Demystifying the Blood Drop A Hematopathology Primer on Peripheral Diagnostics
It appears your patient has a hematologic malignancy.
The CBC screams trouble... severe anemia, thrombocytopenia, and blasts. Immediate blood tests can start answering the urgent questions while the bone marrow biopsy is under way.
In previous Morning Reports, authors walked through bone marrow and blood smear diagnostics. Here, we continue with the blood draw, specifically stat flow cytometry and Karyotyping/FISH.1 These tests provide provisional diagnoses, guide therapy, and let the hematopathologist triage test results efficiently.
The Basics
The laboratory performs four complementary analyses. Together they reveal morphology, immunophenotype, chromosomal architecture, and nucleotide-level lesions... potentially before the bone-marrow biopsy is processed!
| Modality | What It Answers | Typical Turn-Around* |
| Peripheral Smear | Are counts abnormal? Blasts or morphologic dysplasia? |
Instrument = 30 min Manual smear = 2h |
| Flow Cytometry | What lineages are present? Are normal or aberrant antigens present? Is MRD detectable? |
Same day = 4 to 8h |
| FISH/Karyotype | Are there hallmark translocations or aneuploidies? | FISH = 24 to 4h kKaryotype = 5 to 7d |
| PCR/NGS Panel | Which mutations or fusion transcripts are present? | RT-PCR = 4 to 6h NGS = 7 to 14d |
*Times vary by laboratory/blood draw timing but reflect common benchmarks.
Flow Cytometry (FC)
If the CBC & smear suggest blasts, FC provides rapid immunophenotyping and identifies cells that appear morphologically similar.2 This process involves lysing RBCs, staining leukocytes with 8 to 12 fluorochrome-conjugated antibodies, and acquiring data for ~200,000 cells on a cytometer.3 Dot-plot or histogram analysis identifies cell lineage, maturation, and aberrant antigen patterns. Depending on the timing of the blood draw, turn-around is typically 6-8 hours for STAT leukemia panels and ≤24 hours for comprehensive phenotyping. FC requires viable, unfixed cells in suspension and therefore provides no architectural context.
Figure 2. Two-parameter FACS plots.
A) CD45 vs. side-scatter (SSC) separating blasts (blue) & lymphocytes (red).
B) Myeloid panel showing CD34⁺/CD117⁺ blasts.
FC rapidly answers critical questions including:
- Lineage & maturation stage: distinguishes myeloid vs. lymphoid blasts, or clonally restricted B cells.
- Aberrant antigen expression: guide targeted therapy (e.g., CD33 for gemtuzumab).
- Minimal residual disease (MRD): detect leukemic cells down to 0.01%.
Quick tip: Low blood volume and prolonged transit are common causes of “insufficient event” reports and repeat phlebotomy.
FISH and Karyotype
Confirmation of abnormal blasts by FC leads to questions about chromosome appearance and underlying drivers of disease. Two assays provide answers at different magnifications:
- Conventional karyotype involves culturing leukocytes for 24 to 48 h, halting the cells in metaphase, G-banding the spreads, and assembling a 24-panel karyogram. This allows for viewing of balanced translocations, aneuploidy, and complex genomes (CMLs, many ALLs, constitutional studies)4
- Fluorescence in situ hybridization (FISH) skips culture entirely. The buffy coat is fixed to slides, fluorescent probes hybridize overnight, and dual-color signals light up target lesions (myeloid, CLL, myeloma)5
Figure 3. Cytogenetic and FISH detection of the BCR:ABL1 translocation
A) G-banded metaphase showing the Philadelphia chromosome t(9;22).
B) Dual-fusion BCR::ABL1 FISH in interphase nuclei.
Image credit: Dr. Lina Shao, MD, PhD
Why order both?
- FISH can quickly confirm a cryptic t(15;17) within hours, allowing immediate ATRA in APL, while karyotype reveals additional abnormalities that may up-stage risk.
- Karyotype may uncover unexpected anomalies that a limited FISH panel would miss (e.g., complex monosomal karyotype in AML).
Quick tip: Send an extra 3-5 mL of heparinized blood if CML/AAL is suspected to ensure enough metaphases are collected... mitotic yield drops sharply after 24 hours!
Summary
A single blood draw can provide a detailed work-up for suspected hematologic malignancy. The peripheral blood smear offers an immediate morphologic snapshot; flow cytometry adds rapid immunophenotyping within hours; FISH and karyotype map chromosomal architecture in one to seven days; and PCR/NGS pinpoint driver mutations that influence risk stratification and targeted therapy. These modalities compensate for one another’s blind spots and can yield a provisional diagnosis.
- Koutsi A, Vervesou EC. Diagnostic molecular techniques in haematology: recent advances. Ann Transl Med. 2018 Jun;6(12):242. doi: 10.21037/atm.2018.05.30. PMID: 30069444; PMCID: PMC6046288.
- Fang, H., Wang, S.A., Hu, S., Konoplev, S.N., Mo, H., Liu, W., Zuo, Z., Xu, J., Jorgensen, J.L., Yin, C.C., El Hussein, S., Jelloul, F.Z., Tang, Z., Medeiros, L.J., Wang, W., 2022. Acute promyelocytic leukemia: Immunophenotype and differential diagnosis by flow cytometry. Cytometry Part B: Clinical Cytometry 102, 283–291.. https://doi.org/10.1002/cyto.b.22085
- Betters DM. Use of Flow Cytometry in Clinical Practice. J Adv Pract Oncol. 2015 Sep-Oct;6(5):435-40. doi: 10.6004/jadpro.2015.6.5.4. Epub 2015 Sep 1. PMID: 27069736; PMCID: PMC4803461.
- Ozkan E, Lacerda MP. Genetics, Cytogenetic Testing And Conventional Karyotype. In: StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2025 Jan-. Available from: https://www.ncbi.nlm.nih.gov/books/NBK563293/
- 5. Gonzales PR, Mikhail FM. Diagnostic and Prognostic Utility of Fluorescence In situ Hybridization (FISH) Analysis in Acute Myeloid Leukemia. Curr Hematol Malig Rep. 2017 Dec;12(6):568-573. doi: 10.1007/s11899-017-0426-6. PMID: 29064023.