Additional information

Factor number	Name
I	Fibrinogen
II	Prothrombin
III	Tissue factor
IV	Calcium
V	Proaccelerin or labile factor
VII	Stable factor or proconvertin
VIII	Antihemophilic factor A
IX	Antihemophilic factor B or Christmas factor
X	Stuart-Prower factor
XI	Plasma thromboplastin antecedent
XII	Hageman factor
XIII	Fibrin stabilizing factor
XIV	Prekallikrein (Fletcher factor)
XV	HMWK (Fitzgerald factor)

Colony stimulating factors (CSFs)

• They are glycoproteins involved in proliferation and differentiation of hematopoietic progenitor cells.
• Include granulocyte colony-stimulating factor (G-CSF), macrophage colony–stimulating factor (M-CSF, CSF-1), granulocyte-macrophage colony-stimulating factor (GM-CSF), erythropoietin, thrombopoietin and promegapoietin.
• CSFs bind to receptor proteins on the surfaces of hematopoietic stem cells, inducing them to proliferate.
• Inflammatory mediators such as LPS, TNF-alpha, IFN-beta, VEGF, IL-17 and IL-1 may induce production of CSFs.
• G-CSF stimulates the production of polymorphonuclear leukocytes
• M-CSF induces the differentiation of macrophage precursors
• GM-CSF induces maturation and differentiation of granulocytes and monocytes.
• Multiple-CSF/IL-3 stimulates bone marrow stem cells to differentiate into myeloid progenitor cells and stimulates the proliferation of mature granulocytes, monocytes, and dendritic cells
• Erythropoietin (EPO) is produced mainly by kidneys; binds to receptors on erythroid progenitor cells, promoting their survival, proliferation and differentiation; increase RBC mass
• Thrombopoietin (TPO) is mainly synthesized in the liver;binds to TPO receptors on circulating platelets, megakaryocytes and megakaryocyte precursors; increased megakaryocyte growth and decreased apoptosis; increase in platelet count
• Promegapoietin‐1a (PMP‐1a) is an agonist of IL3 and Mpl receptors; stimulates proliferation and differentiation of hematopoietic stem and progenitor cells; prevents apoptosis

Thrombopoietin (TPO) production, function, and homeostasis

TPO is mainly produced by liver parenchymal cells and by endothelial cells of the liver sinusoids. TPO production can also occur in proximal tubule cells of the kidney and in bone marrow stromal cells. The production is mainly constitutive (with no change in TPO mRNA levels), and regulation is mediated by negative feedback of the TPO-R-expressing platelet pool that internalizes and destroys free circulating TPO. IL-6 is able to induce hepatic TPO mRNA production in reactive/inflammatory thrombocythemia. Binding of old desialylated platelets to the hepatic Ashwell–Morell receptor (AMR) has been shown to promote liver TPO mRNA production. TPO plays a dual role in hematopoiesis, with an inhibiting role (red arrow) that occurs at the two extremes of the hematopoietic process, keeping stem cells quiescent and promoting a cell cycle arrest in late megakaryocytes (MKs), and a positive role in stimulating immature precursors (CMP and MEP) and promoting growth of very early progenitors of megakaryopoiesis (BFU-MK and CFU-MK)

In clinical practice, the level of arterial oxygenation can be measured either directly by blood gas sampling to measure partial pressure (PaO2) and percentage saturation (SaO2) or indirectly by pulse oximetry (SpO2).

Oxygen content or concentration: Depends on Hb levels, saturation of Hb with oxygen, affinity of Hb to oxygen and type of Hb, ventilation, gas-exchange, and partial pressure of oxygen. Hb level is the most important factor determining O2 content. SaO2 (arterial saturation with oxygen) is normally above 95%. Oxygen carrying capacity of blood is 20 ml/100 ml of blood Oxygen delivery to tissues depends on cardiac output and oxygen content Normally mixed venous blood oxygen saturation (MVO2) is 75% at rest; exercise, hypotension, reduced cardiac output will increase the tissue extraction of O2 from blood, causing reduced MVO2.

