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Q1 — Blood Components: Preparation, Indication and Uses (20 marks)

Introduction

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1. Whole Blood

• Massive haemorrhage with simultaneous loss of red cells, plasma proteins, and platelets (trauma, military settings)
• Exchange transfusion in neonatal hyperbilirubinaemia
• Exsanguinating hemorrhage when components not immediately available

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2. Packed Red Blood Cells (PRBCs)

• Whole blood centrifuged at 3,000-5,000 rpm for 5-7 minutes (hard spin)
• Plasma supernatant removed
• Residual volume ~200-250 mL; haematocrit 55-80%
• Stored in additive solutions (AS-1/Adsol, AS-3/Nutricel, AS-5/Optisol) extending shelf life to 42 days at 2-6°C
• One unit raises Hb by ~1 g/dL (or haematocrit by ~3%) in a 70 kg adult

• Symptomatic anaemia (Hb <8 g/dL in stable patients; Hb <10 g/dL in cardiac disease)
• Restrictive transfusion trigger: Hb 7-8 g/dL (TRICC trial, Villanueva trial)
• Perioperative blood loss
• Haemolytic disease of newborn
• Acute blood loss >30% blood volume

• ICU/stable: Hb <7 g/dL
• Cardiac disease/ACS: Hb <9-10 g/dL
• Acute GI bleed (no CAD): maintain Hb ≥8 g/dL (Rosen's Emergency Medicine)

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3. Platelet Concentrates (PC)

• Soft spin (2,000-3,000 rpm, 3 minutes) → platelet-rich plasma (PRP) separated
• Hard spin of PRP → platelet button + platelet-poor plasma (PPP)
• Each unit: 5.5×10¹⁰ platelets in 40-70 mL plasma
• 4-6 units pooled = adult therapeutic dose (equivalent to 1 SDP)
• Storage: 20-24°C with continuous agitation for 5 days (risk of bacterial growth)

• Automated apheresis from one donor
• Yield: >3×10¹¹ platelets per collection; equivalent to 6 RDP units
• Advantages: reduced donor exposures, HLA-matched possible, leuco-reduced
• Storage: 20-24°C with agitation, 5 days

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4. Fresh Frozen Plasma (FFP)

• Plasma separated within 6-8 hours of collection (to preserve labile factors V and VIII)
• Frozen at -30°C or colder within 1 hour of separation
• Volume: 200-280 mL; contains all coagulation factors at near-normal levels (1 unit/mL)
• Shelf life: 12 months at -30°C (or 24 months at -65°C); once thawed, use within 24 hours (stable at 1-6°C for up to 5 days as "thawed plasma")

• Solvent/Detergent (S/D) treated plasma: Inactivated viruses (HIV, HCV, HBV); lacks cellular components
• Methylene Blue Photoinactivated Plasma (MBPI): Single-unit pathogen reduction

• Coagulopathy with active bleeding and PT/APTT >1.5× normal
• Massive transfusion protocol (1:1:1 ratio with PRBCs and platelets)
• Reversal of warfarin (when PCC unavailable)
• Liver disease with bleeding
• DIC with active haemorrhage
• TTP (plasma exchange vehicle/replacement)
• Deficiency of single coagulation factor when specific concentrate unavailable (Factor V, XI)
• Hereditary angioedema (C1-esterase inhibitor deficiency) if specific concentrate unavailable

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5. Cryoprecipitate (Cryo)

• FFP thawed slowly at 1-6°C (usually overnight or 12-24 hours)
• Cold-insoluble precipitate forms; supernatant (cryo-depleted plasma) removed
• Cryo resuspended in small volume (~10-20 mL) of plasma
• Re-frozen at -30°C; shelf life 12 months
• Once thawed: use within 6 hours (single unit) or 4 hours (pooled)

• Fibrinogen (~150-300 mg per unit; normal plasma: 250-350 mg/dL)
• Factor VIII (~80-100 IU per unit)
• von Willebrand factor (vWF)
• Factor XIII
• Fibronectin

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6. Granulocyte Concentrates

• Apheresis from stimulated donor (G-CSF + steroids given 12 hours prior)
• Yield: >1×10¹⁰ granulocytes
• Storage: 20-24°C without agitation; must be transfused within 24 hours
• Always irradiated to prevent TA-GvHD

• Severe neutropenia (<0.5×10⁹/L) with documented bacterial/fungal infection unresponsive to antibiotics
• Neonatal sepsis with neutrophil dysfunction

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7. Plasma Derivatives (Fractionated Products)

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8. Transfusion Reactions (brief, for completeness)

• Acute Haemolytic: ABO incompatibility - fever, chills, haemoglobinuria, renal failure
• Febrile Non-Haemolytic (FNHTR): Cytokines in stored blood or anti-leukocyte antibodies; prevented by leuco-reduction
• Transfusion-Related Acute Lung Injury (TRALI): Anti-HLA/anti-HNA antibodies in donor plasma; within 6 hours; managed with O2/mechanical ventilation
• TACO (Transfusion-Associated Circulatory Overload): Volume overload; common in elderly/cardiac patients
• Transfusion-Associated GvHD (TA-GvHD): Donor lymphocytes engraft and attack host; prevented by irradiation
• Delayed Haemolytic Transfusion Reaction (DHTR): 3-10 days post-transfusion; anamnestic response to minor antigens (Kidd, Duffy, Kell)

