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PA16.3 | Acquired Haemolytic Anaemias & Smear Morphology — SDL Guide (Part 2)

The Direct Antiglobulin Test (DAT / Direct Coombs) — Principle & Interpretation

Diagram showing how the direct antiglobulin test detects IgG or C3d bound to patient red blood cells by AHG-mediated agglutination and interprets IgG-positive, C3d-positive, and mixed results. — **Panel A:** Patient red blood cells coated in vivo with bound IgG antibodies and complement C3d; labels for patient RBC, bound IgG, bound C3d, and in vivo sensitization.. **Panel B:** Washed patient red cells incubated with anti-human globulin reagent; labels for washed patient RBCs, AHG reagent, and detection of human IgG/C3d.. **Panel C:** DAT result comparison showing AHG cross-linking with agglutination as positive DAT versus separated cells without agglutination as negative DAT.. **Panel D:** Interpretation flow showing DAT positive followed by monospecific testing for IgG positive, C3d positive, or IgG plus C3d positive patterns with common clinical associations..

The direct antiglobulin test (DAT) detects antibodies or complement (C3d) bound to the patient's own circulating red cells.

Principle:
1. Patient's washed red cells (already coated in vivo) are incubated with anti-human globulin (AHG) reagent — a polyclonal antibody that cross-links human IgG and C3d.
2. Cross-linking of IgG or C3d on adjacent red cells → agglutination = positive DAT.
3. Specific reagents distinguish IgG-positive from C3d-positive results.

Interpretation grid:

DAT result	Most likely type
IgG positive, C3d negative	Warm AIHA (IgG alone)
IgG positive, C3d positive	Warm AIHA (IgG + complement activation)
IgG negative, C3d positive	Cold AIHA (IgM dissociated, C3d remains)
Negative	Non-immune haemolysis (MAHA, PNH, mechanical, etc.)

Indirect Coombs test: Detects free antibody in the patient's serum against test red cells — used in cross-matching, not primary haemolysis diagnosis.

Three-panel diagram showing Direct Antiglobulin Test steps: IgG-coated red cells, agglutination with AHG reagent, and negative control. — **Panel A:** Step 1 showing patient RBC with surface-bound IgG antibodies. **Panel B:** Step 2 showing AHG reagent bridging IgG-coated cells causing positive agglutination. **Panel C:** Negative control showing uncoated RBCs with no agglutination despite AHG addition.

CLINICAL PEARL

DAT-negative haemolysis does NOT rule out immune-mediated disease. Approximately 5–10% of warm AIHA cases are DAT-negative (antibody titre too low for the assay, IgA or IgM antibodies missed by standard reagent, or complement-only coating). If haemolysis is brisk and the smear shows spherocytes, request a more sensitive gel-card DAT or flow cytometric Coombs before dismissing immune cause.

Microangiopathic Haemolytic Anaemia (MAHA)

⚑ AI image — pending faculty review (auto-QA score 8/10; best of 3 attempts)

Diagram showing MAHA as mechanical non-immune red cell fragmentation by fibrin strands, platelet microthrombi, or turbulent flow, producing schistocytes and clinical syndromes such as TTP, HUS, and DIC. — **Panel A:** Narrowed microvessel, fibrin strands, platelet microthrombi, red blood cells, mechanical shearing, schistocytes, direction of blood flow. **Panel B:** Peripheral blood smear, normal red cells, schistocyte, helmet cell, triangular red cell fragment, microspherocyte. **Panel C:** Fibrin strands, platelet microthrombi, turbulent prosthetic valve flow, red cell shearing, schistocyte formation, intravascular haemolysis, anaemia, raised LDH. **Panel D:** TTP with ADAMTS13 deficiency and ultra-large vWF multimers; HUS with Shiga toxin endothelial injury and renal microthrombi; DIC/prosthetic valve with fibrin thrombi or turbulent shear.

Microangiopathic haemolytic anaemia (MAHA) is mechanical, non-immune fragmentation haemolysis caused by red cells colliding with pathological intravascular surfaces.

Mechanism: Fibrin strands, platelet microthrombi, or turbulent flow (prosthetic valves) physically shear red cells → schistocytes (helmet cells, triangular fragments, microspherocytes).

