Molecular Hematology and Coagulation Physiology

Specimen and question. Bone marrow aspirate at diagnosis often supplies dividing cells and a high proportion of the abnormal clone, although hemodilution or marrow fibrosis can reduce yield. Conventional chromosome culture requires viable nucleated cells collected in the laboratory's validated transport system. EDTA blood or marrow is common for nucleic acid testing. Heparin can inhibit amplification and is used only when the method is validated for it. Peripheral blood is suitable when abnormal cells circulate and for many serial molecular measurements. Fixed tissue supports selected FISH and sequencing assays, with fixation and sectioning artifacts addressed during validation. Before reporting, confirm specimen identity, cellularity or nucleic acid quality, target abundance, assay sensitivity, and whether the diagnostic target matches the planned follow-up assay.

Amplification control. High-copy amplicons can contaminate later reactions, so keep reagent preparation, specimen extraction, and post-amplification work physically separated, with one-way movement, aerosol-resistant tips, and validated decontamination procedures. A no-template control monitors reagent contamination, a negative extraction control monitors cross-contamination, a positive control confirms target detection, and an internal amplification control can reveal inhibition or extraction failure. The required controls and acceptance criteria are assay-specific. Uracil-N-glycosylase can prevent amplification of carryover only when prior amplicons contain uracil. It does not remove contaminating native DNA.

Quantitation. Real-time PCR records fluorescence during amplification. A lower quantification cycle, often labeled Cq or Ct, corresponds to more starting target only within the same validated assay and comparable amplification efficiency. A standard curve can estimate copy number. A reference gene can normalize a transcript result to the quantity and quality of amplifiable material. A result between the limit of detection and the limit of quantification may be reported as detected, without a numeric value. Report a quantitative result only within the validated measuring interval. Release a "not detected" result only with acceptable controls, and state the achieved limit of detection or specimen-specific sensitivity when it is clinically relevant. Digital PCR partitions a specimen into many reactions and estimates concentration from the positive partitions. It still requires validated thresholds, controls, and enough target molecules.

FISH patterns and limitations

FISH probes hybridize to complementary sequences in fixed interphase nuclei or metaphase chromosomes. A centromere enumeration probe assesses chromosome copy number. A locus-specific probe can detect a deletion or amplification, often with a control probe for comparison. A break-apart probe places two colors on opposite sides of one gene. An intact locus gives adjacent or fused signals. Separated signals support a rearrangement involving that locus. A break-apart result does not identify the partner gene. A dual-fusion probe targets two specified partner genes and can demonstrate their genomic proximity in a classic reciprocal translocation pattern. FISH does not establish that a functional fusion transcript is expressed.

Interphase FISH works without cell division and can provide a rapid result from marrow, blood, touch preparations, or paraffin sections when validated for that material. Metaphase FISH can localize a probe signal to a chromosome. Conventional chromosome analysis adds a survey of the whole chromosome complement and the context of derivative chromosomes. FISH has greater resolution at its selected targets. A finite cell count can miss a low-level clone, and each FISH probe set has a validated cutoff for each specimen type. Score nuclear truncation in tissue, overlapping nuclei, weak or split signals, signal colocalization, and atypical rearrangements with method-specific scoring and interpretive rules. A negative result applies only to abnormalities covered by the probe design.

Copy number, allele fraction, and transcript identity

A copy-number microarray detects gains and losses of DNA. A balanced rearrangement can preserve dosage, so a normal array result can coexist with an abnormal karyotype or targeted FISH result. An array containing single-nucleotide polymorphism probes also assesses allelic differences. Copy-neutral loss of heterozygosity means that a region retains its copy number but loses the distinction between its alleles. In a neoplastic clone, this can leave two copies carrying the same variant.^{22 Shao L, et al. Chromosomal microarray analysis, including constitutional and… 2021.}

Variant allele fraction (VAF) = variant copies ÷ all copies at that locus. VAF is not the percentage of abnormal cells. In a simple diploid mixture, 40 variant-bearing cells among 100 cells contribute 40 of 200 locus copies if each carries one variant copy. The expected VAF is 20%. Two variant copies in each of those cells contribute 80 of 200, or 40%, without changing total copy number. This calculation assumes equal DNA contribution, two locus copies in every cell, and no variant in the remaining cells. Copy-number changes, subclones, and specimen admixture change the relationship. Sequencing alone cannot establish the underlying model.^{22 Shao L, et al. Chromosomal microarray analysis, including constitutional and… 2021.}

Chromosome structure, DNA dosage, and expressed RNA are complementary findings. For a fusion, DNA FISH supports a genomic rearrangement. It does not identify the expressed transcript. A validated reverse-transcription assay supplies that information, so the monitoring assay can target the patient's transcript.^{33 Apperley JF, et al. 2025 European LeukemiaNet recommendations for the management of… 2025.}

Cytogenomic nomenclature

The short chromosome arm is p and the long arm is q. Regions and bands are numbered outward from the centromere. Thus, 9q34.1 identifies chromosome 9, long arm, region 3, band 4, sub-band 1. A karyotype begins with the chromosome count and sex-chromosome complement, followed by abnormalities:

• 46,XX and 46,XY describe 46-chromosome complements with XX or XY sex chromosomes.
• 47,XY,+8 describes a karyotype with an XY complement and trisomy 8.
• 46,XX,t(9;22)(q34.1;q11.2) describes a reciprocal translocation between the stated bands.
• 46,XX,del(5)(q13q33) describes an interstitial deletion between two breakpoints on 5q.
• der(22)t(9;22)(q34.1;q11.2) names the derivative chromosome 22, which retains the chromosome 22 centromere.
• A bracketed number such as [20] records the number of cells carrying the preceding result.