Anemia: oxygen content decreased; SaO2 normal; PaO2 normal

Methemoglobinemia: Spo2 by pulse oximeter normal;oxygen content low; decreased SaO2; normal PaO2

CO poisoning: Spo2 by pulse oximeter normal; Pao2 normal; SaO2 deceased; oxygen content low

SaO2 can be measured directly by ABG or indirectly by pulse oximetry/ spectrophotometer; SaO2 < 80% causes cyanosis

The delivery of oxygen by arterial blood to the tissues of the body has a number of critical determinants including blood oxygen concentration (content), saturation (SO2) and partial pressure, haemoglobin concentration and cardiac output,

Pulse oximetry can be error prone in poor peripheral circulation, nail varnish, methemoglobinemia, carboxyhemoglobinemia.

Venous blood sampling for ABG can be taken from peripheral veins, central venous catheters, or mixed venous blood (pulmonary artery catheter). Venous pH is slightly lower, and PCO2 is slightly higher than arterial values.

PvO2 35-45 mmHg; PvCO2 45 mmHg// PaO2 80-100 mmHg ; PaCO2 40 mmHg.; normal SvO2 is 65-75%; SaO2 > 95%

PaO2 is determined by dissolved O2 in plasma.

Causes of microcytic anemia

IDA, Thalassemia, lead toxicity, chronic inflammation, anemia of chronic disease, sideroblastic anemia, H.pylori infection, Celiac disease

Differential diagnosis of microcytic anemias*

Test	IDA	Thalassemia	AOCD#	Sideroblastic Anemia
Serum Iron	Low	Normal to high	Normal to low	Normal to high
Ferritin	Low	Normal to high	Normal to high	Normal to high
Marrow iron stores**	Very low or absent	High	Normal to high	High
TIBC	High	Normal	Low	Normal
Transferrin saturation	Low	Normal to high	Normal to low	Normal to high
RDW	High	Normal to high	Normal	High
FEP	High	Normal	Normal	Normal
Serum TfR	High	Normal	Normal	Normal

* Typically MCV, MCH and MCHC are decreased in all hypochromic microcytic anemias.

** Hemosiderin and ferritin

# May be hypochromic or normochromic

Transferrin receptor (TfR): It is a transmembrane protein present on cell surfaces which is involved in iron absorption into the cell. It is upregulated in IDA. Transferrin-bound iron binds to and forms a complex with TfR , which is then internalized, iron is released and utilized for hemoglobin synthesis or stored. Serum levels of TfR increase in IDA whereas levels remain the same in AOCD.

RBC indices

MCV or mean corpuscular volume	Estimates RBC size
MCH or mean corpuscular hemoglobin	Estimates amount of Hb per RBC
MCHC or mean corpuscular hemoglobin concentration	Estimates amount of Hb per unit volume
RDW, related to anisocytosis	Estimates variability in RBC sizes; increased in anisocytosis
PCV or hematocrit	Compares RBC volume to plasma volume. Reduced in anemias

Schilling test

The Schilling test was used to differentiate between the various causes of megaloblastic anemia. The test is done in two stages and includes the oral and parenteral administration of radio-cobalt-labelled Vit B12 followed by monitoring the urinary excretion of the same. In stage 1, the patient is given oral radiolabelled Vit B12, followed 1 hour later by unlabelled intramuscular B12. Urine is tested for B12 levels. If results point towards B12 deficiency, then initial oral dose is repeated with B12 plus intrinsic factor in stage 2.

