Hematology Lecture Notes - Hemoglobin, Anemia, WBCs & Leukemias

Hemoglobin Determination and Analysis

Hemoglobinometry Overview:
- Hemoglobinometry is the quantitative measurement of hemoglobin concentration in blood specimens.
- Historical techniques have largely been superseded by modern automated and reference methods.
- Cyanmethemoglobin (HiCN) Method:
- Recognized universally as the reference method for hemoglobin determination.
- Utilized in automated analyzers including Beckman-Coulter, Abbott Cell-Dyn, and Siemens Advia.
- Exception: Sysmex analyzers do not use the HiCN method; they utilize the Sodium Lauryl Sulfate (SLS) method.
- Chemical Principle of the HiCN Method: $\text{Hemoglobin (Fe}^{2+}\text{)} + \text{K}_3\text{Fe(CN)}_6 \rightarrow \text{Methemoglobin (Fe}^{3+}\text{)} + \text{KCN} \rightarrow \text{Cyanmethemoglobin}$
- Drabkin's Reagent major components:
  - Potassium Ferricyanide ( $\text{K}_3\text{Fe(CN)}_6$ ): Converts the ferrous iron ( $\text{Fe}^{2+}$ ) of hemoglobin into ferric iron ( $\text{Fe}^{3+}$ ), forming methemoglobin.
  - Potassium Cyanide ( $\text{KCN}$ ): Provides cyanide ions ( $\text{CN}^-$ ) that bind with methemoglobin to produce stable cyanmethemoglobin.
- Complete conversion of hemoglobin to cyanmethemoglobin requires $10\,\text{minutes}$ at room temperature.
- Storage Note: Drabkin's reagent is light-sensitive and must be stored in a brown glass bottle or kept in a dark location.
- Spectrophotometric Measurement:
- Absorptance is measured using a spectrophotometer set at a wavelength of $540\,\text{nm}$ .
- Governed by Beer-Lambert's Law: States that the absorbance ( $A$ ) of monochromatic light passing through a solution is directly proportional to the concentration ( $c$ ) of the absorbing substance and the path length ( $l$ ) of the sample medium ( $A \propto c \cdot l$ ).
- Spectrophotometric detection modules are integrated internally within high-throughput automated hematology analyzers.
- All forms of hemoglobin are converted and measured by the HiCN method except Sulfhemoglobin.
- Alternative Hemoglobin Determination Methods:
- Sodium Lauryl Sulfate (SLS) Method:
  - Transforms hemoglobin into SLS-methemoglobin.
  - Advantage: Does not generate toxic cyanide waste products.
  - Implemented in Sysmex series automated analyzers.
- HemoCue System:
  - Handheld point-of-care system that converts hemoglobin to azidemethemoglobin.
  - Read photometrically at dual wavelengths: $570\,\text{nm}$ and $880\,\text{nm}$ .
  - Frequently used in the Blood Bank section for screening potential blood donors to determine eligibility.
Sources of Error in Cyanmethemoglobin Measurement:
- Spectrophotometric measurement requires a clear solution; sample turbidity causes light scattering, leading to falsely elevated hemoglobin values. | Source of Error / Turbidity Cause | Diagnostic Threshold / Trigger | Corrective Laboratory Action | | :--- | :--- | :--- | | High WBC Count | $> 20 \times 10^9/\text{L}$ | Centrifuge the reagent-sample mixture; measure the absorbance of the clear supernatant. | | High Platelet Count | $> 700 \times 10^9/\text{L}$ | Centrifuge the reagent-sample mixture; measure the absorbance of the clear supernatant. | | Lipemia | Postprandial fatty meals | Add $0.01\,\text{mL}$ of patient plasma to $5\,\text{mL}$ of cyanmethemoglobin reagent to prepare a custom reagent blank. | | Hemoglobin S and C | Presence of Hb S or Hb C | Prepare a $1:2$ dilution using distilled water ( $1\text{ part diluted sample} + 1\text{ part } \text{H}_2\text{O}$ ); multiply the concentration read from the standard curve by $2$ . | | Abnormal Globulins | Plasma cell myeloma, Waldenström macroglobulinemia | Add $0.1\,\text{g}$ of potassium carbonate ( $\text{K}_2\text{CO}_3$ ) to the reagent. Modern commercial reagents contain $\text{KH}_2\text{PO}_4$ salt to prevent protein precipitation. |
Hemoglobin Electrophoresis:
- Principles: Migration of charged protein molecules in an electric field based on differences in net surface electrical charge.
- Hemoglobin is an amphoteric molecule; its net charge depends on the pH environment to which it is exposed.
- At alkaline pH, hemoglobin carries a net negative charge and migrates toward the positive electrode (anode).
- At acidic pH, hemoglobin carries a net positive charge and migrates toward the negative electrode (cathode).

Cellulose Acetate Electrophoresis Migration

Cellulose Acetate Electrophoresis:
- Operating pH: $\text{pH } 8.4\text{–}8.6$ (alkaline buffer).
- Role: Primary screening procedure for detecting hemoglobin variants.
- Point of Application (POA): Located near the cathode ( $-$ ).
- Migration order from origin/cathode ( $-$ ) toward anode ( $+$ ):
  - Slowest hemoglobins (remain near POA/cathode): Hb C, Hb $A_2$ , Hb E, Hb $\text{C}_{\text{Harlem}}$ , Hb $\text{O}_{\text{Arab}}$ .
  - Intermediate hemoglobins: Hb S, Hb D, Hb G (co-migrate in the same band position).
  - Fastest normal hemoglobin: Hb A (Hb $A_1$ ).
  - Fast abnormal hemoglobins: Hb Bart's, Hb H, Hb I (migrate past Hb A toward the anode).
- Limitations: Cannot distinguish hemoglobins that co-migrate to identical positions (e.g., Hb S vs Hb D vs Hb G, or Hb C vs Hb $A_2$ vs Hb E).

