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Laboratory Investigations of Myocardial Infarction

Diagnostic Framework

• Symptoms of myocardial ischemia
• New ischemic ECG changes
• Development of pathologic Q waves
• Imaging evidence of new loss of viable myocardium or new regional wall motion abnormality
• Identification of a coronary thrombus by angiography or autopsy (Type 1 MI)

Symptom to Diagnosis: An Evidence-Based Guide, 4th Ed. | Harrison's Principles of Internal Medicine, 22nd Ed.

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1. Cardiac Troponins (Primary Biomarker)

Biochemistry

• Troponin C - calcium-binding subunit (not cardiac-specific - same isoform as slow-twitch skeletal muscle)
• Troponin I (cTnI) - inhibitory subunit; cardiac-specific due to a unique 31-amino-acid N-terminal extension not present in skeletal muscle isoforms
• Troponin T (cTnT) - tropomyosin-binding subunit; cardiac-specific due to an 11-amino-acid amino-terminal residue

Tietz Textbook of Laboratory Medicine, 7th Ed.

Release Mechanism

Kinetics

• cTnI and cTnT are normally undetectable in serum
• Persist elevated for 7-10 days, allowing diagnosis even when presentation is delayed
• Serial measurements over 3-6 hours improve sensitivity for early presentations

Robbins & Kumar Basic Pathology | Robbins, Cotran & Kumar Pathologic Basis of Disease

Threshold

• Diagnosis requires at least one value above the 99th percentile URL of a healthy reference population
• Modern high-sensitivity cTn (hs-cTn) assays allow detection at much lower concentrations, enabling 0h/1h or 0h/2h rapid rule-out protocols
• Sex-specific URL values are recommended (women have lower 99th percentile thresholds)

Tietz Textbook of Laboratory Medicine, 7th Ed. | Harrison's Principles, 22nd Ed.

Causes of Troponin Elevation Other Than MI

• Myocarditis, myocardial trauma
• Heart failure, pulmonary embolism
• Renal failure (reduced clearance), sepsis
• These causes do not follow the characteristic rise-and-fall pattern of acute MI

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2. Creatine Kinase - MB Isoform (CK-MB)

• No longer recommended as the primary marker for initial diagnosis of NSTEMI (Washington Manual of Medical Therapeutics)
• May still be used to detect reinfarction (since troponin remains elevated for 7-10 days, CK-MB's shorter window makes a second rise detectable)
• Occasionally used in perioperative settings (e.g., post-cardiac surgery)

Washington Manual of Medical Therapeutics | Robbins & Kumar Basic Pathology

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3. Myoglobin

• A heme-containing oxygen-binding protein found in both cardiac and skeletal muscle
• Earliest to rise after MI onset (detectable within 1-3 hours)
• Returns to normal within 24 hours - shortest window
• Very low specificity - released by any muscle injury (skeletal, cardiac)
• Not recommended as a standalone diagnostic test but historically used as an early sensitive marker
• Useful as a negative predictor if levels are normal very early after symptom onset

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4. Other Routine Lab Investigations

Washington Manual of Medical Therapeutics

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5. BNP / NT-proBNP (Natriuretic Peptides)

• BNP (B-type natriuretic peptide) is a biomarker of myocardial wall stress and ventricular dysfunction
• Elevated BNP in the setting of ACS indicates:
  • Larger infarction size
  • Worse outcomes / higher mortality
  • Possible large infarction requiring urgent angiography (if severely elevated in a patient without known HF)
• Aids in distinguishing cardiac vs. pulmonary cause of dyspnea

Washington Manual of Medical Therapeutics | Tietz Textbook of Laboratory Medicine, 7th Ed.

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6. Universal MI Classification and Biomarker Thresholds

Harrison's Principles of Internal Medicine, 22nd Ed. | Sabiston Textbook of Surgery

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Summary Table: Biomarker Kinetics

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Robbins, Cotran & Kumar Pathologic Basis of Disease | Tietz Textbook of Laboratory Medicine, 7th Ed. | Symptom to Diagnosis: An Evidence-Based Guide, 4th Ed. | Washington Manual of Medical Therapeutics

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Functional Tumors of the Ovary

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Classification

Berek & Novak's Gynecology, Table 39-8

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1. Granulosa Cell Tumors (GCT)

Overview

• Most common type of malignant sex cord-stromal tumor
• Account for 2-5% of all ovarian malignancies
• Considered low-grade malignancies
• Almost always unilateral (bilateral in only 2%)
• Tumors that are hormonally active show yellow coloration on cut surface (due to intracellular lipids)

Subtypes

Histology - Call-Exner Bodies (Pathognomonic)

Hormone Secretion & Clinical Effects

• Prepubertal girls: isosexual precocious puberty (breast development, premature menarche)
• Reproductive age women: menstrual irregularities, secondary amenorrhea, infertility
• Postmenopausal women: abnormal uterine bleeding (most common presenting symptom)
• Endometrial effects: cystic hyperplasia → endometrial hyperplasia → endometrial carcinoma in 10-15%; low-grade endometrial cancer co-exists in at least 5% of adult GCTs; endometrial hyperplasia in 25-50%
• Proliferative breast disease

Tumor Markers

• Inhibin A and B - secreted by granulosa cells; inhibin B more frequently elevated. Useful for:
  • Diagnosis (elevated inhibin in premenopausal woman with amenorrhea/infertility)
  • Monitoring treatment response
  • Detecting recurrence - lead time of ~12 months before clinical recurrence
• Anti-Müllerian Hormone (AMH) - elevated in 75% of adult GCTs preoperatively; >90% sensitivity for detecting recurrence