Interpretation of Schilling test results

Test Results	Interpretation
Normal stage 1#	Dietary deficiency, partial gastrectomy, malabsorption
Abnormal stage 1, Normal stage 2	Pernicious anemia, gastrectomy
Both stage 1 and 2 abnormal	Ileal resection,small bowel bacterial overgrowth syndrome, Crohn’s disease, renal insufficiency, D.latum infestation

# Adequate levels of radiolabeled B12 excreted in urine after oral and parenteral doses.

Common findings on peripheral smear

Finding	Significance
Red blood cells	Biconcave disks, smaller than small lymphocytes, central pallor equal to up to 45% of diameter, normocytic and normochromic
Macrocytes	Large RBCs, seen in macrocytic anemias, aplastic anemia, chronic liver disease
Microcytes	Small RBCs, seen in microcytic anemias, hemolytic anemia
Anisocytosis	Abnormal variability in RBC size
Poikilocytosis	Abnormal variability in RBC shape, seen in anemias, microangiopathic hemolytic anemia
Polychromatophilia/chromasia	Dense staining, bluish grey RBCs, due to increase in immature RBCs or reticulocytes
Spherocytes	Round, uniformly stained RBCs, no central pallor, seen in hereditary spherocytosis, AIHA, ABO hemolytic disease of newborn
Stomatocytes	Elliptical area of central pallor, appearing like a “stoma” or mouth; seen in hereditary stomatocytosis, chronic alcoholism
Target cells (codocytes)	RBC appears like “target”, due to increased RBC membrane; liver disease, IDA, thalassemia, splenectomy, Hb C disease
Sickle cells (drepanocytes)	Crescent or “sickle” shaped cells, seen in sickle cell anemia, Hb C-Harlem
Elliptocytes (ovalocytes)	Oval shaped RBCs; seen in hereditary elliptocytosis, megaloblastic anemia
Teardrop cells (dacrocytes)	RBCs shaped like “tear drops” with one rounded end and the other pointed end; marrow infiltration by fibrosis, malignancies, disorders of the spleen, megaloblastic anemia, thalassemia
Acanthocytes (spur cells)	Spiculated RBCs with multiple surface projections or spicules which are irregularly placed; liver disease, splenectomy
Echinocytes or burr cells	Spiculated RBCs with relatively evenly spaced and pointed spicules; uremia, pyruvate kinase deficiency, hemolytic anemias, hypomagnesemia, hypophosphatemia, burns, liver disease; may be an artifact
Bite cells (degmacytes) and Heinz bodies	Bite cells are RBCs with irregular membrane that results from removal of denatured Hb (Heinz bodies) from RBC cytoplasm by splenic macrophages; seen in G6PD deficiency
Schistocytes or helmet cells or horn cells	Fragmented RBCs, variable morphologies; TTP, HUS, artificial heart valves, burns, DIC, HELLP syndrome
Howell-Jolly bodies	RBCs with basophilic inclusions of DNA remnants; seen post-splenectomy, sickle cell anemia, megaloblastic anemia, MDS, hereditary spherocytosis, severe hemolytic anemia
Basophilic stippling or punctate basophilia	Multiple, coarse or fine blue cytoplasmic granules in RBCs; negative Perls’ reaction; due to aggregates of ribosomes or RNA; seen in lead poisoning, megaloblastic anemia, thalassemia, defective Hb, infections, liver disease
Pappenheimer bodies	RBCs with cytoplasmic granules of iron; stain with iron stains like Prussian blue; positive Perls’ reaction; typically seen in bone marrow; sideroblastic anemia, hemochromatosis,hemolytic anemia, MDS, lead poisoning, sickle cell disease,
Ringed sideroblasts	Erythroblasts with perinuclear ring of blue iron containing granules due to iron-loaded mitochondria; sideroblastic anemia, lead poisoning, MDS, pyridoxine deficiency, drugs like isoniazid, linezolid, cycloserine, copper deficiency, excess zinc,
Toxic granules	Large, prominent, basophilic granules in neutrophil cytoplasm seen in bacterial infections, aplastic anemia, inflammation, myelofibrosis
Dohle bodies	Blue inclusions in neutrophil cytoplasm; consist of ribosomes and endoplasmic reticulum; seen in bacterial infections, inflammation, burns, pregnancy, cyclophosphamide
Nucleated RBCs	Severe hemolysis, hemorrhage, hypoxia, myelofibrosis.
Pelger-Huet anomaly	WBCs with bilobed, “peanut” or “dumb-bell” ” shaped nuclei; hereditary, myeloblastic and proliferative disorders
Hypersegmented neutrophils	Neutrophils with six or more nuclear lobes; Vit B12 and folate deficiency, myeloblastic and proliferative disorders
Reactive lymphocytes	Larger than small lymphocytes, cytoplasmic vacuoles, dark blue cytoplasm, “kidney-bean” shaped or cleaved nuclei; viral infections like infectious mononucleosis, hepatitis, CMV, HIV etc. phenytoin.
Megathrombocytes	Large platelets, >3 micrometer in diameter; thrombocytopenia, DIC, myelofibrosis, megaloblastic anemia, Bernard Soulier disease
Microthrombocytes	Small platelets; seen in Wiskott Aldrich syndrome