Citrate Agar Electrophoresis:
- Operating pH: $\text{pH } 6.0\text{–}6.2$ (acidic buffer).
- Role: Confirmatory/definitive procedure following abnormal cellulose acetate screening.
- Point of Application (POA): Located near the center of the gel medium.
- Hemoglobins assume distinct positive or negative charges at acidic pH.
- Definitive separation: Specifically differentiates Hb S from Hb D and Hb G, and separates Hb C from Hb E, Hb $\text{O}_{\text{Arab}}$ , and Hb $\text{C}_{\text{Harlem}}$ .
- Hemoglobin Biosynthesis:
Heme Synthesis (Ferroprotoporphyrin IX):
- Primary sites: Mitochondria and cytoplasm of erythroid precursors in bone marrow (accounts for $85\%$ of daily synthesis) and hepatocytes.
- Key enzyme: Ferrochelatase (Heme Synthetase) catalyzes the insertion of ferrous iron ( $\text{Fe}^{2+}$ ) into the protoporphyrin IX ring.
- Step-by-step Biosynthetic Pathway:
  1. Mitochondrion: Succinyl-CoA + Glycine $\rightarrow$ $\delta$ -Aminolevulinic Acid (ALA) (catalyzed by ALA synthase).
  2. Cytosol: $\delta$ -ALA exits mitochondrion $\rightarrow$ Porphobilinogen $\rightarrow$ Hydroxymethylbilane $\rightarrow$ Uroporphyrinogen III $\rightarrow$ Coproporphyrinogen III.
  3. Mitochondrion: Coproporphyrinogen III re-enters mitochondrion $\rightarrow$ Protoporphyrinogen IX $\rightarrow$ Protoporphyrin IX.
  4. Mitochondrion: Protoporphyrin IX + $\text{Fe}^{2+} \xrightarrow{\text{Ferrochelatase}} \text{Heme}$ .
Globin Synthesis:
- Site: Cytoplasmic ribosomes of normoblasts.
- Genetic Control:
  - Chromosome 16: Dictates the synthesis of $\alpha$ (alpha) and $\zeta$ (zeta) globin chains. Each chain contains exactly 141 amino acids.
  - Chromosome 11: Dictates the synthesis of $\beta$ (beta), $\epsilon$ (epsilon), $\delta$ (delta), and $\gamma$ (gamma) globin chains. Each chain contains exactly 146 amino acids.
- Forms of Hemoglobin Throughout Development:
Embryonic Hemoglobins (Present exclusively during embryonic development; $0\%$ in newborns and adults):
- Hb Portland: Composition = $2\zeta, 2\gamma$
- Hb Gower 1: Composition = $2\zeta, 2\epsilon$
- Hb Gower 2: Composition = $2\alpha, 2\epsilon$
Fetal Hemoglobin:
- Hb F: Composition = $2\alpha, 2\gamma$ . Predominant hemoglobin during fetal life and in newborns ( $80\%$ in newborns; $<1\%$ in healthy adults).
Adult Hemoglobins:
- Hb $A_1$ (Hb A): Composition = $2\alpha, 2\beta$ . Predominant adult form ( $20\%$ in newborns; $97\%$ in adults).
- Hb $A_2$ : Composition = $2\alpha, 2\delta$ ( $<0.5\%$ in newborns; $2.5\%$ in adults).
- Note: Hb F ( $<1\%$ ) is classified as both fetal hemoglobin and a normal minor adult hemoglobin component.
- Hemoglobin Derivatives:
Functional Hemoglobins (Capable of reversible oxygen transport):
- Oxyhemoglobin ( $\text{HbO}_2$ ): Ferrous iron ( $\text{Fe}^{2+}$ ) bound to molecular oxygen ( $\text{O}_2$ ). Predominates in arterial blood; imparts a bright red color. Exists in the Relaxed (R) state.
- Deoxygenated Hemoglobin: Ferrous iron ( $\text{Fe}^{2+}$ ) unbound to oxygen. Predominates in venous blood; imparts a dark red color. Exists in the Tense (T) state.
- Phlebotomy Scope Restriction: Medical technologists are strictly prohibited from performing arterial punctures. Arterial collections are restricted to licensed physicians.
Dyshemoglobins (Non-functional hemoglobins incapable of normal oxygen transport):
- Carboxyhemoglobin (HbCO):
  - Hemoglobin bound to carbon monoxide ( $\text{CO}$ ).
  - $\text{CO}$ exhibits a 240-fold greater affinity for hemoglobin than oxygen.
  - Blood and skin appearance: Cherry red.
  - Major source: Automobile exhaust fumes, combustion gas.
- Methemoglobin (Hi / Ferrihemoglobin / Hemiglobin):
  - Iron oxidized to the ferric state ( $\text{Fe}^{3+}$ ); incapable of binding oxygen.
  - Blood appearance: Chocolate brown.
- Sulfhemoglobin (SHb):
  - Mixture of oxidized, partially denatured hemoglobin containing a sulfur atom inserted into the porphyrin ring.
  - Irreversible modification; cannot be converted to cyanmethemoglobin (cannot be quantitated by the HiCN method).
  - Blood appearance: Mauve-lavender.
  - Etiologies: Chronic constipation, enterogenous cyanosis, Clostridium perfringens bacteremia, exposure to sulfonamides/phenacetin.