Molecular Genetics

• FOXL2 mutation (C134W) in 97% of adult GCTs - encodes a transcription factor important in granulosa cell development; nearly pathognomonic
• Molecular FOXL2 testing improves diagnostic accuracy (almost 30% of adult GCTs were misdiagnosed historically)
• FOXL2 mutation is absent in juvenile GCTs

Malignant Potential & Prognosis

• All GCTs are potentially malignant - impossible to predict biologic behavior from histology alone
• Malignant behavior (recurrence, extension): 5-25%
• Characteristically indolent course - recurrences may appear 10-20 years after original tumor removal
• 10-year survival: ~90-95% for Stage I

Robbins, Cotran & Kumar Pathologic Basis of Disease | Berek & Novak's Gynecology

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2. Thecomas and Fibromas

Thecoma

• Composed of plump spindle cells with lipid droplets
• Represents theca cell differentiation
• Almost always benign (tumors composed predominantly of theca cells are almost never malignant)
• Hormonally active - typically estrogenic; produce endometrial hyperplasia, abnormal bleeding
• Usually unilateral, postmenopausal women

Fibroma

• Composed of well-differentiated fibroblasts with scant collagenous stroma
• Not usually hormonally active, but may occasionally be
• Unilateral in ~90% of cases; solid, hard, gray-white mass
• Account for ~4% of all ovarian tumors
• Meigs Syndrome - classic triad of:
  • Ovarian fibroma (or thecoma)
  • Ascites
  • Right-sided pleural effusion
  • (Resolves after tumor removal)
• Associated with Basal Cell Nevus Syndrome (Gorlin syndrome)

Robbins, Cotran & Kumar Pathologic Basis of Disease

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3. Sertoli-Leydig Cell Tumors (Androblastoma)

Overview

• Recapitulate testicular architecture (Sertoli cells and Leydig cells)
• Extremely rare - <0.2% of ovarian cancers
• Peak incidence: 2nd and 3rd decades (75% under age 40)
• Unilateral; low-grade malignancies
• Associated with DICER1 gene somatic mutations (encodes endonuclease for micro-RNA processing)

Histology

Differentiation Grades

Hormone Secretion & Clinical Effects

• Predominantly androgenic (testosterone and androstenedione elevated)
• Virilization/defeminization in 70-85% of patients:
  • Oligomenorrhea → amenorrhea
  • Breast atrophy
  • Acne, hirsutism
  • Clitoromegaly
  • Voice deepening
  • Receding hairline
• Rarely - estrogenic effects (isosexual precocity or postmenopausal bleeding)
• Plasma DHEAS normal or only slightly elevated (helps distinguish from adrenal source)

Prognosis

• Recurrence or metastasis: <5% (most are stage I, low grade)
• 5-year survival: 70-90%
• Most fatalities occur with poorly differentiated tumors

Berek & Novak's Gynecology | Robbins, Cotran & Kumar Pathologic Basis of Disease

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4. Leydig Cell (Hilus Cell) Tumors

• Rare, unilateral tumors located in the ovarian hilus
• Composed of large lipid-laden Leydig cells with characteristic Reinke crystalloids (cytoplasmic crystalline inclusions - pathognomonic)
• Androgen-secreting → predominantly testosterone → mild-to-moderate virilization (hirsutism, voice changes, clitoromegaly)
• Milder virilization than Sertoli-Leydig cell tumors
• Almost always benign
• Treatment: surgical excision

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5. Steroid Cell Tumors

• Account for <1% of ovarian neoplasms
• Most associated with virilization (most common)
• Some produce corticosteroids → obesity, hypertension, glucose intolerance (Cushing-like features)
• Rare cases of estrogen secretion and isosexual precocity
• ~20% may metastasize (especially if >8 cm)
• Treatment: surgical removal; no established role for radiation or chemotherapy

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6. Gynandroblastoma and Sex Cord Tumor with Annular Tubules

• Gynandroblastoma: rare; contains both well-differentiated male (Sertoli/Leydig) and female (granulosa/theca) elements; may produce mixed hormonal effects
• Sex cord tumor with annular tubules (SCTAT): distinctive tumor with ring-shaped tubules; strongly associated with Peutz-Jeghers syndrome; may produce progesterone; occasionally malignant

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Summary Table

Robbins, Cotran & Kumar Pathologic Basis of Disease | Berek & Novak's Gynecology | Robbins & Kumar Basic Pathology | Guyton & Hall Textbook of Medical Physiology

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Hemorrhagic Disorders: Classification and Laboratory Diagnosis

Introduction

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Classification

HEMORRHAGIC DISORDERS
├── I.  Vascular (Vessel Wall) Disorders
├── II. Platelet Disorders
│   ├── A. Quantitative (Thrombocytopenia)
│   └── B. Qualitative (Thrombocytopathy)
├── III. Coagulation Factor Disorders
│   ├── A. Hereditary
│   └── B. Acquired
└── IV. Mixed / Combined Defects (e.g., DIC, liver disease)

---

I. Vascular (Vessel Wall) Disorders

A. Hereditary

B. Acquired

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II. Platelet Disorders

A. Quantitative - Thrombocytopenia

• Drug-induced: alcohol, thiazides, cytotoxic drugs
• Infections: measles, HIV
• Nutritional deficiency: B12, folate (megaloblastic states)
• Aplastic anemia
• Bone marrow replacement (leukemia, lymphoma, myeloma, metastases)
• Myelodysplastic syndrome

