Client Bulletin Hemepath


1. The Pathline legacy 8-color flow cytometry assay is now phased out and has been updated to an FDA-approved, 10 color analysis for comprehensive leukemia / lymphoma immunophenotyping.

  • Laboratory workflow is locked down per FDA requirements, resulting in end-to-end assay standardization from wet lab processing to data analysis.
  • Accuracy of antigen expression profiling is significantly improved, compared to non-FDA approved 10-color assays that are less standardized. Specifically, antibodies have been redesigned and engineered to minimize nonspecific immunofluorescence, and manual color compensation is no longer required.
  • New markers included in the analysis, e.g., CD200 and CD123, are especially useful for resolution of traditionally difficult differential diagnoses., e.g., atypical CLL versus Mantle Cell lymphoma/leukemia; regenerative hematogone hyperplasia versus residual B-lymphoblastic leukemia; or Hairy cell leukemia versus other mature B-cell neoplasms, among others.
  • TAT will remain unchanged:
    • <24 hours for liquid samples (peripheral blood, marrow aspirate, etc.).
    • <48 hours for tissue samples.
  • CPT coding remains essentially unchanged: 88184, 88185 x 26, 88189.


2. ZAP-70 flow cytometry is now phased out and no longer available.

  • As per NCCN guidelines, ZAP-70 is no longer recommended for routine clinical evaluation of CLL/SLL; ZAP-70 is an imprecise surrogate for IGHV somatic hypermutation status, and discordant results may be identified in up to 28% of cases (NCCN Guidelines, CLL/SLL, Version 2.2022).
  • Direct assessment of IGHV somatic hypermutation status via gold standard Sanger Sequencing is available for order and is performed in-house at Pathline.



3. FLT3 PCR is now available for in-house testing at Pathline.

  • An expedited PCR assay has been validated for in-house testing at Pathline and is approved by the NY State Dept. of Health for analysis of peripheral blood or marrow aspirate samples.
  • This assay targets FLT3 internal tandem duplication (ITD) and tyrosine kinase domain (TKD) mutations in patients with Acute Myeloid Leukemia (AML). Traditional PCR remains a gold standard methodology for most accurate assessment of FLT3 ITD mutant: wild type allele ratio. Furthermore, PCR is the most expedient testing modality available for FLT3 ITD and TKD mutation detection, especially important given that anti-FLT3 targeted therapy must be administered by day +8 of induction chemotherapy in FLT3-mutated AML.
  • In addition to FLT3 PCR, testing for AML patients is also suggested to include conjunctional Next Generation Sequencing (NGS) myeloid mutation profiling (see below).
  • FLT3 PCR will be performed STAT by default. Pathline TAT currently averages <2 days.
  • FLT3 PCR CPT codes: 81245, 81246.


4. RapidPath™ Myeloid NGS is now available.

  • This assay has been validated and approved by the NY State Dept of Health for peripheral blood or marrow aspirate samples submitted for diagnostic evaluation of clonal myeloid neoplasms, e.g., MDS, MPN, AML, MDS/MPN, etc.
  • RapidPath™ Myeloid NGS panel content is aligned to NCCN Guidelines and expert society recommendations.1-5
  • The following 40 genes are targeted for analysis: ABL1, ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DNMT3A, ETV6, EZH2, FLT3, GATA2, HRAS, IDH1, IDH2, IKZF1, JAK2, KIT, KRAS, MPL, MYD88, NF1, NPM1, NRAS, PHF6, PRPF8, PTPN11, RB1, RUNX1, SETBP1, SF3B1, SH2B3, SRSF2, STAG2, TET2, TPT53, U2AF1, WT1, ZRSR2.
  • Legacy, disease specific NGS panels for MPN, MDS, and AML, will be phased out, to provide more up to date and comprehensive coverage of clinically relevant genes explicitly indicated by NCCN guidelines for acute and chronic myeloid neoplasms.


5. RapidPath™ Myeloid NGS (cont.)

  • All orders submitted to Pathline with “Comprehensive Hematopathology Analysis and Report” checked off on the test requisition will undergo RapidPath™ Myeloid NGS, as deemed relevant by an appropriately board-certified hematopathologist / molecular pathologist.
  • For patients with AML, conjunctional testing for both FLT3 PCR and RapidPath™ myeloid NGS is suggested.
  • TAT: 5 days. Majority (95%) of cases are typically released within 2-3 days.
  • CPT coding remains unchanged: 81450.