Classification and Clinical Pathology of Anemias

General Overview of Anemia:
- Definition: Reduction below normal reference limits in one or more of the following parameters: Red Blood Cell (RBC) count, Hemoglobin concentration, or Hematocrit (Packed Cell Volume).
- Anemia is a clinical manifestation of an underlying pathological process, not a primary disease entity.
Classifications of Anemia:
- Morphologic Classification: Based on erythrocyte indices—Mean Cell Volume (MCV) and Mean Cell Hemoglobin Concentration (MCHC). Clinically most useful.
- Normocytic, Normochromic: Normal MCV ( $80\text{–}100\,\text{fL}$ ), Normal MCHC ( $31\text{–}37\,\text{g/dL}$ ).
- Microcytic, Hypochromic: Low MCV ( $<80\,\text{fL}$ ), Low MCHC ( $<31\,\text{g/dL}$ ).
- Macrocytic, Normochromic: Elevated MCV ( $>100\,\text{fL}$ ), Normal MCHC ( $31\text{–}37\,\text{g/dL}$ ).
Normocytic, Normochromic Anemias:
- Categorized by reticulocyte response:
- Normal or Decreased Reticulocyte Count:
  - Aplastic Anemia
  - Kidney Disease (due to deficient erythropoietin synthesis)
  - Acute Blood Loss (initial phase before reticulocyte response)
- Increased Reticulocyte Count:
  - Paroxysmal Nocturnal Hemoglobinuria (PNH)
  - Paroxysmal Cold Hemoglobinuria (PCH / Donath-Landsteiner Hemolytic Anemia; mediated by Biphasic Hemolysin / Auto anti-P)
  - Sickle Cell Disease
  - Enzyme Deficiencies (G6PD deficiency, Pyruvate Kinase deficiency)
Aplastic Anemia:
- Severe bone marrow failure disorder characterized by pancytopenia (profound reduction in RBCs, WBCs, and platelets), reticulocytopenia, and marked bone marrow hypocellularity with stem cell depletion.
- Acquired Aplastic Anemia ( $80\text{–}85\%$ of cases):
- Idiopathic: No identifiable cause.
- Secondary: Triggered by exposure to benzene (histopathology clearing agent), viruses (Epstein-Barr virus, hepatitis viruses), or drugs (Chloramphenicol—the drug most frequently implicated).
- Inherited Aplastic Anemia ( $15\text{–}20\%$ of cases):
- Fanconi Anemia (FA): Most common inherited aplastic anemia. Chromosome instability disorder characterized by aplastic anemia, cancer predisposition, microcephaly, short stature, skeletal malformations (absent/hypoplastic thumbs), and skin hyperpigmentation/café-au-lait spots.
- Crucial Distinction: Fanconi Anemia is a bone marrow failure syndrome; Fanconi Syndrome is a renal proximal convoluted tubule reabsorption defect.
- Other inherited syndromes: Dyskeratosis congenita, Shwachman-Bodian-Diamond syndrome.
Paroxysmal Nocturnal Hemoglobinuria (PNH / Marchiafava-Micheli Syndrome):
- Acquired clonal stem cell disorder caused by a mutation in the PIGA gene, resulting in a deficiency of GPI-anchored surface proteins, specifically:
- DAF (Decay-Accelerating Factor / CD55)
- MIRL (Membrane Inhibitor of Reactive Lysis / CD59)
- Absence of CD55 and CD59 renders erythrocytes abnormally sensitive to complement-mediated intravascular lysis, particularly during sleep (nocturnal acidosis).
- Diagnostic Assays:
- Ham's Acidified Serum Test: PNH RBCs undergo lysis in acidified serum ( $\text{pH } 6.2$ ).
- Sugar Water Test (Sucrose Hemolysis Test): Screening test demonstrating increased complement susceptibility in low-ionic-strength sucrose.
- Flow Cytometry using FLAER (Fluorescein-labeled proaerolysin): Confirmatory diagnostic standard; detects absent GPI-anchored proteins on blood cells.
Microcytic, Hypochromic Anemias:
- Mnemonic CTAILS:
- C: Chronic blood loss
- T: Thalassemia
- A: Anemia of Chronic Inflammation (ACI)
- I: Iron Deficiency Anemia (IDA)
- L: Lead poisoning (Plumbism / Saturnism)
- S: Sideroblastic anemia
Anemia of Chronic Inflammation (ACI) / Anemia of Chronic Disease (ACD):
- Most common anemia among hospitalized patients. Associated with chronic infections (TB), inflammation (Rheumatoid Arthritis), or malignancies.
- Pathophysiology: Sideropenia in the presence of abundant tissue iron stores.
- Hepcidin: Master regulator peptide hormone/acute-phase reactant produced by the liver. Increased in chronic inflammation.
- Hepcidin binds to and induces degradation of Ferroportin (the transmembrane protein that exports iron from macrophages and enterocytes into plasma).
- Consequence: Stored iron is trapped in tissue macrophages and cannot be released to circulating transferrin for erythropoiesis.
- Protein Distinction: Ferroportin exports iron from tissue stores into blood; Transferrin transports iron within plasma.
Sideroblastic Anemia:
- Caused by abnormal protoporphyrin synthesis or impaired incorporation of iron into protoporphyrin.
- Iron accumulates within erythroblast mitochondria surrounding the nucleus.
- Diagnostic Hallmark: Ring Sideroblasts (nucleated RBC precursors with ring-like arrangements of iron-laden mitochondria visible on Prussian blue stain).
- Peripheral blood demonstrates a dimorphic population (dual presence of normochromic and hypochromic cells).
Iron Deficiency Anemia (IDA):
- Most common cause of anemia worldwide.
- Etiologies: Chronic hemorrhage, inadequate dietary intake, increased demand (pregnancy, lactation, growth spurts), intestinal malabsorption, hookworm infection.

