27 Laboratory Hematology
- CBC w/ differential: screening via automated counters & microscopy
- Flow cytometry: diagnose malignancies & monitor residual disease
- Cytogenetics/molecular: classify neoplasms (sensitivity vs. cost tradeoff)
- Coagulation tests: PT/aPTT limited for bleeding disorders
- Viscoelastic/platelet tests: guide transfusion in OR/trauma; poorly standardized
27.1 Introduction
- Clinical correlation essential: hematology tests require specific clinical context
- Error sources: preanalytical (collection/processing), analytical (test performance), postanalytical (interpretation)
- Common errors: clotted/hemolyzed samples, wrong anticoagulant, mislabeled specimens
27.2 Collection Tubes
| Stopper | Anticoagulant | Use | Notes |
|---|---|---|---|
| Purple | EDTA | CBC/differential | NOT for coagulation—chelates Ca²⁺ |
| Blue | Na citrate | PT, aPTT, fibrinogen, D-dimer | Fill volume critical |
| Yellow | ACD | Special studies | Promotes cell survival |
| Red | Clot activator | Serum chemistry | Fast turnaround |
| Green | Heparin | Plasma studies | Faster results |
27.3 Automated Blood Cell Counting
- CBC & 5-part differential: WBC, RBC, Hgb, Hct, plts + neutrophils, lymphs, monos, eos, basos
- Advanced parameters: reticulocytes, NRBCs, immature granulocytes, MPV
- Methods: impedance (cell size), optical scatter (morphology)
- Operator flags: alert for abnormalities requiring smear review
| Parameter | Normal | Unit |
|---|---|---|
| WBC | 4.8 | 10³/μL |
| RBC | 4.5 | 10⁶/μL |
| Hgb | 13.1 | g/dL |
| Hct | 39.9 | % |
| MCV | 88.6 | fL |
| MCH | 31.6 | pg |
| MCHC | 35.6 | g/dL |
| Plt | 173 | 10³/μL |
| Retic | 0.5-2% | % |
27.4 Reticulocytes & RBC Analysis
- Reticulocytes: immature RBCs w/ residual RNA (normal 1-2%)
- Flow cytometry most accurate
- Reflect marrow erythropoietic response
- RBC indices: MCV, MCH, MCHC, RDW
- Affected by: agglutination, hyperglycemia, leukocytosis
- RDW ↑ w/ ↑ variability
27.5 WBC Differential & Platelet Count
- WBC differential: automated impedance/optical ± flow cytometry
- Relative & absolute counts
- Manual review needed (~200 cells) for verification
- Digital microscopy (CellaVision, Scopio) improves classification
- Platelet enumeration: impedance w/ volume
- Giant plts/clumping → false ↑↑ (8-10× actual)
- RBC fragments misclassified
- MPV: correlate w/ destruction vs. underproduction
- EDTA artifact → recollect in different tube
27.6 Peripheral Blood Smear
- Technique: Wright or May-Grunwald-Giemsa stain
- Scanning (×10): platelet clumps, microaggregates
- Morphology (×100, oil): cell type, abnormalities
27.7 Bone Marrow Aspirate & Biopsy
- Indications: unexplained cytopenia, leukocytosis, staging lymphoma/tumors, infection assessment
- Site: posterior iliac crest (infants: anterior tibia)
- Quality: adequate spicules from perpendicular area
- Flags from analyzers → real-time smear review
- Manual differential limited (n~200 cells); smear verification essential
- Platelet count artifact: EDTA → recollect w/ different anticoagulant
27.8 RBC Morphology
| Abnormality | Appearance | Cause | Association |
|---|---|---|---|
| Schistocytes | Fragmented, 2-3 points | Mechanical trauma | MAHA, DIC, TTP, HUS |
| Spherocytes | Dense, small, round | Membrane loss | HS, AIHA |
| Target cells | Bull’s-eye | ↑ membrane redundancy | Thalassemia, liver dz, HTN |
| Acanthocytes | Irregular spicules | Lipid imbalance | Liver dz, abetalipoproteinemia |
| Ovalocytes | Elliptical | Cytoskeleton defect | Hereditary elliptocytosis |
| Howell-Jolly | Small blue inclusion | Nuclear remnant | Asplenia, HA, MDS |
| Basophilic stippling | Coarse punctate | Ribosomes | Lead toxicity, thalassemia |
| Sickle cells | Pointed crescent | HbS aggregation | Sickle cell dz (not trait) |
| Stomatocyte | Mouth-like cleft | Cation defect | Hereditary stomatocytosis |
27.9 Schistocytes
- Importance: diagnose MAHA (TTP, HUS, DIC, transplant-related)
- Modern analyzers: RBC fragment alerts assist smear review
- Reporting: >1% schistocytes → biotic microaggregate (verify lab threshold)
27.10 Nucleated RBCs (NRBCs)
- Abnormal beyond newborn period: stress or marrow compromise
- Associated w/: hemolytic anemia, myelodysplasia
27.11 Hemostasis Testing Strategy
| Category | Test | Use |
|---|---|---|
| Screening | CBC/diff, PT, aPTT, fibrinogen | Initial assess |
| Diagnostic | Thrombin time, plt aggregation, secretion | Specific bleeding disorder |
| Monitoring | PT/INR (warfarin), aPTT (heparin), TEG, ROTEM | Anticoagulation adequacy |
27.12 Flow Cytometry
- Analysis: single cells by forward/side scatter
- FS ≈ cell size
- SS ≈ complexity (granularity)
- Lymphs (low), monos (intermediate), neutrophils (high)
- Detection: fluorescent-labeled monoclonal Ab (CD45, etc.)