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Q2 — Classification and Laboratory Diagnosis of Hemolytic Anaemias (20 marks)

Definition

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Classification

A. By Site of Haemolysis

• Complement-mediated direct RBC lysis
• Causes: ABO incompatibility, PNH, severe G6PD deficiency, microangiopathic haemolysis, severe malaria, Clostridium septicaemia
• Lab features: haemoglobinaemia, haemoglobinuria, haemosiderinuria (chronic), low haptoglobin (free Hb scavenged), methaemalbuminaemia

• RBCs are phagocytosed by macrophages
• Causes: most immune haemolytic anaemias, hereditary spherocytosis, sickle cell anaemia, thalassaemia
• Lab features: splenomegaly, jaundice (indirect bilirubin), no haemoglobinuria, urobilinogenuria, moderate haptoglobin reduction

B. By Aetiology

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I. INHERITED (Intrinsic/Intracorpuscular) Defects

• Sickle cell anaemia (HbS - Glu→Val at β6): vaso-occlusion, haemolysis
• HbC disease (Glu→Lys at β6): mild haemolysis, target cells
• Unstable haemoglobins: Heinz body haemolytic anaemia

• α-thalassaemia: gene deletions on chromosome 16; range from silent carrier to Hb Bart's hydrops
• β-thalassaemia: point mutations on chromosome 11; β-thal minor, intermedia, major

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II. ACQUIRED (Extrinsic/Extracorpuscular) Defects

• Antibody class: IgG (rarely IgA)
• Optimal reactivity: 37°C
• Complement: usually not fully activated
• Causes: idiopathic, SLE, lymphoproliferative disorders (CLL, lymphoma), drugs
• RBC destruction: extravascular (spleen)

• Cold Agglutinin Disease (CAD): IgM antibody; complement activation at <37°C; intravascular and extravascular; causes: Mycoplasma pneumoniae, EBV, lymphoma
• Paroxysmal Cold Haemoglobinuria (PCH): Donath-Landsteiner IgG antibody; anti-P specificity; biphasic haemolysin; post-viral in children, congenital syphilis in adults
• Mixed-type AIHA: IgG + IgM components

• Hapten mechanism: Penicillin (high-dose) binds RBC membrane; IgG antibody directed at drug-RBC complex
• Immune complex mechanism: Quinidine, rifampicin, cefotetan - drug + antibody form immune complex that deposits on RBC; complement-mediated lysis
• Autoantibody mechanism: Alpha-methyldopa, fludarabine - true warm AIHA induced; DAT positive for IgG only
• Drug-adsorption: Non-immunological protein adsorption to RBC membrane (cephalosporins)

• Haemolytic Transfusion Reaction (acute - ABO; delayed - Kidd, Duffy, Kell)
• Haemolytic Disease of the Fetus and Newborn (HDFN): maternal IgG crosses placenta

• Mechanical fragmentation by fibrin strands in small vessels
• Causes: TTP, HUS (Typical: STEC; Atypical: complement dysregulation), DIC, malignant hypertension, HELLP syndrome, prosthetic heart valves, March haemoglobinuria
• Morphology: schistocytes (>1% diagnostic), helmet cells, fragmented RBCs

• Malaria (Plasmodium falciparum): parasitised + non-parasitised RBC destruction; blackwater fever (intravascular haemolysis)
• Clostridium perfringens septicaemia: phospholipase-C (alpha toxin) destroys membrane
• Bartonella bacilliformis: Oroya fever
• Babesiosis: similar to malaria

• Oxidants: dapsone, nitrites, aniline dyes (methaemoglobinaemia + haemolysis)
• Lead poisoning: inhibits G6PD, ALA dehydratase; basophilic stippling
• Burns (>20% TBSA): thermal denaturation of RBC membrane; microspherocytes

• Acquired clonal disorder: PIGA mutation → deficiency of GPI-anchored proteins (CD55/DAF, CD59/MIRL)
• Complement-mediated intravascular haemolysis
• Classified separately (intrinsic but acquired)
• Triad: haemolysis + thrombosis + cytopenias

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Laboratory Diagnosis of Haemolytic Anaemias

General Evidence of Haemolysis

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Specific Investigations by Type

• Detects IgG and/or complement (C3d) on RBC surface
• Positive DAT: AIHA, DIIHA, HDFN, delayed haemolytic transfusion reactions
• IgG only positive: warm AIHA, methyldopa, high-dose penicillin
• C3d only positive: immune complex mechanism, cold agglutinin disease
• IgG + C3d: warm AIHA with complement activation, some DIIHA
• Indirect Antiglobulin Test (IAT): detects antibodies in patient serum