Clinical Manifestations: Fatigue, pallor, koilonychia (spooning of fingernails), glossitis (atrophic smooth red tongue), angular cheilosis, and pica (craving non-food items; specifically pagophagia—craving ice).
Diagnostic Laboratory Parameters:
- Total Iron-Binding Capacity (TIBC): Indirect measure of transferrin concentration; elevated in IDA.
- Serum Ferritin: Reflects total body tissue iron stores. Most sensitive laboratory test for early iron deficiency; first parameter to become abnormal as stores deplete. | Progression Stage | Hemoglobin | Serum Iron | TIBC | Serum Ferritin | | :--- | :--- | :--- | :--- | :--- | | Normal Iron Status | Normal | Normal | Normal | Normal | | Stage 1: Storage Iron Depletion | Normal | Normal | Normal | Decreased | | Stage 2: Transport Iron Depletion | Normal | Decreased | Increased | Decreased | | Stage 3: Functional Iron Depletion (Frank IDA) | Decreased | Decreased | Increased | Decreased |
- Differential Diagnosis Table of Microcytic, Hypochromic Anemias: | Disease Entity | Serum Iron | TIBC | Serum Ferritin | Free Erythrocyte Protoporphyrin (FEP) | | :--- | :--- | :--- | :--- | :--- | | Thalassemias | High | Normal | High | Normal | | Anemia of Chronic Inflammation | Low | Low | High | High | | Iron Deficiency Anemia | Low | High | Low | High | | Lead Poisoning | Normal to High (Adults) / Low (Children) | Normal | Normal | High | | Sideroblastic Anemias | High | Normal | High | Mixed (High & Low) |
- Macrocytic, Normochromic Anemias:
Differentiated into Megaloblastic and Nonmegaloblastic forms: | Feature / Factor | Megaloblastic Anemia | Nonmegaloblastic Anemia | | :--- | :--- | :--- | | Etiologies | Vitamin $B_{12}$ Deficiency, Folate Deficiency, Pernicious Anemia | Chronic Alcoholism, Liver Disease, Bone Marrow Failure | | Root Mechanism | Impaired DNA synthesis (deficiency of Thymidine Triphosphate - TTP) | Membrane lipid alterations / reticulocytosis | | Hypersegmented Neutrophils ( $\ge 6$ lobes) | Present | Absent | | Erythrocyte Morphology | Oval Macrocytes | Round Macrocytes | | Megaloblasts in Marrow | Present | Absent |
- Vitamin $B_{12}$ (Cobalamin) vs Folate Deficiency:
Vitamin $B_{12}$ :
- Required as a cofactor to convert methyl-THF to THF, generating Thymidine Triphosphate (TTP) for DNA synthesis. Unconverted precursors accumulate as deoxyuridine triphosphate, causing nuclear fragmentation.
- Dietary sources: Animal products (meat, liver, eggs, dairy, shellfish). Synthesized forms: Cyanocobalamin (supplements), Methylcobalamin (natural).
- Strict vegans are uniquely susceptible to dietary $B_{12}$ deficiency.
- Pernicious Anemia: Autoimmune destruction of gastric parietal cells (or anti-Intrinsic Factor antibodies). Parietal cells produce HCl and Intrinsic Factor (IF), which is essential for $B_{12}$ absorption in the terminal ileum.
- Clinical feature: Produces neurologic and neuropsychiatric symptoms (subacute combined degeneration: numbness, tingling, loss of vibration sense, memory loss, psychosis) due to demyelination.
Folate (Folic Acid):
- Dietary sources: Leafy green vegetables, citrus fruits, grains, liver. Heat-labile (destroyed by overcooking).
- Deficiency during pregnancy causes fetal neural tube defects (e.g., spina bifida). | Diagnostic Test | Folate Deficiency | Vitamin $B_{12}$ Deficiency | | :--- | :--- | :--- | | Serum Vitamin $B_{12}$ | Normal | Decreased | | Serum Folate | Decreased | Normal or Increased | | RBC Folate | Decreased | Normal or Decreased | | Serum Methylmalonic Acid (MMA) | Normal | Increased | | Serum Homocysteine | Increased | Increased | | Holotranscobalamin Assay (holoTC) | Normal | Decreased |
Note: Holotranscobalamin (Vitamin $B_{12}$ bound to Transcobalamin) represents the metabolically active form of circulating Vitamin $B_{12}$ .
- Pathophysiologic Classification of Anemias:
Decreased RBC Production: Aplastic anemia, Megaloblastic anemia, Iron deficiency anemia, Thalassemia, Renal disease, Endocrine disorders, ACI, Marrow infiltration (Myelophthisis), Sideroblastic anemia.
Increased RBC Destruction (Hemolytic):
- Intracorpuscular: Membrane defects (Hereditary Spherocytosis, Hereditary Elliptocytosis, Pyropoikilocytosis), Enzyme deficiencies (G6PD deficiency, Pyruvate Kinase deficiency), Globin defects (Sickle Cell Disease, Hb C, Hb SC), PNH.
- Extracorpuscular: Mechanical trauma (MAHA), Infections (Malaria, Babesia, Bartonella, Ehrlichia), Chemical/Physical agents (Burns, toxins, drugs), Immune-mediated (Warm AIHA, Cold Agglutinin Disease).
Blood Loss: Acute hemorrhage (normocytic) vs Chronic hemorrhage (microcytic).

White Blood Cell Development and Morphological Kinetics

General Characteristics of White Blood Cells (Leukocytes):
- Nucleated immune defense cells.
- Reference Ranges:
- Adults (Males & Females): $4.5\text{–}11.0 \times 10^9/\text{L}$ ( $4,500\text{–}11,000/\mu\text{L}$ ).
- Newborns: $13.5\text{–}38.0 \times 10^9/\text{L}$ ( $13,500\text{–}38,000/\mu\text{L}$ ).
- Age-related Predominance:
- Adults: Neutrophils predominate.
- Children $\le 4\text{ years}$ : Lymphocytes predominate (normal relative lymphocytosis).
- Nuclear Chromatin Pattern is the most reliable criterion for assessing morphological cell maturity.
- Functional Groupings:
- Granulocytes: Neutrophils, Eosinophils, Basophils.
- Agranulocytes: Lymphocytes, Monocytes.
- Phagocytes: Neutrophils, Eosinophils, Basophils, Monocytes. (Lymphocytes are the only immunocytes lacking phagocytic function).
Granulocytic Maturation Kinetics and Stages:
1. Myeloblast:
- Diameter: $14\text{–}20\,\mu\text{m}$ . Earliest morphologically recognizable precursor under light microscopy.
- Subtypes:
  - Type I: No primary granules, open chromatin, 2–4 nucleoli.
  - Type II: Contains $\le 20$ primary (azurophilic) granules.
  - Type III: Contains $> 20$ primary granules without obscuring the nucleus.
- Bone marrow transit time: $15\,\text{hours}$ .
1. Promyelocyte:
- Diameter: $16\text{–}25\,\mu\text{m}$ . Largest cell in the granulocytic series (exception to the rule of progressive size reduction).
- Primary granule synthesis stage. Cytoplasm filled with prominent azurophilic granules.
- Contains a cytoplasmic "Hof" (perinuclear clear zone) in normal cells; absent in malignant promyelocytes of APL.
- Bone marrow transit time: $24\,\text{hours}$ .
1. Myelocyte:
- Diameter: $15\text{–}18\,\mu\text{m}$ .
- Last stage capable of mitotic division.
- Synthesis of Secondary (Specific) Granules. Morphological indicator = "Dawn of Neutrophilia" (pink granule patches appearing near the Golgi apparatus).
- Bone marrow transit time: $4.3\,\text{days}$ .
1. Metamyelocyte (Juvenile Cell):
- Diameter: $14\text{–}16\,\mu\text{m}$ . Non-mitotic. Synthesizes tertiary (gelatinase) granules.
- Nuclear indentation initiates: Kidney bean or peanut shape (nuclear indentation is $< 1/2$ of nuclear width).
1. Band Cell (Stab / Staff Cell):

 - Diameter:  $9\text{–}15\,\mu\text{m}$ . Synthesizes secretory vesicles.
 - Nucleus: Elongated, curved, sausage-shaped with parallel margins (indentation is ** $> 1/2$  of nuclear width**).
 - **Youngest granulocytic precursor normally present in peripheral blood**.
 - *CLSI Standard*: Recommends counting band cells combined within total neutrophil counts rather than reporting them as an isolated separate category.

Mature Granulocytes:
- Segmented Neutrophil ( $9\text{–}15\,\mu\text{m}$ ): $3\text{–}5$ nuclear lobes connected by thin chromatin filaments.
  - Neutrophilia Causes: Bacterial infections, inflammation, corticosteroids, acute hemorrhage, and Physiologic Neutrophilia (Pseudoneutrophilia) caused by marginated cell pool shifting into circulating pool due to exercise, stress, rage, or panic.
  - Neutropenia Causes: Overwhelming sepsis, bone marrow aplasia, chemotherapy, benzene, viral infections. Agranulocytosis = Extreme neutropenia ( $<0.5 \times 10^9/\text{L}$ ).
- Eosinophil ( $9\text{–}15\,\mu\text{m}$ ): Bilobed nucleus, cytoplasm filled with large spherical reddish-orange specific granules.
  - Eosinophilia: Associated with helminthic parasitic infections (Trichinella spiralis causes the highest counts), asthma, allergic reactions, eczema, psoriasis.
- Basophil ( $10\text{–}16\,\mu\text{m}$ ): Unregularly lobed nucleus obscured by coarse, dark purple-black granules containing histamine and heparin.
  - Basophilia: Immediate hypersensitivity reactions, hypothyroidism, ulcerative colitis, chronic myelogenous leukemia (CML).