Normal hemoglobins: Physiologically occurring hemoglobins are HbA (95-98%) composed of alpha 2 beta 2 chains; HbA2 (2-3%) composed of alpha 2 delta 2 and HbF (1% in adults; 60-80% in fetuses) composed of alpha 2 gamma 2.

Hemoglobin Constant Spring (Hb CS): It is an abnormal Hb caused by a mutation at the termination codon of α2-globin gene. This mutation leads to the synthesis of unstable and elongated α-globin chains with 172 instead of 141 amino acid residues. It is more common in Southeast Asian and Chinese people. Homozygosity of Hb CS could be associated with a thalassemia intermedia phenotype with mild anemia, jaundice and hepatosplenomegaly. It increases the risk of HbH disease.

Folate supplementation is needed in thalassemia due to increased demand. Iron supplementation, though, is contraindicated except if iron deficiency is also present concurrently.

Anemia in lead poisoning: Lead inhibits enzymes delta aminolevulinic acid dehydrogenase and ferrochelatase. It shows microcytic hypochromic anemia, basophilic stippling of RBCs, Pappenheimer bodies and ringed sideroblasts.

Clinical box: Blood group testing is complicated in the presence of AIHA. Blood group should be confirmed by using washed RBCs before typing, saline or LISS suspended RBCs, serum dilution, adsorption studies, chloroquine elution testing and RBC genotyping.

Direct and indirect antiglobulin tests

• Direct antiglobulin test or DAT or Coombs test is used to determine immunoglobulins and/or complement that are coating RBCs (mainly IgG and C3)
• Indirect antiglobulin or indirect Coombs test or IAT is used to determine anti-RBC antibodies in the serum
• In positive DAT, coated patient RBCs + Coomb’s reagent (antihuman globulin) = clumping
• Scaled from 0 to 4, with 0 being no clumping.
• DAT will be positive in AIHA, transfusion related hemolysis, hemolytic disease of the newborn, some cases of DIHA
• DAT will be negative in non-immune hemolysis, some cases of DIHA and hemolysis due to IgA or IgM.
• IAT is used in cross matching before blood transfusions and to detect Rh isoimmunization
• In positive IAT, patient serum + test RBCs, followed by adding Coomb’s reagent in the second step, results in clumping.
• Positive IAT is seen in sensitized Rh negative mother

Genes associated with Hereditary Spherocytosis

Gene	Role
ANK1	Ankyrin -1 gene; protein attaches to membrane proteins; role in maintaining structure, stability and flexibility of RBC
EPB42	Erythrocyte membrane protein band 4.2 , ATP binding protein, regulates association of protein 3 with ankyrin; Autosomal recessive; alternate splicing defects.
SLC4A1	Codes for anion exchanger 1 or AE1; transports anions across RBC membrane (Cl-/HCO3- exchanger)
SPTA1	Codes for spectrin; scaffold protein in RBC membrane; connects to actin
SPTB	Codes for spectrin; associated with spherocytosis type 2

Types of Sickle cell disease

Type	Characteristics
HbSS or SCA (sickle cell anemia)	Homozygous for HbS, also called sickle cell anemia, most severe
HbSC	Heterozygous, carry one HbS and other HbC, milder disease
HbS beta thalassemia	Heterozygous, carry one HbS and other beta thalassemia gene
HbSD, HbSE and HbSO	Double heterozygous, one HbS , other either abnormal Hb D,E or O.