• RBCs exposed to decreasing NaCl concentrations (0.9% to 0)
• HS: increased fragility (lysis at higher NaCl concentrations) - left-shifted curve
• After 24-hour incubation: increases sensitivity for HS
• Iron deficiency/thalassaemia: decreased fragility (right-shifted curve)
• EMA binding test (flow cytometry with eosin-5'-maleimide): more sensitive and specific for HS; band 3 protein quantification

• Fluorescent spot test (screening): NADPH fluoresces under UV; negative in G6PD deficiency
• G6PD enzyme quantitative assay: definitive; express as IU/g Hb
• Note: enzyme may be normal during haemolytic crisis (young reticulocytes have higher activity); test 3 months after episode
• G6PD variants: G6PD A- (Africa), G6PD Mediterranean, G6PD Canton

• Alkaline electrophoresis (pH 8.6): separates HbA, HbS, HbC, HbF, HbA2
• Acid electrophoresis (pH 6.2): confirmatory
• HPLC (High-Performance Liquid Chromatography): gold standard; simultaneous quantification of HbA, HbA2, HbF, HbS, HbC; detects haemoglobin variants; used in thalassaemia diagnosis
• Sickling test (sodium metabisulphite): screening for HbS
• Solubility test: HbS insoluble in deoxygenated state

• Ham's test (acidified serum lysis): acidification activates complement; PNH RBCs lyse; historical test
• Sugar water test (sucrose lysis): screening test; poor specificity
• Flow cytometry: gold standard; FLAER (fluoraescein-labelled aerolysin) + CD55/CD59 on RBCs and granulocytes; quantifies clone size

• Cold agglutinin titre at 4°C: significant if >1:64 at 4°C
• Thermal amplitude: pathological if agglutination occurs at >30°C
• Donath-Landsteiner test for PCH: biphasic haemolysin; serum + P-positive RBCs incubated at 0-4°C (sensitisation), then 37°C (complement activation lysis)

• Erythroid hyperplasia with M:E ratio <1:1
• Dyserythropoiesis in thalassaemia
• Ringed sideroblasts in sideroblastic anaemia

• DNA analysis: β-globin mutations (ARMS-PCR, sequencing), alpha-globin deletions (gap-PCR, MLPA)
• Next-generation sequencing: hereditary haemolytic anaemias with atypical presentations
• G6PD gene mutation analysis in females (X-linked)

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Q3 — Plasma Cell Dyscrasias and Laboratory Approach (20 marks)

Definition

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Classification

A. Multiple Myeloma (MM)

B. Smouldering Multiple Myeloma (SMM)

C. Monoclonal Gammopathy of Undetermined Significance (MGUS)

D. Waldenström's Macroglobulinaemia (WM)

E. Light Chain (AL) Amyloidosis

F. Heavy Chain Diseases (HCD)

G. Plasma Cell Leukaemia (PCL)

H. Solitary Plasmacytoma (Bone and Extramedullary)

I. POEMS Syndrome

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1. Multiple Myeloma (MM)

• C - HyperCalcaemia (>11 mg/dL)
• R - Renal insufficiency (creatinine >2 mg/dL)
• A - Anaemia (Hb <10 g/dL)
• B - Bone lesions (lytic, punched-out; "rain-drop skull")

• Clonal bone marrow plasma cells ≥10% OR biopsy-proven plasmacytoma
• PLUS one of: CRAB criteria OR myeloma-defining biomarkers (MDE):
  • Clonal BMPC ≥60%
  • Serum FLC ratio ≥100 (involved:uninvolved)

  • 1 focal lesion on MRI

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2. Smouldering Multiple Myeloma (SMM)

• M-protein ≥30 g/L (serum) or urinary M-protein ≥500 mg/24h
• AND/OR clonal BMPC 10-60%
• No CRAB criteria or MDE
• Risk of progression to MM: ~10% per year (first 5 years); "20-2-20" risk model

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3. MGUS

• M-protein <30 g/L
• Clonal BMPC <10%
• No CRAB criteria
• Prevalence: ~3% in adults >50 years; ~5% in >70 years
• Risk of progression: 1% per year (overall)
• Higher risk if: non-IgG MGUS, M-protein >15 g/L, abnormal FLC ratio

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4. Waldenström's Macroglobulinaemia

• IgM M-protein production by lymphoplasmacytic lymphoma (LPL)
• MYD88 L265P mutation present in >90%
• Clinical: hyperviscosity syndrome, cryoglobulinaemia, cold agglutinin disease, peripheral neuropathy (anti-MAG antibody)
• Diagnosis: IgM M-protein + LPL on bone marrow biopsy

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5. AL (Light Chain) Amyloidosis

• Free light chains (kappa or lambda, usually lambda) misfold → amyloid fibrils
• Organ deposition: kidneys (nephrotic syndrome), heart (restrictive cardiomyopathy), liver (hepatomegaly), nerves, tongue (macroglossia)
• Diagnosis: Congo red staining (apple-green birefringence under polarised light); LC-MS/MS for typing
• Differentiate from other amyloid types: AA (SAA-derived, reactive), ATTR (transthyretin), Aβ2M (dialysis)