Morphological Characteristics of Neutrophil Precursors: | Stage | N:C Ratio | Chromatin Pattern | Cytoplasm Color | | :--- | :--- | :--- | :--- | | Myeloblast | $4:1$ | Reticular / Fine | Medium Blue | | Promyelocyte | $3:1$ | Smooth / Open | Moderate Blue | | Myelocyte | $2:1 \text{ to } 1:1$ | Slightly Clumped | Blue-Pink | | Metamyelocyte | $1:1$ | Clumped | Pink | | Band Cell | $1:1$ | Very Clumped | Pink | | Segmented Neutrophil | $1:1$ | Densely Packed | Pink |
Agranulocytic Series:
- Lymphocytic Series:
- Lymphoblast ( $15\text{–}20\,\mu\text{m}$ , N:C $4:1$ ) $\rightarrow$ Prolymphocyte ( $15\text{–}18\,\mu\text{m}$ ) $\rightarrow$ Mature Lymphocyte.
- Small Lymphocyte: Diameter $6\text{–}9\,\mu\text{m}$ . High N:C ratio ( $4:1$ to $3:1$ ) with scanty cytoplasm. The nucleus of a normal small lymphocyte serves as an internal size benchmark ( $6\text{–}8\,\mu\text{m}$ ) when evaluating erythrocyte size.
- Large Lymphocyte: Diameter $17\text{–}20\,\mu\text{m}$ , abundant pale blue cytoplasm.
- Plasma Cells:
  - Fully differentiated antigen-secreting B lymphocytes ( $10\text{–}28\,\mu\text{m}$ ).
  - Nucleus: Small, round, eccentrically located with coarse, clumped chromatin presenting a "cartwheel", "clock-face", or "tortoise shell" pattern.
  - Cytoplasm: Intense cornflower blue with a prominent perinuclear halo (Golgi apparatus).
  - May contain spherical cytoplasmic inclusions packed with immunoglobulins termed Russell Bodies.
- Monocytic Series:
- Monoblast ( $12\text{–}20\,\mu\text{m}$ ) $\rightarrow$ Promonocyte ( $12\text{–}20\,\mu\text{m}$ ) $\rightarrow$ Mature Monocyte ( $12\text{–}18\,\mu\text{m}$ ).
- Monocytes circulate temporarily in peripheral blood before emigrating into tissue spaces to transform into macrophages.
- Tissue Macrophages (Histiocytes):
- Diameter: $40\text{–}50\,\mu\text{m}$ . Macrophages are the most abundant cell type in the entire human body.
- Functions: Phagocytosis, Nitric Oxide synthesis (microbicidal activity), Interleukin-1 (IL-1) secretion (induces fever and inflammatory response), and Transcobalamin production.
- Tissue-Specific Macrophage Nomenclature: | Tissue / Anatomical Site | Macrophage Cell Designation | | :--- | :--- | | Peripheral Blood | Monocyte | | Liver | Kupffer Cells | | Lungs | Alveolar Macrophages (Dust Cells) | | Kidneys | Mesangial Cells | | Brain / Central Nervous System | Microglial Cells | | Skin (Epidermis) | Langerhans Cells (distinct from Islets of Langerhans in Pancreas) | | Spleen | Littoral Cells / Splenic Macrophages | | Synovial Membranes | Type A Synoviocytes | | Bone Tissue | Osteoclasts | | Placenta | Hofbauer Cells | | Lymph Nodes | Dendritic Cells |

Manual and Automated Leukocyte Quantification and Differential

Manual Hemocytometry:
- Standard Chamber: Levy Chamber with Improved Neubauer Ruling.
- Chamber Dimensions:
- Total ruled area = $3\,\text{mm} \times 3\,\text{mm} = 9\,\text{mm}^2$ .
- Depth (distance between floor and heavy coverglass) = $0.1\,\text{mm}$ .
- Total volume of chamber = $0.9\,\text{mm}^3$ ( $\mu\text{L}$ ).

Grid Layout for Cell Counts:
- 4 Corner Large Squares (labeled 'W'): Each square is $1\,\text{mm}^2$ and subdivided into 16 small squares. Used for WBC counting (Total area counted = $4\,\text{mm}^2$ ).
- Central Large Square: $1\,\text{mm}^2$ , subdivided into 25 medium squares. Used for RBC and Platelet counting.
Thoma Pipet Characteristics: | Feature | RBC Thoma Pipet | WBC Thoma Pipet | | :--- | :--- | :--- | | Etched Markings | 0.5, 1, 101 | 0.5, 1, 11 | | Internal Bulb Bead Color | Red | White / Colorless | | Bulb Volume Factor | 100 | 10 | (Note: Plain microhematocrit capillary tubes are standard modern alternatives for charging hemocytometers).
- WBC Diluting Fluids:
Solutions: $1\%$ Ammonium Oxalate, $3\%$ Acetic Acid, or $1\%$ Hydrochloric Acid.
Function: Lyses non-nucleated erythrocytes to prevent cell overlap. Nucleated RBCs (NRBCs) resist lysis and remain intact.
Standard Dilution: $1:20$ (e.g., $25\,\mu\text{L}$ blood + $475\,\mu\text{L}$ diluting fluid).
- Counting Rules and Calculations:
Preparation: Allow diluted mixture to sit for $10\,\text{minutes}$ to complete RBC lysis. Charge chamber and let settle in a moist chamber for $10\,\text{minutes}$ .
Boundary Line Inclusion Rule:

- **TLC (Top and Left = COUNTED)**: Count cells touching top or left inner boundary lines.
- **BRI (Bottom and Right = IGNORED)**: Do not count cells touching bottom or right boundary lines.