Key causes and distinguishing features:

Cause	Key pathology	Distinguishing features
TTP (thrombotic thrombocytopenic purpura)	ADAMTS13 deficiency → ultra-large vWF multimers → platelet microthrombi	Pentad: MAHA + thrombocytopenia + fever + renal failure + neurological symptoms
HUS (haemolytic uraemic syndrome)	Shiga toxin (STEC O157:H7) → endothelial injury → renal microthrombi	Post-diarrhoeal, predominantly renal failure, children
DIC	Widespread fibrin deposition in vessels	Prolonged PT, APTT, ↓fibrinogen, ↑D-dimer; often in sepsis/obstetric emergency
Mechanical valve	Turbulent flow + prosthetic surface	Isolated MAHA, no thrombocytopenia, audible valve click

Peripheral smear hallmark: Schistocytes (>1% of RBCs diagnostic) + polychromasia. No spherocytes (different mechanism).

Peripheral blood smear showing multiple helmet-shaped schistocytes and red blood cell fragments characteristic of thrombotic thrombocytopenic purpura with reduced platelets visible. — **Single Panel:** Schistocytes (helmet cells), crescent fragments, microspherocytes, normal RBCs, reduced platelets, 100× oil immersion Leishman stain.

DAT: Negative (non-immune).

SELF-CHECK

A 35-year-old woman presents with sudden confusion, petechiae, and anaemia. CBC shows Hb 7 g/dL, platelets 18 × 10⁹/L. Peripheral smear: numerous helmet-shaped RBC fragments, polychromasia. Creatinine: 1.8 mg/dL. DAT: negative. Which diagnosis fits BEST?

A. Warm autoimmune haemolytic anaemia

B. Hereditary spherocytosis with aplastic crisis

C. Thrombotic thrombocytopenic purpura (TTP)

D. G6PD deficiency after oxidative stress

Reveal Answer

Answer: C. Thrombotic thrombocytopenic purpura (TTP)

The triad of MAHA (schistocytes on smear), severe thrombocytopenia, and neurological symptoms (confusion) with negative DAT is the classic presentation of TTP. ADAMTS13 deficiency leads to ultra-large vWF multimers, spontaneous platelet microthrombi throughout the microvasculature, and mechanical shearing of red cells. HUS would be more renal-dominant and post-diarrhoeal. Spherocytes and a positive DAT would point to AIHA. G6PD produces bite cells, not schistocytes.

Paroxysmal Nocturnal Haemoglobinuria (PNH)

Four-panel diagram explaining PNH as PIG-A mutation causing loss of GPI-anchored CD55 and CD59, complement-mediated intravascular haemolysis, clinical triad, and flow cytometry diagnosis. — **Panel A:** Normal RBC with GPI anchors, CD55, CD59, restrained complement, PNH RBC with absent GPI anchors, absent CD55/CD59, C5b-9 membrane attack complex, and intravascular haemolysis. **Panel B:** Acquired clonal haematopoietic stem cell disorder, somatic PIG-A mutation, defective GPI-anchor biosynthesis, loss of CD55/CD59, complement-mediated haemolysis. **Panel C:** Nocturnal acidosis, sleep-to-morning haemolysis, morning haemoglobinuria, haemolytic anaemia, venous thrombosis, Budd-Chiari syndrome, cerebral venous sinus thrombosis, cytopenias. **Panel D:** Flow cytometry showing reduced or absent CD55 and CD59 on red cells and granulocytes, DAT negative, Ham acidified serum test obsolete.

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal haematopoietic stem cell disorder caused by a somatic mutation in the PIG-A gene (X-linked), which is essential for GPI-anchor biosynthesis.

Pathogenesis:
1. Absence of GPI-anchored complement regulatory proteins — particularly CD55 (decay-accelerating factor) and CD59 (protectin) — on the red cell surface.
2. Without CD55/CD59 to brake complement, the membrane attack complex (C5b-9) forms freely on PNH red cells.
3. Result: intravascular haemolysis, especially during sleep (due to relative acidosis of nocturnal apnoea) — classic morning haemoglobinuria.

Clinical triad: Haemolytic anaemia + venous thrombosis (especially unusual sites: hepatic vein → Budd-Chiari, cerebral venous sinus) + cytopenias.

Smear: Often normocytic/normochromic or mildly macrocytic (due to haemolysis + folate use). No specific morphological hallmark.

Diagnosis: Flow cytometry — reduced or absent CD55 and CD59 on red cells and granulocytes (high sensitivity). The older Ham's acidified serum test has been replaced.

DAT: Negative (complement, not antibody).