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6. POEMS Syndrome

• Polyneuropathy, Organomegaly, Endocrinopathy, M-protein (usually IgG or IgA lambda), Skin changes
• Associated: papilloedema, ascites, pleural effusion, thrombocytosis/polycythaemia
• Pathogenesis: elevated VEGF (vascular endothelial growth factor) levels are central
• Diagnosis: mandatory features: Polyneuropathy + M-protein; minor criteria as above + elevated VEGF

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Laboratory Approach to Plasma Cell Dyscrasias

Step 1: Screening Tests

• Normochromic normocytic anaemia (most common)
• Rouleaux formation on peripheral smear (parallel stacking of RBCs; high ESR)
• Leucopenia and thrombocytopenia (marrow infiltration)
• Plasmacytes in peripheral blood (plasma cell leukaemia if >20% or >2×10⁹/L)

• Markedly elevated (often >100 mm/1st hour)
• Due to paraprotein coating of RBCs increasing rouleaux

• Rouleaux formation
• Circulating plasma cells (PCL)
• Anaemia morphology
• Thrombocytopenia in advanced disease

Step 2: Protein Studies (Most Diagnostic)

• Shows discrete narrow M-spike (monoclonal band) in γ, β, or α2 region
• Identifies M-protein type and concentration
• Background immunoglobulins: reduced in other regions (immunoparesis)
• Pattern interpretation:
  • Tall, narrow spike: monoclonal (myeloma, WM)
  • Broad diffuse increase: polyclonal (infection, chronic inflammation, liver disease)
  • Decreased γ-globulin: hypogammaglobulinaemia (CLL, non-secretory myeloma)

• Gold standard for M-protein characterisation
• Identifies heavy chain class (IgG, IgA, IgM, IgD, IgE) and light chain type (kappa or lambda)
• More sensitive than SPEP (detects M-protein as low as 0.1 g/L)
• IgG myeloma most common (52%), then IgA (21%), light chain only (16%), IgD (<2%), IgM (<0.5%), IgE (rare), biclonal (<1%), non-secretory (<1%)

• 24-hour urine collection
• Bence Jones proteins (BJP) = free monoclonal light chains
• Heat test (obsolete): BJP precipitates at 56°C, redissolves at 100°C
• Urine IFE: identifies BJP type (kappa or lambda)
• BJP indicates light chain myeloma or light chain disease

• 200 mg/24h BJP: significant

• Quantifies free kappa and lambda light chains in serum
• Normal kappa:lambda ratio: 0.26-1.65
• Abnormal ratio (>1.65 or <0.26): suggests clonal plasma cell disorder
• FLC ratio >100: MDE for myeloma
• Used for monitoring response in non-secretory/oligosecretory myeloma
• Highly sensitive; detects disease not visible on SPEP

• Total protein, M-protein quantification
• Proteinuria pattern: nephrotic range in AL amyloidosis; tubular in cast nephropathy (myeloma kidney)

Step 3: Bone Marrow Examination

• Quantification of plasma cells (% of nucleated cells)
• Clonal plasma cells: increased size, eccentric nucleus, clock-face/cartwheel chromatin, perinuclear hof (Golgi zone), basophilic cytoplasm
• Special forms: flame cells (IgA myeloma), Mott cells (multiple Russell bodies), Russell bodies (intracytoplasmic Ig deposits), Dutcher bodies (intranuclear)
• Plasma cell aggregates/sheets

• Architecture: diffuse vs. focal/paratrabecular infiltration
• Reticulin fibrosis
• Vascular pattern (increased in POEMS)
• Essential when aspirate is dry/hypocellular (fibrotic marrow)

• CD138 (syndecan-1): plasma cell marker; highlights myeloma cells
• CD38: plasma cells (also NK cells)
• Kappa/Lambda light chain restriction: monoclonality confirmed when one light chain predominates
• CD56: aberrant expression in myeloma (helps distinguish from reactive plasmacytosis - reactive cells are CD56 negative)
• CD19: negative in myeloma; positive in normal plasma cells and WM

• Normal plasma cells: CD38+, CD138+, CD19+, CD45+, CD56-
• Myeloma plasma cells: CD38+, CD138+, CD19-, CD45-/dim, CD56+, CD117+
• Light chain restriction: demonstrates monoclonality
• EuroFlow panel: standardised 8-colour panel for MRD assessment
• Minimal Residual Disease (MRD) by flow: sensitivity 10⁻⁵; prognostic importance

Step 4: Cytogenetics and Molecular Studies

• Low yield due to low proliferative index
• Monosomy 13/del(13q): adverse prognosis
• Hyperdiploidy (trisomies): standard risk, better prognosis

• t(4;14)(p16;q32): FGFR3/MMSET rearrangement; adverse prognosis
• t(14;16)(q32;q23): MAF rearrangement; adverse prognosis
• t(14;20): adverse
• t(11;14)(q13;q32): Cyclin D1; standard risk; positive in 15% - VENETOCLAX sensitive
• del(17p)/TP53 deletion: worst prognosis; aggressive disease
• del(1p) / amp(1q): adverse/intermediate
• Classification: Standard Risk (SR) vs. High Risk (HR) FISH

