL-IKZF1 is an in vitro diagnostic (IVD)1 or a research use only (RUO) semi-quantitative assay2 for the detection of deletions of the IKZF1 gene for stratification of patients with acute lymphoblastic leukemia (ALL) into prognostic subgroups. The SALSA MLPA Probemix P335 ALL-IKZF1 is a RUO assay2 for the detection of deletions or duplications in B-cell differentiation and cell cycle control genes (EBF1, CDKN2A/B, PAX5, ETV6, BTG1 and RB1) and in the PAR1 region. This assay is for use on genomic DNA isolated from human peripheral whole blood and bone marrow specimens.

Copy number alterations (CNAs) detected with P335 ALL-IKZF1 should be confirmed with a different technique. In particular, CNAs detected by only a single probe always require confirmation by another method. In the majority of patients, defects in the IKZF1 gene are deletions, but point mutations can occur which will not be detected by MLPA. It is therefore recommended to use this assay in combination with sequence analysis of the IKZF1 gene.

Assay results are intended to be used in conjunction with other clinical and diagnostic findings, consistent with professional standards of practice, including confirmation by alternative methods, clinical genetic evaluation, and counselling, as appropriate. The results of this test should be interpreted by a clinical molecular geneticist or equivalent.
This device is not intended to be used for standalone diagnostic purposes or population screening.

1 Please note that this probemix is for in vitro diagnostic (IVD) use in the countries specified at the end of this product description. In all other countries, the product is for research use only (RUO).
2 To be used in combination with a SALSA MLPA Reagent Kit and Coffalyser.Net analysis software.

Note that the clinical relevance of some genes in the P335 ALL-IKZF1 probemix is not yet fully established. Therefore, the CE mark for diagnostic use only applies to the IKZF1 gene, and all other genes in the probemix are meant to be used in a research setting only.

Clinical background
The overall incidence rate of ALL amounts to 1.6 in 100,000 per year (Malard and Mohty 2020). The peak incidence lies in childhood at an age of less than 5 years; thereafter, the incidence rate declines continually until the age of 50 years. After that, incidence slightly rises a second time. In patients over 50 years it rises a second time and reaches another peak at the age of over 80 years). There is a slight predominance of males (1.2:1). B-cell ALL accounts for 75% of all cases of ALL and T-cell ALL accounts for the remaining 25% of cases (

Partial or complete deletions of the IKZF1 (IKAROS family zinc finger 1) gene are frequently detected in ALL cases (Mullighan et al. 2008), especially in those patients who also carry the BCR-ABL1 gene fusion (Philadelphia chromosome). IKZF1 deletions can be identified in approximately 70% of the children with Philadelphia chromosome-positive (Ph+) ALL (2-4% of all paediatric ALL cases), in 10-15% of Philadelphia chromosome-negative (Ph−) paediatric ALL, and in 40% of adult B-cell precursor ALL cases (Bernt and Hunger 2014; Lejman et al. 2022; van der Sligte et al. 2015). Deletion of IKZF1 is associated with a poor prognosis in B-ALL patients (Mullighan et al. 2009a) and a higher chance of relapse (Kuiper et al. 2010).

Several other (partial) gene deletions and duplications, such as in PAX5, ETV6, RB1, BTG1, EBF1 and CDKN2A/2B, have also been described in ALL patients. Prognostic profiles combining these aberrations, such as the IKZF1plus profile (IKZF1 deletions that co-occur with deletions in CDKN2A, CDKN2B, PAX or PAR1 in the absence of ERG deletion; Stanulla et al. 2018), have been described in recent years. See Table 2 for more information on all genes and regions covered in this probemix.

Probemix content
The SALSA MLPA Probemix P335-C2 ALL-IKZF1 contains 57 MLPA probes with amplification products between 120 and 504 nucleotides (nt). This includes one probe for each of the eight exons of the IKZF1 gene (7p12.2). Furthermore, this probemix also contains seven probes for PAX5 (9p13.2), six probes for ETV6 (12p13.2), five probes for RB1 (13q14.2), four probes for BTG1 and the BTG1 downstream region (12q21.33), four probes for EBF1 (5q33.3), three probes for CDKN2A/CDKN2B (9p21.3) and five probes for the Xp22.33 region (PAR1 region; SHOX area, CRLF2, CSF2RA, IL3RA and P2RY8 genes). In addition, one probe at Yp11.31 (ZFY) and one probe at 9p24.1 (JAK2) have been included to help determine the extent of a deletion/duplication detected in patient samples. See page 6 and 7 for more information about interpretation of results of the ZFY probe. Finally, 13 reference probes are included that target relatively copy number stable regions in ALL. Complete probe sequences and the identity of the genes detected by the reference probes are available in Table 3 and online (

This probemix contains nine quality control fragments generating amplification products between 64 and 105 nt: four DNA Quantity fragments (Q-fragments), two DNA Denaturation fragments (D-fragments), one Benchmark fragment, and one chromosome X and one chromosome Y-specific fragment. More information on how to interpret observations on these control fragments can be found in the MLPA General Protocol and online at

Order Items


Item no.
SALSA MLPA Probemix P335 ALL-IKZF1 – 25 rxn
€ 281.00
SALSA MLPA Probemix P335 ALL-IKZF1 – 50 rxn
€ 550.00
SALSA MLPA Probemix P335 ALL-IKZF1 – 100 rxn
€ 1075.00

Required Reagents (Sold Separately)

Item no.
SALSA MLPA Reagent Kit – 100 rxn – FAM
€ 341.00
SALSA MLPA Reagent Kit – 100 rxn – Cy5
€ 341.00
SALSA MLPA Reagent Kit – 500 rxn – FAM
€ 1571.00
SALSA MLPA Reagent Kit – 500 rxn – Cy5
€ 1571.00
SALSA MLPA Reagent Kit – 2000 rxn – FAM
€ 6037.00

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One PAX5 specific probe has been replaced.


CE-marked products are for In Vitro Diagnostic (IVD) use only in EU (candidate) member states and members of the European Free Trade Association (EFTA), and the UK.


IVD-registered in Colombia.


IVD-registered in Costa Rica.


IVD-registered in Israel.