• Immune Thrombocytopenic Purpura (ITP) - most important:
  • Autoantibodies (IgG) against platelet membrane glycoproteins (GpIIb/IIIa)
  • Acute form: children, post-viral; Chronic form: adult women
  • Megakaryocytes increased in bone marrow (compensatory)
  • Lab: low platelet count, normal PT/PTT, normal bleeding time only if severe
• Thrombotic Thrombocytopenic Purpura (TTP):
  • Deficiency of ADAMTS13 (metalloprotease that cleaves ultra-large vWF multimers)
  • Pentad: thrombocytopenia, microangiopathic hemolytic anemia (MAHA), fever, renal failure, neurologic changes
  • Lab: fragmented RBCs (schistocytes) on peripheral smear, elevated LDH, low ADAMTS13 activity
• Hemolytic Uremic Syndrome (HUS): E. coli O157:H7 (Shiga toxin), complement dysregulation; mainly renal involvement
• Drug-induced immune thrombocytopenia: heparin (HIT), quinine

• Hypersplenism (any cause of splenomegaly)

• Massive blood transfusion with stored blood (low platelets)

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B. Qualitative - Thrombocytopathy (Platelet Function Disorders)

• Aspirin / NSAIDs: irreversible cyclooxygenase inhibition → impaired thromboxane A2 synthesis → impaired aggregation
• Uremia: complex defects in adhesion, secretion, and aggregation (requires dialysis or desmopressin for correction)
• Dysproteinemias (myeloma): paraproteins coat platelet surface

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III. Coagulation Factor Disorders

A. Hereditary Coagulation Factor Deficiencies

• X-linked recessive; affects males; carrier females may occasionally bleed
• Accounts for 80% of hemophilias
• Severity based on factor VIII level:
  • Severe: <1% activity → spontaneous bleeding into joints (hemarthrosis), muscles, CNS
  • Moderate: 1-5% → bleeding with minor trauma
  • Mild: 5-25% → bleeding with surgery/major trauma
• Lab: ↑PTT, Normal PT, Normal TT, Normal platelet count and bleeding time; specific factor VIII assay confirms

• X-linked recessive; clinically identical to Hemophilia A
• Lab: ↑PTT, Normal PT - identical pattern to Hemophilia A
• Distinguished only by specific factor assay

• Autosomal dominant (Type 1, 2); autosomal recessive (Type 3)
• vWF defect impairs BOTH platelet adhesion AND factor VIII stability

---

B. Acquired Coagulation Disorders

• Vitamin K required for carboxylation (activation) of factors II, VII, IX, X and anticoagulant proteins C and S
• Causes: malabsorption (biliary obstruction, celiac), newborns (hemorrhagic disease of newborn), warfarin therapy, prolonged antibiotic use
• Lab: ↑PT (first affected - Factor VII has shortest half-life), then ↑PTT; Normal TT; Normal platelet count

• Liver synthesizes most clotting factors (I, II, V, VII, VIII partially, IX, X, XI)
• Results in multiple factor deficiencies + thrombocytopenia (portal hypertension-related splenomegaly) + ↓ clearance of activated factors
• Lab: ↑PT, ↑PTT, ↓fibrinogen, thrombocytopenia, ↑TT

• Acquired inhibitors against specific factors (commonly Factor VIII) - rare
• Lab pattern mimics deficiency but does not correct on mixing study (1:1 mix of patient + normal plasma)

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IV. Disseminated Intravascular Coagulation (DIC)

Pathophysiology

1. Triggers → systemic activation of coagulation (via tissue factor release)
2. Widespread fibrin deposition in microcirculation → organ ischemia + microangiopathic hemolytic anemia (RBC shearing through occluded vessels)
3. Consumption of platelets and clotting factors → coagulopathy
4. Secondary fibrinolysis activated → generates fibrin degradation products (FDPs/D-dimers) which further inhibit thrombin, platelet aggregation, and fibrin polymerization → hemorrhage

Common Triggers

• Sepsis (most common), Gram-negative endotoxemia
• Obstetric complications: amniotic fluid embolism, abruptio placentae, dead fetus syndrome
• Massive trauma, burns, extensive surgery
• Malignancy: acute promyelocytic leukemia (APL), adenocarcinomas (lung, pancreas, colon, stomach)
• Transfusion reactions, snake bites, Kasabach-Merritt syndrome

Lab Findings in DIC (Tintinalli Emergency Medicine Table 233-7)

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Screening Laboratory Tests in Evaluation of Bleeding

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Differential Patterns at a Glance

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Clinical Pearl: Type of Bleeding Localizes the Defect

Robbins, Cotran & Kumar Pathologic Basis of Disease | Goldman-Cecil Medicine, International Ed. | Henry's Clinical Diagnosis and Management by Laboratory Methods | Tintinalli's Emergency Medicine | Berek & Novak's Gynecology (for ITP classification)

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Quality Assurance in the Hematology Laboratory

Definition and Overview

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The Testing Cycle: Three Phases of Error

PRE-ANALYTICAL  →  ANALYTICAL  →  POST-ANALYTICAL
(before testing)    (during testing)   (after testing)

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I. Pre-Analytical Phase

1. Patient Preparation

• Correct patient identification (two identifiers: name + date of birth or ID number)
• Appropriate timing: fasting state, time of collection relative to medication
• Avoiding diurnal variation (e.g., eosinophil counts higher in morning)
• Position effects: WBC and platelet counts can vary slightly with posture