• MGUS/SMM: somatic mutation profiling
• WM: MYD88 L265P PCR; CXCR4 mutation
• Next-generation sequencing: emerging for prognostication and treatment selection

Step 5: Imaging

Step 6: Other Studies

• Serum calcium: hypercalcaemia (PTHrP-like activity, osteoclast activation)
• Creatinine/eGFR: myeloma cast nephropathy
• Uric acid: hyperuricaemia
• LDH: elevated = adverse prognosis
• Beta-2 microglobulin (β2M): major component of ISS staging

• International Staging System (ISS):
  • Stage I: β2M <3.5 mg/L + Albumin ≥3.5 g/dL
  • Stage II: Neither I nor III
  • Stage III: β2M ≥5.5 mg/L
• Revised ISS (R-ISS): adds LDH and high-risk FISH to ISS

• Serum viscosity >4 cP: symptomatic hyperviscosity
• Plasmapheresis/leukapheresis indicated for symptomatic hyperviscosity

• Type I (monoclonal IgM): WM, myeloma; precipitates at <37°C
• Type II/III (mixed): HCV, SLE, infections

• Congo red staining of abdominal fat aspirate (sensitivity ~70-80%) or bone marrow biopsy
• Laser capture microdissection/mass spectrometry (LC-MS/MS): amyloid typing gold standard
• Cardiac biomarkers: troponin T/I, NT-proBNP (cardiac staging for AL)
• Echocardiography: granular sparkling appearance, restrictive filling pattern

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Q4 — Short Notes

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Q4(a) — Different Blood Group Systems with a Note on Bombay Blood Group (10 marks)

Introduction

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Major Blood Group Systems

• Discovered by Karl Landsteiner in 1900; most clinically important
• Antigens: A and B glycoprotein antigens on RBC surface; encoded by ABO gene on chromosome 9q34
• A antigen: N-acetylgalactosamine added to H antigen
• B antigen: D-galactose added to H antigen
• Naturally occurring IgM antibodies (isohemagglutinins) present from birth (6 months)
• Rule of Landsteiner: person lacks the antigen they carry the antibody for

• Group O universal donor (RBCs); Group AB universal recipient; Group AB universal plasma donor
• Subgroups: A1 (strongest), A2 (weaker); A2 has anti-A1 in ~1-2% cases (clinically significant)

• Discovered by Landsteiner and Wiener in 1940; second most important
• 54 antigens; most important: D, C, c, E, e
• D antigen: most immunogenic after ABO; 85% of population is Rh D-positive
• Rh genes: RHD (encodes D antigen) and RHCE (encodes C/c/E/e) on chromosome 1p34-36
• Weak D: quantitatively reduced D antigen expression; now classified by molecular testing
• Partial D: qualitative deficiency of D antigen epitopes; can form anti-D despite typing Rh+
• Antibodies: IgG (immune); clinically significant; cause HDFN and delayed HTR
• du/weak D typing: weak reactivity by IAT; clinical significance depends on genotype
• Anti-D prophylaxis: Rh-D negative mothers receive anti-D immunoglobulin to prevent sensitisation

• 36 antigens; most important: K (Kell) and k (cellano)
• K antigen: found in 9% of Caucasians; highly immunogenic
• Anti-K: IgG; causes severe HDFN and haemolytic transfusion reactions
• McLeod phenotype: absence of Kx protein; associated with McLeod neuroacanthocytosis syndrome
• Kp^a, Kp^b, Js^a, Js^b: other clinically significant antigens

• Fy^a and Fy^b antigens on DARC protein (Duffy Antigen Receptor for Chemokines)
• Receptor for Plasmodium vivax and Plasmodium knowlesi invasion
• Fy(a-b-): Duffy-null phenotype; common in sub-Saharan Africans; resistant to P. vivax malaria
• Anti-Fy^a: IgG; causes HDFN and delayed HTR

• Jk^a and Jk^b on Kidd glycoprotein (urea transporter, HUT11)
• Anti-Jk^a: notorious for: immune evasion (antibody levels fluctuate, often undetectable), causing severe delayed HTR, rare acute HTR
• Jk(a-b-) phenotype: "null" Kidd; resistant to urea-induced RBC lysis (clinical test)

• Le^a and Le^b antigens: NOT intrinsic to RBC; adsorbed from plasma
• Lewis genes: FUT3 (Le gene) and FUT2 (Se secretor gene)
• Most anti-Lewis antibodies are IgM, clinically insignificant (cold-reactive, complement activating)
• Le(a+b-): Le gene present, non-secretor; Le(a-b+): Le gene + secretor; Le(a-b-): no Le gene

• M, N, S, s antigens on glycophorin A (GPA) and B (GPB)
• Anti-M and Anti-N: usually IgM, clinically insignificant
• Anti-S and Anti-s: IgG; clinically significant; cause HDFN and HTR
• En(a-): rare; absence of GPA; associated with Miltenberger antibodies