6. Plasma Cell Isolation for Myeloma FISH is now launched and applied on all cases submitted for workup of plasma cell neoplasms.

  • The practice of plasma cell enrichment for myeloma FISH is highly variable among laboratories in the US, typically performed in only a minority of samples.7
  • To maximize diagnostic sensitivity, plasma cell isolation will be performed at Pathline on all myeloma FISH orders, provided that marrow aspirate volume is adequate.
  • Preferred minimum sample input remains as 1 ml marrow aspirate, although testing may still be attempted with 0.5 ml.
  • TAT for Pathline myeloma FISH with plasma cell isolation remains unchanged: ~2-3 days.
  • CPT coding remains unchanged: 88368, 88377×5.


7. Legacy 1-off mutation testing for JAK2 V617F, CALR, MPL, and/or JAK2 exon 12 analysis, is no longer recommended for evaluation of myeloproliferative neoplasms. Multi-gene NGS panel testing is now strongly recommended.

  • Multi-gene NGS panels provide broader coverage and greater alignment to NCCN guidelines recommendations for clinical evaluation of MPNs. NGS also shows significantly increased analytic sensitivity and specificity compared to traditional methodologies.
  • Compared to traditional Sanger Sequencing, NGS has a significantly lower limit of detection for disease defining JAK2 exon 12 or MPL mutations, i.e., <5% mutant allele frequency using NGS, versus ~20% by Sanger Sequencing. In addition to greater analytic sensitivity, NGS testing is also more specific and can exclude false positive, benign variants in CALR that are otherwise known to confound traditional PCR/fragment analysis results. Pathogenic “Type-1-like” or “Type-2-like” mutations in CALR are also more readily identified by NGS.
  • NCCN guidelines now recommend evaluation for “HMR” (high molecular risk) mutations in MPNs. For example, according to the MIPSS-70 algorithm for prognostic risk stratification in primary myelofibrosis, pathogenic mutations among ASXL1, EZH2, SRSF2, U2AF1, and/or IDH1/2 may be associated with extremely poor clinical outcomes, with 5-year overall survival as low as 7% in high-risk disease (i.e., >2 HMR mutations detected), versus 91% in low-risk disease. Extended mutations detected by NGS are also explicitly recommended in NCCN Guidelines as useful diagnostic adjuncts that can serve as clonal markers of disease in diagnostically challenging cases, e.g., “triple negative MPNs”.3,6
  • Therefore, RapidPath™ Myeloid NGS will be performed at default, for all cases of known or suspected MPN, if “Comprehensive Hematopathology Analysis and Report” is checked off on the requisition.
  • An additional reflex assay, performed entirely by NGS, will also be made available: “MPN NGS reflex”, i.e., JAK2 V617F, if negative, reflex to → CALR, if negative, reflex to → MPL, if negative, reflex → JAK2 exon 12, if negative, → reflex to full NGS myeloid panel.
  • Compared to traditional cascaded testing, TAT for multigene NGS profiling is significantly improved, i.e., <5 days via NGS (traditional testing typically >7-10 days).
  • CPT code for NGS assay testing is 81450. Cost is only marginally increased in comparison to the sum of charges for cascaded 1-off testing using traditional methodologies.



  1. NCCN Guidelines, Myelodysplastic Syndrome. Version 3.2022.
  2. Li et al. Clinical, molecular, and prognostic comparisons between CCUS and lower-risk MDS: a study of 187 molecularly annotated patients. Blood Adv. 2021 Apr 27;5(8):2272-2278. PMID: 33904893
  3. NCCN Guidelines, Myeloproliferative Neoplasms. Version 1.2022.
  4. NCCN Guidelines, Acute Myeloid Leukemia. Version 1.2022.
  5. McClure et al. Clinical Significance of DNA Variants in Chronic Myeloid Neoplasms: A Report of the Association for Molecular Pathology. J Mol Diagn 2018 Nov;20(6):717-737. PMID: 30138727.
  6. Tefferi et al. MIPSS70: Mutation-Enhanced International Prognostic Score System for Transplantation-Age Patients with Primary Myelofibrosis. J Clin Oncol. 2018 Feb 1;36(4):310-318.
  7. Yu et al. Variability in Cytogenetic Testing for Multiple Myeloma: A Comprehensive Analysis from Across the United States. JCO Oncol Pract 2020 Oct;16(10):e1169-e1180. PMI