2. Specimen Collection

• Correct anticoagulant: EDTA (K₂EDTA or K₃EDTA) tubes (lavender/purple top) are standard for CBC, differential, and platelet count
  • EDTA chelates calcium, preventing coagulation without affecting cell morphology
  • Citrate tubes (blue top) used for coagulation studies - 9:1 ratio of blood to citrate is critical
• Correct fill volume: Under-filled tubes change the blood:anticoagulant ratio
  • Under-filled EDTA: EDTA excess causes RBC shrinkage → falsely low MCV and hematocrit
  • Under-filled citrate: excess citrate → falsely prolonged PT/PTT
• Order of draw: Prevents cross-contamination between additives
• Venepuncture technique: Avoid prolonged tourniquet (>1 minute → haemoconcentration), traumatic venepuncture (haemolysis, platelet activation), and IV line contamination

3. Specimen Labeling and Transport

• Labels must be applied immediately after collection at the bedside
• Barcoding enables positive patient and specimen identification through the entire pre-analytical, analytical, and post-analytical journey; 2D barcodes (QR-type) preferred over 1D barcodes for error correction capability
• Transport at room temperature; avoid excessive heat, cold, or agitation
• Coagulation specimens must be tested within 4 hours at room temperature; if delayed, plasma should be separated and frozen at -20°C

4. Common Pre-Analytical Errors in Hematology

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II. Analytical Phase

A. Instrumentation and Calibration

• Electrical impedance (Coulter principle): Cell counting and sizing
• Light scattering (laser/optical): Differential counting, reticulocyte analysis, platelet indices
• Fluorescence flow cytometry: Nucleated RBC, immature cell fractions

• Calibrate when installing new instrument, after major maintenance, when QC indicates systematic drift
• Calibrators must be commutable with patient samples

B. Internal Quality Control (IQC)

• Commercially prepared, stabilized blood-based controls (e.g., XE-Alpha N/L/H levels for Sysmex analyzers)
• Three levels typically used: low, normal, and high - to monitor performance across the analytical measuring interval
• Same lot should be used for extended periods (≥1 year) to establish reliable interpretive criteria

• Mean (target value): Established by running the control ≥20 times
• Standard Deviation (SD): Reflects random imprecision; ±1 SD = 68.3%, ±2 SD = 95.4%, ±3 SD = 99.7% of all values
• Coefficient of Variation (CV%) = (SD/Mean) × 100: Allows comparison of imprecision across different analytes
• Bias: Systematic deviation from the true value; detected by calibration drift

C. Levey-Jennings Chart

• X-axis: Time (sequential run number or date)
• Y-axis: QC result value
• Lines drawn at: Mean, ±1 SD, ±2 SD, ±3 SD

• Results within ±2 SD: in control (acceptable)
• Results outside ±2 SD: warning (evaluate with rules)
• Results outside ±3 SD: rejection (immediate action)

D. Westgard Rules

• 1₂s is the initial warning trigger
• If warning triggered, apply all other rules
• If any rejection rule is violated → do not release results → investigate and correct
• Trends (gradual drift toward one side) indicate reagent deterioration, calibration drift, or temperature changes
• Shifts (sudden jump to one side) indicate calibration change, reagent lot change, or instrument malfunction

E. Sigma Metrics and Six Sigma in the Laboratory

• TEa = Total allowable error (based on clinical requirements, e.g., CLIA, biological variation)
• Bias = Systematic error (estimated from EQA)
• SD = Imprecision

F. Moving Average / Bull's Algorithm (Patient-Based QC)

• If the moving average drifts outside ±3% of target, it signals analyzer malfunction
• Advantage: detects systematic errors in real time between scheduled QC runs
• Limitation: assumes patient population is stable; unreliable during population shifts (e.g., post-transfusion, ICU patients)

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III. Post-Analytical Phase

1. Reference Intervals

• Validated reference intervals specific to the laboratory's population (age, sex, ethnicity)
• Pediatric values differ markedly from adult values (e.g., neonatal Hb 14-20 g/dL)
• Must be reviewed when changing analyzers or reagent lots

2. Critical Values (Panic Values)

• Predefined thresholds requiring immediate clinician notification:

• All critical value notifications must be documented (time, person notified, person reporting)

3. Delta Checks

• Comparison of current result with previous result for the same patient
• Flags results that differ by more than a defined threshold (delta limit)
• Detects: specimen identification errors (wrong patient), analytical errors, genuine acute clinical changes
• Example: Hb delta check >2 g/dL → verify specimen identity before releasing result

4. Result Review and Smear Preparation

• Automated analyzers flag abnormal results (morphology flags, immature cell flags) requiring manual peripheral blood smear review
• ICSH (International Council for Standardization in Haematology) criteria define when smear review is mandatory
• Examples of flags requiring smear: blasts, atypical lymphocytes, left shift, NRBC, platelet clumps

5. Turnaround Time (TAT)

• Defined targets for routine and urgent/STAT testing
• TAT monitoring is a key performance indicator (KPI)
• Common benchmark: STAT CBC reported within 60 minutes of receipt

6. Result Reporting and Transcription

• Laboratory Information System (LIS) integration minimizes transcription errors
• Verified, authorized results only
• Corrected reports must be issued with documentation when errors are found after release

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IV. External Quality Assessment (EQA) / Proficiency Testing (PT)