- Uses: stem cells, residual disease, fetal vs. adult RBCs
27.12.1 Specimen Allocation
| Problem | Techniques |
|---|---|
| Pancytopenia | FC (LGL, hairy cell, PNH, MDS, AML); cyto (AML, MDS); molecular |
| AML | FC (phenotyping, MRD); cyto/FISH (classification); NGS |
| Lymphoproliferation | FC (phenotyping, MRD); cyto/FISH; IHC |
| MPN | Cyto/FISH (diagnosis); molecular (diagnosis, prognosis) |
| Plasma cell | FC, IHC, FISH; molecular |
27.13 Hemostasis Testing
- Primary hemostasis: platelet aggregation
- Secondary hemostasis: coagulation cascade (factors)
27.13.1 Prothrombin Time (PT)
- Measures: extrinsic & common pathways (TF → fibrin)
- Monitors: warfarin (reported as INR)
- INR: standardizes monitoring across labs & reagents
27.13.2 Activated Partial Thromboplastin Time (aPTT)
- Measures: intrinsic & common pathways (contact factors, F IX → fibrin)
- Prolonged aPTT: factor deficiency (F VIII, IX, XI, XII), inhibitors, anticoagulants
- F XII/PK/HMWK: rare, aPTT >100 sec, no bleeding
- F VIII/IX: X-linked (hemophilia A/B), normal <40 IU/dL
- F XI: Ashkenazi Jewish, variable bleeding risk
- Threshold: ~30-40 IU/dL for clinical effect
PT variability w/ reagent → INR standardizes warfarin monitoring
27.13.3 Thrombin Time (TT)
- Measures: fibrinogen functionality (exogenous thrombin → fibrin)
- Affected by: fibrinogen abnormality, heparin, FDP, direct thrombin inhibitors
27.13.4 DOAC Interference on Coagulation Tests
| Test | Factor Xa Inhibitor | Factor IIa Inhibitor | Note |
|---|---|---|---|
| PT/aPTT | Unaffected | + Bias | Risk missing AT deficiency |
| Anti-Xa | + Bias | Unaffected | Risk missing AT deficiency |
| Anti-IIa | Unaffected | + Bias | Risk missing AT deficiency |
| F V, VIII, II | – Bias | – Bias | Inhibitor pattern |
| LAC | Abnormal | Abnormal | False LAC |
| PC/PS | + Bias | + Bias | False deficiency |
| 1:1 mix (PT/aPTT) | Prolonged | Prolonged | Inhibitor pattern |
| TT | Prolonged | Unaffected | |
| Fibrinogen | – Bias | Unaffected | Method-dependent |
27.14 Specialized Coagulation Tests
Viscoelastic Tests (VETs): - Utility: limited for bleeding disorder diagnosis - Use: guide transfusion in OR/trauma - Methods: TEG, ROTEM measure kinetics (fibrin formation, thrombin generation, fibrinolysis)
Platelet Function Tests: - Challenge: complex, require fresh samples, poorly standardized - PFA-100/200: high-shear primary hemostasis - Collagen-coated aperture (ADP or epinephrine) - Closure time (CT) = platelet adhesion - Indications: vWD, mild-to-moderate bleeding - Studies: aggregation, secretion, genetic testing
27.15 Hemoglobin Electrophoresis
- Methods: separate normal (A, A₂, F) & abnormal Hb by charge difference
- Isoelectric focusing (IF)
- High-performance liquid chromatography (HPLC) ← most effective
- Capillary electrophoresis
- Alkaline gel: normal Hb & most abnormal
- Acid gel: separates specific variants
- Application: Hb S vs. C, thalassemia screening
- HPLC & capillary electrophoresis most effective for Hb variants
- α-thalassemia & immunohemoglobinopathies: different electrophoretic patterns
27.16 G6PD Testing