Sickle cell trait or SCT: Heterozygotes that inherit one HbS and other normal Hb. They are asymptomatic, but their offspring may suffer from SCD. They may develop pain crises under conditions of increased atmospheric pressure such as scuba diving, low atmospheric oxygen at high altitudes, flying, mountain climbing, extreme exercise or dehydration. People with SCT are at increased risk of heat stroke, renal medullary carcinoma, chronic renal disease, thromboembolism, complications in pregnancy such as eclampsia, maternal infections, IUGR and fetal loss. It is diagnosed by sickle solubility testing (for screening; outdated; HbS is insoluble) while hemoglobin electrophoresis, high-performance liquid chromatography (HPLC), and isoelectric focusing (IEF) can be used as screening and confirmatory tests. DNA sequencing and genotyping for the single-nucleotide polymorphism (SNP) causing mutation are done for research purposes. Sodium metabisulphite can induce RBC sickling (positive sickling test) in SCT. Electrophoresis shows 35-40% of total Hb as HbS. 25. Pancytopenia: It is a disorder characterized by reduced RBC , platelet and WBC counts.

Causes of pancytopenia

Chemotherapy, radiation therapy, Fanconi anemia, aplastic anemia (multiple causes), myelodysplastic syndromes, hypersplenism, megaloblastic anemia, copper deficiency, zinc excess, myelofibrosis bone marrow infiltration from malignancy, leukemias and lymphomas, Gaucher’s disease, PNH, SLE, Shwachman-Diamond syndrome etc.

Fanconi Anemia

• Inherited bone marrow failure syndrome; AR or rarely X-linked recessive
• More common in persons of Ashkenazi Jewish descent, Roma population of Spain and black South Africans
• Mutations in genes involved in DNA repair such as FANCA, FANCC, FANCG which results in insufficient repair of DNA interstrand cross-links by FA core complex that normally activates repair proteins FANCD2 and FANC1
• Presents with aplastic anemia, fatigue, MDS, cafe-au-lait spots, short stature, skeletal anomalies like absent radius, missing or extra thumb/fingers, absent kidneys, eye and ear defects, heart defects, infertility, hydrocephalus, microcephaly, etc.
• Increased risk of AML, cancers of the head, neck, skin, GIT, GUT
• Rare AD form occurs from mutation in RAD51, as a de-novo mutation

Diamond-Blackfan Syndrome

• Presents with anemia, microcephaly, low frontal hairline, wide-set eyes, ptosis, flat bridge of nose, small lower jaw , low set ears, cleft lip, webbed neck, short stature, absent thumbs, cataracts, glaucoma, strabismus, renal and heart defects, hypospadias
• Caused by mutations in genes coding for ribosomal proteins including RPS19, RPL5, RPL11, RPL35A, RPS10, RPS17, GATA1 etc.
• Inherited as AD or de-novo mutations; rarely X-linked
• Defective regulation of cell division; abnormal enhanced apoptosis; anemia is from reduced RBC count
• Increased risk of MDS, AML, osteosarcoma, adrenal insufficiency, hypogonadism, hypothyroidism
• Curative treatment is bone marrow transplantation

Left shift of neutrophils: The presence of immature neutrophils like band forms, myelocytes, metamyelocytes and promyelocytes in the peripheral smear is called “left shift” of neutrophils. Leukocyte alkaline phosphatase will increase in a “left shift”. It is the effect of cytokines IL1 and TNF which accelerate the release of WBCs from the bone marrow pool. It is seen in bacterial infections.

Von Willebrand Factor or vWF: It is a glycoprotein that has binding domains for FVIII, platelets and connective tissue. It protects FVIII from degradation by activated protein C and helps in clot formation. Thrombin releases activated FVIII from vWF. vWF is synthesized by endothelial cells and megakaryocytes, and stored as Weibel-Palade bodies in endothelial cells. It is also present in alpha granules of platelets. Histamine, oestrogens, thrombin and fibrin regulate the release of vWF from endothelial cells into the circulation. vWF can be released from platelets by ADP, collagen and thrombin. It helps platelets adhere to vascular endothelium. vWF binds to platelets at platelet receptor GPIb.