• Assesses trueness (systematic bias of a method)
• Allows inter-laboratory and inter-method comparison
• Identifies outliers and systematic problems not detectable by IQC
• Required for laboratory accreditation (ISO 15189, CAP, NABL)

1. External agency (e.g., UK NEQAS, CAP, RCPAQAP) sends lyophilized or stabilized samples
2. Laboratory tests them like routine samples
3. Results returned to agency; laboratory receives performance report
4. Unacceptable results trigger root cause analysis and corrective action

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V. Personnel Competency Assessment

• Six elements of competency must be documented for each employee for each test:
  1. Direct observation of routine patient test performance
  2. Monitoring of recording and reporting of results
  3. Review of intermediate test results, QC records, and PT results
  4. Direct observation of preventive maintenance and function checks
  5. Assessment of problem-solving skills
  6. Testing of unknown specimens (where applicable)
• Competency must be documented within the first 6 months of testing, then annually, and whenever methodology or instruments change

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VI. Hematology-Specific QA Considerations

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VII. Laboratory Accreditation Standards

• ISO 15189:2022 - International standard for medical laboratory quality and competence
• CLSI (Clinical and Laboratory Standards Institute) - H20 series for hematology QC
• CAP (College of American Pathologists) - Accreditation checklists
• NABL (National Accreditation Board for Testing and Calibration Laboratories) - India
• CLIA (Clinical Laboratory Improvement Amendments) - USA federal regulation

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Summary: Key Components of QA in Hematology

QA IN HEMATOLOGY LABORATORY
│
├── PRE-ANALYTICAL
│   ├── Patient identification (barcoding, 2 identifiers)
│   ├── Correct specimen collection (EDTA, fill volume, order of draw)
│   ├── Transport and storage conditions
│   └── Rejection criteria (clotted, haemolysed, wrong tube)
│
├── ANALYTICAL
│   ├── Calibration (traceable to reference methods)
│   ├── IQC (3-level controls, Levey-Jennings chart)
│   ├── Westgard Rules (1₃s, 2₂s, R₄s, 4₁s, 10ₓ)
│   ├── Bull's Algorithm (XB moving average for RBC indices)
│   └── Sigma metrics (TEa - bias / SD)
│
├── POST-ANALYTICAL
│   ├── Reference intervals (validated, population-specific)
│   ├── Critical values (immediate notification + documentation)
│   ├── Delta checks (intra-patient comparison)
│   ├── Peripheral smear review (flagged samples)
│   └── TAT monitoring
│
└── EXTERNAL
    ├── EQA/Proficiency Testing (independent agency)
    ├── Personnel competency documentation (6 elements, annual)
    └── Accreditation (ISO 15189, CAP, NABL, CLIA)

Tietz Textbook of Laboratory Medicine, 7th Ed. (Chapters 2 & 6) | Henry's Clinical Diagnosis and Management by Laboratory Methods | Quick Compendium of Clinical Pathology, 5th Ed.

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Transfusion Transmitted Infections (TTIs) in Blood Banking

Introduction

1. Donor selection and deferral (questionnaire-based)
2. Laboratory testing of donated units
3. Pathogen reduction/inactivation technologies
4. Hemovigilance and post-donation surveillance

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The Window Period - Central Concept

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Mandatory Testing (Per National Blood Safety Policy - India/WHO)

1. HIV (Types 1 and 2)
2. Hepatitis B virus (HBV)
3. Hepatitis C virus (HCV)
4. Syphilis (Treponema pallidum)
5. Malaria (Plasmodium species)

Park's Textbook of Preventive and Social Medicine

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Residual Risk and Window Periods (Goldman-Cecil Medicine, Table 162-3)

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I. Viral Transfusion Transmitted Infections

1. Human Immunodeficiency Virus (HIV)

• Antibody detection: Chemiluminescent immunoassay (ChLIA) for anti-HIV-1 (groups M, N, O) and anti-HIV-2; detects antibodies ~3 weeks post-infection
• NAT (HIV-1 RNA): Reduces window period to ~9-10 days
• Combined antigen/antibody (4th generation) assays detect both p24 antigen and antibodies

• High-risk behaviors: MSM within 3 months, sex workers, IV drug users
• History of positive HIV test → permanent deferral
• Recipients of antiretroviral PrEP/PEP → deferred for 3 months after last dose

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2. Hepatitis B Virus (HBV)

• HBsAg (Hepatitis B surface antigen): Window period 35-40 days; first introduced 1972
• Anti-HBc (core antibody): Can be positive as early as 1 week post-infection; high false-positive rate (~1% of donors); used to detect occult HBV infection (OBI)
• HBV NAT (DNA): Reduces window to 15-34 days (individual testing) or 40-50 days (mini-pool)
• Confirmatory: HBsAg neutralization

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3. Hepatitis C Virus (HCV)

• Anti-HCV antibody (EIA/ChLIA): Window period 51-70 days post-infection
• HCV NAT (RNA): Shortens window to 7-8 days - most significant advance in HCV blood safety
• NAT introduced for HCV in blood banking in 2000

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4. Human T-Lymphotropic Virus (HTLV-I/II)

• HTLV-I: Adult T-cell leukemia/lymphoma (ATL); HTLV-associated myelopathy/tropical spastic paraparesis (HAM/TSP)
• HTLV-II: Generally less pathogenic; no significant disease in immunocompetent individuals
• Both: lymphadenopathy

• Window period: ~80 days
• Residual risk: ~1 in 2-3.3 million units
• Donors testing reactive → no re-entry protocol (permanent deferral)

