Sakura’s   Tissue-Tek   Xpress® x120   is   a   powerful   instrument    that    minimises    the    time needed    for    processing  tissue  and  maximises  a  lab’s  workflow. Prof. Herbst and Dr Kriese both work at the Vivantes Klinikum  Neukölln  in  Berlin,  and  are  happy  to  reveal that  processing  tissue  with  this  instrument  does  not  influence their ability to carry out molecular diagnostic assays. 

Vivantes   Hospital   Group   is   the   largest   state-owned   healthcare  group  in  Germany,  and  has  been  accredited  by  the  College  of  American  Pathologists  (CAP),  making  it  the  largest  accredited  CAP  institution  in  Europe.  Vivantes’  main Pathology department, out of five, is the Institute of Pathology at Vivantes Klinikum Neukölln. Here, they focus on  high-throughput  technologies,  immunohistochemistry  and   molecular   pathology.   Vivantes   Klinikum   Neukölln   receives  more  than  2,000  molecular  pathology  cases  per  year for DNA and RNA isolation techniques.

For   the   most   part,   Vivantes’   molecular   pathology   lab   analyses  tissue  from  formalin-fixed,  paraffin-embedded (FFPE) blocks. As all assays performed depend on the DNA and RNA quality and ultimately on the PCR amplification performance, it is vital that the FFPE material received is of high quality. Vivantes uses two tissue processing methods: the conventional method and continuous processing, which is where Sakura’s Tissue-Tek Xpress x120 comes in.  

Director of the Institute of Pathology at Vivantes Klinikum Neukölln  in  Berlin,  Professor  Herbst  has  been  working  for  Vivantes  Hospital  Group  for  over  16  years.  His  direct  colleague,   Doctor   Kriese,   is   Head   of   the   Molecular   Pathology laboratory, and is in her 11th year of working for Vivantes. 

In this interview, Prof. Herbst and Dr Kriese explain that the two different types of tissue processing, conventional and continuous, have no effect on the ability to determine clear diagnoses. They start with explaining how important quality is  and  how  it  is  assured  in  their  lab  before  talking  about  the  two  processing  methods  and  the  future  of  molecular  pathology.  

Dr Kriese (left) and Prof. Herbst (right)

The   importance   of   on-going   accreditation   and   the   material’s source

“The CAP ... accreditation process,” tells Prof. Herbst, “is a review of procedures, quality and qualification of people involved.” CAP accreditation takes place every other year. In  between CAP  assessments, Vivantes performs and records  their  own  assessments.  “We  also  participate  in  proficiency tests for the German Quality Assurance Initiative for Pathology (QuIP).” On-going profi  ciency tests are useful as they keep laboratory quality and output high.

When  it  comes  to  FFPE  blocks  and  block  creation,  most  blocks  are  from  the  labs  of  the  Vivantes  Hospital  Group.  They do not regularly receive blocks from external sources. With  blocks  from  internal  sources,  a  lot  of  information  is  provided  and  it  is  possible  to  estimate  the  duration  of  fixation. Prof. Herbst explains that this is actually required for CAP accreditation. Regarding the blocks received from external sources, not much information is known. However, the  DNA extraction processes  are  very safe. “In  most cases,” explains Prof. Herbst, “we get DNA that can be used for PCR amplification purposes ... and for Next Generation Sequencing (NGS), the main technology that we are using.” Dr  Kriese  adds,  “we  always  do  a  quality  assessment  with  PCR  and  we  look  at  the  integrity  of  the  nucleic  acids  we  isolate.  This  way  we  can  assess  beforehand  how  well  the  amplification process and NGS could be.” 

Conventional  versus  continuous  processing  methods

The   two   methods   of   processing   tissue   and   making   an  FFPE block – conventional and continuous – do not seem to   influence  quality  or  downstream  molecular  testing. According to Dr Kriese, there are no significant differences that affect the  tests they perform in  the  lab.  For  the tests, isolated  DNA  needs  to  amplify  to  up  to  600  base  pairs.  Concerning DNA amplification  above 600 base pairs,Dr  Kriese  tells: “This is not necessary because none of ourmolecular diagnostic tests needs DNA amplicons longer than 600 base pairs.”

“There are no significant differences that affect the tests that we do in the laboratory.” –Dr Kriese  

“What  is  critical,”  Prof.  Herbst  notes,  “is  the  quality  of  the  formalin. It has to be neutral buffered formalin.”  Additionally, temperature of the fixative is an important factor. Heated formalin will show a significant increase in the amount of formic acid, rendering DNA and RNA almost useless.

“What is critical, is the quality of the formalin.” – Prof. Herbst

Prof.  Herbst explains  that  both  fixative and  fixation length play  crucial  roles.  “If  the  chemicals  are  of  higher  quality and are treated and stored properly, then everything should be  fine.”. Luckily, poor  DNA  integrity  happens  rarely. In most cases, those blocks have come from external sources or  may  have  already been  damaged  before the  fixation process has begun. Prof. Herbst: “If the material is not fixed properly  and  autolytic,  the  DNA  strands  will  be  damaged  and RNA might be gone completely.”  

The future of molecular diagnostics

Prof. Herbst sees a future in which the molecular pathologyfield will become more automated. In his view, in 5-10 years, technicians won’t merely perform a simple NGS diagnosticspanel, but a whole exome sequencing. A pathology institute could have a molecular pathology lab as large as the routinehistopathology  part,  or  it  could  even  outgrow  this  part.  “Over the last 10 years ... there [has been] an exponential growth in the molecular diagnostic lab and I think this willcontinue. I think it is very important to keep histopathologyconnected to molecular tests in one institute.”

“Over the last 10 years ... there [has been] an exponential growth in the moleculardiagnostic lab and I think this will continue.” – Prof. Herbst