• P1 antigen (polylactosamines on glycolipids)
• Anti-P1: IgM, clinically insignificant
• p (null phenotype): very rare; anti-P+P1+P^k (anti-Tja); strong; cause spontaneous abortions and HTR
• Donath-Landsteiner antibody: anti-P specificity (PCH)

• i antigen (linear polylactosamine) converted to I (branched) during first 18 months of life
• Anti-I: cold IgM antibody; warm-reactive anti-I associated with Mycoplasma pneumoniae infections and cold agglutinin disease
• i antigen: retained in adult red cells in congenital dyserythropoietic anaemia type II (HEMPAS) and thalassaemia major

• Lu^a and Lu^b; IgG anti-Lu^a: can cause mild HDFN
• Inhibited by In(Lu) phenotype (dominant gene inhibits Lutheran expression)

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Bombay Blood Group (Oh / H-null Phenotype)

• Normal ABO synthesis requires the H antigen as a precursor
• H antigen is synthesised by FUT1 (H gene) enzyme (alpha-2-L-fucosyltransferase) on chromosome 19q13
• FUT1 transfers fucose to the terminal galactose of the precursor carbohydrate chain on RBCs
• Bombay phenotype results from homozygous mutations in FUT1 (se/se, h/h) → complete absence of H antigen
• Without H antigen, neither A nor B antigens can be synthesised (even if ABO genes are present)
• Blood type appears as group "O" on routine testing but lacks H antigen

• RBC antigens: No H, No A, No B
• Serum antibodies: Anti-H + Anti-A + Anti-B (all three, IgM + IgG)
• Anti-H: potent IgM + IgG; haemolytic; reacts at all temperatures
• Forward grouping: Appears as group O
• Reverse grouping: Reacts with all cells including O cells (group O cells express abundant H antigen)
• "Discrepancy" in ABO grouping alerts to possibility of Bombay

• Lectin tests: Ulex europaeus lectin (anti-H): agglutinates normal O/A/B/AB red cells; DOES NOT agglutinate Bombay RBCs
• H antigen: absent on RBCs and in secretions (even in secretors)
• Serological: serum reacts with all donor cells including O cells

• India: 1 in 10,000
• Europe/USA: 1 in 1,000,000
• Maharashtra (India): higher incidence due to consanguinity
• Para-Bombay: weaker variant; H absent from RBCs but present in secretions; FUT1 mutant, FUT2 intact

• Extremely difficult to transfuse: Anti-H reacts with ALL human red cells (including O cells); universal incompatibility
• Patients can only be safely transfused with Bombay-type blood
• Autologous blood donation before elective surgery is recommended
• Family members should be tested (identical phenotype sibling donors)
• Misidentification: If not recognised, group O blood is given - catastrophic haemolytic transfusion reaction (anti-H destroys all O cells)
• Anti-H titre: Can be dangerous even in pregnancy though HDFN rarely reported (fetal cells express low H)

• Mutations in FUT1 but FUT2 intact: H antigen absent from RBCs but present in saliva and other secretions
• Less severe clinical significance but still incompatible with most blood

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Q4(b) — Semen Analysis (10 marks)

Introduction

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Sample Collection

• Abstinence period: 2-7 days (WHO 2021; earlier WHO: 2-5 days)
• Collection method: masturbation into sterile wide-mouthed polypropylene container
• Special Silastic condoms available if masturbation not possible
• Deliver to lab within 60 minutes of collection; maintain at body temperature (20-37°C) during transport
• Minimum 2 specimens, 2-7 weeks apart, for reliable assessment
• Incomplete specimens not analysed

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WHO 2021 Reference Values (Lower Reference Limits - 5th Centile)

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Macroscopic Examination (after 20-60 min liquefaction)

• Normal: complete within 60 minutes at room temperature (usually 15-20 min)
• Failure to liquefy: prostate insufficiency; poor semen quality

• Normal: grey-white opalescent
• Yellow: pyospermia, prolonged abstinence
• Brown/rust: haematospermia (seminal vesicle bleeding)

• Normal: ≥1.4 mL (WHO 2021)
• Hypospermia (<1.4 mL): retrograde ejaculation, ejaculatory duct obstruction, congenital bilateral absence of vas deferens (CBAVD), androgen deficiency
• Aspermia: absent ejaculate; retrograde ejaculation, anejaculation, obstruction
• Hyperspermia (>6 mL): seminal vesicle hypersecretion

• Normal: ≥7.2
• Low pH (<7.0): acidic fluid from prostate dominates; absent/blocked seminal vesicles or ejaculatory ducts; accompanies azoospermia in CBAVD (fructose negative)
• High pH (>8.0): acute infection, prostatitis

• Assess by allowing sample to drop from pipette: droplets form normally
• Hyperviscosity: poor semen quality, anti-sperm antibodies; may affect motility assessment

• Produced by seminal vesicles; absent in CBAVD, ejaculatory duct obstruction, seminal vesicle agenesis
• Fructose negative + azoospermia + low pH + small volume = obstructive azoospermia at/below seminal vesicle level