- Indication: nonimmune hemolytic anemia
- Methods: dye decolorization, fluorescent spot (NADPH-dependent)
- Use: prevent oxidant hemolysis (areas w/ ↑ prevalence)
27.17 Hereditary RBC Disorders
- RBC flexibility: lipid bilayer + α₂-spectrin scaffold
- Osmotic fragility ↑, blood viscosity altered
- Ethnic variation: inheritance patterns & prevalence vary
27.18 ESR
- Use: assess systemic inflammation (limited utility)
- Avoid: asymptomatic screening
- Reference ranges: ↑ w/ age, especially women
| Stain | Positive Cells | Use |
|---|---|---|
| MPO | Neutrophil primary, eosinophil secondary, monocyte lysosomal | Myeloid differentiation |
| Naphthol AS-D | Neutrophil, eosinophil | Monocytic/granular diff |
| ACP/TRAP | Intracellular glycogen, mucopolysaccharides | ALL, Gaucher cells |
| PAS | Glycogen, neutral mucopolysaccharides | ALL, not AML |
| Iron (Prussian blue) | Hemosiderin in NRBC (sideroblasts) | Sideroblastic anemia |
| GMS | Fungal organisms | Fungal ID |
| Acid-fast (Ziehl-Neelsen) | Mycolic acid-rich (mycobacteria) | TB, MAC |
27.19 Immunohistochemical Stains
- IHC: enzyme-linked antigen-Ab detection on biopsies/tissue
- Advantages: alternative to FC; can phenotype undifferentiated tumors
- Applications: blast enumeration, lymphoproliferation, small infiltrates
- Tissues: lymph node, BM biopsy, other tissues
- Specific Ab: EBV, CMV
27.20 Bone Marrow Sample Prep
- EDTA: FC & molecular analysis
- Heparin: cytogenetics
- Paraffin-embedded: IHC
27.21 Cytogenetics
Conventional Cytogenetics: - Method: mitotic cells, chromosomal banding patterns - Clone: ≥2 metaphase cells w/ identical abnormality - TAT: 4-10 days (need dividing cells) - Limitation: difficult to detect clones <5-10 cells
FISH (Fluorescence In Situ Hybridization): - Method: fluorescent-labeled DNA probes on interphase nuclei - Advantage: faster, narrow regions of interest - Limitation: fixed specimen only
Chromosome Microarray: - Method: fluorescent oligonucleotide probes - Detects: copy number variations, copy-neutral rearrangements - Limitation: cannot detect balanced rearrangements - Use: BM & tissue biopsies
27.22 Bibliography
Hereditary Thrombocytopenias
Norn P, Pecci A. Hereditary thrombocytopenias: a growing list of disorders. Hematology. 2017;2017(1):385-399.
Nurden AT, Nurden P. Inherited thrombocytopenias: history, advances and perspectives. Haematologica. 2010;95(5):839-3819.
Pecci A, Balduini CL. Inherited thrombocytopenias: an update. Hematology. 2013;48:1984.
Acquired Disorders of Platelet Function
Abrams CS, Shattil SJ, Brewer JS. Acquired qualitative platelet disorders. In: Kaushansky K, Lichtman MA, Beutler E, Kipps TJ, Seligsohn UI, Prchal JT, eds. Williams Hematology. 8th ed. McGraw-Hill; 2010:1971-1991.
Díaz-González F, López JA. Acquired disorders of platelet function. Semin Hematol. 2013;50:S5-S12.
Wisloff F, Amundsen E, Duckert F, Malasky M, et al. Laboratory hemostasis approach to diagnosis of mild von Willebrand disease: validation method for test reference samples and characterization based on clinical assessment before and after clinical diagnosis*