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5. Cytomegalovirus (CMV)

• Immunocompetent recipients: Usually asymptomatic or mild self-limited illness
• High-risk recipients (neonates, HIV patients, organ/stem cell transplant recipients): Severe multiorgan failure - hepatitis, pneumonitis, retinitis, GI disease, marrow failure

• Use of CMV-seronegative blood products for at-risk patients
• Leukoreduction (LR): Significantly reduces transmission risk (from 1-3% to 0.023%); reduces but does not eliminate risk completely

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6. West Nile Virus (WNV)

• Mosquito-borne flavivirus; emerged in USA 1999; peak epidemic 2002-2003 (23 transfusion-transmitted cases)
• Clinical: Mild febrile illness in most; encephalitis/meningitis in immunosuppressed
• NAT testing introduced in 2003 dramatically reduced cases to only 13 since 2003
• Testing strategy: Mini-pool NAT; switch to individual donor NAT during seasonal outbreaks

---

7. Other Viruses

---

II. Bacterial Contamination

1. Donor bacteremia at time of donation (transient, e.g., from dental procedures, skin infections)
2. Skin flora introduced at venepuncture - most common; skin organisms (Staphylococcus, Bacillus species) are drawn into collection tubing with the initial blood flow

1. Diversion pouch: First 20-40 mL of blood diverted into a separate chamber to exclude skin-plug contamination
2. Skin antisepsis: Rigorous cleaning with chlorhexidine-isopropanol
3. Sterile collection technique
4. Culture-based testing of platelets (mandatory in USA since 2004):
   • BACTEC blood culture system: 8 mL sampled ≥24h after collection
   • Growth detected by pH change, CO₂ production
5. Point-of-issue rapid testing (prior to platelet transfusion):
   • Colorimetric peptidoglycan detection
   • Lateral flow immunoassay for lipoteichoic acid/LPS
6. Pathogen inactivation (see below)

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III. Parasitic Transfusion Transmitted Infections

1. Malaria (Plasmodium species)

• Most important parasitic TTI worldwide (especially in endemic regions - Sub-Saharan Africa, South Asia)
• Mandatory screening in India - microscopical examination of all donor blood + malarial antibody ELISA
• Donor deferral: Travel to malaria-endemic areas (12 months deferral); history of malaria (3 years deferral)
• RBCs stored at 4°C: Plasmodium trophozoites can survive for the shelf life of blood
• Clinical consequence: Transfusion-transmitted malaria can be fatal, especially in non-immune recipients

2. Chagas Disease (Trypanosoma cruzi)

• Endemic in Latin America; significant concern with immigration patterns
• Testing with Chagas antibody EIA now performed in USA and many European countries
• 7 reported cases in USA prior to testing; none since testing implemented
• Trypanosomes survive in refrigerated RBCs for weeks

3. Babesiosis (Babesia microti)

• Tick-borne protozoan; endemic in northeastern USA
• Causes hemolytic anemia; life-threatening in asplenic/immunocompromised patients
• NAT testing now required in endemic US states
• Donor deferral: History of Babesia → deferred; re-entry possible after 2 years if testing negative

4. Toxoplasma gondii

• Rare TTI; mainly in immunocompromised recipients
• No routine screening; managed through donor history questionnaire

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IV. Prion Diseases

Variant Creutzfeldt-Jakob Disease (vCJD)

• Caused by abnormal prion proteins (PrPSc); related to bovine spongiform encephalopathy (BSE/"mad cow disease")
• UK epidemic in 1980s-1990s; 4 confirmed transfusion-transmitted cases in the UK
• No available screening test for prions in blood
• Managed entirely through donor deferral:
  • Residence in UK >3 cumulative months (1980-1996) → permanent deferral
  • Residence in France/Ireland >5 years (1980-present) → permanent deferral
• Fatal neurodegenerative disease; incubation period years to decades
• Prions cannot be inactivated by standard pathogen reduction technologies

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V. Pathogen Reduction / Inactivation Technologies (PRT)

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VI. Donor Selection: The First Line of Defense

Key Deferral Categories:

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VII. Summary: Multi-Layered Blood Safety Strategy

BLOOD SAFETY PYRAMID
│
├── 1st Layer: Voluntary non-remunerated donors (lowest risk population)
│
├── 2nd Layer: Donor History Questionnaire (DHQ) - behavioral/travel deferral
│
├── 3rd Layer: Mandatory Serological Screening (ELISA/ChLIA)
│             HIV 1/2, HBsAg, anti-HCV, anti-HBc, anti-HTLV, RPR/TPHA,
│             Malaria antibody, Chagas Ab, WNV NAT (seasonal)
│
├── 4th Layer: NAT (Nucleic Acid Testing - PCR/TMA)
│             HIV RNA, HCV RNA, HBV DNA, WNV RNA
│             → Dramatically shortens window period
│
├── 5th Layer: Pathogen Inactivation (for platelets, plasma)
│             INTERCEPT, Mirasol systems
│
├── 6th Layer: Leukoreduction (for CMV, HTLV risk reduction)
│
└── 7th Layer: Hemovigilance (post-transfusion surveillance, lookback)

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Key Table: Comparison of TTIs

Tietz Textbook of Laboratory Medicine, 7th Ed. | Barash, Cullen & Stoelting's Clinical Anesthesia, 9th Ed. | Goldman-Cecil Medicine, International Ed. | Park's Textbook of Preventive and Social Medicine

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Paroxysmal Nocturnal Hemoglobinuria (PNH)