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Microscopic Examination

• Using Improved Neubauer hemocytometer or Makler chamber (1:20 or 1:5 dilution)
• At least 400 cells counted per chamber
• Azoospermia: no sperm in pellet after centrifugation (2000g, 15 min)
• Cryptozoospermia: rare sperm on fresh examination, but present after centrifugation

• WHO categories:
  • PR (Progressive): moves actively in straight line or large circles
  • NP (Non-Progressive): all other non-progressive movement
  • IM (Immotile): no movement
• Normal: Total motility (PR+NP) ≥42%; Progressive ≥30%
• Asthenozoospermia: reduced motility
• Immotile cilia syndrome (Kartagener): all sperm immotile; absent dynein arms on EM; situs inversus, bronchiectasis

• Based on Tygerberg strict criteria: only "perfect" forms counted as normal
• Normal sperm: oval head (3-5 µm length, 2-3 µm width), smooth acrosome covering 40-70% of head, intact midpiece, single straight tail
• Normal: ≥4% (WHO 2021); teratozoospermia if <4%
• Abnormal forms:
  • Head defects: macrocephaly, microcephaly, round head (globozoospermia - no acrosome), double head, vacuoles
  • Midpiece defects: thick/irregular/absent midpiece, cytoplasmic droplets
  • Tail defects: coiled, short, multiple, bent tails
• Special staining: Papanicolaou, Diff-Quik, Shorr stain

• Eosin-nigrosin exclusion test: live sperm exclude eosin (unstained); dead sperm take up eosin (pink)
• Hypoosmotic swelling (HOS) test: live sperm swell and tail coils; important for ICSI
• Normal: ≥54% live

• Distinguish from round spermatogenic cells
• Peroxidase staining (leukocytes stain brown) or immunocytochemistry (CD45)

• 1×10⁶/mL: pyospermia; indicates infection (prostatitis, seminal vesiculitis, epididymitis)

• Culture for gonorrhoea, Chlamydia, aerobic/anaerobic organisms

• Sperm clumping head-to-head, tail-to-tail, mixed
• Suggests anti-sperm antibodies
• Grade 1 (<10 sperm per clump) to Grade 4 (all agglutinated)

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Advanced/Additional Tests

• Mixed Agglutination Reaction (MAR) test: IgG and IgA ASA on live motile sperm; performed with PRBC coated with IgG or IgA
• Direct Immunobead test (IBT): immunobeads coated with anti-IgG, IgA, IgM bind to sperm; positive if ≥50% motile sperm with beads
• Significant: >50% sperm bound; associated with post-vasectomy, genital tract obstruction, testicular torsion

• Automated video-microscopy analysis
• Provides: VCL (curvilinear velocity), VSL (straight-line velocity), VAP (average path velocity), ALH (amplitude of lateral head displacement), BCF (beat cross frequency), LIN (linearity), STR (straightness)
• Distinguishes hyperactivated sperm (required for fertilisation)
• Quality control for ART: IVF, ICSI

• Alpha-glucosidase (neutral): epididymal marker; low in epididymal obstruction
• Fructose: seminal vesicle marker
• Zinc/Citric acid: prostatic marker; low in prostatitis
• Acrosin activity: acrosomal function

• TUNEL assay, SCSA (Sperm Chromatin Structure Assay), SCD (Sperm Chromatin Dispersion) test
• DFI >25-30%: impairs fertilisation, embryo development, recurrent miscarriage
• Normal: DFI <15%

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Nomenclature Summary

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Q4(c) — Interpretation of Scattergram and Histogram in Automated Haematology Analysers (10 marks)

Introduction

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Histograms

• X-axis: Cell size (volume in femtolitres, fL)
• Y-axis: Relative frequency (number of cells)
• Each peak/curve represents a cell population

• Three regions: Lymphocyte peak (~35-100 fL), Mononuclear peak (~100-200 fL), Granulocyte peak (~200-450 fL)

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Scattergrams (Cytograms)

• Light scatter: Forward scatter (FSC) = size; Side scatter (SSC) = granularity/internal complexity
• Fluorescence: Nucleic acid dyes (e.g., polymethine dyes, oxazine) for RNA/DNA content
• Impedance (Coulter): Volume

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• Y-axis: Side fluorescence (SFL) - RNA/DNA content
• X-axis: Side scatter (SSC) - granularity

• Reticulocytes stained with fluorescent dye (polymethine/oxazine) - RNA-rich
• Y-axis: SSC (Side scatter/size)
• X-axis: SFL (Fluorescence intensity, RNA content)

• RBC: Low SFL, scattered (no RNA)
• Reticulocytes: SFL positive; subclassified by RNA content:
  • LFR (Low Fluorescence Reticulocytes): mature retics, slightly elevated SFL
  • MFR (Middle Fluorescence Reticulocytes): intermediate
  • HFR (High Fluorescence Reticulocytes): immature retics, high RNA = Immature Reticulocyte Fraction (IRF)
• PLT-O (Optical Platelets): Low SFL, low SSC
• RPC (Reticulocyte Platelet Count): Reticulated/immature platelets detected here

