Laboratory Techniques

Flow cytometry: principles and gating

• Principle: detection of cell-surface or cytoplasmic proteins using fluorescent-labeled monoclonal antibodies, plus assessment of DNA content using DNA-binding dyes. Used to phenotype cell populations and to enumerate early progenitors for stem cell work.
• Gating identifies the cells of interest within a mixed population. The three major leukocyte populations (lymphocytes, monocytes, neutrophils) can be defined by light scatter:
  • Forward scatter (FS, low angle): measures cell size.
  • Side scatter (SS): measures internal granularity.
  • Lymphocytes have the lowest FS and SS; monocytes are intermediate; neutrophils have high SS with slightly lower FS.
• SS vs CD45 gating (the most common method): cells separate by staining intensity (bright vs dim):
  • Lymphocytes: bright CD45, low SS.
  • Neutrophils: dim to moderately bright CD45, high SS.
  • Monocytes: bright CD45, intermediate SS.
  • Blasts: low SS with dim-to-negative CD45 (dimmer CD45 is more typical of lymphoid-lineage blasts).
• NK cells: express CD2, CD7, CD16, CD56, with variable CD57 and CD8; they do NOT express CD3, which distinguishes them from T cells.
• ZAP-70 in CLL: a prognostic marker. ZAP-70 positivity correlates strongly with unmutated IgVh (CLL from pre-germinal-center cells) and decreased overall survival. CD38 positivity also correlates with unmutated IgVh but less strongly than ZAP-70.

Flow cytometry: lineage maturation markers

• Progenitors: uncommitted hematopoietic progenitors are CD34+ and CD38-negative; acquisition of CD38 marks lineage commitment.
• Myeloid maturation: CFU-GEMM (CD34+, MHC class II+, CD33 variable), then CFU-GM (CD34+, MHC class II+, CD33+, CD13 variable, CD15 variable). Neutrophil precursors progressively lose MHC class II and CD33 and gain CD11b, CD16, CD32. Monocytes retain MHC class II and CD33 and gain CD14 and CD64.
• Erythroid maturation: gain of CD71 with loss of CD34 and CD33 and decreased CD45. With further differentiation, CD71 decreases, glycophorin increases, and CD45 disappears.
• Megakaryocytic differentiation: expression of GPIIb (CD41); GPIIIa (CD61) rises as CD34 falls; GPIb (CD42b) at the promegakaryocyte stage; GPV (CD42d) rises with differentiation. Differential CD41/CD42b/CD61 expression helps diagnose Glanzmann thrombasthenia and Bernard-Soulier syndrome.
• B-cell differentiation: early precursors coexpress CD19 and CD10; as B cells mature they lose CD34 and CD10 and express surface IgM with mature markers (CD20, CD21, CD22, CD79b). Mature B cells express an Ig heavy chain and either a κ or λ light chain. Predominant expression of one light-chain type (light-chain restriction) indicates a monoclonal process.
• T-cell differentiation: precursors express TdT, HLA-DR, CD34; then cytoplasmic CD3 and CD7, followed by CD2 and CD5. Common thymocytes also express CD1a, CD4, CD8. Mature helper cells: CD2, CD3, CD4, CD5 (+/- CD7); mature cytotoxic cells: CD2, CD3, CD5, CD8 (+/- CD7). Aberrant T-cell antigen patterns can flag T-cell neoplasms.

Flow cytometry: red cell and PNH applications

• PNH: PNH cells lack GPI-anchored membrane proteins, including two complement regulators, CD55 (DAF) and CD59 (MIRL). Their absence on RBCs is detected with anti-CD55 and anti-CD59. PNH granulocytes are detected by absent GPI-anchor binding using FLAER (an Alexa-488-labeled aerolysin variant), the most sensitive approach.
• Other RBC uses: flow can discriminate fetal from adult RBCs and RhD-positive from RhD-negative RBCs in pregnancy and postpartum, and can identify RBC cytoskeletal disorders such as hereditary spherocytosis using eosin-5-maleimide (EMA) binding.