Quality Control: Calculate percentage difference between the two sides of the chamber: $\% \text{ Difference} = \frac{|V_1 - V_2|}{\left(\frac{V_1 + V_2}{2}\right)} \times 100$
- The difference between side counts must be $< 10\%$ . If $\ge 10\%$ difference, discard and repeat procedure.
General Hemocytometer Calculation Formula: $\text{Cells}/\mu\text{L} = \frac{\text{Average Cells Counted} \times \text{Dilution Factor}}{\text{Area Counted (mm}^2\text{)} \times \text{Depth (0.1 mm)}}$
- Example: For $150\text{ cells}$ counted in 4 corner squares ( $4\,\text{mm}^2$ ) at a $1:20$ dilution: $\text{WBC}/\mu\text{L} = \frac{150 \times 20}{4 \times 0.1} = 7,500/\mu\text{L}$
- Correction for Nucleated Red Blood Cells (NRBCs):
Performed when $\ge 5\text{ NRBCs}$ are observed per $100\text{ WBCs}$ during peripheral blood smear examination.
Formula: $\text{Corrected WBC Count} = \text{Uncorrected WBC Count} \times \frac{100}{100 + \text{NRBC Count}}$
Rounding Rule: Results of corrected WBC counts must always be rounded to the nearest hundred.
Worked Example: Uncorrected WBC = $26,708/\mu\text{L}$ ; NRBCs observed = $15\text{ per } 100\text{ WBCs}$ . $\text{Corrected WBC} = 26,708 \times \frac{100}{100 + 15} = 23,224.3/\mu\text{L} \xrightarrow{\text{Round to nearest hundred}} 23,200/\mu\text{L}$
- WBC Differential Count Protocols:
100-Cell Differential: Routine method on Wright-stained smears.
200-Cell Differential: Indicated when WBC count is $>40 \times 10^9/\text{L}$ , or when abnormal distributions are noted (Eosinophils $\ge 10\%$ , Basophils $>2\%$ , Monocytes $>11\%$ , or Lymphocytes outnumber Neutrophils in adults). Divide final raw tally by 2.
300- or 400-Cell Differential: Indicated when WBC count is $>100 \times 10^9/\text{L}$ . Divide totals by 3 or 4 respectively.
50-Cell Differential: Indicated in severe leukopenia ( $\text{WBC} < 1.0 \times 10^9/\text{L}$ ) using a concentrated buffy coat smear. Multiply counts by 2.
- Differential Reporting Parameters and Reference Intervals: | Leukocyte Cell Type | Relative Reference Interval (%) | Absolute Reference Interval ( $/\mu\text{L}$ ) | | :--- | :--- | :--- | | Segmented Neutrophils | $51\text{–}67\%$ | $1,600\text{–}7,260/\mu\text{L}$ | | Lymphocytes | $25\text{–}33\%$ | $960\text{–}4,400/\mu\text{L}$ | | Monocytes | $2\text{–}6\%$ | $180\text{–}880/\mu\text{L}$ | | Eosinophils | $1\text{–}4\%$ | $45\text{–}440/\mu\text{L}$ | | Basophils | $0\text{–}1\%$ | $45\text{–}110/\mu\text{L}$ |
Absolute Count Calculation: $\text{Absolute Count} (/\mu\text{L}) = \text{Relative Count (decimal)} \times \text{Total WBC Count} (/\mu\text{L})$
- Leukocyte Kinetic Terminology:
Shift to the Left (STTL): Increase in circulating immature granulocytic precursors (bands, metamyelocytes).
- Degenerative Shift to the Left: Elevated immature cells with Normal or Low Total WBC count (e.g., Tuberculosis). Represents marrow exhaustion.
- Regenerative Shift to the Left: Elevated immature cells with Elevated Total WBC count (e.g., Acute Appendicitis). Represents functional marrow response.
Shift to the Right (STTR): Increase in hypermature, hypersegmented neutrophils (e.g., Pernicious Anemia).
Leukoerythroblastic Reaction: Simultaneous presence of immature granulocytes, nucleated RBCs, and teardrop erythrocytes (dacrocytes) in blood smears. Associated with bone marrow infiltration (Myelophthisis) and Primary Myelofibrosis (PMF).
- Non-Counted Peripheral Smear Artifacts and Cells (Report in commentary/remarks):
Smudge Cells: Nuclear remnants of fragile lymphocytes resembling thumbprints. Associated with Chronic Lymphocytic Leukemia (CLL).
Basket Cells: Nuclear remnants of granulocytes with netlike chromatin.
Necrotic Cells: Pyknotic apoptotic granulocytes caused by prolonged EDTA exposure.
Endothelial Cells: Large ( $20\text{–}30\,\mu\text{m}$ ) cells lining vessels, accidentally scraped during venipuncture.
- Comparison of Principles Across Automated Hematology Analyzers: | Parameter | Beckman Coulter UniCel DxH 800 | Sysmex XN Series | Abbott CELL-DYN Sapphire | Siemens ADVIA 2120i | | :--- | :--- | :--- | :--- | :--- | | WBC | Impedance, VCS (Volume, Conductivity, Scatter) | Fluorescent staining & light scatter | Primary light scatter & impedance | Light scatter & absorption | | Hemoglobin | Modified HiCN ( $525\,\text{nm}$ ) | Sodium Lauryl Sulfate (SLS) ( $555\,\text{nm}$ ) | Modified HiCN ( $540\,\text{nm}$ ) | Modified HiCN ( $546\,\text{nm}$ ) | | Hematocrit | $\frac{\text{RBC} \times \text{MCV}}{10}$ | Cumulative pulse height detection | $\frac{\text{RBC} \times \text{MCV}}{10}$ | $\frac{\text{RBC} \times \text{MCV}}{10}$ | | Differential Method | VCS Technology | Fluorescent flow cytometry | MAPSS Technology | Peroxidase reaction & laser scatter |
Acroynms: VCS = Volume, Conductivity, Scatter; MAPSS = Multi-Angle Polarized Scatter Separation.

Morphological Anomalies and Inclusion Pathology of Leukocytes

Lipid Storage Diseases:
- Niemann-Pick Disease (NPD):
- Deficiency of Sphingomyelinase.
- Autosomal recessive; predilection for Ashkenazi Jewish populations. Marked hepatosplenomegaly.
- Pathognomonic Cell: Pick's Cell (Foam Cell)—large tissue macrophage with cytoplasm packed with uniform lipid droplets, giving a foamy or bubbly appearance.
- Gaucher Disease:
- Deficiency of $\beta$ -Glucocerebrosidase (most common lipidosis).

- Pathognomonic Cell: **Gaucher Cell**—large bone marrow macrophage with a small eccentric nucleus and wrinkled cytoplasm presenting a characteristic **"crumpled tissue paper"** or **"onion skin"** appearance.
- *Pseudo-Gaucher Cells*: Non-specific macrophages resembling Gaucher cells seen in CML, ALL, Thalassemia, and lymphomas.