Definition

• Incidence: 2-5 per million (annual)
• Age: Any age; most common between 10-50 years; mean age at diagnosis ~34 years
• Sex: Equal male:female ratio; no family history (acquired)

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Pathogenesis / Molecular Basis

Step 1: The PIGA Mutation

• PIGA is located on the short arm of the X chromosome (Xp22.1)
• PIGA encodes an enzyme essential for the early step in GPI (Glycosylphosphatidylinositol) biosynthesis
• Because PIGA is X-linked, a single mutation in the active X allele is sufficient to abolish GPI synthesis in that cell and all its clonal descendants
• The mutation affects cells of all hematopoietic lineages (red cells, white cells, platelets)
• ~150 different PIGA mutations have been identified, most causing total loss of GPI production

Step 2: GPI Anchor Deficiency

Step 3: Loss of Complement Regulatory Proteins

Step 4: Complement-Mediated Lysis

• Sleep (mild respiratory acidosis → slight drop in pH → increased complement activation)
• Infection (activates complement via classical pathway)
• Surgery, physical stress, blood transfusion
• Acidosis, hypoxia

PNH Erythrocyte Types (Henry's)

Why Does the PNH Clone Expand?

• PNH is frequently associated with aplastic anemia (AA), which has an autoimmune basis
• T cells destroying GPI-positive stem cells spare GPI-negative (PNH) stem cells → selective clonal expansion
• This explains the clinical overlap and transitions between PNH and AA

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Clinical Features

1. Hemoglobinuria (Classic but not the most common presentation)

• Dark, port wine-colored urine (classically in the morning after overnight sleep)
• Only occurs in ~25% of patients during paroxysms
• Results from the mild acidosis during sleep activating complement
• Hemosiderinuria (urinary iron loss as hemosiderin) is almost constantly present - more reliable sign

2. Chronic Intravascular Hemolytic Anemia

• The dominant feature; usually mild to moderate
• Normocytic or hypochromic microcytic (due to chronic iron loss in urine)
• Reticulocytosis less than expected for degree of anemia
• Direct Antiglobulin Test (Coombs) is NEGATIVE - cardinal feature distinguishing PNH from autoimmune hemolytic anemia

3. Thrombosis - Leading Cause of Death

• Occurs in ~40% of patients; venous in 85% of cases
• Unusual sites:
  • Budd-Chiari syndrome (hepatic vein thrombosis) → hepatomegaly, ascites
  • Portal vein thrombosis
  • Cerebral vein thrombosis
  • Mesenteric vein thrombosis
• Mechanism: CD59-deficient platelets → phosphatidylserine externalization → prothrombinase complex formation; free hemoglobin scavenges nitric oxide (NO) → platelet aggregation and endothelial dysfunction

4. Smooth Muscle Dystonia (due to NO scavenging by free hemoglobin)

• Dysphagia (esophageal spasm)
• Abdominal pain/colic (~1/3 of patients)
• Erectile dysfunction in males

5. Bone Marrow Failure

• Pancytopenia: Neutropenia (in 3/5 patients) and thrombocytopenia (in 2/3 patients) are common
• Bone marrow may be hypocellular or show erythroid hyperplasia
• ~1/3 of cases evolve into aplastic anemia

6. Iron Deficiency

• Chronic urinary iron loss (hemosiderin in urine → hemosiderinuria)
• Coombs-negative hemolytic anemia + iron deficiency = suspect PNH

7. Transformation to Myeloid Neoplasm

• ~5% of patients eventually develop AML or myelodysplastic neoplasm

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Diagnosis

1. Gold Standard: Flow Cytometry (Current Standard)

• Red cells: CD59 (most sensitive for RBCs)
• Granulocytes: CD24, CD16, CD66b (best for small clones - not affected by hemolysis)
• Monocytes: CD14
• FLAER (Fluorescent Aerolysin Variant): Bacterial protein that binds directly to GPI anchor; most reliable and sensitive reagent for studying GPI-linked antigens on leukocytes; preferred for diagnosing PNH on granulocytes and monocytes

2. Historical Tests (Now Superseded)

3. Routine Laboratory Findings

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PNH Classification (Clinical Categories)

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Treatment

1. Complement Inhibitors (Disease-Modifying Therapy)

• Binds complement component C5, preventing cleavage to C5a+C5b → blocks MAC formation
• Eliminates intravascular hemolysis; reduces thrombosis risk by up to 90%
• Reduces/eliminates transfusion requirements
• Dose: 600 mg IV weekly × 4 weeks → 900 mg at week 5 → 900 mg every 2 weeks
• Side effect: Increased risk of Neisseria meningitidis infection (MAC normally defends against encapsulated organisms)
• Pre-treatment requirement: Vaccination against Meningococcus A, B, C (and Y/W) + prophylactic oral penicillin

2. Supportive Treatment

• Iron and folate supplementation (to counter urinary losses and increased erythropoiesis)
• Blood transfusions (leukoreduced; for severe anemia)
• Anticoagulation for thrombotic episodes (LMWH/warfarin/DOACs)
• Corticosteroids (short-term during acute hemolytic crises - debated benefit)
• Immunosuppression (for AA component - anti-thymocyte globulin + cyclosporine)

3. Curative Therapy

• Allogeneic hematopoietic stem cell transplantation (HSCT) - the only cure
• Reserved for: aplastic anemia with PNH clone, transformation to AML/MDS, recurrent life-threatening thrombosis despite complement inhibition, young patients with HLA-matched sibling