Ristocetin cofactor assay: It is used to assess the activity of vWF. It is based on the principle that ristocetin facilitates binding of platelet GPIb to vWF. Patient’s plasma is added to a suspension of washed platelets and ristocetin. The rate of platelet aggregation is measured. Normal values are between 50-200 IU/dl. VWD patients typically have levels much lower than that.

RIPA or ristocetin-induced platelet aggregation is similar to the ristocetin cofactor assay with the exception of using patient’s platelets. A positive test results in platelet aggregation. Negative test means that there is a defect in vWF and/or platelet GPIb. Hyperactive agglutination in RIPA but absence of agglutination in ristocetin cofactor assay is seen in type 2B and pseudo-vWD. Negative RIPA and presence of aggregation on ristocetin cofactor assay is seen in Bernard-Soulier disease.

Hemophilia presents more commonly with joint bleeding and vWD with mucocutaneous bleeding.

Cryoprecipitate: It is derived from plasma and it contains clotting factors such as fibrinogen, Factor VIII, Factor XIII, and vWF. A single unit of cryoprecipitate typically has a volume between 10 to 15 ml. It has a higher concentration of clotting factors than FFP. It may transmit blood-borne pathogens as it is pooled from multiple donors.

Fresh frozen plasma or FFP: It is derived from whole blood or plasma. Each unit of FFP is derived from a single donor and has a volume of 250-300 ml. It is composed of clotting factors. Each unit increases clotting factor level by 3%. FFP does not contain platelets. It should be of the same ABO type as the recipient. Adverse effects include allergic reactions, anaphylaxis, transfusion related lung injury, hemolysis, blood-borne infections (rare) and fluid overload. It is indicated in end-stage liver disease, DIC, multiple factor deficiencies, vit K deficiency, TTP, HUS, acute blood loss and for immediate reversal of warfarin therapy.

Red blood cells used in transfusion

Packed red cells

• Concentrated RBCs
• Each unit is 200ml and increases hematocrit by 3% and Hb by 1gm/dl

Washed red cells

• RBCs are washed with saline
• Each unit is 180 ml

Irradiated red cells

• RBCs are gamma irradiated to kill lymphocytes
• Prevents development of GVHD in susceptible patients

Leukoreduced red cells

• Packed RBCs with most WBCs removed
• Decrease risk of febrile reactions and blood-borne viral pathogens like EBV, CMV

Each unit of platelet transfusion increases the platelet count by 5-8000/mm3 in adults. 36.

Primary thrombotic microangiopathy (TMA) syndromes

• TTP
• ST-HUS
• Complement-mediated or atypical HUS
• Drug induced (gemcitabine, quinine, ticlopidine, clopidogrel)
• Metabolic in homozygous mutations in MMACHC (involved in Vit B12 metabolism)
• Coagulation mediated homozygous mutations in DGKE, PLG (plasminogen gene) and THBD

Infections, cancers, vitamin deficiencies and autoimmune disorders can present with similar symptoms as TMA.