ISCN 2024 is the current standard for karyotype, FISH, chromosomal microarray, and other cytogenomic descriptions.^{44 Hastings RJ, et al. ISCN 2024: An International System for Human Cytogenomic… 2024.} Its print edition has three published errata. The latest was published online in December 2025. Use the corrected online text, or apply all published errata to the print edition.^{5,6,75 Erratum. Cytogenet Genome Res. 2025.6 Erratum. Cytogenet Genome Res. 2025.7 Erratum. Cytogenet Genome Res. 2026.}

Recurrent abnormalities

BCR::ABL1. The classic Philadelphia chromosome is the shortened derivative chromosome 22 created by t(9;22)(q34.1;q11.2). The amino-terminal portion of BCR joins the carboxyl-terminal ABL1 tyrosine kinase domain, producing a constitutively active kinase. The common e13a2 and e14a2 transcripts encode p210, the characteristic CML fusion protein. e1a2 encodes p190 and is common in BCR::ABL1-positive B-lymphoblastic leukemia. e19a2 encodes p230 and occurs in uncommon CML presentations. Each transcript can occur outside its usual association, so classification integrates morphology, immunophenotype, cytogenetics, and molecular findings.

The 2025 European LeukemiaNet recommendations call for bone marrow morphology and chromosome banding plus reverse-transcription PCR on peripheral blood or marrow at CML diagnosis. FISH helps detect cryptic or variant rearrangements. Identifying the transcript type at diagnosis is essential because the monitoring assay must recognize it. For the common e13a2 and e14a2 transcripts, quantitative reverse-transcription PCR or validated reverse-transcription digital PCR results are reported on the BCR::ABL1 International Scale. Major molecular response is 0.1% or lower. A deep molecular response requires the stated ratio and enough reference-gene molecules to support the achieved sensitivity. Atypical transcripts require a validated personalized assay and are reported relative to the patient's baseline because the International Scale does not apply.^{33 Apperley JF, et al. 2025 European LeukemiaNet recommendations for the management of… 2025.}

JAK2. The JAK2 p.V617F variant supports a clonal myeloproliferative neoplasm in the appropriate blood count, marrow, and clinical setting. It occurs in most polycythemia vera cases and in substantial fractions of essential thrombocythemia and primary myelofibrosis. When polycythemia vera is still suspected after a negative V617F result, the next step is validated JAK2 exon 12 testing. Distinguishing polycythemia vera, essential thrombocythemia, and primary myelofibrosis still requires the blood counts, marrow findings, and full diagnostic criteria. Variant allele fraction, detection limit, specimen cellularity, and low-level clonal hematopoiesis affect interpretation.

Acute myeloid leukemia. The fifth-edition WHO classification (WHO5) lists AML types defined by PML::RARA, RUNX1::RUNX1T1, CBFB::MYH11, DEK::NUP214, RBM15::MRTFA, or BCR::ABL1 fusion; rearrangement of KMT2A, MECOM, or NUP98; and mutation of NPM1 or CEBPA. AML with CEBPA mutation includes biallelic mutations or a single mutation in the basic leucine zipper region. Most AML types with defining genetic abnormalities can be diagnosed with fewer than 20% blasts when increased blasts and clinicopathologic correlation show that the abnormality drives the disease. AML with BCR::ABL1 and AML with CEBPA mutation require at least 20% blasts. KMT2A, MECOM, and especially NUP98 rearrangements may be cryptic on a conventional karyotype. AML, myelodysplasia-related requires at least 20% blasts plus specified myelodysplasia-related cytogenetic or molecular abnormalities, a history of a myelodysplastic neoplasm (MDS) or myelodysplastic/myeloproliferative neoplasm (MDS/MPN), or both. Multilineage dysplasia alone no longer establishes the category. The final classification integrates clinical history, morphology, immunophenotype, cytogenetics, and molecular results.^{88 Khoury JD, et al. The 5th edition of the World Health Organization Classification… 2022.}

Clonal hematopoiesis during AML monitoring

A persistent acquired variant does not necessarily indicate residual acute myeloid leukemia (AML). DNMT3A, TET2, and ASXL1 variants can mark clonal hematopoiesis that persists during remission. The 2021 European LeukemiaNet measurable residual disease (MRD) recommendations exclude these variants from AML MRD interpretation. Leukemia-specific targets such as an established NPM1 mutation and validated flow cytometry provide more informative monitoring. Keep the sequencing finding in the report, and explain its limited specificity for residual leukemia. Negative leukemia-specific results are limited by specimen quality and the achieved assay sensitivity.^{99 Heuser M, et al. 2021 update on MRD in acute myeloid leukemia. 2021.}