Standard Document
Second Edition
Molecular Diagnostics

CLSI MM05

Nucleic Acid Amplification Assays for Molecular Hematopathology

This CLSI guideline provides essential guidance on nucleic acid-based testing for diagnosing and managing hematopoietic neoplasms. It covers key applications such as clonality analysis, chromosomal translocation detection, micromutation identification, and chimerism quantification in post-transplant monitoring.

The document details molecular techniques, including PCR, reverse-transcriptase PCR, real-time fluorescence-based PCR, DNA sequencing, FISH, and microarray assays. It also includes recommendations for specimen collection, validation, reporting, and quality assurance.

March 30, 2012
Daniel E. Sabath, MD, PhD

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Abstract

Analysis of nucleic acids is playing an increasing role in the diagnosis and management of patients with hematopoietic neoplasms. The tests include those for detection of clonality by analysis of gene rearrangements in the antigen receptor genes or detection of nonrandom inactivation of the X chromosome, detection and quantification of junctions formed by chromosomal translocations, detection of micromutations, quantification of chimerism after allogeneic hematopoietic transplantation, and quantification of normal DNA or RNA sequences. The methods used in clinical molecular hematopathology include end-point PCR, reverse-transcriptase PCR, real-time fluorescence-based PCR, DNA sequencing, FISH, and hybridization-based microarray and microbead assays. Clinical and Laboratory Standards Institute document MM05-A2—Nucleic Acid Amplification Assays for Molecular Hematopathology; Approved Guideline—Second Edition addresses the needs of the laboratory by providing recommendations on a variety of laboratory tests based on analysis of nucleic acids. It addresses preexamination and examination issues affecting assay performance, reporting of laboratory results, and QA. The guideline is designed to assist a molecular diagnostic laboratory in acquiring a new assay or new technology, as well as serve as a refresher for those already experienced with a particular area of analysis.

Scope

The use of molecular methods in clinical diagnosis is now essential in pathology, especially in hematopathology. These methods help identify clonal cell proliferations, chromosomal translocations, abnormal RNA, somatic mutations, and quantify abnormal nucleic acids. Molecular pathologists have worked to determine when these tests are needed and to improve their quality. This guideline is designed for laboratory directors, surgical pathologists, medical technologists, hematopathologists, hematologists, oncologists, and regulatory personnel. It aims to help laboratories implement nucleic-acid-based hematology assays with proper controls and to determine what materials and records to save. 

The document covers:

 • Indications for molecular diagnostic testing 

• Specimen collection, transport, and processing 

• Assessment of specimen adequacy 

• Conduct of molecular hematology assays, and sensitivity, specificity, controls, and artifacts 

• Quality assurance (QA) 

• Interpretation of results 

This document does not cover genetic testing for inherited mutations. Refer to CLSI document MM01 for genetic testing guidelines. This document also does not cover procedures for validation of molecular diagnostic tests. For information on validation of molecular diagnostic tests, refer to CLSI documents C28, EP05, EP06, EP09, EP12, EP15, EP17, MM01, MM03, and MM06. In addition, this document does not discuss next-generation sequencing; therefore, discussion of sequencing or direct sequencing is in reference to Sanger sequencing.

Product Details
MM05A2E
1-56238-791-X
108
Additional Details

This document is available in electronic format only.

The U.S. Food and Drug Administration (FDA) has evaluated and recognized this approved-level consensus standard for use in satisfying a regulatory requirement.

This archived document is no longer being reviewed through the CLSI Consensus Document Development Process. However, this document is technically valid and because of its value to the laboratory community, it is being retained in CLSI’s library.

Authors
Daniel E. Sabath, MD, PhD
Rajyalakshmi Luthra, PhD
Elizabeth Algar, MSc, PhD, mHGSA, FFSc, RCPA
Richard D. Press, MD, PhD
Pritish K. Bhattacharyya, MD
Paul G. Rothberg, PhD, FACMG
Karen E. Bijwaard, MS, RAC, MB(ASCP)
Rana Domiati Saad, PhD
Tao Hong, PhD
Zeba N. Singh, MD
Neal Lindeman, MD
Emily S. Winn-Deen, PhD
Abstract

Analysis of nucleic acids is playing an increasing role in the diagnosis and management of patients with hematopoietic neoplasms. The tests include those for detection of clonality by analysis of gene rearrangements in the antigen receptor genes or detection of nonrandom inactivation of the X chromosome, detection and quantification of junctions formed by chromosomal translocations, detection of micromutations, quantification of chimerism after allogeneic hematopoietic transplantation, and quantification of normal DNA or RNA sequences. The methods used in clinical molecular hematopathology include end-point PCR, reverse-transcriptase PCR, real-time fluorescence-based PCR, DNA sequencing, FISH, and hybridization-based microarray and microbead assays. Clinical and Laboratory Standards Institute document MM05-A2—Nucleic Acid Amplification Assays for Molecular Hematopathology; Approved Guideline—Second Edition addresses the needs of the laboratory by providing recommendations on a variety of laboratory tests based on analysis of nucleic acids. It addresses preexamination and examination issues affecting assay performance, reporting of laboratory results, and QA. The guideline is designed to assist a molecular diagnostic laboratory in acquiring a new assay or new technology, as well as serve as a refresher for those already experienced with a particular area of analysis.

Scope

The use of molecular methods in clinical diagnosis is now essential in pathology, especially in hematopathology. These methods help identify clonal cell proliferations, chromosomal translocations, abnormal RNA, somatic mutations, and quantify abnormal nucleic acids. Molecular pathologists have worked to determine when these tests are needed and to improve their quality. This guideline is designed for laboratory directors, surgical pathologists, medical technologists, hematopathologists, hematologists, oncologists, and regulatory personnel. It aims to help laboratories implement nucleic-acid-based hematology assays with proper controls and to determine what materials and records to save. 

The document covers:

 • Indications for molecular diagnostic testing 

• Specimen collection, transport, and processing 

• Assessment of specimen adequacy 

• Conduct of molecular hematology assays, and sensitivity, specificity, controls, and artifacts 

• Quality assurance (QA) 

• Interpretation of results 

This document does not cover genetic testing for inherited mutations. Refer to CLSI document MM01 for genetic testing guidelines. This document also does not cover procedures for validation of molecular diagnostic tests. For information on validation of molecular diagnostic tests, refer to CLSI documents C28, EP05, EP06, EP09, EP12, EP15, EP17, MM01, MM03, and MM06. In addition, this document does not discuss next-generation sequencing; therefore, discussion of sequencing or direct sequencing is in reference to Sanger sequencing.

Additional Details

This document is available in electronic format only.

The U.S. Food and Drug Administration (FDA) has evaluated and recognized this approved-level consensus standard for use in satisfying a regulatory requirement.

This archived document is no longer being reviewed through the CLSI Consensus Document Development Process. However, this document is technically valid and because of its value to the laboratory community, it is being retained in CLSI’s library.

Authors
Daniel E. Sabath, MD, PhD
Rajyalakshmi Luthra, PhD
Elizabeth Algar, MSc, PhD, mHGSA, FFSc, RCPA
Richard D. Press, MD, PhD
Pritish K. Bhattacharyya, MD
Paul G. Rothberg, PhD, FACMG
Karen E. Bijwaard, MS, RAC, MB(ASCP)
Rana Domiati Saad, PhD
Tao Hong, PhD
Zeba N. Singh, MD
Neal Lindeman, MD
Emily S. Winn-Deen, PhD