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Key Concepts Summary

PIGA mutation (somatic, X-linked)
        ↓
GPI anchor deficiency
        ↓
Loss of CD55, CD59, C8-BP on ALL blood cell lineages
        ↓
┌───────────────────────────────────────────────┐
│ Uncontrolled complement activation on RBCs    │
│ MAC (C5b-9) assembly → Intravascular hemolysis│
│ Hemoglobinuria + Hemosiderinuria              │
└───────────────────────────────────────────────┘
        ↓
┌──────────────────────────────────────────────┐
│ Free Hb → NO scavenging → smooth muscle      │
│ dystonia (dysphagia, abdo pain, erectile dys)│
└──────────────────────────────────────────────┘
        ↓
┌──────────────────────────────────────────────┐
│ CD59-deficient platelets → Thrombosis        │
│ (Budd-Chiari, cerebral, portal vein)         │
│ → LEADING CAUSE OF DEATH                     │
└──────────────────────────────────────────────┘

DIAGNOSIS: Flow cytometry (CD55, CD59, FLAER)
            Coombs NEGATIVE
TREATMENT: Eculizumab/Ravulizumab (anti-C5)
            HSCT (only cure)

Robbins, Cotran & Kumar Pathologic Basis of Disease | Harrison's Principles of Internal Medicine, 22nd Ed. | Goldman-Cecil Medicine, International Ed. | Henry's Clinical Diagnosis and Management by Laboratory Methods

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Myelodysplastic Syndrome (MDS): Classification and Diagnostic Criteria

Definition

• Ineffective hematopoiesis - dysplastic (morphologically abnormal) maturation in one or more blood cell lineages
• Peripheral blood cytopenias (despite a usually normo- or hypercellular bone marrow)
• Variable risk of transformation to Acute Myeloid Leukemia (AML)

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Pathogenesis (Brief)

• Spliceosome components: SF3B1 (20-25%), SRSF2 (10-15%), U2AF1 (10%) - strongly associated with ring sideroblasts when SF3B1 is mutated
• DNA methylation/epigenetics: TET2 (20-25%), DNMT3A (10-15%), ASXL1 (15-20%)
• Transcription factors: RUNX1 (10-15%), ETV6 (3%)
• Tumor suppressors: TP53 (5-10%) - poor prognosis
• RAS pathway: NRAS, KRAS (≤10%)

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WHO Classification of MDS

WHO MDS Subtypes (Goldman-Cecil Medicine, Table 162)

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1. MDS with Single Lineage Dysplasia (MDS-SLD)

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2. MDS with Ring Sideroblasts (MDS-RS)

• MDS-RS-SLD - single lineage dysplasia
• MDS-RS-MLD - multilineage dysplasia

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3. MDS with Multilineage Dysplasia (MDS-MLD)

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4. MDS with Excess Blasts-1 (MDS-EB-1)

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5. MDS with Excess Blasts-2 (MDS-EB-2)

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6. MDS with Isolated del(5q) - "5q- Syndrome"

• Predominantly affects middle-aged women
• Lenalidomide is specifically effective for this subtype
• Auer rods must be absent; del(5q) + monosomy 7 = excluded from this category (worse prognosis)

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7. MDS, Unclassifiable (MDS-U)

• Persistent cytopenias with <10% dysplastic cells but a characteristic MDS-associated cytogenetic abnormality
• Cases with 1% peripheral blood blasts on two occasions
• Cases with single lineage dysplasia + pancytopenia

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Additional WHO 2022 Categories

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Diagnostic Criteria

Minimal Diagnostic Criteria (Goldman-Cecil Medicine)

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Step-by-Step Diagnostic Work-Up

1. Clinical Suspicion

• Older patient with unexplained cytopenias (especially macrocytic anemia not explained by B12/folate deficiency, drugs, or alcohol)
• Fatigue, dyspnea, recurrent infections, mucosal bleeding

2. Peripheral Blood Examination

• Hb <10 g/dL (or <13 g/dL in males)
• ANC <1.8 × 10⁹/L
• Platelets <100 × 10⁹/L

3. Bone Marrow Examination (Essential)

4. Cytogenetics (Essential)

5. Molecular/Genetic Testing (Next-Generation Sequencing)

• SF3B1 mutation → confirms MDS-RS even with <15% ring sideroblasts (≥5% sufficient)
• TP53 biallelic mutations → distinct entity; very poor prognosis
• RUNX1 → thrombocytopenic prodrome
• GATA2 → mycobacterial infections, monocytopenia

6. Flow Cytometry

• Useful for diagnosing PNH clone (in hypoplastic MDS/AA overlap)
• Immunophenotyping can support diagnosis but should not be used to count blast percentage

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Prognostic Scoring: IPSS-R

1. Cytogenetics (5 categories: very good → very poor)
2. Marrow blast %
3. Hemoglobin
4. Platelet count
5. ANC

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Differential Diagnosis

• B12 / folate deficiency (megaloblastic anemia)
• Copper deficiency (especially if ring sideroblasts)
• Iron deficiency
• HIV infection
• Drug effects (cytotoxic chemotherapy, methotrexate, azathioprine)
• Heavy alcohol use
• Congenital sideroblastic anemias (ALAS2 mutations)
• Aplastic anemia
• T-cell large granular lymphocyte (T-LGL) leukemia

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Summary Table

Goldman-Cecil Medicine, International Ed. | Henry's Clinical Diagnosis and Management by Laboratory Methods