Complete blood count (CBC) interpretation

• Hemoglobin / hematocrit: Hb (g/dL) measures total oxygen-carrying capacity; Hct (%) is the RBC volume fraction. Hct is approximately 3 × Hb in normal RBCs.
• MCV (mean corpuscular volume): average RBC size. Normal 80 to 100 fL.
• MCH and MCHC: MCH: mean Hb mass per RBC (pg). MCHC: mean Hb concentration in RBCs (g/dL; Hb/Hct). ↑ MCHC is seen in hereditary spherocytosis and AIHA (concentration rises in dehydrated cells).
• RDW (red cell distribution width): a measure of anisocytosis. ↑ early in iron deficiency, B12/folate deficiency, post-transfusion, and MDS.
• Reticulocyte count: raw count or absolute reticulocyte count (ARC). Corrected retic % = retic % × (patient Hct / 45). The reticulocyte production index (RPI) accounts for shift cells in severe anemia.
• Platelet indices: MPV (mean platelet volume) is ↑ in destructive thrombocytopenia (ITP, TTP), Bernard-Soulier (giant platelets), and MYH9-related disease; ↓ in marrow failure and Wiskott-Aldrich (small platelets).

Peripheral blood smear: red cell clues

• Schistocytes (helmet cells, fragments): microangiopathic hemolytic anemia (TTP, HUS, DIC, HELLP), mechanical valve, severe hypertension.
• Spherocytes: hereditary spherocytosis, warm AIHA, ABO hemolytic disease of the newborn.
• Sickle cells, target cells: sickle cells in HbS sickling disorders (HbSS, HbSC); target cells in SCD, HbC, thalassemia, liver disease, post-splenectomy.
• Bite cells / blister cells with Heinz bodies: G6PD oxidative hemolysis.
• Tear-drop cells (dacrocytes) with leukoerythroblastic picture: marrow infiltration or myelofibrosis.
• Howell-Jolly bodies (nuclear remnants): hyposplenism (SCD, post-splenectomy, celiac).
• Heinz bodies: oxidative damage on supravital staining (G6PD, unstable Hb).
• Basophilic stippling: lead poisoning, thalassemia, sideroblastic anemia.
• Pappenheimer bodies (iron granules): sideroblastic anemia, post-splenectomy.
• Rouleaux: ↑ ESR, plasma cell dyscrasia, infection.
• Spur cells (acanthocytes): severe liver disease, abetalipoproteinemia.
• Echinocytes (burr cells): uremia, pyruvate kinase deficiency, artifact.
• Polychromasia: reticulocytosis, suggesting recent hemorrhage or hemolysis.
• nRBCs in adults: marrow stress, infiltration, myelofibrosis, severe hemolysis.

Peripheral blood smear: white cell clues

• Hypersegmented neutrophils (≥6 lobes, or >5% with 5 lobes): B12 or folate deficiency.
• Pelger-Huet cells (bilobed neutrophils): MDS (acquired pseudo-Pelger-Huet), congenital LBR mutation; can mimic post-chemotherapy changes.
• Toxic granulation, Dohle bodies, vacuolization: severe infection or sepsis.
• Auer rods: AML (especially APL, with multiple Auer rods or "faggot cells").
• Smudge cells: CLL (fragile lymphocytes).
• Hairy cells (cytoplasmic projections): hairy cell leukemia.
• Reactive lymphocytes (Downey cells): EBV, CMV, other viral infections.
• Atypical lymphocytes / Sezary cells (cerebriform nuclei): cutaneous T-cell lymphoma / Sezary syndrome.
• Plasmacytoid lymphocytes: lymphoplasmacytic lymphoma / Waldenstrom macroglobulinemia.
• Blasts: leukemia (≥20% in blood or marrow is diagnostic; lower thresholds apply to AML with defining genetic abnormalities: no cutoff for most per WHO 2022, ≥10% per ICC 2022 for most; BCR::ABL1 still requires ≥20%; TP53 or MDS-related with 10 to 19% blasts = MDS/AML).