Inherited Leukocyte Disorders:
- Chédiak-Higashi Syndrome:
- Rare autosomal recessive defect in the Golgi complex (LYST gene), impairing vesicle fusion and granule assembly.
- Characterized by giant peroxidase-positive lysosomal granules in phagocytes and giant peroxidase-negative granules in lymphocytes.
- Clinical triad: Partial albinism (silvery hair, photophobia), recurrent pyogenic infections, and bleeding tendencies.
- Wiskott-Aldrich Syndrome (WAS):
- X-linked recessive disorder presenting with classic triad: Thrombocytopenia, Immunodeficiency, and Eczema.
- Key feature: Microthrombocytes (abnormally small, dysfunctional platelets lacking dense granules).
- May-Hegglin Anomaly:

- Autosomal dominant mutation in *MYH9* gene.
- Characterized by triad: **Döhle body-like inclusions** (blue spindle-shaped RNA inclusions in granulocytes), **Giant Platelets**, and variable leukopenia/thrombocytopenia.

Pelger-Huët Anomaly (PHA):
- Autosomal dominant mutation in the Lamin $\beta$ -receptor gene, leading to failure of normal nuclear segmentation.
- True PHA neutrophils function normally with normal cytoplasmic granulation.
- Heterozygous Form: Neutrophils demonstrate hyposegmentation with bilobed "pince-nez" or spectacle-shaped nuclei.
- Homozygous Form: All neutrophils possess unsegmented round or ovoid nuclei.
- Pseudo-Pelger-Huët Anomaly (Acquired): Hyposegmented, hypogranular neutrophils seen in AML, Myelodysplastic Syndromes (MDS), or severe infections.
- Morphological Variants and Specific Diagnostic Cells:
Hypersegmented Neutrophils: Neutrophils possessing $\ge 6$ nuclear lobes. Pathognomonic for Megaloblastic Anemias; also seen in MDS and Myelokathexis.
Lupus Erythematosus (LE) Cell: Neutrophil that has phagocytized an antibody-coated, denatured nuclear mass from another leukocyte. In vitro phenomenon characteristic of Systemic Lupus Erythematosus (SLE).
Tart Cell: Monocyte that has ingested an intact lymphocyte or nuclear nucleus. Associated with drug hypersensitivity reactions.

Rieder Cell: Malignant lymphocyte exhibiting a deeply notched, lobulated, or cloverleaf-like nucleus. Associated with Chronic Lymphocytic Leukemia (CLL).
Grape Cell (Mott Cell / Morula Cell): Abnormal plasma cell with cytoplasm completely filled with spherical Russell bodies (immunoglobulins). Pathognomonic for Plasma Cell Myeloma.
- Plasma Cell Myeloma CRAB Criteria: C = Hypercalcemia, R = Renal insufficiency, A = Anemia, B = Bone lesions. Urinary excretion of Bence-Jones Proteins (free immunoglobulin light chains).
Hairy Cells: Malignant B lymphocytes featuring fine hair-like cytoplasmic projections. Characterized by strong positive staining with Tartrate-Resistant Acid Phosphatase (TRAP) due to abundant isoenzyme 5 production.

Reed-Sternberg Cell: Giant binucleated malignant cell with prominent eosinophilic nucleoli imparting an "owl's eyes" appearance. Definitive histologic hallmark of Hodgkin's Lymphoma.
Flower Cells: Leukemic T lymphocytes featuring flower-like lobulated nuclei. Pathognomonic for Adult T-Cell Leukemia/Lymphoma (ATLL).
Popcorn Cells (L&H Cells): Multi-lobated lymphocytic and histiocytic variants seen in Nodular Lymphocyte-Predominant Hodgkin Lymphoma (NLPHL).
Sézary Cells: Malignant T lymphocytes displaying cerebriform (brain-like) folded nuclei. Pathognomonic for Mycosis Fungoides / Sézary Syndrome.

Alder-Reilly Inclusions: Coarse purple-red cytoplasmic granules composed of precipitated mucopolysaccharides. Present across ALL leukocyte types (neutrophils, eosinophils, basophils, monocytes, lymphocytes). Associated with mucopolysaccharidoses (Hurler, Hunter, and Maroteaux-Lamy syndromes).
Toxic Granulations: Altered, dark blue-black primary granules present exclusively in neutrophils. Associated with severe bacterial infections and lead poisoning.
Auer Rods: Red-staining linear crystalline fused primary azurophilic granules present in myeloblasts and promyelocytes of Acute Myelogenous Leukemia (AML).

- **Faggot Cells**: Leukemic cells containing **dense clusters or bundles of Auer rods** in their cytoplasm. Pathognomonic for **Acute Promyelocytic Leukemia (APL / AML M3)**.

Döhle Bodies vs. May-Hegglin Anomaly (MHA) Inclusions: | Parameter | Döhle Bodies | May-Hegglin Inclusions | | :--- | :--- | :--- | | Composition | Parallel rows of ribosomal RNA (rRNA) | Messenger RNA (mRNA) | | Morphology | Small, round/oval pale blue inclusions | Large, spindle-shaped blue inclusions | | PAS Reaction | Positive | Negative | | Associated Findings | Toxic granulation, cytoplasmic vacuolation | Giant Platelets, thrombocytopenia | | Etiology | Severe infections, burns, pregnancy | Inherited MYH9 gene mutation |