• WNR channel (WBC/NRBC): differentiates NRBCs from WBCs using nucleic acid staining
• WDF channel: Full 5-part differential
• WPC channel (White cell Precursor Channel): detects myeloid blasts and immature cells

• Optical platelet counting using fluorescent dye
• More accurate in thrombocytopenia (avoids confusion with debris, microcytic RBCs)
• Detects IPF (Immature Platelet Fraction) = reticulated platelets

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Clinical Utility of Histogram/Scattergram Flags

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Q4(d) — Automated Reticulocyte Parameters and Their Clinical Utility (10 marks)

Introduction

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Standard Reticulocyte Parameters

• Absolute number of reticulocytes per unit volume (×10⁹/L or ×10¹²/L)
• Normal: 20-100 ×10⁹/L
• More clinically meaningful than percentage (which is affected by the total RBC count)

• Normal: 0.5-2.5%
• Elevated: Haemolytic anaemia, haemorrhage, treatment response (iron/B12/folate)
• Decreased: Aplastic anaemia, bone marrow failure, nutritional deficiency

• Corrects for anaemia-related "stress" release of reticulocytes
• Formula: RPI = (Retic% × Patient Hct/Normal Hct) / Maturation Factor
• Maturation factor: 1 (Hct >35%), 1.5 (25-35%), 2 (15-25%), 2.5 (<15%)
• Interpretation:
  • RPI >3: Adequate marrow response (haemolytic/blood loss)
  • RPI <2: Inadequate response (hypoproliferative anaemia - iron deficiency, aplasia)

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Advanced Reticulocyte Parameters (Sysmex XN nomenclature)

• Also called Reticulocyte Maturity Index (RMI) on some platforms
• Represents the proportion of highly RNA-rich (immature) reticulocytes among all reticulocytes
• Subfractions:
  • LFR (Low Fluorescence Reticulocytes): mature reticulocytes, near-mature
  • MFR (Medium Fluorescence Reticulocytes)
  • HFR (High Fluorescence Reticulocytes): most immature, highest RNA content
  • IRF = (MFR + HFR) / Total Reticulocytes
• Normal IRF: 2-17%
• Elevated IRF: earlier indicator of marrow recovery than total reticulocyte count; haemolysis; also called "stress reticulocytes"

• Bone marrow engraftment monitoring: IRF rises 2-3 days before total reticulocyte count after stem cell transplant; earliest indicator of engraftment
• Haemolysis: High IRF indicates active haemolysis with rapid marrow response
• Treatment response: IRF rises first after iron/EPO therapy, before RBC count improves
• Aplastic anaemia: IRF low in aplasia; rising IRF signals recovery

• Reticulocyte Haemoglobin equivalent (Ret-He on Sysmex; CHr - Cellular Haemoglobin in Reticulocytes on Beckman)
• Measures the haemoglobin content of individual reticulocytes (pg/cell)
• Reflects current (real-time) iron availability for erythropoiesis (reticulocytes are 1-2 days old)
• Normal Ret-He: 30-35 pg; CHr: 28-35 pg

• Ret-He is superior to conventional indices (serum ferritin, TSAT) in detecting functional iron deficiency in dialysis patients
• Cut-off for functional iron deficiency in haemodialysis: Ret-He <32 pg (KDIGO guidelines)
• ADVANTAGE: Not affected by inflammation (unlike ferritin, serum iron, TSAT)

• Volume of reticulocytes (larger than mature RBCs normally)
• Elevated in macrocytic states (B12/folate deficiency) before MCVr changes
• Low in microcytic conditions (iron deficiency, thalassaemia)
• Earlier indicator than MCV (reflects most recent RBC production)

• Reticulocyte MCHC
• Elevated MCHCr: hereditary spherocytosis

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Integrated Clinical Applications

• Ret-He used to guide iron supplementation in EPO-treated patients
• Ret-He <29 pg during EPO therapy → IV iron indicated (absolute or functional iron deficiency)
• More timely than waiting for Hb drop

• IRF rises on Day +10 to +12 post-transplant (earliest sign of engraftment)
• Total reticulocyte count rises Day +12 to +15
• Absolute neutrophil count recovery Day +14 to +21
• Rising IRF with low absolute reticulocyte count = early marrow recovery

• Post-chemotherapy nadir: IRF low (marrow suppressed)
• Recovery phase: IRF rises first (immature retics released) → then reticulocyte count → then Hb

• Reticulocyte crisis (peak): Day 7-10 after starting iron therapy in IDA
• IRF and Ret-He normalise before Hb rises fully
• Failure of Ret-He to normalise despite iron therapy → consider concurrent thalassaemia, ACD, or non-compliance

• Elevated IRF at baseline; spikes further during crisis
• Ret-He often low (functional iron deficiency component, sickling effect on erythropoiesis)
• Post-transfusion: Ret-He suppressed (exogenous RBCs suppress erythropoiesis)

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Summary Table: Key Automated Reticulocyte Parameters

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