Coagulation testing

• PT/INR: extrinsic plus common pathway. ↑ in factor VII, X, V, II deficiency; warfarin; vitamin K deficiency; early liver disease.
• aPTT: intrinsic plus common pathway. ↑ in deficiency of XII (no bleeding), XI, IX, VIII, X, V, II; unfractionated heparin; severe vWD; lupus anticoagulant.
• Mixing study: a 1:1 mix of patient and normal pooled plasma; if PT/aPTT corrects on the immediate 1:1 mix it suggests factor deficiency, but add an incubated mix (~2 h at 37C): a time-dependent factor VIII inhibitor (acquired hemophilia A) can correct immediately yet fail after incubation, if it does NOT correct it is an inhibitor (lupus anticoagulant or a factor inhibitor).
• Thrombin time (TT): prolonged with heparin contamination, dysfibrinogenemia, hypofibrinogenemia, or a direct thrombin inhibitor.
• Reptilase time: normal with heparin contamination (heparin-resistant) but prolonged in dysfibrinogenemia, distinguishing the two.
• Fibrinogen (Clauss): <100 mg/dL in DIC, severe liver disease, hypofibrinogenemia.
• D-dimer: cross-linked fibrin breakdown product. ↑ in DVT/PE, DIC, malignancy, infection, pregnancy; high negative predictive value in low pretest probability.
• Anti-Xa level: monitors LMWH and fondaparinux; some labs use it for unfractionated heparin (vs aPTT).
• TEG / ROTEM (viscoelastic): real-time clot formation and strength; used in trauma, cardiac surgery, and liver transplant.
• Specific factor assays: confirm a deficiency identified by a mixing study.
• Bethesda assay: quantifies factor inhibitor titer (in Bethesda units).

Direct antiglobulin test (DAT / Coombs)

• Direct test (DAT): detects IgG and/or C3 already on patient RBCs. Positive in autoimmune hemolytic anemia.
• Indirect test (IAT): detects circulating anti-RBC antibodies in plasma; used in transfusion crossmatch and prenatal screening.
• IgG-only DAT+: warm AIHA pattern.
• C3-only DAT+: cold agglutinin disease pattern.
• IgG + C3: common warm AIHA pattern (or drug-induced); mixed AIHA only if a clinically significant cold agglutinin is also present.
• DAT-negative AIHA (~5 to 10%): low-titer IgG, IgA-mediated, or IgM-mediated; consider alternative testing.

Specialized hematology testing

• Hemoglobin electrophoresis / HPLC: identifies and quantifies HbA, HbA2, HbF, HbS, HbC, HbE, HbH.
• Flow cytometry (PNH): FLAER on granulocytes (most sensitive); CD55/CD59 deficiency on RBCs and granulocytes; can detect clones as small as >0.01%.
• Osmotic fragility / EMA-binding flow: hereditary spherocytosis (EMA is more sensitive).
• Ham test, sucrose lysis: historical tests for PNH (replaced by FLAER).
• Donath-Landsteiner test: paroxysmal cold hemoglobinuria (biphasic IgG anti-P).
• ADAMTS13 activity and inhibitor: TTP diagnosis (activity <10%).
• FISH / cytogenetics: heme malignancy diagnosis (CML t(9;22), APL t(15;17), etc.).
• NGS panels: somatic mutations in heme malignancy; germline testing for inherited bone marrow failure and hereditary thrombophilia.
• BCR::ABL1 quantitative PCR (IS): CML monitoring on the international standard scale.
• Flow MRD (~10 to the minus 4 conventional, 10 to the minus 5 to 10 to the minus 6 with next-generation flow), NGS clonoSEQ MRD (10 to the minus 6): leukemia and myeloma minimal residual disease.
• Ristocetin cofactor assay (vWF:RCo) / GPIbM: vWF activity.
• Light transmission aggregometry (LTA): platelet function testing.
• JAK2 V617F, CALR, MPL: MPN driver testing.
• BM aspirate + biopsy + flow + cytogenetics + molecular: cornerstone of the heme malignancy workup.

High yield

• Pseudothrombocytopenia: EDTA-induced platelet clumping causes a falsely low platelet count. Repeat in a citrate or heparin tube; the smear shows clumps.
• Mixing study that does NOT correct: an inhibitor. Differential: lupus anticoagulant (paradoxical thrombosis) vs acquired factor VIII inhibitor (acquired hemophilia A, bleeding).
• Heparin contamination: suspect if TT is prolonged but reptilase time is normal.
• Lupus anticoagulant paradox: prolongs aPTT in vitro but causes thrombosis in vivo.
• Severe bleeding with normal PT/aPTT: consider factor XIII deficiency, alpha-2-antiplasmin deficiency, dysfibrinogenemia, or platelet dysfunction.
• Hb electrophoresis pearls: β-thalassemia trait shows ↑ HbA2 (>3.5%); HbH disease shows ↑ HbH (β4); sickle trait shows an HbAS pattern; HbS/β0-thalassemia mimics HbSS.
• Light-chain restriction on flow (predominant κ or λ) indicates a monoclonal B-cell process.