Diseases associated with changes in platelet size

Small platelets

• Wiskott-Aldrich syndrome
• X-linked thrombocytopenia

Large platelets

• Bernard Soulier syndrome
• May-Hegglin syndrome
• Di-George syndrome

FAB (French-American-British) classification of AML

Class	Features
M0 Undifferentiated	Peroxidase and Sudan black negative; CD34+, TdT negative; resistant to chemotherapy, associated with trisomy 13
M1 AML without maturation	Granular staining with Sudan black B; <10% cells are mature beyond promyelocyte stage;
M2 AML with maturation	More mature than M1, promyelocytes, myelocytes seen; more granular cells; prominent Auer rods; associated with t(8;21) resulting in AML-ETO fusion gene whose product can be detected by RT-PCR; better prognosis
M3 Acute promyelocytic leukemia	Promyelocytes show heavily granulated cytoplasm with azurophilic granules; Multiple Auer rods seen (Faggot cells); t(15;17); Sudan black B and MPO +; CD33+; HLA DR negative; associated with DIC, ICH
M4 Acute myelomonocytic leukemia (Naegeli type)	Mix of myeloid and monocytic elements; >20% of leukemic cells in bone marrow are monocytic; extramedullary sites may be involved; hyperleukocytosis, dysplastic eosinophils may be seen; associated with inversion of Chr 16; hypokalemia may occur
M5 Acute monocytic leukemia (Schilling type)	>80% bone marrow blasts are myelocytes; folded nuclei may be seen; more common in older adults; extramedullary involvement like gum hypertrophy, infiltration of the skin, GIT, CNS, eyes etc. ; high lysozyme levels, hypokalemia; hyperleukocytosis; erythrophagocytosis may occur; less responsive to chemotherapy
M6 Acute erythroleukemia (Di Guglielmo’s syndrome)	Myeloblasts plus erythroid precursor changes like increased number of megaloblasts, karyorrhexis, increased mitosis, ringed sideroblasts; antibodies to glycophorin A seen; PAS +; less responsive to chemotherapy
M7 Acute megakaryocytic leukemia	Dysplastic megakaryocytes with cytoplasmic budding; thrombocytosis; PAS and acid phosphatase positive; Sudan black B, MPO, esterase negative; platelet antigens positive like CD41 OR GpIIB/IIIa; platelet peroxidase +

Retinoic acid role in treatment of M3 promyelocytic leukemia (PML): Translocation t(15;17) is seen in PML. It affects the function of RAR alpha or retinoic acid receptor alpha gene on chromosome 17 and the function of PML gene on chromosome 15. This translocation results in the formation of a fusion gene that blocks transcription of genes controlled by RAR alpha. Administration of all-trans retinoic acid removes the block in transcription which helps the blast cell to differentiate into mature cells and induces remission of leukemia. Anthracyclines are added to the regimen for curative therapy.

FAB classification of ALL*

Class	Features
L1	Small cells with scant basophilic cytoplasm, small nucleoli and homogenous chromatin
L2	Large cells with moderate cytoplasm, large nucleoli, nuclear clefts, large nucleoli
L3**	Medium to large cells, moderate cytoplasm, stippled chromatin, prominent cytoplasmic vacuoles

* Not clinically relevant nowadays

**L3 is now Burkitt’s lymphoma

WHO classification of ALL: Most ALL arise from B lymphocytes while about 15% arise from T lymphocytes. Accordingly, ALL is classified into three groups - Precursor B cell not otherwise specified; Precursor B cell with recurrent cytogenetic abnormalities such as t(9;22), hyperdiploidy, hypodiploidy, t(12;21) etc; and Precursor T cell class also called T lymphoblastic leukemia/lymphoma. WHO classification of ALL is relevant for accurate diagnosis and treatment and is done on the basis of immunophenotyping.

Immunophenotyping of B-cell lineage ALL: TdT+, CD19+, CD10, surface and cytoplasmic immunoglobulin negative is early - cell-precursor ALL. TdT+, CD19+, CD10+, cytoplasmic Ig + but surface Ig negative is Pre-B cell ALL while TdT negative, CD19+, CD10+, cytoplasmic and surface Ig + cells are seen in B-cell ALL.

Immunophenotyping of T-cell lineage ALL: TdT+, surface CD3 negative, CD4+ and CD8 negative is seen in early or precursor-T cell ALL. TdT+, surface CD3+, CD4 and CD8 variable is seen in T-cell ALL.

Reed-Sternberg cells: They are malignant, CD 15+, CD 30+ B cells classically seen in Hodgkin’s lymphoma. They show inhibition of transcription factor NF-kB and hence, escape apoptosis. They are large cells, with bilobar or multilobar nuclei (owl eye nucleus) with perinuclear halo and abundant slightly basophilic cytoplasm. Mononuclear variants of Reed-Sternberg cells are called Hodgkin cells.

B symptoms: They are systemic symptoms of lymphomas and include fever, drenching night sweats and weight loss. It is a bad prognostic marker and indicates distant spread of the malignancy.