Clinical Hematology of Leukemias and Cytochemistry

Comparison Between Leukemias and Lymphomas:
- Leukemia: Neoplastic proliferation of hematopoietic cells originating primarily within the bone marrow and spilling into peripheral blood. M:E ratio often increases to $10:1$ .
- Lymphoma: Solid malignant tumors originating within secondary lymphoid organs (lymph nodes, spleen).
Acute vs. Chronic Leukemias:
- Acute Leukemias: Rapid clinical onset, severe symptoms, predominance of immature blast forms ( $\ge 20\%$ blast threshold), and normocytic normochromic anemia.
- Chronic Leukemias: Insidious onset, prolonged clinical course, predominance of mature, differentiated leukocytes.
French-American-British (FAB) Subclassifications:
- Acute Lymphoblastic Leukemia (ALL):
- ALL L1: Homogeneous population of small blasts. Predominant form in childhood ALL ( $70\%$ ).
- ALL L2: Heterogeneous population of large blasts. Predominant form in adult ALL ( $70\%$ ).
- ALL L3 (Burkitt-Type Leukemia): Homogeneous large blasts displaying prominent nuclear and cytoplasmic vacuoles. Immunologically express surface immunoglobulin (sIg).
- Cytochemistry of ALL: Myeloperoxidase (MPO) negative, Sudan Black B (SBB) negative, Periodic Acid-Schiff (PAS) positive (block-like positivity in L1/L2), TdT positive in $90\%$ of cases.
- Chronic Lymphocytic Leukemia (CLL):
- Most common leukemia in the elderly. Characterized by persistent mature lymphocytosis and abundant Smudge Cells and Rieder Cells on blood smears.
FAB Classification of Acute Myelogenous Leukemia (AML): | Subtype | Official Designation | Key Morphological / Diagnostic Features | | :--- | :--- | :--- | | AML M0 | AML with Minimal Differentiation | Blasts negative for MPO and SBB by light microscopy; requires immunophenotyping. | | AML M1 | AML Without Maturation | High blast count ( $\ge 90\%$ ); Auer rods may be present. MPO/SBB positive. | | AML M2 | AML With Maturation | Most common subtype of AML. Blasts show maturation past myeloblast stage; Auer rods present. | | AML M3 | Acute Promyelocytic Leukemia (APL) | Predominance of abnormal promyelocytes packed with Auer rods (Faggot Cells). High risk of fatal Disseminated Intravascular Coagulation (DIC). | | AML M3V | Microgranular Variant of APL | Hypogranular promyelocytes with "butterfly", "bowtie", or "coin-on-coin" bilobed nuclei. | | AML M4 | Acute Myelomonocytic Leukemia (AMML) | Also called Naegeli Monocytic Leukemia. Dual myeloid and monocytic differentiation. | | AML M4E | AMML with Increased Bone Marrow Eosinophils | Contains abnormal bone marrow eosinophils with basophilic granules. | | AML M5 | Acute Monocytic Leukemia (AMoL) | Also called Schilling Leukemia. Monocytic lineage ( $>80\%$ ). Subtypes: M5a (poorly differentiated; monoblasts predominate; children) and M5b (well differentiated; promonocytes/monocytes predominate; adults). | | AML M6 | Acute Erythroleukemia | Also called Di Guglielmo's Syndrome. Neoplastic proliferation of erythroid and myeloid precursors. Erythroid cells show strong PAS positivity. Presents with macrocytic, normochromic anemia. | | AML M7 | Acute Megakaryocytic Leukemia | Proliferation of megakaryoblasts. Requires immunocytochemistry: Factor VIII positive, CD41/CD42/CD61 positive. | | AML M8 | Acute Basophilic Leukemia | Rare subtype with basophilic differentiation. |
Chronic Myelogenous Leukemia (CML) and Philadelphia Chromosome:
- Philadelphia Chromosome ( $\text{Ph}^1$ ):
- Reciprocal translocation between the long arms of chromosome 9 and chromosome 22: $t(9;22)(q34;q11)$ .
- Translocates the ABL1 proto-oncogene from chromosome 9 to the BCR gene on chromosome 22, creating the $BCR\text{-}ABL1$ fusion gene, which encodes a constitutively active tyrosine kinase.
- Present in $>90\%$ of CML cases. Presence of $\text{Ph}^1$ confers a favorable prognosis.
- Clinical Phases: Chronic Phase $\rightarrow$ Accelerated Phase $\rightarrow$ Blast Crisis Phase ( $\ge 20\%$ blasts).
Differentiation: CML vs. Leukemoid Reaction (LR):
- Leukemoid Reaction: Reactive hyperleukocytosis ( $\text{WBC} > 50 \times 10^9/\text{L}$ ) resembling leukemia, triggered by severe infections or inflammation.
- Leukocyte Alkaline Phosphatase (LAP / NAP) Test:
- LAP enzyme is present within secondary granules of normal mature neutrophils.
- Kaplow's Method: Substrate = Sodium $\alpha$ -naphthyl phosphate; Fixative = Formalin/Methanol; Counterstain = Mayer's Hematoxylin.
- LAP Scoring: Evaluate 100 mature neutrophils/bands on a $0\text{ to }4+$ precipitation scale. Maximum score = 400. Normal reference score = 15 to 100. | Diagnostic Parameter | Chronic Myelogenous Leukemia (CML) | Leukemoid Reaction (LR) | | :--- | :--- | :--- | | LAP Score | Markedly Decreased ( $<15$ ) | Markedly Increased ( $>100$ ) | | Philadelphia Chromosome ( $\text{Ph}^1$ ) | Present ( $>90\%$ ) | Absent | | Eosinophils & Basophils | Increased | Normal or Decreased | | Toxic Granulation / Döhle Bodies | Absent | Present | | Splenomegaly | Prominent | Absent or Mild |
Flow Cytometry Immunophenotyping Markers: | Lineage Category | Monoclonal Antibody Lineage Markers | | :--- | :--- | | Immature / Hematopoietic Stem Cell | CD34, CD117, TdT | | Granulocytic / Monocytic | CD13, CD14, CD15, CD33 | | Erythroid | CD71, Glycophorin A | | Megakaryocytic | CD41, CD42, CD61 | | B Lymphocyte | CD19, CD20, CD22, Surface Immunoglobulin (sIg), $\kappa$ and $\lambda$ Light Chains | | T Lymphocyte | CD2, CD3, CD4, CD5, CD7, CD8 |
Summary of Diagnostic Cytochemical Stains:
- Myeloperoxidase (MPO): Stains peroxidase enzymes in primary granules. Differentiates AML (positive) from ALL (negative).
- Sudan Black B (SBB): Stains lipids, sterols, and phospholipids. Most sensitive stain for granulocytic precursors. Parallels MPO reactivity.
- Specific Esterase (Naphthol AS-D Chloroacetate Esterase - CAE): Positive in myeloid cells (neutrophils, myeloblasts); negative in monocytic cells.
- Nonspecific Esterase ( $\alpha$ -Naphthyl Acetate / $\alpha$ -Naphthyl Butyrate Esterase - NSE): Strongly positive in monocytic cells. Monocytic positivity is inhibited by Sodium Fluoride (NaF).
- Periodic Acid-Schiff (PAS): Stains cytoplasmic glycogen. Produces coarse block-like positivity in ALL lymphoblasts and intense positivity in neoplastic erythroblasts of AML M6 (normal erythroid precursors are PAS negative).
- Terminal Deoxynucleotidyl Transferase (TdT): Intranuclear DNA polymerase marker present in primitive lymphoblasts. Positive in $90\%$ of ALL cases; negative in AML.